Synthesis of ester prodrugs of 9-(S)-[3-Hydroxy-2-(phosphonomethoxy)propyl] -2,6-diaminopurine (HPMPDAP) as anti-poxvirus agents

Article abstract of DOI:10.1021/jm901828c

9-(S)-[3-Hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine (HPMPDAP) and its cyclic form were selected for further evaluation as potential drug candidates against poxvirus infections. To increase bioavailability of these compounds, synthesis of their structurally diverse ester prodrugs was carried out: alkoxyalkyl (hexadecyloxypropyl, octadecyloxyethyl, hexadecyloxyethyl), pivaloyloxymethyl (POM), 2,2,2-trifluoroethyl, butylsalicylyl, and prodrugs based on peptidomimetics. Most HPMPDAP prodrugs were synthesized in the form of monoesters as well as the corresponding cyclic phosphonate esters. The activity was evaluated not only against vaccinia virus but also against different herpes viruses. The most potent and active prodrugs against vaccinia virus were the alkoxyalkyl ester derivatives of HPMPDAP, with 50% effective concentrations 400-600-fold lower than those of the parent compound. Prodrugs based on peptidomimetics, the 2,2,2-trifluoroethyl, the POM, and the butylsalicylyl derivatives, were able to inhibit vaccinia virus replication at 50% effective concentrations that were equivalent or ～10-fold lower than those observed for the parent compounds.

Full text of DOI:10.1021/jm901828c

J. Med. Chem. 2010, 53, 6825–6837 6825
DOI: 10.1021/jm901828c
Synthesis of Ester Prodrugs of 9-(S)-[3-Hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine
(HPMPDAP) as Anti-Poxvirus Agents
‡
†
Marcela Krecmerova,*,† Antonı
ꢁ †
ꢀ
Milena Masojıdkova, Martin Dracı
´ ´
´
n Holy, Graciela Andrei, Karel Pomeisl, Tomas Tichy, Petra Brehova,
ꢀ
ꢁ
ꢁ †
ꢁꢀ
ꢁ †
nsky, Radek Pohl, Genevieve Laflamme, Lieve Naesens, Hon Hui, Tomas Cihlar,
Johan Neyts,‡ Erik De Clercq,‡ Jan Balzarini,‡ and Robert Snoeck‡
ꢀ
ꢁ †
†
§
‡
§
§
ꢁ †
†Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Gilead Sciences and IOCB Research Centre,
‡
Flemingovo nam. 2, CZ-166 10, Prague 6, Czech Republic, Rega Institute for Medical Research, Katholieke Universiteit Leuven,
ꢁ
Minderbroedersstraat 10, B-3000 Leuven, Belgium, and §Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, California 94404
Received December 11, 2009
9-(S)-[3-Hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine (HPMPDAP) and its cyclic form
were selected for further evaluation as potential drug candidates against poxvirus infections. To increase
bioavailability of these compounds, synthesis of their structurally diverse ester prodrugs was carried out:
alkoxyalkyl (hexadecyloxypropyl, octadecyloxyethyl, hexadecyloxyethyl), pivaloyloxymethyl (POM),
2,2,2-trifluoroethyl, butylsalicylyl, and prodrugs based on peptidomimetics. Most HPMPDAP prodrugs
were synthesized in the form of monoesters as well as the corresponding cyclic phosphonate esters. The
activity was evaluated not only against vaccinia virus but also against different herpes viruses. The most
potent and active prodrugs against vaccinia virus were the alkoxyalkyl ester derivatives of HPMPDAP,
with 50% effective concentrations 400-600-fold lower than those of the parent compound. Prodrugs
based on peptidomimetics, the 2,2,2-trifluoroethyl, the POM, and the butylsalicylyl derivatives, were able
to inhibit vaccinia virus replication at 50% effective concentrations that were equivalent or∼10-fold lower
than those observed for the parent compounds.
Introduction
disease eradicated. The last naturally occurring outbreak of
smallpox was in Somalia in 1977. After the disease was
eliminated from the world, routine vaccination against small-
pox among the general public was stopped because it was no
longer necessary for prevention. By 1983, all known stocks of
VARV were kept in two WHO collaborating centers: the U.S.
Center for Disease Control and Prevention (CDC) in Atlanta
and the Russian State Research Center of Virology and
Biotechnology in Novosibirsk. However, in the aftermath of
the events of September 2001, there is increasing concern that
undeclared stocks of VARV might exist and that they might
be used as a bioterrorist weapon. The discontinuation of
routine vaccination against smallpox has rendered the human
populationmoresusceptible tothisdisease. Moreover, VARV
is highly stable and retains its infectivity for relatively long
periods of time outside the host. For all of these reasons,
special attention has been paid to precautions for dealing with
a smallpox outbreak. Onthe other hand, VACVvaccinationis
supposed to cause several complications, especially among
immunocompromised patients. Therefore, there are increased
demands for anti-poxvirus effective drugs to treat acute
infections. Despite of progressive investigation in this area,
and the number of compounds in preclinical and/or early
clinical stage of development, no FDA approved drugs are
currently available on the market.2,3 Only three drugs have
been used in the clinic for the treatment of complications
associated with vaccination against smallpox.4 These include
the acyclic nucleoside analogue cidofovir and its oral prodrug,
hexadecyloxypropyl ester (HDP-CDV), which inhibit viral
DNA replication and compound ST-246, an orally bioavail-
able compound that targets orthopoxvirus morphogenesis.5
The family of Poxviridae represents a group of enveloped,
double-stranded DNA viruses with unique morphology and
cytoplasmic site of replication.1 They are very large (about
300ꢀ 200nm) andbrick-shaped. Poxvirus-associateddiseases
are a serious problem for human health as well as veteri-
nary medicine. The most dangerous human pathogen of this
group is a member of the Orthopoxvirus genus: variola virus
(VARV),a the causative agent of smallpox. Smallpox was one
of the main causes of morbidity and mortality until recent
times. In the 20th century alone, smallpox deaths worldwide
numbered in the millions. In 1980, after an intensive program
of immunization with a related but relatively nonpathogenic
virus, i.e., vaccinia virus (VACV), the WHO declared the
*Corresponding author: phone, þ420 220183475; fax, þ420 220183560;
e-mail, marcela@uochb.cas.cz.
aAbbreviations: ANP, acyclic nucleoside phosphonate; bis(POC)-
PMPA, bis(isopropyloxycarbonyloxymethyl) ester of 9-(R)-[2-(phos-
phonomethoxy)propyl]adenine; bis(POM)-PMEA, bis(pivaloyloxymethyl)
ester of 9-[2-(phosphonomethoxy)ethyl]adenine; DCC, N,N0-dicyclohexyl-
carbodiimide; HATU, O-7-(azabenzotriazol-1-yl)-N,N,N0,N0-tetramethy-
luronium hexafluorophosphate; HCMV, human cytomegalovirus; HDP-
CDV, hexadecyloxypropyl ester of cidofovir; HOBt, 1-hydroxybenzotria-
zole; HPMPA, 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine;
HPMPC, 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine
(cidofovir); HPMPDAP, 9-(S)-[3-hydroxy-2-(phosphonomethoxy)-
propyl]-2,6-diaminopurine; HSV-1, herpes simplex virus type 1;
HSV-2, herpes simplex virus type 2; iPr, 2-propyl; POM, pivaloyloxy-
methyl; PyBOP, (benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate; PyBroP, bromotripyrrolidinophosphonium
hexafluorophosphate; TEA, triethylammonium; TEAB, triethylam-
monium hydrogen carbonate; VACV, vaccinia virus; VARV, variola
virus.
r
2010 American Chemical Society
Published on Web 09/01/2010
pubs.acs.org/jmc

