N4-Acyl derivatives as lipophilic prodrugs of cidofovir and its 5-azacytosine analogue, (S)-HPMP-5-azaC: Chemistry and antiviral activity

Article abstract of DOI:10.1016/j.bmc.2014.03.031

Even number fatty acid residues - docosanoyl (behenoyl) and stearoyl were selected for introduction to the N⁴-position of (S)-1-[3-hydroxy-2- (phosphonomethoxy)propyl]cytosine) (HPMPC, cidofovir), and its 5-azacytosine counterpart, (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine) (HPMP-5-azaC) with the aim to prepare a new type of lipophilic prodrugs. The study on the influence of these modifications to the stability and biological activity of both antivirals was performed. Different reactivity of both systems towards acylation reactions was also found: the 4-NH₂ group of cidofovir was more reactive compared to that of HPMP-5-azaC. In 5-azacytosine derivatives, we found mostly a destabilizing effect of the N⁴-acylation but this could be compensated by a positive influence of the esterification of the phosphonate group. Chemical stability of the 5-azacytosine moiety in the HPMP series is increasing in the following order: HPMP-5-azaC < cyclic HPMP-5-azaC < HPMP-5-azaC esters. From the view of prodrug development, the best chemical stability was observed in case of the double prodrug 7: the N ⁴-behenoyl derivative of the hexadecyloxyethyl ester of cyclic HPMP-5-azaC. The free phosphonic acid (N⁴-behenoyl-HPMPC) appeared to be a more potent and selective inhibitor of herpesvirus replication than the parent HPMPC derivative.

Full text of DOI:10.1016/j.bmc.2014.03.031

Accepted Manuscript
N 4-Acyl derivatives as lipophilic prodrugs of cidofovir and its 5-azacytosine
analogue, (S)-HPMP-5-azaC: Chemistry and antiviral activity
Marcela Krečmerová, Radek Pohl, Milena Masojídková, Jan Balzarini, Robert
Snoeck, Graciela Andrei
PII:
S0968-0896(14)00216-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.03.031
BMC 11478
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 December 2013
24 February 2014
18 March 2014
Please cite this article as: Krečmerová, M., Pohl, R., Masojídková, M., Balzarini, J., Snoeck, R., Andrei, G., N 4-
Acyl derivatives as lipophilic prodrugs of cidofovir and its 5-azacytosine analogue, (S)-HPMP-5-azaC: Chemistry
and antiviral activity, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.03.031
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Graphical abstract:
N4-Acyl derivatives as lipophilic prodrugs of
cidofovir and its 5-azacytosine analogue, (S)-HPMP-
5-azaC: Chemistry and antiviral activity
Marcela Krečmerováa,*, Radek Pohla, Milena Masojídkováa, Jan Balzarinib, Robert Snoeckb, and
Graciela Andreib
aInstitute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i.,
Flemingovo nám. 2, CZ-166 10, Prague 6, Czech Republic; bRega Institute for Medical Research, KU
Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
1