6826 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Krecꢀmerovaꢁ et al.
Scheme 1a
aConditions: (a) cesium carbonate, DMF, 100 °C; (b) dimethylformamide dimethylacetal; (c) BrCH2P(O)(OiPr)2, NaH, DMF, room temperature;
(d) NH4OH (H2O-MeOH); (e) 80% AcOH, reflux; (f) (CH3)3SiBr, CH3CN, room temperature.
Until recently, the search for small molecule compounds
with anti-orthopoxvirus activity was focused on the area
of nucleoside analogues using VACV as a model system for
testing. However, new classes of molecules able to selectively
inhibit orthopoxvirus replication have been recently described.6
Several nucleoside analogues have been shown to inhibit
VACV replication in vitro, and according to their mechanism
of action they can be divided into several categories: S-adeno-
sylhomocysteine hydrolase inhibitors, inosine monophosphate
dehydrogenase inhibitors (ribavirin), orotidine 50-monopho-
sphate decarboxylase inhibitors, cytidine triphosphate synthe-
tase inhibitors, thymidylate synthase inhibitors, nucleoside
analogues, and acyclic nucleoside phosphonates targeted at
viral DNA synthesis.7-9
One of the most promising groups of potent and selective
anti-poxvirus agents is acyclic nucleoside phosphonates
(ANPs) of the HPMP series,10 i.e., (S)-[3-hydroxy-2-(phos-
phonomethoxypropyl)] derivatives of diverse nucleobases:
cytosine (HPMPC), 5-azacytosine (HPMP-5azaC),11 adenine
(HPMPA), 2,6-diaminopurine (HPMPDAP),12 7-deazaade-
nine (HPMP-7-deazaA), 3-deazaadenine (HPMP-3-deazaA),
and 8-azaadenine (HPMP-8-azaA).13 In a series of “open-
ring” analogues (also called the second generation of ANPs),
a remarkable anti-poxvirus activity has been described for
(R)-{2,4-diamino-3-hydroxy-6-[2-(phosphonomethoxy)-
propoxy]}pyrimidine, (R)-HPMPO-DAPy.14 Cidofovir, licensed
for the treatment of cytomegalovirus retinitis in AIDS patients,
is the only approved acyclic nucleoside analogue with anti-
poxvirus activity. Currently, it is recommended by the U.S.
Centers for Disease Control and Prevention for the treatment
of severe adverse effects following smallpox vaccination.15,16
However, the useof cidofovir is limited partly by renal toxicity
and partly by its poor oral bioavailability, implying it can only
be administered by intravenous injections. To improve oral
bioavailability of ANPs, there is a tendency for their trans-
formation to lipophilic ester prodrugs. In case of cidofovir,
so far the most successful drug candidate blocking smallpox
virus reproduction is its hexadecyloxypropyl ester (HDP-
cidofovir).17-20 Other ANPs have been subjected to esterifica-
tion with alkoxyalkyl groups such as HDP or ODE. In the case
of HPMPDAP, the ODE derivative has been shown to be a
potent and selective inhibitor of herpes virus and orthopoxvirus
replication.21 In spite of that, the necessity of search for other
drug candidates remains still timely.
In our approach, several ANPs of the (S)-HPMP series
(HPMPA, HPMPC, HPMP-azaC, 3- and 7-deaza-HPMPA,
HPMPDAP), (R)-HPMPO-DAPy, and the corresponding
cyclic phosphonates were subjected to detailed in vitro and in
vivo investigations. The selected drug candidate had to fulfill
the following requirements: high inhibitory activity against
poxviruses including drug-resistant virus mutants, low cyto-
toxicity, low potential for in vivo nephrotoxicity, and
sufficient metabolic stability. Based on these criteria, (S)-
HPMPDAP and its cyclic form were selected for further
evaluation22 including the synthesis and characterization of
their structurally diverse ester prodrugs. These esters were
subjected to a comparative study concerning their antiviral
activity and selectivity.
Chemistry
The originally described synthesis of 9-(S)-[3-hydroxy-2-
(phosphonomethoxy)propyl]-2,6-diaminopurine (HPMPDAP, 1)
is an eight-step process based on nucleophilic opening of an
oxirane ring in (R)-glycidol butyrate with 2,6-diaminopurine
and introduction of a phosphonomethyl residue using diisopro-
pyl tosyloxymethylphosphonate and is accompanied by several
protection-deprotection steps with an overall yield of 11% of
the final (S)-HPMPDAP (calculated to starting 2,6-diamino-
purine).12 For our purpose of further development of the
compound including prodrugs for animal experiments, first of
all we needed to optimize the synthesis of (S)-HPMPDAP.
The new approach is described in Scheme 1. At present,
synthetic approaches started by nucleophilic opening of (2S)-
2-[(trityloxy)methyl]oxirane (2) with nucleobases belong to
the most utilized methodology in preparations of (S)-HPMP
derivatives. In this case, the overall yield of 1 was 40%
(calculated to starting 2,6-diaminopurine); most reaction
steps can be performed without isolation and purification
of intermediates (intermediates 4-6). The method is appro-
priate especially for large-scale syntheses.
For an introduction of alkoxyalkyl groups, the starting 1
was transformed to its cyclic form 7 prepared from 1 in
quantitative yield by its heating with N,N0-dicyclohexylcarbo-
diimide and N,N0-dicyclohexyl-4-morpholinecarboxamidine

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6827
in DMF. Alkylation of 7 with alkoxyalkyl bromides was
performed via its tetrabutylammonium salt; the resulting
alkoxyalkyl esters of cHPMPDAP 8 were used directly for
anti-poxvirus screening or cleaved to the corresponding
monoesters 9 (Scheme 2).
was performed by HPLC technique. Diastereoisomers were
distinguished by their characteristic 31P chemical shifts and by
comparing H,H, H,P, and C30,P coupling constants in chair
conformation of the cyclic phosphonate esters that have
characteristic values for each diastereoisomer. Determination
of the relative configuration of cyclic phosphonate esters from
NMR spectra (H,H-ROESY or comparison of 31P (1H dec)
NMR spectra) is explained in detail in ref 25. Generally, 31P
nucleus in less polar trans-diastereoisomers resonates at high-
er magnetic field than in polar minor cis-diastereoisomers.
Structures of both diastereoisomers are outlined in Figure 1.
Structures of other diastereoisomeric cyclic phosphonates
(8a-c) are analogous. Concerning absolute configuration,
all trans-isomers have (S,R) configuration, cis-isomers have
(S,S) configuration.
Another type of appropriate prodrug form for phosphonic
acid based compounds is 2,2,2-trifluoroethyl esters. These
esters are successfully used for example in development
of effective anti-HBV agents, 2-amino-6-arylthio-9-[2-(phos-
phonomethoxy)ethyl]purines.26,27 These phosphonates were
transformed to the corresponding bis(2,2,2-trifluoroethyl)
esters which were then partially hydrolyzed to the correspond-
ing monoesters in plasma and liver. In our case of HPMPDAP,
we made an effort to synthesize all three possible types of
trifluoroethyl ester prodrugs: bis(2,2,2-trifluoroethyl) ester, a
corresponding monoester, and 2,2,2-trifluoroethyl ester of
cyclic HPMPDAP. Esterification of (S)-HPMPDAP and its
cyclic form was performed with trifluoroethanol in the pre-
sence of ethyldiisopropylamine (Hunig’s base) after acti-
vation of a phosphonic acid residue with some hexafluoro-
phosphate condensation reagents. Such reagents, e.g., HATU,
PyBOP, or PyBroP [bromotripyrrolidinophosphonium hexa-
fluorophosphate], are currently used especially as coup-
ling reagents in peptide chemistry.28 Transformation of (S)-
HPMPDAP and its cyclic form to the corresponding trifluo-
roethyl esters is outlined in Scheme 4. Esterification of 1 using
PyBroP as coupling reagent afforded exclusively 2,2,2-trifluo-
roethyl monoester 12; no formation of bis(trifluoroethyl) ester
or ester of cyclic phosphonate was observed. Analogously,
esterification of cyclic form 7 was performed successfully with
trifluoroethanol after activation of a phosphonic acid residue
with HATU, i.e., O-7-(azabenzotriazol-1-yl)-N,N,N0,N0-tetra-
methyluronium hexafluorophosphate. The resulting cyclic
phosphonate 13 was isolated and tested as a diastereoisomeric
mixture. We also tried to find some alternative approaches to
13, but these experiments were mostly unsuccessful (e.g.,
reaction of tetrabutylammonium salt of 7 with 2,2,2-trifluoro-
ethyl iodide under conditions described for 8 or transformation
of 7to corresponding chloridate by the action of oxalyl chloride
followed by reaction with trifluoroethanol).
With respect to a generally high bioavailability and
optimal pharmacokinetic properties, neutral diesters of
HPMPDAP, e.g., bis(POM) derivatives, are expected to
be optimal prodrugs. However, as we found, transforma-
tion to such type of esters is appropriate only for phospho-
nates lacking a free hydroxyl group in an aliphatic side
chain, e.g., PMEA, which is transformed easily to bis-
(POM)PMEA23 or (R)-PMPA (tenofovir) clinically used
in the form of its bis(isopropyloxycarbonyloxymethyl) ester
(bis(POC)-PMPA).24 Unfortunately, this procedure is not
possible to use in HPMP series where phosphonic acid
diesters are not sufficiently chemically stable due to the
presence of a neighboring hydroxyl function.
In the case of (S)-HPMPDAP, we successfully synthesized
two types of POM esters: POM monoester 10 and POM ester
of cyclic (S)-HPMPDAP 11 (Scheme 3). Transformation of 1
to the corresponding POM monoester was performed by the
action of chloromethyl pivalate in DMF in the presence of N,
N0-dicyclohexyl-4-morpholinecarboxamidine. Reaction con-
ditions were optimized so as no cyclization or formation of
decomposition products occurred. This procedure was found
to be useful also for preparation of other POM HPMP
monoesters (e.g., HPMPC).
Transformation of cyclic (S)-HPMPDAP to POM ester 11
was carried out by the treatment of its tetrabutylammonium
salt with chloromethyl pivalate in refluxing dioxane.
The ratio of diastereoisomers in 11 was 6:1 in favor of
the less polar trans-isomer; separation of diastereoisomers
Scheme 2a
aConditions: (a) DCC, N,N-dicyclohexyl-4-morpholinecarboxami-
dine, DMF, 100 °C; (b) tetrabutylammonium hydroxide; (c) alkoxyalkyl
bromide, DMF, 100-120 °C; (d) 2 M NaOH, 80 °C, 1 h.
Scheme 3a
aConditions: (a) chloromethyl pivalate, N,N-dicyclohexyl-4-morpholinecarboxamidine, DMF, room temperature, 24 h; (b) tetrabutylammonium
hydroxide; (c) chloromethyl pivalate, dioxane, reflux 3 h.

6828 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Krecꢀmerovaꢁ et al.
Figure 1
Scheme 4a
aConditions: (a) PyBroP, DMF, ethyldiisopropylamine, room temperature; (b) CF3CH2OH, room temperature f 90 °C in 3 h; (c) HATU,
ethyldiisopropylamine, room temperature; (d) CF3CH2OH, room temperature f 90 °C, 3 h.
Scheme 5a
Scheme 6a
aConditions: (a) (CH3)3SiBr, CH3CN, room temperature; (b) oxalyl
chloride, dichloromethane (þDMF); (c) CF3CH2OH, dichloromethane,
Et3N.
Some attempts to bis(trifluoroethyl) esters were also stu-
died. Regarding the fact that direct esterification of 1 with
trifluoroethanol afforded monoester 12 only, we decided to
work out preparation of bis(trifluoroethyl) tosyloxymethyl-
phosphonate (15) as alkylating reagent for direct phosphony-
lation of 3 or 4, respectively. So far, it revealed that such
reactions would require special (milder) conditions compared
to an analogous process with diisopropyl tosyloxymethylpho-
sphonate (14) often used for preparation of ANPs. Such
reactions are usually carried out in strongly alkaline condi-
tions12 (sodium hydride), generally not appropriate for fluori-
nated compounds. Utilization of synthon 15 for preparations
of bis(trifluoroethyl) esters is currently intensively studied.
Its synthesis from diisopropyl tosyloxymethylphosphonate is
described in Scheme 5.
aConditions: (a) PyBOP, DMF, ethyldiisopropylamine, sonication,
room temperature;(b) butyl salicylate, room temperature in 6 days, soni-
cation; (c) [CH(Cl)dNþ(Me)2]Cl-, DMF; room temperature f 100 °C;
(d) sodium salt of butyl salicylate, room temperature f 100 °C.
stereochemistry on a phosphorus atom: trans (equatorial)
isomers displayed poor chemical stability; for further bio-
logical evaluation cis-isomers were preferable. As to oral bio-
availability, the best results were found in the case of the
butylsalicylyl ester.32 This type of salicylyl ester was also selec-
ted for preparation of another structural type of HPMPDAP
prodrug, compound 16. Unfortunately, synthesis of 16 ac-
cording to an original procedure described for cidofovir31
(reaction of free HPMP derivative with Vilsmeier reagent
followed by the action of sodium salt of butyl salicylate)
repeatedly failed. As expected, activation and subsequent
cyclization of a phosphonate moiety into the intermediate
17 otherwise successfully proceeded (Scheme 6). However,
reactivity of thus formed phosphonate with salicylate sodium
salt was probably insufficient as observed by TLC monitoring
Another type of phosphonate ester prodrugs is aryl esters:
phenyl and its substituted analogues. In a study in PME series
(adefovir, anti-hepatitis B agent), these esters [e.g., bis(2-
ethoxyphenyl)PMEA] revealed very good oral bioavailability
together with sufficient stability in intestinal homogenate and
plasma and enzymatic degradationtothe parentcompoundin
liver homogenate.29,30 In the HPMP series aryl esters were
developed for the cyclic form of HPMPC (cidofovir). Besides
simple phenyl ester and its substituted analogues a large series
of alkyl salicylate esters was also developed as a very promis-
ing group of prodrugs with a high bioavailability.31,32 Varia-
tions of alkyls on a carboxylic function allow resulting
modification of lipophilicity, solubility, and enzymatic stabi-
lity of the prodrug. Physicochemical properties and stability
of salicylates of cyclic HPMPC are strongly dependent on

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6829
Scheme 7a
of the reaction mixture. Therefore, we developed an alter-
native procedure using PyBOP as a more suitable mild
coupling agent (see Scheme 6). In the presence of Hunig’s
baseandbutyl salicylate, the phosphonate1 primarilycyclized
into 7. After a few days of stirring at room temperature and
sonication of the reaction mixture, a small quantity of the
favored cyclic butyl salicylyl ester 16 was finally observed and
separated while the unreacted cyclic HPMPDAP (7) could be
recovered for further utilization.
Special types of prodrugs represent esters of amino acids,
peptides, and peptidomimetics. Their development was in-
itiated with the aim to find prodrugs releasing in target tissue
an active compound together with a completely nontoxic and
natural accompanying material as byproduct. One of the first
examples of such approach was the development of valacy-
clovir, an L-valine ester of acyclovir.33,34 Development of
orally active peptide drugs is limited by their unfavorable
physicochemical properties (size, charge, hydrogen bond
formation) preventing them from permeation through a cell
membrane as well as their instability toward enzymes present
in intestinal mucosa. However, appropriate structural mod-
ificationsofpeptides toformpeptidomimetics openthe way to
circumvent the metabolic enzymes. Modifications increasing
the hydrophobicity but simultaneously decreasing the hydro-
gen-bonding potential are promising strategies for improving
the oral bioavailability of peptide drugs.35 In the field of
ANPs, successful utilization of peptidomimetics was de-
scribed by McKenna’s group for design of prodrugs of cyclic
cidofovir. The original strategy, based on ethylene glycol-
linked amino acid conjugates, was replaced later by esterifica-
tion of cCDV with a side-chain hydroxyl group of some
serine-containing dipeptides.36,37 Promising pharmacokinetic
properties were found especially in the case of Val-Ser dipep-
tide with a valine carboxyl function esterified by an iso-
propyl group: Val-Ser-COOiPr cHPMPC.36 Therefore, this
peptidomimetic group was selected finally for esterification
of HPMPDAP to prepare additional types of HPMPDAP
prodrugs for anti-poxvirus screening. The synthesis was per-
formed employing above-described conditions using PyBOP,
Hunig’s base, and protected Val-Ser block possessing a free
hydroxyl group. The dipeptide moiety was prepared from
Boc-L-valine and L-serine isopropyl ester hydrochloride using
carbodiimide-mediated coupling. The Boc group of the con-
jugate 19 was then cleaved by the action of hydrogen chloride
in dioxane (Scheme 7).
a Conditions: (a) EDC, HOBt, Et3N, CH2Cl2, room temperature;
(b) PyBOP, iPr2EtN, DMF, room temperature in 2 days, sonication;
(c) HCl/dioxane, room temperature.
observedfor(S)-HPMPC(cidofovir), whilethey didnot affect
cell morphology or growth of HEL cells measured as res-
pectively the minimum cytotoxic concentration (MCC) or the
50% cytostatic concentration (CC50), up to a concentration
of >314 μM ((S)-HPMPDAP) or >333 μM (cyclic-(S)-
HPMPDAP), the highest concentrations tested (Table 1). In
the case of HSV-1, HSV-1 ACVr, HSV-2, and VZV, EC50
values for (S)-HPMPC, (S)-HPMPDAP, and its cyclic form
were similar (Table 2). In contrast, (S)-HPMPC was more
inhibitory toward HCMV than (S)-HPMPDAP or its cyclic
form (Table 1).
The ester prodrugs of (S)-HPMPDAP, i.e., hexadecylox-
ypropyl (compound 9a), octadecyloxyethyl (compound 9b),
and hexadecyloxyethyl (compound 9c) derivatives, emerged
as the most potent and selective compounds against VACV
with EC50 values consistently in the range of 0.00074-0.0012
μM (Table 1). Althoughthe ester prodrugs of (S)-HPMPDAP
proved more toxic than the parent compound, either for HEL
cell morphology or HEL cell growth (except for compound 9a
that had a CC50 g161 μM), they were more selective than (S)-
HPMPDAP, with selectivity indices (ratio = CC50/EC50)
higher than 10000 compared to >625 for (S)-HPMPDAP.
The ester prodrugs of the cyclic form of (S)-HPMPDAP [i.e.,
compounds 8a, 8b (cis and trans), and 8c (cis and trans)]
inhibited VACV replication with EC50 values that were 10-
fold (compound 8b-trans), 48-fold (compound 8a), 100-fold
(compound 8b-cis), and 100-200-fold(compound8c-cis and -
trans) lower than that of cyclic (S)-HPMPDAP (Table 1). The
EC50’s obtained for the alkoxyalkyl ester prodrugs of cyclic
(S)-HPMPDAP were about 3-70-fold higher against vacci-
nia virus than the corresponding ester prodrugs of (S)-
HPMPDAP, and they had selectivity indices of 1200-8000.
In a previous study, Valieaeva and collaborators reported
the antiviral activity of different ODE esters of (S)-3-hydroxy-
2-(phosphonomethoxy)propyl nucleosides against herpes
Biological Activity
The antiviral activity of the different compounds was
evaluated against various DNA viruses, including poxviruses
[i.e., vaccinia virus (VACV)] and herpes viruses [i.e., herpes
simplex virus type 1 (HSV-1) and type 2 (HSV-2), thymidine
kinase-deficient HSV-1 (acyclovir-resistant, ACVr), varicella-
zoster virus (VZV), human cytomegalovirus (HCMV), and
human herpes virus 6 (HHV-6)] (Tables 1 and 2). Some of the
compounds were also evaluated against retroviruses [i.e.,
human immunodeficiency virus type 1 (HIV-1) and type 2
(HIV-2)]. All compounds were also examined against several
RNA viruses, including vesicular stomatitis virus, Coxsackie
B4, respiratory syncytial virus (RSV), parainfluenza virus
type 3, reovirus-1, Sindbis virus, and Punta Toro virus. None
of the compounds showed activity against any of the RNA
viruses tested at nontoxic concentrations (data not shown).
(S)-HPMPDAP and its cyclic form inhibited VACV repli-
cation with EC50 values that were ∼10-fold lower than those

6830 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Krecꢀmerovaꢁ et al.