marcela@uochb.cas.cz; mailto: graciela.andrei@rega.kuleuven.ac.be
*Corresponding author: Tel.: +420 220183475; fax: +420 220183560; e-mail: marcela@uochb.cas.cz
___________________________________________________________________________________
Abstract: Even number fatty acid residues - docosanoyl (behenoyl) and stearoyl were selected for
introduction to the N4-position of (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine) (HPMPC,
cidofovir), and its 5-azacytosine counterpart, (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine)
(HPMP-5-azaC) with the aim to prepare a new type of lipophilic prodrugs. The study on the influence
of these modifications to the stability and biological activity of both antivirals was performed. Different
reactivity of both systems towards acylation reactions was also found: the 4-NH2 group of cidofovir was
more reactive compared to that of HPMP-5-azaC. In 5-azacytosine derivatives, we found mostly a
destabilizing effect of the N4-acylation but this could be compensated by a positive influence of the
esterification of the phosphonate group. Chemical stability of the 5-azacytosine moiety in the HPMP
series is increasing in the following order: HPMP- 5-azaC < cyclic HPMP-5-azaC < HPMP-5-azaC
esters. From the view of prodrug development, the best chemical stability was observed in case of the
double prodrug 7: the N4-behenoyl derivative of the hexadecyloxyethyl ester of cyclic HPMP-5-azaC.
The free phosphonic acid (N4-behenoyl-HPMPC) appeared to be a more potent and selective inhibitor of
herpesvirus replication than the parent HPMPC derivative.
________________________________________________________________________________
Keywords: Acyclic nucleoside phosphonates; Cidofovir; 5-Azacytosine; HPMP-5-azaC; Prodrug;
Phosphonate Ester ; Antivirals
1. Introduction
Cidofovir (HPMPC, VistideTM), (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine), a
compound developed originally at the Institute of Organic Chemistry and Biochemistry in Prague,
belongs to the most effective anti-DNA virus agents available (Fig. 1).1 The compound was approved
for the treatment of HCMV retinitis in AIDS patients in the form of intravenous injections.2 “Off
2

label” cidofovir is also used as a topical gel in the treatment of acyclovir-resistant HSV and for the
treatment of human papilloma virus (HPV) and poxvirus infections.3 On the other hand, the low
bioavailability of cidofovir and its side-effects, especially nephrotoxicity, remain the limiting factors
in its widespread use. To improve its pharmacological properties, the main attention is currently paid
to the development of its prodrugs, especially alkoxyalkyl esters. One of them, the HDP
(hexadecyloxypropyl) ester, i.e. CMX001, was advanced to clinical trials by Chimerix Inc.4 The
randomized double-blind placebo controled study, CMX001-102 in healthy volunteers recently has
concluded that CMX001 is orally bioavailable and can be well tolerated up to a dose of 140 mg in
adults. This is the first demonstration of the use of phospholipid conjugation technology to achieve
plasma drug concentrations that are expected to result in activity against multiple double-stranded
DNA viruses.5
In our group, the search for new cidofovir derivatives was investigated in two directions: substitutions
in the cytosine base (N4-alkyl derivatives6, C-5 alkyl/aryl derivatives7) and substitution of the cytosine
moiety by a triazine conterpart.8 Other data published on the modification of the cytosine moiety are
rather scarce in literature.9 While the first two substitutions lead to a complete loss of antiviral activity,
preparation of a triazine (5-azacytosine) analogue of cidofovir, HPMP-5azaC, opened the way to a
new class of acyclic phosphonate analogs that are extraordinary active against a variety of DNA
viruses with decreased toxicity compared to cidofovir.8
Figure 1
In contrast to cidofovir, HPMP-5-azaC has a more complicated metabolic profile due to its chemical and
enzymatic instability. In aqueous solutions ring opening between C-6 and N-1 of the triazine moiety
occurs
and
HPMP-5-azaC
is
successively
degraded
to
2-{[(2S)-3-hydroxy-2-
(phosphonomethoxy)propyl]carbamoylguanidine. This final decomposition product has no cytotoxicity
in vitro but is antivirally inactive. The hydrolytic degradation of the 5-azacytosine moiety is initiated by
3