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6831

6832 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Krecꢀmerovaꢁ et al.
virus and orthopoxviruses, emerging ODE-(S)-HPMPDAP
as the most selective compound (selectivity index =3500)
against VACV.38 In this study, the authors reported EC50
values for ODE-(S)-HPMPDAP of 0.02, 0.03, and 21.2 μM,
for respectively VACV, cowpox virus, and ectromelia virus,
resulting in selectivity indices in the range of 100-3500
[SI = 104 (ectromelia virus), 2330 (cowpox virus), and 3500
(VACV). Assays for VACV and cowpox virus were per-
formed in human foreskin fibroblasts while for ectromelia
virus they were done in the monkey cells (i.e., BSC-1 cells).
The alkoxyalkyl ester prodrugs of (S)-HPMPDAP and its
cyclic form proved also remarkably potent and selective
against HSV-1, HSV-2, HSV-1 ACVr, VZV, and HCMV,
with EC50 values in the range of 0.007-0.0008 μM (Tables 1
and 2). Lower activities of the alkoxyalkyl ester prodrugs of
cyclic (S)-HPMPDAP than of the corresponding alkoxyalkyl
ester prodrugs of (S)-HPMPDAP were also observed against
HSV and VZV (Table 2). The alkoxyalkyl ester prodrugs
also showed potent activity against HCMV, with EC50 values
at least 225-fold lower than those for the corresponding
parent compound (Table 1). The alkoxyalkyl ester prodrugs
proved rather toxic against the lymphoblast HSB-2 cells, and
only compounds 8a and 9a, the HDP derivatives of (S)-
HPMPDAP and cyclic (S)-HPMPDAP, respectively, showed
selective activity against HHV-6 at nontoxic concentrations
(Table 1).
Although the POM (compounds 10 and 11-cis and -trans),
2,2,2-trifluoroethyl(compounds12and 13), andbutylsalicylyl
(compound 16) derivatives, and prodrugs based on peptido-
mimetics (compound 21-cis and -trans), proved less active
than the alkoxyalkyl ester prodrugs, they appeared to be
less cytotoxic and cytostatic (Tables 1 and 2). In contrast to
alkoxyalkyl ester prodrugs that affected cell morphology in
the concentration range of 0.5-40 μM (HSB-2 cells) and
6-40 μM (HEL cells), the POM, 2,2,2-trifluoroethyl, butyl-
salicylyl, and peptidomimetic prodrugs showed MCC values
ofg60μM (HSB-2 cells) andg100μM(HEL cells). The POM
derivate of cyclic (S)-HPMPDAP (compound 11), either the
diastereoisomeric mixture or the trans-isomer, was able to
inhibit VACV replication with EC50 values similar to those
observed with the parent compounds, while compounds 10,
12, 13, and 21 showed EC50 values 4-16-fold higher than the
parent compounds (Table 1). The POM, 2,2,2-trifluoroethyl,
and butylsalicylyl derivatives and prodrugs based on pep-
tidomimetics proved also active against HSV, VZV, and
HCMV, with EC50’s similar or 10-fold higher than those
of the parent drugs (Tables 1 and 2). Compounds 10 and 11
also displayed potent and selective activity against HHV-6
(Table 1). The EC50 values and the selectivity indices for these
compounds against HHV-6 were equivalent to those observed
for (S)-HPMPDAP.
DNA. It should be noted, however, that the activities of POM
derivatives of (S)-HPMPDAP and of its cyclic form are
substantially weaker compared to similar prodrugs of other
acyclic nucleoside phosphonates such as adefovir (i.e., adefovir
dipivoxil) and tenofovir (i.e., tenofovir disoproxil fumarate).
On the other hand, the HDP ester of tenofovir (CMX-157),
reported to be 267-fold more active than tenofovir against
HIV-1 and HIV-2,41 appears to be significantly more active
than the current compounds 11, 11 (trans), and 21.
Conclusion
In conclusion, a series of ester prodrugs of (S)-HPMPDAP
(1) was synthesized for further evaluation against a variety of
viruses, in particular VACV, and a general methodology for
their preparation was worked out. Besides standard synthetic
approaches, special attention was paid also to utilization of
hexafluorophosphate coupling reagents for the synthesis of
some ANP ester prodrugs. The most active prodrugs against
VACV, HSV, VZV, and HCMV were the alkoxyalkyl ester
derivatives of HPMPDAP and its cyclic form. Prodrugs based
on peptidomimetics, the 2,2,2-trifluoroethyl, the butylsali-
cylyl, and the POM prodrugs were able to inhibit VACV
replication with 50% inhibitory concentrations that were
equivalent or ∼10-fold higher than those observed for the
parent compounds. The different prodrugs displayed potent
and selective activity against different herpesviruses (i.e., HSV,
VZV, and HCMV), although only the POM and HDP ester
prodrugs were able to selectively inhibit the replication of
HHV-6. The 2,2,2-trifluoroethyl, the butylsalicylyl, the piva-
loyloxymethyl, and peptidomimetic prodrugs were less potent
but also less toxic than the alkoxyalkyl ester produgs, as
measured by alteration of cell morphology or cell growth. In
conclusion, the alkoxyalkyl and POM prodrugs of both
HPMPDAP and its cyclic form are promising candidates for
further exploration as candidates for anti-poxvirus therapy.
Experimental Section
Unless stated otherwise, solvents were evaporated at 40 °C/2
kPa, andcompounds weredried at 13Pa. Purification of products
(except 16) by reverse-phase HPLC technique was performed on
a Waters Delta 600 instrument with a Waters 2487 dual
λ absorbance detector using preparative columns, Luna Pheno-
menex C-18 (10 μm, 21 ꢀ 250 mm, flow 12 mL/min). HPLC
purification of 16 was performed on a column 300 ꢀ 50 mm; RP
C18 TESEC; flow rate 45 mL/min. Solvent systems are given in
the text. Column chromatography was performed on silica gel
60 μm (Fluka). TLC was performed on silica gel 60 F254 plates
(Merck KGaA, Darmstadt, Germany). 1H and 13C NMR spectra
were measured on a Bruker Avance 600 spectrometer (1H at
600 MHz, 13C at 151 MHz) or Bruker Avance 500 spectrometer
(1H at 500 MHz, 13C at 125.7 MHz). 31P NMR spectra were
measured on a Bruker Avance 500 spectrometer (202.3 MHz) in
CDCl3 using H3PO4 as external standard. Mass spectra were
measured on a ZAB-EQ (VG Analytical) spectrometer using
FAB (ionization with xenon, accelerating voltage 8 kV, glycerol
matrix) or by ESI technique. The purity of compounds was
g95%, and it was determined from elemental analyses (except
hygroscopic materials 20 and 21 which purity was determined
by HPLC technique using above-mentioned Waters Delta 600
instrument, an analytical column, Nova Pack C18, 3.9 ꢀ 150 mm,
with flow rate 1 mL/min and solvent system methanol-
phosphate buffer).
Several compounds showed activity against HIV-1 and
HIV-2 although with somewhat narrow selectivity (i.e., 11
(EC50, 7.4-17.6 μM; CC50, 95 μM), 11 (trans) (EC50, 30-32
μM; CC50, 109 μM), and 21 (cis) (EC50, 70-138 μM;
CC50, >177 μM)) at subtoxic concentrations. It was generally
assumed that HPMP derivatives were unable to inhibit HIV
in cell culture. However, recent reports have shown that
ODE-(S)-HPMPA and ODE-(S)-HPMPDAP can inhibit
HIV replication in vitro.39,40 Probably the increased uptake
of prodrugs into the target cells may eventually release higher
levels of the active metabolites than those that can be achieved
by administration of parent drug, resulting in inhibition of the
HIV reverse transcriptase and/or incorporation into the viral
Materials and Solvents. Most of the chemicals and ion-
exchange resins (Dowex 1X2-400) were purchased from Sigma-
Aldrich (Czech Republic). (2S)-2-[(trityloxy)methyl]oxirane (2)

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6833
was purchased from DAISO Co. Ltd. (Japan). Dimethylforma-
mide and dioxane were dried by distillation from CaH2 (DMF in
vacuo) and stored over molecular sieves, 4 A (DMF), or sodium
(dioxane). Alkoxyalkyl bromides were prepared from the cor-
responding alcohols by the action of carbon tetrabromide and
triphenylphosphine.18a,42
3.0 mmol) in absolute methanol (100 mL) and the mixture
stirred for 10 min in an ultrasound bath and evaporated. The
residue was coevaporated with toluene (50 mL), dissolved in
appropriate solvent (20 mL, DMF for alkoxyalkyl esters or
dioxane for POM esters), and stirred with alkoxyalkyl bromide
(8 mmol) at 100-115 °C for 3 h or refluxed with chloromethyl
pivalate (5 mmol) for 3 h (TLC control). The reaction mixture
was cooled to room temperature, diluted with methanol (2 mL),
and evaporated. The residue was chromatographed on a column
of silica gel (50 mL) in system chloroform-methanol (85:15). To
remove the rest of tetrabutylammonium salts, final purification
of products was performed by crystallization or by HPLC
technique.
3-(Hexadecyloxy)propyl Ester of 9-{[(5S)-2-Hydroxy-2-oxi-
do-1,4,2-dioxaphosphinan-5-yl]methyl}-2,6-diaminopurine (8a).
Crystallization from acetone afforded 600 mg (33%) of 8a as
a diastereoisomeric mixture (trans:cis, 1.34:1.0). Product con-
tained in mother liquors was additionally purified by pre-
parative reverse-phase HPLC: gradient 50-100% methanol in
25 min eluted impurities and tetrabutylammonium salts, elution
with 100% methanol afforded 8a-trans (150 mg), followed by
8a-cis (50 mg). Overall yield: 800 mg (46%) of a white solid.
1H NMR (500.0 MHz, CDCl3) for trans-isomer: 0.87 (t, 3H,
Jvic = 7.0, CH3), 1.28 (m, 26H, CH2), 1.54 (m, 2H, CH2), 1.97
9-(S)-[3-Hydroxy-2-(phosphonomethoxy)propyl]-2,6-diamino-
purine (1). Cesium carbonate (1.3 g) was added to a stirred mix-
ture of 2,6-diaminopurine (8.46 g, 56.3 mmol) and (S)-tritylgly-
cidol (2, 17.8 g, 56.3 mmol) in DMF (110 mL) at 100 °C, and the
heating was continued for additional 8 h. The mixture was
evaporated and the residue coevaporated with toluene and
chromatographed on a column of silica gel (900 mL) in system
chloroform-methanol (9:1) to give 24 g (91%) of compound 3
as a yellowish foam. This intermediate was dissolved in chloro-
form (130 mL) and treated with dimethylformamide dimethy-
lacetal (50 mL) at 25 °C for 24 h, and the solution was then
evaporated and dried in vacuo. DMF (200 mL) was added,
followed by 60% oil suspension of NaH (4 g, 100 mmol) and
BrCH2P(O)(OiPr)2 (14.8 g, 57 mmol). After being stirred at
25 °C for 24 h, the mixture was neutralized with acetic acid to pH
7 and evaporated and the residue coevaporated with toluene. A
mixture of methanol (250 mL) and 25% aqueous ammonia
(65 mL) was added and the solution set aside for 24 h at room
temperature and evaporated. The residue was refluxed with
80% acetic acid (270 mL) for 1 h, then evaporated, and parti-
tioned between water (800 mL) and ether (400 mL). An aqueous
layer was concentrated to approximately 200 mL and applied
onto a column of Dowex 50 (Hþ form, 300 mL) and elution
performed by aqueous ammonia (1 L), followed by water (1 L)
and 2.5% aqueous ammonia. Product containing ammonia
fractions was evaporated and dried in vacuo. Acetonitrile
(250 mL) was added, followed by bromotrimethylsilane
(40 mL), and the mixture was stirred in the dark for 24 h. After
evaporation, the mixture of 50% aqueous ethanol and triethy-
lamine (20:1) was added to neutral reaction, the mixture evapo-
rated, and the residue crystallized from water. The solid product
1 was filtered off, washed with water, followed by acetone and
ether, and dried in vacuo. Mother liquors were desalted on
Dowex 50 (Hþ form, 150 mL) as described above; UV absorbing
ammonia fractions were evaporated, and the residue was dis-
solved in water and applied onto a column of Dowex 1 (acetate
form, 100 mL). Elution was performed with a linear gradient of
acetic acid (0-1 M, 2 L), and compound 1 was eluted with 0.5 M
acetic acid. Appropriate fractions were evaporated and coeva-
porated with water (3 ꢀ 50 mL), and the product was crystal-
lized from water. Overall yield: 7 g (39%). Spectral data are in
agreement with literature.12
(p, 2H, Jvic = 6.2, CH2), 3.38 (m, 2H, OCH2), 3.48 (t, 2H, Jvic
=
6.1, OCH2), 3.91 (dd, Jgem = 14.8, JH,P = 0.5, CHaHbP), 3.99
(dd, 1H, Jgem = 14.8, JH,P = 6.9, CHaHbP), 4.08 (m, 1H, H-20),
4.28-4.13 (m, 4H, H-30 and P-OCH2), 4.38 (m, 2H, H-10), 5.10
(br s, 2H, NH2-2), 6.10 (br s, 2H, NH2-6), 7.58 (s, 1H, H-8). 1H
NMR (500.0 MHz, CDCl3) for cis-isomer: 0.88 (t, 3H, Jvic = 7.0,
CH3), 1.26 (m, 26H, CH2), 1.54 (m, 2H, CH2), 1.94 (p, 2H, Jvic
=
6.2, CH2), 3.38 (t, 2H, Jvic = 6.7, OCH2), 3.48 (t, 2H, Jvic = 6.1,
OCH2), 3.86 (dd, 1H, Jgem = 14.3, JP,H = 3.3, CHaCHbP), 4.12
(dd, 1H, Jgem = 14.3, JP,H = 0.9, CHaCHbP), 4.13 (m, 1H, H-20),
4.28-4.18 (m, 4H, H-30 and P-OCH2), 4.42 (m, 2H, H-10), 4.76
(br s, 2H, NH2), 5.53 (br s, 2H, NH2), 7.56 (s, 1H, H-8). 31P (1H
dec) NMR (202.3 MHz, CDCl3): 11.08 (trans), 12.79 (cis). Anal.
(C28H51N6O5P) C, H, N, P. FABMS: 583 (MH)þ (65). HRMS
(FAB): for C28H52N6O5P (MHþ) calculated, 583.3737; found,
583.3746.
2-(Octadecyloxy)ethyl Ester of 9-{[(5S)-2-Hydroxy-2-oxido-
1,4,2-dioxaphosphinan-5-yl]methyl}-2,6-diaminopurine (8b). Over-
all yield: 800 mg (45%, trans:cis, 3:1). Separation of diastereo-
isomers by column chromatography afforded 600 mg (34%) of
8b-trans (mp 100-104 °C, methanol) and 200 mg (11%) of 8b-cis
(mp 110-114 °C, methanol). Anal. (C29H53N6O5P) C, H, N, P.
ESIMS(-): 595 (M - H)- (100). HRMS (þESI): for C29H54N6-
O5P (MH)þ calculated, 597.3888; found, 597.3876.
9-{[(5S)-2-Hydroxy-2-oxido-1,4,2-dioxaphosphinan-5-yl]-
methyl}-2,6-diaminopurine (7). A mixture of 1 (4.8 g, 15 mmol),
N,N0-dicyclohexyl-4-morpholinecarboxamidine(4.7 g, 16mmol),
and DCC (3.5 g, 17 mmol) in DMF (50 mL) was heated at 100 °C
for 3 h. After being cooled to room temperature, the solid was
filtered off and washed with water, and combined filtrates were
applied onto a column of Dowex 1 (acetate form, 150 mL).
Elution was performed with methanol (2 L), followed by water
(1 L) and a linear gradient of acetic acid (0-0.5 M, 2 L). Elution
of 7 started at 0.2 M AcOH. Product containing fractions were
evaporated, and the residue was coevaporated with water (3 ꢀ
100 mL) and finally recrystallized from water. Yield: 3.2 g
(71%) of trans-isomer. White crystals. Mp >297 °C (dec). 1H
2-(Hexadecyloxy)ethyl Ester of 9-{[(5S)-2-Hydroxy-2-oxido-
1,4,2-dioxaphosphinan-5-yl]methyl}-2,6-diaminopurine (8c). Yield
after column chromatography: 882 mg (52%) of a diastereoiso-
meric mixture (trans:cis, 3:1), amorphous solid. Separation of
diastereoisomers was performed by HPLC technique using gra-
dient water-MeOH 50-100% in 40 min, followed by 100%
MeOH. Elution with 100% MeOH afforded 420 mg (25%) of 8c-
trans followed by 190 mg (11%) of 8c-cis (amorphous solid).
Anal. (C27H49N6O5P) C, H, N, P. ESIMS: 569.4 (MH)þ (100).
HRMS (ESI): for C27H50N6O5P (MH)þ calculated, 569.3575;
found, 569.3560.
Pivaloyloxymethyl Ester of 9-{[(5S)-2-Hydroxy-2-oxido-
1,4,2-dioxaphosphinan-5-yl]methyl}-2,6-diaminopurine (11). Yield
after chromatography: 534 mg (43%) of a white foam. Final
purification and separation of diastereoisomers were performed
by preparative reverse-phase HPLC using gradient 20-100%
MeOH: 11 (trans:cis 6:1) was eluted with 95-100% MeOH (200
mg), followed by pure 11-cis eluted with 100% MeOH (70 mg).
1H NMR (500.0 MHz, CDCl3) for trans-isomer, 7.48 (s, 1H,
H-8), 5.72 and 5.69 (2 ꢀ dd, 2H, JP,CH = 5.3, Jgem = 12.2,
OCH2O), 5.60 (br s, 1H, NH2), 4.81 (br s, 1H, NH2), 4.41-
4.13 (m, 4H, NCH2, OCH2), 4.07 (m, 1H, H-20), 3.97 (dd, 1H,
NMR (500.0 MHz, D2O): 3.75 (dd, 1H, Jgem = 14.1, JH,P
=
2.2, CHaHbP), 3.99 (dd, 1H, Jgem = 14.1, JH,P = 8.8,
CHaHbP), 4.12 (m, 3H, H-20, H-30), 4.23 and 4.32 (2 ꢀ m,
2 ꢀ 1H, H-10), 7.78 (s, 1H, H-8). ESIMS: 323 (M þ Na)þ (10),
301.1 (MH)þ. HRMS (ESI): for C9H14N6O4P (MH)þ calcu-
lated, 301.0809; found, 301.0816.
Transformation of 7 to Cyclic Phosphonate Esters. General
Procedure. One molar methanolic tetrabutylammonium hydro-
xide (3 mL, 3 mmol) was added to a solution of 7 (900 mg,