the nucleophilic attack of a hydroxyl ion in position 6 whose electron density is much lower compared
to cytosine. On the other hand, we found a large difference between hydrolytic instability of the 5-
azacytosine free phosphonic acids and their corresponding esters. While HPMP-5-azaC is degraded
rapidly under slightly basic (physiological) or neutral conditions, a targeted degradation of its esters
(e.g. the diisopropyl ester) represents a several days lasting process requiring treatment with
concentrated ammonia solution.10 A stabilizing effect of the ester groups was observed also in a series
of cyclic phosphonate esters of HPMP-5-azaC. Generally, in the HPMP series, the stability of the 5-
azacytosine ring was found to decrease in the following order: esters of cyclic HPMP-5-azaC > cyclic
HPMP-5-azaC > HPMP-5azaC. A detailed study of chemical degradation of diverse 5- acyclic
azacytosine nucleoside phosphonates at different pH values was performed using NMR spectroscopy.11
Besides chemical decomposition, HPMP-5-azaC undergoes also enzymatic deamination in cell culture,
similarly like cytosine derivatives.12
To improve the stability towards the deamination process we tried to transform HPMP-5azaC in diverse
N4-acyl prodrugs on the level of free phosphonic acids as well as on the level of some ester prodrugs.
As appropriate acyl groups we selected even number fatty acid residues (e.g. docosanoyl, stearoyl).
Corresponding N4-acyl derivatives were prepared also from HPMPC (cidofovir) and its esters. Thus far,
the most important example of the use of N4-acyl derivatives in cytosine-based drugs is N4-behenoyl-1-
(β-D-arabinofuranosyl)cytosine.13 The compound was developed as a lipophilic prodrug of the
commonly known cytostatic cytosine arabinoside (araC) drug.
Different reactivity of both systems towards the acylation process and the influence of N4- acyl groups
on the stability and biological activity of both types of ANP (the cidofovir and its 5-azacytosine
analogue) will be also studied.
Acylation of 4-NH2 group in cytosine derivatives is known as a simple process generally used in nucleic
acid chemistry. In contrast, acylation of their 5-azacytosine counterparts is very difficult and it can be a
real synthetic challenge. As witnessed by a literature search, from the outset of 5-azacytosine chemistry
in 1960s (ref.14) there are only two references regarding this reaction. One of them concerns preparation
4

of N4-acetyl-5-azacytidine by nucleosidation reaction from acetylated 5-azacytosine and tri-O-benzoyl-
D-ribosyl chloride followed by deprotection of benzoyl groups. The mentioned compound was unstable
and spontaneously hydrolyzed to N-acetyl-N´-formyl-amidourea.15 The second hit is N4-phenoxyacetyl-
2´-deoxy-5-azacytidine described as a precursor in preparation of oligonucleotide analogues
incorporating 5-azacytosine.16 To understand the different reactivity of both systems, it is necessary to
realize the fact that in aromatic amines, electrons are always withdrawn from the amino group into the
aromatic ring with a strong decrease of basicity in the NH2 group. In pyrimidine systems, e.g. in
cytosine, it is primarily nitrogen atom N-3 that serves as the electron acceptor. As a consequence, this
nitrogen becomes more basic than the –NH2 group and is the major site of protonation. In 5-azacytosine,
the presence of an additional nitrogen atom in the ring (N-5) thus causes further decrease of basicity of
NH2 group. Therefore, the reactivity of 5-azacytosine amino group towards acylation is much lower
compared to 4-NH2 group of cytosine. Besides this complication, we had to take in account also the fact
that acylation of 5-azacytosine 4-NH2 group decreases electron density on C-6 which can contribute to
destabilization of the ring. From our previous studies, we know that the factor playing a key role in
destabilization of the ring is a presence of free hydroxyl group(s) in a molecule. It concerns both
nucleosides and acyclic derivatives including acyclic nucleoside phosphonates.11 Moreover, also
hydroxy groups of the phosphonate function were found to influence stability of the ring opening as
witnessed by higher stability of cyclic phosphonates and esters compared to a free phosphonic acid.10,11
Considering all these facts, we came to a conclusion that synthesis of some types of satisfactory stable
N4-acylated 5-azacytosine derivatives as prodrugs could be realizable, at least on a level of cyclic
phosponates or phosphonate esters.
2. Chemistry
For the study of the N4-acylation of HPMP-5-azaC derivatives we selected its trityl protected
diisopropyl ester 1, an intermediate easy to obtain in synthesis starting from (2S)-2-
[(trityloxy]methyl]oxirane.8 This compound is hydrolytically stable; moreover, as mentioned above,
5