6834 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Krecꢀmerovaꢁ et al.
Jgem = 14.4, JP,CHax = 7.1, PCHax), 3.92 (br d, 1H, Jgem = 14.4,
PCHeq), 1.21 (s, 9H, t-Bu). 1H NMR (500.0 MHz, CDCl3) for cis-
isomer: 7.50 (s, 1H, H-8), 6.28 (br s, 1H, NH2), 5.74 (dd, 1H,
for C15H25N6O7NaP (M þ Na)þ calculated, 455.1420; found,
455.1413.
(b) An alternative purification was performed on a column
of DEAE-Sephadex A25 (1.5 ꢀ 6 cm) activated with 0.02 M
triethylammonium hydrogen carbonate (TEAB) using elution
with water (250 mL) followed by a linear gradient of TEAB
(0-0.5M, 400 mL). Collected UV absorbing fractions were
evaporated at 25 °C and finally purified by preparative HPLC
technique using the following conditions: 10% MeOH (0-
10 min), gradient 10-100% MeOH (10-25 min). Elution of 10
occurred at concentration 85-95% MeOH. Yield after HPLC:
250 mg (25%, TEA salt).
2,2,2-Trifluoroethyl Ester of 9-(S)-[3-Hydroxy-2-(phosphono-
methoxy)propyl]-2,6-diaminopurine (12). PyBroP (2.8 g, 6 mmol)
was added to a suspension of 1 (1 g, 3.14 mmol) in DMF (12 mL)
and stirred at 25 °C until dissolution (0.5-1 h). Ethyldiisopro-
pylamine (1.6 mL, 9.0 mmol) was added after 1 h followed by
trifluoroethanol (2 mL) and an additional amount of ethyldiiso-
propylamine (1 mL). The mixture was stirred at 90 °C for 3 h. The
reaction course was monitored by TLC in system 2-propanol-
25% ammonia-water (7:1:2). In case of insufficient conversion,
DCC was added (660 mg, 3.2 mmol) and the heating continued
for an additional 1 h. The mixture was evaporated in vacuo and
the residue chromatographed on a short column of silica gel
(height 4 cm, width 5 cm) in system chloroform-methanol (4:1).
The crude product was additionally chromatographed in system
ethyl acetate-acetone-ethanol-water (15:3:4:3). Final purifica-
tion of 12 was performed by preparative HPLC technique using a
linear gradient of methanol in water (5-100% MeOH in 60 min);
elution of 12 occurred at 40% MeOH. A product contain-
ing fraction was evaporated and the pure product lyophilized
from water. Yield: 335 mg (27%) of a white solid. Anal.
(C11H16N6O5F3P) C, H, N, P. ESIMS: 401.1(MH)þ (15). HRMS
(ESI): for C11H17N6O5F3P (MHþ) calculated, 401.0945; found,
401.0946.
2,2,2-Trifluoroethyl Ester of 9-{[(5S)-2-Hydroxy-2-oxido-
1,4,2-dioxaphosphinan-5-yl]methyl}-2,6-diaminopurine (13). Ethyl-
diisopropylamine (0.79 mL, 4.5 mmol), followed by HATU (1.7 g,
4.5 mmol), was added under stirring in an ultrasound bath to a
suspension of 7 (670 mg, 2.23 mmol) in DMF (10 mL). Trifluoro-
ethanol (4 mL) was added after 15 min and the mixture stirred for
1 h at 25 °C and then heated to 90 °C during 3 h. The resulting dark
brown solution was evaporated and the residue chromatographed
on a silica gel column (100 mL) in ethyl acetate-acetone-
ethanol-water (15:3:4:3) followed by additional chromatography
in chloroform-methanol (4:1) to give TLC pure 13 as a yellow
foam (450 mg). Final purification of 13 was performed by
preparative HPLC using a gradient of MeOH (5-100% MeOH
in 40 min, retention time 20 min). Yield: 300 mg (35%) of
a diastereoisomeric mixture (white solid). Anal. (C11H14N6O4-
F3P.2H2O) C, H, N, P. ESIMS: 383.1 (MH)þ (100). HRMS
(ESI): for C11H15N6O4F3P (MHþ) calculated, 383.0839; found,
383.0840.
Bis(2,2,2-Trifluoroethyl) Tosyloxymethylphosphonate (15).
Bromotrimethylsilane (25 mL, 187 mmol) was added to a
solution of 14 (10 g, 28.6 mmol) in acetonitrile (150 mL). After
standing for 48 h, the solution was evaporated and the residue
coevaporated gradually with solvents in the following order,
acetonitrile, water, absolute ethanol, and dichloromethane, and
dried in vacuo. DMF (1 mL) and dichloromethane (400 mL)
were added, the solution was cooled to 0 °C, and oxalyl chloride
(16 mL, 180 mmol) was added dropwise under stirring. The
mixture was heated to room temperature and then refluxed for
4 h. The solution was evaporated, the residue dissolved in
dichloromethane (300 mL), and pyridine (10 mL) added at
0 °C. The resulting solution was added to a mixture of trifluoro-
ethanol (5 mL, 70 mmol), triethylamine (46 mL), and dichloro-
methane cooled to -25 °C. The resulting black solution was
stirred at -25 °C for 2 h and then 2 days at room temperature
and evaporated. The residue was chromatographed on a silica
JP,CH = 5.6, Jgem = 12.3, OCH2O), 5.60 (dd, 1H, JP,CH = 5.1,
OCH2O), 5.13 (br s, 1H, NH2), 4.45-5.03 (m, 5H, H-10, OCH2,
H-20), 3.94 (br d, 1H, JP,CHax e 1.0, Jgem = 14.4, PCHax), 3.85
(dd, 1H, Jgem = 14.4, JP,CHeq = 3.2, PCHeq), 1.19 (s, 9H, t-Bu).
Anal. (C15H23N6O6 P.H2O) C, H, N, P. ESIMS: 850.5 (2M þ
Na)þ (41), 437.0 (M þ Na)þ (10), 414.9(6) (MH)þ. HRMS (ESI):
for C15H23N6O6Na P (M þ Na)þ calculated, 437.1314; found,
437.1305.
Transformation of Cyclic Phosphonate Esters to Monoesters.
General Procedure. A mixture of 8 (1 mmol) and 2 M NaOH
(20 mL) was heated at 80 °C for 1 h, then cooled to room
temperature, neutralized with glacial acetic acid to pH 5, and set
aside at 4 °C for 12 h. A precipitated product was filtered off,
washed with water, followed by acetone and ether, and dried in
vacuo. Final purification for in vivo experiments was performed
by preparative HPLC technique using gradient 50-100%
MeOH (0-18 min), followed by 100% MeOH; monoesters 9
were eluted with 100% MeOH with retention time 20-25 min.
Another possibility of purification is column chromatography on
silica gel (100 mL) in system ethyl acetate-acetone-ethanol-
water (15:3:4:3), followed by crystallization of the product from
the same system.
3-(Hexadecyloxy)propyl Ester of 9-(S)-[3-Hydroxy-2-(phos-
phonomethoxy)propyl]-2,6-diaminopurine Sodium Salt (9a).
Yield: 338 mg (54%) of a white solid. H NMR (500.0 MHz,
1
CD3OD): 7.89 (s, 1H, H-8), 4.64 (m, 1H, H-20), 4.31 (dd, 1H,
J1 a,2 = 4.0, Jgem = 14.6, H-10a), 4.24 (dd, 1H, J1 b,2 = 6.0,
0
0
0
0
H-10b), 3.93 (m, 2H, OCH2), 3.69 (dd, 1H, JP,CH = 9.3, Jgem
=
13.6, PCHa), 3.66 (dd, 1H, JP,CH = 9.3, PCHb), 3.64 (dd, 1H,
J3 a,2 = 4.2, Jgem = 12.4, H-30a), 3.51 (dd, 1H, J3 b,2 = 4.6,
H-30b), 3.49 (t, 2H, J = 6.5, OCH2), 3.38 (t, 2H, J = 6.7, OCH2),
1.81 (pent, 2H, CH2), 1.53 (m, 2H, CH2), 1.30 (m, 26H, CH2),
0.91 (t, 3H, JCH3,CH2 = 7.2, CH3). Anal. (C28H52N6O6 NaP) C,
H, N, P. FABMS: 645 (M þ Na)þ (51), 623 (MH)þ (40). HRMS
(FAB): for C28H53N6O6NaP (MHþ) calculated, 623.3662; found,
623.3673.
0
0
0
0
2-(Octadecyloxy)ethyl Ester of 9-(S)-[3-Hydroxy-2-(phos-
phonomethoxy)propyl]-2,6-diaminopurine Sodium Salt (9b).
Yield: 330 mg (52%) of a white solid. Anal. (C29H55N6O6 NaP)
C, H, N, P. ESIMS: 637 (MH)þ (30), 615 (M - Na þ 2H)þ
(100). HRMS (QTOF): for C29H55N6O6NaP (MHþ) calculated,
637.3818; found, 637.3826.
2-(Hexadecyloxy)ethyl Ester of 9-(S)-[3-Hydroxy-2-(phos-
phonomethoxy)propyl]-2,6-diaminopurine Sodium Salt (9c).
Yield: 416 mg (68%) of a white solid. Anal. (C27H50N6O6 NaP)
C, H, N, P. ESIMS: 587.4 (MH)þ (100) - phosphonic acid, 609.3
(30) (MH)þ - sodium salt. HRMS (ESI): for C27H51N6O6NaP
(MHþ) calculated, 609.3500; found, 609.3508.
Pivaloyloxymethyl Ester of 9-(S)-[3-Hydroxy-2-(phosphono-
methoxy)propyl]-2,6-diaminopurine (10). (a) Chloromethyl piva-
late (1.3 mL, 9 mmol) was added to a mixture of 1 (600 mg,
1.88 mmol) and N,N0-dicyclohexyl-4-morpholinecarboxamidine
(1.32 g, 2.25 mmol) inDMF (10mL), and the mixture was strirred
for 24 h at 25 °C. Methanol (2 mL) was added, the solution
evaporated, and the residue chromatographed on a column of
silica gel (150 mL) in system ethyl acetate-acetone-ethanol-
water (15:3:4:3). Crystallization from dry ether afforded 5 in yield
350 mg (43%, free monoester). 1H NMR (500.0 MHz, CD3OD):
7.88 (s, 1H, H-8), 5.54 (dd, 1H, JP,OCH = 5.4, Jgem = 12.2,
OCH2O), 5.50 (dd, 1H, JP,OCH = 4.9, OCH2O), 4.60 (m, 1H,
H-20), 4.28 (dd, 1H, J1 a,2 = 4.1, Jgem = 14.5, H-10a), 4.20 (dd,
0
0
0
0
1H, J1 b,2 = 6.4, H-1 b), 3.71 (dd, 1H, JP,CHa = 9.3, Jgem = 13.0,
0
0
0
=
PCHa), 3.65 (dd, 1H, JP,CHb = 9.1, PCHb), 3.60 (dd, 1H, J3 a,2
4.6, Jgem = 12.4, H-30a), 3.49 (dd, 1H, J3 b,2 = 4.9, H-30b), 1.15
(s, 9H, tert-butyl). Anal. (C15H25N6O7 P.1/2H2O) C, H, N, P.
FABMS:455 (Mþ Na)þ (0.2), 433 (MH)þ (0.15). HRMS (FAB):
for C15H26N6O7P (MHþ) calculated, 433.1600; found, 433.1606;
0
0