targeted ring-opening in it is rather difficult, requiring harder conditions.10 Introduction of fatty acid
acyl groups was performed by reaction of 1 with appropriate even number acyl chlorides in pyridine:
docosanoyl (behenoyl) chloride and stearoyl chloride. In both cases the desired N4-acyl derivatives 2
and 3, were formed exclusively in good preparative yield (Scheme 1). Preservation of the triazine ring
is proven by the presence of typical H-6 signals in the 1H NMR spectrum (singlet at 9.18 or 9.45 ppm,
respectively) and C-6 in the 13C NMR spectrum (163.30 ppm, 161.10 ppm). Interestingly, in contrast
to 5-azacytosine compounds with a free amino group, the molecular peaks in the mass spectra of 2 and
3 are very small while the main peaks belong to their corresponding adduct with water (M+H2O)
whose supposed structure is shown in Fig. 2. This structure can be understood as the first step leading
to decomposition of a triazine ring before its opening to a N-formyl derivative.11 It witnesses a
decreased stability of both derivatives, even during the ionization process in MS.
Scheme 1
Figure 2
Deprotection of the ester and trityl groups was performed with bromotrimethylsilane in acetonitrile
under standard conditions used for acyclic nucleoside phosphonates.6-8 In contrast to this procedure
applied to 1 and leading successfully to HPMP-5-azaC, the reaction starting from N4-behenoyl
derivative 2 afforded a free phosphonic acid containing always a degraded triazine ring 4. A similar
result was found at the N4-stearoyl derivative 3. In this case we tried first to quickly remove a trityl
group under mild conditions using trifluoroacetic acid, still before deprotection of the ester groups.
Unfortunately, decrease of electron density at the C-6 by acyl groups was found so critical, that,
instead of a detritylated 5-azacytosine phosphonate, we obtained only the fully protected open-ring
derivative 5, together with a small amount of its detritylated counterpart 6 (Scheme 1). The ring-
opening reaction proved to be the fastest reaction step. Preparation of N4-acyl derivatives of HPMP-5-
azaC was thus found practically impossible without parallel ring-opening reactions.
6

The situation was slightly better when the free HPMP-5-azaC was substituted by its cyclic form
(cHPMP-5-azaC) or substantially improved when some ester prodrugs of the cyclic form, e.g. the
alkoxyalkyl ester of cHPMP-5-azaC, was used. These facts are in agreement with previously observed
increased stability of cyclic forms11. Increased stability of the cyclic HPMP-5-azaC compared to
HPMP-5-azaC is done by the absence of a free hydroxy group in the aliphatic chain which was found
to participate in the ring-opening reaction. In our previous study of decomposition of acyclic 5-
azacytosine nucleoside analogs we showed decomposition of HPMP-5-azaC as a process involving
formation of the N-formiate intermediate which was not observed in the decomposition reaction of
other derivatives. The explanation for this different reaction pathway is that the intermediary
formylamido derivative can react in a transformylation reaction with a spatially close hydroxy group to
form an O-formiate (Scheme 2). As a result, we can say that triazine can serve as a formylating agent
when a free hydroxyl group is in a suitable close spatial arrangement.11
Scheme 2
Acylation of the alkoxyalkyl (hexadecyloxyethyl) ester of the cyclic HPMP-5-azaC with behenoyl
chloride proceeded without any difficulties to give compound 7, the derivative designed as a double
prodrug of HPMP-5-azaC (Scheme 3). The aim of its synthesis was to prove an influence of the
introduction of an additional lipophilic chain to the antiviral activity: the starting HDE cHPMP-5-azaC
is known as a compound with extraordinary activity against a broad variety of of DNA viruses.17
Acylation of free cHPMP-5-azaC using stearoyl chloride was more or less successful, affording the
desired N4-substituted derivative 8. Unfortunately, this product is not sufficiently stable and, as
witnessed by NMR spectra, it is accompanied also by two decomposition products. Both by-products
are cHPMP-5-azaC bearing an opened 5-azacytosine moiety: one of them still containing stearamide
moiety, one of them containing a free 4-NH2 group. Open forms were deduced from missing C-6 in
13C NMR of both by-products.
7

Scheme 3
Similarly, N4-acyl compounds were prepared also from HPMPC (cidofovir) and some of its esters.
Different reactivity of both systems (cytosine and 5-azacytosine) was observed: cidofovir was always
much more reactive towards acylation reactions due to a higher basicity of its 4-NH2 group. Despite this
fact, direct acylation of a free cidofovir with behenoyl (or stearoyl) chloride was not successful. It is
caused by its total insolubility in solvents usual for acylations (pyridine, acetonitrile, etc.) due to a
highly polar character of a phosphonic acid residue. A more advantageous synthetic route was started
from the fully protected phosphonate ester 9 using either carbonyldiimidazol mediated acylation with
behenic acid, or classic acylation using acyl (behenoyl) chloride in pyridine (Scheme 4). Deprotection of
the trityl group with trifluoroacetic acid, followed by the treatment with bromotrimethylsilane gave the
free phosphonic acid (N4-behenoyl-HPMPC) 12 or its monoisopropylester 13, dependent on the reaction
conditions: the reaction performed at 0°C afforded selectively monoester 13. To preserve the N4-acyl
group during work-up and the purification process, utilization of Dowex 1 in CH3COO- form, an anion
exchanger used commonly for purification of ANPs must be strictly avoided.
The different reactivity of cytosine vs 5-azacytosine base moiety was observed also in preparation of
other types of prodrugs, e.g. pivaloyloxymethyl (POM) derivatives. Whereas cHPMP-5-azaC treated
with chloromethyl pivalate afforded exclusively the POM ester of cHPMP-5-azaC17, an analogous
reaction performed with cyclic cidofovir afforded its POM ester bearing additionally the N4-pivaloyl
group 14 as a main reaction product (Scheme 5). This additional N4 acylation is caused the strongly
basic character of the cytosine N4-amino group. Despite the fact that formation of the POM derivative of
cHPMPC 15 is mentioned briefly in a patent, the course of reaction, yields or formation of other
products are not described and full characterization of the product(s) is there also missing.18 Anyway,
promising antiviral activities of both POM derivatives, along with a low toxicity, moved us to include
compounds 14 and 15 to this study and make comparison with their long-chain acylated counterparts.
8

3. Antiviral activity
The antiviral activity of the N4-acyl derivatives was evaluated against various DNA viruses, including
different herpesviruses [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), and human
cytomegalovirus (HCMV) (Table 1). The compounds were also evaluated against several poxviruses,
encompassing vaccinia virus (VACV), cowpox virus, camelpox virus and orf virus. Reference
antiherpesvirus drugs (i.e. acyclovir, ganciclovir and brivudin) as well as cidofovir (HPMPC), HPMP-5-
azaC and their cyclic forms were included in the antiviral and cytostatic assays. The N4-acyl derivatives
evaluated for their antiviral properties included compounds 7 [HDE ester (N4-behenoyl-cHPMP-5-
azaC)], 12 [free phosphonic acid (N4-behenoyl-HPMPC), 13 [monoisopropylester (N4-behenoyl-
HPMPC)] and 14 [POM ester (N4-pivaloyl-cHPMPC)] as well as the POM ester of cHPMPC
(compound 15). Both cyclic phosphonates 14 and 15 were tested as diastereoisomeric mixtures.
Compound 12 emerged as the most potent anti-HCMV compound evaluated in the present study with
20-30 fold lower EC50 values compared to HPMPC and an >11- to >17-fold gain in selectivity. In the
case of HSV and VZV, the free phosphonic acid (N4-behenoyl-HPMPC) was only 2- to 4.6-fold (HSV)
and 4- to 6-fold (VZV) more active than the parent compound HPMPC. The monoisopropylester (N4-
behenoyl-HPMPC), i.e. compound 13, proved to be 3- to 7-fold (HCMV) and 2- to 8-fold (VZV) less
active than the free phosphonic acid (N4-behenoyl-HPMPC). In the case of HSV, the difference in
activity between these N4-behenoyl-HPMPC derivatives was not higher than 2-fold. The activity of the
HDE ester (N4-behenoyl-cHPMP-5-azaC) did not significantly differ from the parent compound
cyclicHPMP-5-azaC among the different herpesviruses.
When the N4-acyl derivatives and the POM ester (cyclic-HPMPC) were evaluated against different
poxviruses, their activities were not markedly different from those of the parent drugs, with a maximum
of 4-fold decrease in EC50 value for compound 13 compared to HPMPC against the orf virus.
9