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6835
gel column (1 L) in a gradient of toluene-ethyl acetate (3:1 to
1:1). Rf of 15: 0.6 in toluene-ethyl acetate (1:1). Yield: 3.5 g
(28%) of colorless syrup crystallizing after standing in a refrig-
erator. Anal. (C12H13F6O6PS) C, H, F, P, S. ESIMS: 452.91
(M þ Na)þ (100), 430.8 (MH)þ (5). HRMS (ESI): for C12H13-
F6O6PSNa (M þ Na)þ calculated, 452.9967; found, 452.9963.
Butylsalicyl Ester of 9-{[(5S)-2-Hydroxy-2-oxido-1,4,2-diox-
aphosphinan-5-yl]methyl}-2,6-diaminopurine (16). A mixture of 1
(2.09 g, 6.96 mmol) and PyBOP (9.01 g, 17.32 mmol) in DMF
(240 mL) was sonicated in an ultrasonic bath for 45 min.
N-Diisopropylethylamine (4.45 g, 34.44 mmol) and butyl salicy-
late (2.14 g, 11.01 mmol) were added, and the resulting mixture
was sonicated for 15 min. The suspension was stirred for 6 days at
room temperature as long as the conversion of 1 to 16 proceeded
(monitoring of the reaction mixture was performed by TLC in
chloroform-methanol 4:1). During this reaction, sonication was
repeated several times. The mixture was evaporated in vacuo to
dryness, and the residue was chromatographed on a column
of silica gel (360 mL) in chloroform-methanol (95:5, followed
by 4:1). Fractions containing the mixture of compound 16 (Rf =
0.62) and PyBOP byproducts (Rf = 0.56) were combined and
evaporated in vacuo. The residue was subsequently purified by
preparative HPLC technique using gradient 40-100% MeOH/
H2O (0-30 min). Product was collected in 80-100% MeOH/
H2O. Final purification was performed by preparative TLC
chromatography in chloroform-methanol (5:1). The product
16 (trans:cis 12:88) was obtained after lyophilization as a white
amorphous solid. Yield: 522 mg (15%). Anal. (C20H25N6-
to dryness, and the residue was chromatographed on a column of
silica gel (400 mL) in CHCl3-MeOH (9:1). Fractions containing
20-cis or 20-trans contaminated with PyBOP related products
were combined and evaporated in vacuo. Fractions containing
20, mixture of cis and trans, were chromatographed again. Frac-
tions containing 20-cis or -trans were subsequently purified by
preparative HPLC technique using gradient 30-100% MeOH/
H2O (0-25 min, 10 mL/min). Product was collected in 75-85%
MeOH/H2O. Final purification was performed by means of
crystallization from EtOH-Et2O. Total yield: 0.48 g (21%).
ESIMS: 629.0, 630.0 [M þ H]þ, 651.1, 652.1 [M þ Na]þ.
Isopropyl-(L-valyl)-L-serin-O3-yl Ester of 9-{[(5S)-2-Hydroxy-
2-oxido-1,4,2-dioxaphosphinan-5-yl]methyl}-2,6-diaminopurine
(21). Boc derivative 20-cis or 20-trans (135 mg, 0.215 mmol) was
treated with HCl in dioxane (4 M, 1 mL) at 0 °C. Mixture
was allowed to reach room temperature 30 min later. Conversion
was monitored (TLC in CHCl3-MeOH, 5:1). The mixture was
evaporated and codistilled (dioxane) when the starting com-
pound disappeared. Crude product was purified by prepara-
tive HPLC technique using gradient 20-60% MeOH/H2O (0-
25 min, 10 mL/min). Product was collected in 35-50% MeOH/
H2O. Yield: 101 mg (83%) of amorphous solid. ESIMS: 528.9,
529.9 [M þ H]þ, 551.0, 552.1 [M þ Na]þ. HRMS (ESI): for C20-
H34O7N8P [M þ H]þ calculated, 529.2283; found, 529.2288.
Antiviral Activity Assays. The compounds were evalua-
ted 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, human herpes virus 6
subtype A (HHV-6A) strain GS, vaccinia virus Lederle strain,
human immunodeficiency virus (HIV) type 1 (IIIB) and type 2
(ROD), respiratory syncytial virus (RSV) strain Long, vesicular
stomatitis virus (VSV), Coxsackie B4, Parainfluenza 3, Reo-
virus-1, Sindbis, and Punta Toro. The antiviral assays, other
than HIV, were based on inhibition of virus-induced cytopathi-
city or plaque formation in human embryonic lung (HEL)
fibroblasts, African green monkey cells (Vero), human epithelial
cells (HeLa), or human T-lymphoblasts HSB-2, according to
previously established procedures.16 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 (PFU). After 1-2 h
adsorption period, residual virus was removed, and the cell
cultures were incubated in the presence of varying concentra-
tions 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 concentration (expressed in micromolar) required for
reducing virus-induced cytopathogenicity or viral plaque for-
mation by 50%. The methodology of the anti-HIV assays was as
follows: human CEM (∼3 ꢀ 105 cells/cm3) were infected with
100 CCID50 of HIV-1 (IIIB) or HIV-2 (ROD)/mL and seeded in
200 μL wells of a microtiter plate containing appropriate dilu-
tions of the test compounds. After 4 days of incubation at 37 °C,
HIV-induced CEM giant cell formation was examined micro-
scopically.
O6P 0.6H2O) C, H, N, P. ESIMS: 477 (M þ H)þ. HRMS
3
(ESI): for C20H26O6N6P (M þ H)þ calculated, 477.1646; found,
477.1646.
L-Serine Isopropyl Ester Hydrochloride (18). Thionyl chloride
(120 mL) was added dropwise to cooled (∼-30 °C) 2-propanol
(300 mL). Addition rate and cooling should be set up in such a
way that maximum temperature of reaction mixture did not
violate 0 °C. The solution was stirred an additional 1 h at 0 °C;
then L-serine (10 g, 95 mmol) was added. The suspension was
kept to reach room temperature. Gas was evolved during several
hours. Gas production was controlled by means of subsequent
slow heating up to 80 °C (additional several hours). When the
mixture was completely dissolved and no gas evolved, 2-propa-
nol was partly distilled off, and the product was crystallized
from a distillation residue (∼100 mL). A crude product was
crystallized from 2-propanol again. Yield 9.0 g (52%). Anal.
(C6H14O3NCl) C, H, N, Cl.
Isopropyl tert-Butyloxycarbonyl-L-valyl-L-serinate (19). Boc-
Val-OH (2.09 g, 9.60 mmol), compound 18 (1.76 g, 9.60 mmol),
and hydroxybenzotriazole (1.30 g, 9.62 mmol) were suspended
in CH2Cl2 (30 mL). The mixture was cooled (0 °C), and
triethylamine (1.34 mL, 9.61 mmol) and EDC (1.78 mL, 10.0
mmol) were added. The mixture was stirred for 1 h at 0 °C and
then overnight at room temperature. The solution was diluted
with CH2Cl2 and washed with a solution of NaHCO3, citric
acid, and NaHCO3 again. The organic layer was dried with
MgSO4 and evaporated, and the residue was chromatographed
on a silica gel column (250 mL) in hexane-ethyl acetate (3:2 f
2:3). The syrupy product was precipitated from Et2O-hexane.
Yield: 2.73 g (82%). Anal. (C16H30O6N2) C, H, N.
Isopropyl-(tert-butyloxycarbonyl-L-valyl)-L-serin-O3-yl Ester of
9-{[(5S)-2-Hydroxy-2-oxido-1,4,2-dioxaphosphinan-5-yl]methyl}-
2,6-diaminopurine (20). A mixture of 1 (1.15 g, 3.62 mmol) and
PyBOP (4.81 g, 9.25 mmol) in DMF (120 mL) was sonicated in an
ultrasonic bath for 45 min. N-Diisopropylethylamine (3.25 mL,
18.55 mmol) and 19 (1.38 g, 3.98 mmol) were added, and the
resulting mixture was sonicated for 15 min. The suspension was
stirred for 2 daysat roomtemperatureas longasthe conversionof
1 to 20 proceeded (monitoring of the reaction mixture was per-
formed by TLC in CHCl3-MeOH (5:1), Rf(20) ∼ 0.5, two spots
close to each other). During this reaction a process of sonication
was repeated several times. The mixture was evaporated in vacuo
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 con-
centrations of the test compounds was added. After 3 days
of incubation at 37 °C, the cell number was determined with a
Coulter counter. The cytostatic concentration (expressed in
micromolar) was calculated as the CC50 or the compound
concentration required for reducing 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