Table 1. Antiviral activity of some N4-acyl derivatives against herpesviruses in HEL cells compared to cidofovir, HPMP-5-azaC and
their cyclic forms as well as to reference antiherpesvirus drugs.
Antiviral activity: EC50 (µM)a
Cytotoxicity (µM)
(Selectivity index: ratio CC50 to EC50)
VZV
Herpes simplex virus
HSV-2
HSV-1 TK–
Cell
morphology
Human cytomegalovirus
Cell growth
Compounds
TK+ strains
TK– strains
HSV-1
KOS
c
YS
OKA
1.21 0.18
>97
07/1
YS/R
0.36 0.14
>327
G
KOS ACVr
1.2 0.2
>100
AD-169
Davis
0.16 0.10
>737
MCCb
20
CC50
0.52 0.09
>225
0.30 0.13
>384
1.4 0.2
>83
1.4 0.3
>83
0.21 0.04
>570
>117
>166
7
0.04 0.01
>4162
0.33
0.13 0.27
>1241
0.02 0.02
>6961
0.013 0.003
>12973
0.08
0.33 0.71
>499
0.33 0.71
>499
0.33 0.71
>499
0.027 0.009
>6243
0.046 0.079
>3632
20
12
≥31
0.27 0.31
>584
0.05
0
0.47 0.22
>333
0.93 0.44
>166
0.70 0.11
>222
0.20 0.15
>768
0.15 0.05
>1050
>155
13
14
>475
>3219
0.11 0.08
>885
>1996
0.04
0.15 0.07
>680
N.D.
1.4 0.8
>71
0.87
>115
0
0.98 0.15
>102
0.48 0.05
>211
0.24
>100
>100
>357
>381
≥1270
>336
>1776
≥1200
≥1210
>100
>2500
0.2
>417
0.036 0.030
1296
0.023 0.023
2044
N.D.
1.6 0.38
29
0.93 0.19
49
2.1
22
0
0.42
110
0
0.4
46
15
230
115
266 ± 251
254 ± 155
298 207
247 184
≥1123 ± 406
≥842 377
355 ± 392
0.22 0.18
1232
0.35 0.28
753
0.42 0.41
634
0.09
2981
N.D.
0.80 0.45
333
0.94 0.39
283
0.88 0.45
301
0.42 0.17
629
0.65 0.64
411
HPMP-5-azaC
cHPMP-5-azaC
HPMPC
cHPMPC
Acyclovir
Brivudin
Ganciclovir
0.35
0.52 0.37
0.39 0.30
1.3 0.9
1.7 1.4
1.4 0.7
0.20 0.15
0.27 0.13
732
485
656
202
146
178
1241
954
0.18 0.19
0.30 0.30
0.06 0.08
0.06
4693
1.5 1.1
2.1 1.2
1.4 1.0
0.81 0.34
0.92 0.64
1632
0.43 0.21
580
1002
0.54 0.37
458
4880
0.34 0.37
731
198
2.0 1.2
124
143
2.7 1.6
92
207
1.8 1.3
139
368
0.62 0.39
397
325
0.93 0.36
267
0.13
1835
3.3 1.7
≥338
2.4 1.5
≥465
53 31
≥21
62 35.8
≥18
0.20 0.12
≥5668
0.34 0.09
≥3326
57 35.6
≥20
N.D.
N.D.
0.025 0.029
≥33815
N.D.
0.019 0.016
≥44362
N.D.
95.5 60.0
≥9
140 17
≥6
0.028 0.004
≥30618
121 78
≥7
35 25
≥24
N.D.
N.D.
N.D.
N.D.
0.070 0.067
0.060 0.058
4.2 3.5
8.3 4.7
7.1 3.7
5071
5917
85
43
50
a Effective concentration required to reduce virus-induced cytopathicity by 50%; b Minimum cytotoxic concentration required to cause a
microscopically detectable alteration of cell morphology; c Cytotoxic concentration required to reduce cell growth by 50%; N.D. not determined.
11

Table 2. Antiviral activity of some N4-acyl derivatives against herpesviruses in HEL cells compared to cidofovir and HPMP-5-azaC
Antiviral activity: EC50 (µM)a
Cytotoxicity (µM)
(Selectivity index: ratio CC50 to EC50)
Cell
Vaccinia virus
Lederle
Cowpox virus
Camelpox virus
Orf virus
Cell growth
morphology
Compounds
c
WR
Brighton
N.D.
CML-1
CML-14
N.D.
NZ2
N.D.
MCCb
CC50
8.2 3.3
>14
N.D.
N.D.
≥23
33
>117
>166
7
2.7 1.8
>63
10
7
5.5 3.1
>30
2.6 1.5
>65
0.53 0.88
>312
12
>16
>24
5.2 1.8
>30
6.2
0
5.2 1.5
>30
5.5 2.0
>28
2.3 0.7
>67
0.25
≥31
>155
13
14
>25
7.2 5.3
>14
>624
16 15
>6
8.1 6.5
>12
11 7.1
>9
5.7 4.3
>18
1.0
>100
>100
>357
>381
≥1270
>336
>100
>98
12
10 0.6
4
13 1.8
4
13 13.8
3
7.9 5.3
6
0.83
46
15
4
57
5.8 1.2
46
9.0
29
0
12 3.7
23
7.2 4.1
37
5.9 2.5
45
0.88 0.32
302
266 ± 251
254 ± 155
298 207
247 184
HPMP-5-azaC
cHPMP-5-azaC
HPMPC
cHPMPC
7.6 5.4
7.9 0.6
12 4.0
8.1 4.2
4.9 2.4
0.7 0.5
33
32
20
32
52
360
9.0 6.1
14 5.2
16 3.7
8.0 3.8
6.1 4.1
1.0 0.6
33
21
18
15 2.9
16
37
8.9 5.3
28
49
4.8 2.6
52
290
1.1 0.7
221
12
21
5
16
16
8
a Effective concentration required to reduce virus-induced cytopathicity by 50%; b Minimum cytotoxic concentration required to cause a microscopically
detectable alteration of cell morphology; c Cytotoxic concentration required to reduce cell growth by 50%; N.D. not determined.
12

4. Conclusions
N4-Acyl derivatives as lipophilic prodrugs of cidofovir and its 5-azacytosine analogue (S)-HPMP-5-
azaC have been prepared and their antiviral properties against herpes and poxviruses were evaluated in
vitro. The free phosphonic acid (N4-behenoyl-HPMPC) proved to be more potent and selective than its
parent compound HPMPC against herpesviruses. Although the monoisopropylester (N4-behenoyl-
HPMPC) was less active than the free phosphonic acid (N4-behenoyl-HPMPC), it inhibited herpesvirus
replication with lower EC50 values than HPMPC. The potencies of the POM derivatives of cHPMPC
and of N4-pivaloyl-cHPMPC as well as that of the HDE ester with 5-azaC moiety (N4-behenoyl-
cHPMP-5-azaC ester, 7) were not strikingly different than those of the parent compounds. Also the
activities and selectivities of the N4-acyl derivatives and the POM ester of cyclic-HPMPC against
poxviruses were not markedly different compared to their respective parent drugs. In the 5-azacytosine
series, N4-acyl derivatives of free phosphonic acids (acyclic or cyclic) revealed as not appropriate
prodrugs due to their low stability towards degradation of a triazine ring.
5. Experimental
5.1. General
Unless stated otherwise, solvents were evaporated at 40 °C/2 kPa and 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 text. Column chromatography was performed on silica gel 60 µm (Fluka). Preparative
reverse phase HPLC separations were performed on a Waters Delta 600 instrument with a Waters
®
2487 Dual λ Absorbance Detector using a column Luna Phenomenex C-18 (10 µm, 10x150 mm),
flow 5 mL/min. Optical rotations were measured on Autopol IV polarimeter (Rudolph Research
13