6836 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Krecꢀmerovaꢁ et al.
(percentage of control) as a function of the concentration of the
test compounds. Alternatively, cytotoxicity of the test com-
pounds was expressed as the minimum cytotoxic concentration
(MCC) or the compound concentration that caused a micro-
scopically detectable alteration of cell morphology.
Triazine analogs of 1-(S)- [3-hydroxy-2-(phosphonomethoxy)-
propyl]cytosine (cidofovir) and related compounds. J. Med. Chem.
2007, 50, 1069–1077.
ꢁ
(12) Holy, A. Syntheses of enantiomeric N-(3-hydroxy-2-phos-
phonomethoxypropyl) derivatives of purine and pyrimidine
bases. Collect. Czech. Chem. Commun. 1993, 58, 649–674.
ꢁ
ꢁ
´
ꢁ
(13) Holy, A.; Dvorakova, H.; Jindrich, J.; Masojıdkova, M.;
Budesinsky, M.; Balzarini, J.; Andrei, G.; De Clercq, E. Acyclic
ꢁ
Acknowledgment. This study was performed as a research
project of the Institute of Organic Chemistry and Biochemistry,
Academy of Sciences of the Czech Republic (0Z40550506), and
the Center for New Antivirals and Antineoplastics (1M0508)
by the Ministry of Education, Youth and Sports of the Czech
Republic. We gratefully acknowledge the financial support
of the NIH (Grant 1UC1 AI062540-01), Grant ME10040 by
the Ministry of Education, Youth and Sports of the Czech
Republic, Gilead Sciences, Inc. (Foster City, CA), the
“Geconcerteerde Onderzoeksacties” (GOA), Krediet nr. 05/
19 of the K. U. Leuven, and Fonds voor Wetenschappelijk
Onderzoek;Vlaanderen (FWO), Grant G.0680.08. The
authors’ thanks are also due to the staff of mass spectrometry
nucleotide analogs derived from 8-azapurines: synthesis and anti-
viral activity. J. Med. Chem. 1996, 39, 4073–4088.
(14) (a) De Clercq, E.; Andrei, G.; Balzarini, J.; Leyssen, P.; Naesens,
ꢁ
L.; Neyts, J.; Pannecouque, C.; Snoeck, R.; Ying, C.; Hockova, D.;
Holy, A. Antiviral potential of a new generation of acyclic nucleo-
ꢁ
side phosphonates, the 6-[2-(phosphonomethoxy)alkoxy]-2,4-dia-
minopyrimidines. Nucleosides, Nucleotides Nucleic Acids 2005, 24,
ꢀ
ꢁ
331–341. (b) Duraffour, S.; Snoeck, R.; Krecmerova, M.; van Den
Oord, J.; De Vos, R.; Holꢁy, A.; Crance, J.-M.; Garin, D.; De Clercq, E.;
Andrei, G. Activities of several classes of acyclic nucleoside phospho-
nates against camelpox virus replication in different cell culture models.
Antimicrob. Agents Chemother. 2007, 51, 4410–4419.
(15) Centers for Disease Control and Prevention. Smallpox vaccine
adverse events monitoring and response system for the first stage of
the smallpox vaccination program. Morb. Mortal. Wkly. Rep.
2003, 52, 88–89.
(16) De Clercq, E. Clinical potential of the acyclic nucleoside phospho-
nates cidofovir, adefovir and tenofovir in treatment of DNA virus
and retrovirus infections. Clin. Microbiol. Rev. 2003, 16, 569–596.
(17) Buller, R. M.; Owens, G.; Schriever, J.; Melman, L.; Beadle, J. R.;
Hostetler, K. Y. Efficacy of oral active ether lipid analogs of
cidofovir in a lethal mousepox model. Virology 2004, 318, 474–481.
(18) (a) Kern, E. R.; Hartline, C.; Harden, E.; Keith, K.; Rodriguez, N.;
Beadle, J. R.; Hostetler, K. Y. Enhanced inhibition of orthopox-
virus replication in vitro by alkoxyalkyl esters of cidofovir and
cyclic cidofovir. Antimicrob. Agents Chemother. 2002, 46, 991–995.
(b) Keith, K. A.; Wan, W. B.; Ciesla, S. L.; Beadle, J. R.; Hostetler,
K. Y.; Kern, E. R. Inhibitory activity of alkoxyalkyl and alkyl esters of
cidofovir and cyclic cidofovir against orthopoxvirus replication in vitro.
Antimicrob. Agents Chemother. 2004, 48, 1869–1871.
ꢀ
(Dr. J. Cvacka, Head) and analytical departments of the
ꢀ
ꢁ
ꢀ
ꢁ
Institute (Dr. S. Matejkova, Head) and Ms. Julie Krelinova
forexcellent technical assistance. We alsothank Anita Camps,
Steven Carmans, Frieda De Meyer, Leentje Persoons, Leen
Ingels, Wim Van Dam, and Lies Van den Heurck for excellent
technical assistance with the antiviral assays and Christiane
Callebaut for invaluable editorial assistance.
Supporting Information Available: 1H and 13C NMR spectral
data of compounds, characterization of intermediates in pre-
paration of HMPMDAP, and elemental analyses and HPLC of
tested compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
(19) Morris, K. Oral drug and old vaccine renew smallpox bioterror
debate. Lancet Infect. Dis. 2002, 2, 262–262.
(20) Bradbury, J. Orally available cidofovir derivative active against
smallpox. Lancet 2002, 359, 1041–1041.
(21) Valiaeva, N.; Prichard, M. N.; Buller, R. M.; Beadle, J. R.; Hartline,
C. B.; Keith, K. A.; Schriewer, J.; Trahan, J.; Hostetler, K. Y.
Antiviral evaluation of octadecyloxyethyl esters of (S)-3-hydroxy-
2-(phosphonomethoxy)propyl nucleosides against herpesviruses and
orthopoxviruses. Antiviral Res. 2009, 84, 254–259.
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