1
13
31
Analytical, U.S.A.) at 20 °C, [ ]D values are given in 10-1 deg cm2 g-1. H, C and P NMR spectra
were measured on a Bruker Avance 500 spectrometer (1H at 500.0 MHz, 13C at 125.7 MHz and 31P at
202.3 MHz) in CD3OD, d6-DMSO or CDCl3. The spectra were referenced to TMS or residual solvent
system (δ 3.31 and 49.0 for CD3OD, δ 77.0 for CDCl3, 2.5 and 39.7 for d6-DMSO). 31P NMR spectra
were referenced to H3PO4 as an external standard. The assignment of chemical shifts was done with
the help of 2D NMR experiments (H,H-COSY, H,C-HSQC and H,C-HMBC). Diastereoisomers of
31
cyclic phosphonates were distinguished by their characteristic P chemical shifts and by comparing
H,H; H,P and C3´,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.17. Mass spectra were measured on LCQ classic instrument using
electrospray ionization (ESI) or ZAB-EQ (VG Analytical) spectrometer using FAB (ionization with
xenon, accelerating voltage 8 kV, glycerol matrix). Most of chemicals and ion-exchange resins
(Dowex 1X2-400) were purchased from Sigma-Aldrich.
5.2.
1-{(2S)-2-[(Diisopropoxyphosphoryl)methoxy-3-(triphenylmethoxy)]propyl}-N4-docosa-
noyl-5-azacytosine (2). Behenoyl chloride (1 g, 2.8 mmol) was added to a solution of 1 (900 mg,
1.5 mmol) in pyridine (5 mL), the mixture stirred for 2 days at room temperature and quenched by
addition of methanol (1 mL). The solution was evaporated, the residue coevaporated with toluene
and partitioned between water and ethyl acetate (150 mL each). The organic layer was dried over
sodium sulfate, evaporated and chromatographed on a column of silica gel (200 mL) in system
_
toluene – ethyl acetate (1:1). Yield: 750 mg (55 %) of 2 as a white foam. [α]D 11.9 (c 0.298, CHCl3).
1H NMR (CDCl3, ppm) δ: 0.89 (t, 3H, JCH3,CH2 = 7.0, CH3), 1.28 (m, 16H, CH2), 1.69 (m, 2H, CH2),
2.46 (t, 2H, JCH2,CH2 = 7.6, CH2), 1.33, 1.34, 1.37 and 1.38 (4xd, 12H, JCH3,CH = 6.1, CH3), 4.68 (m,
1H, P-OCH), 4.78 (m, 1H, P-OCH), 3.30 (dd, 1H, JP,CHa = 11.1, Jgem = 13.2, PCHa), 3.81 (dd, 1H,
JP,CHb= 9.7, Jgem = 13.2, PCHb), 3.10 (dd, 1H, J3´a,2´ = 2,5, Jgem = 13.1, H-3´a), 3.56 (dd, 1H, J3´b,2´
=
14

4,0, Jgem = 13.1, H-3´b), 4.11 (dd, 1H, J1´a,2´ = 7.2, Jgem = 14.8, H-1´a), 4.21 (dd, 1H, J1´b,2´ = 3,3, Jgem
= 13.1, H-1´b), 4.24 (m, 1H, H-2´), 7.21 (t, 3H, H-arom.), 7.27 (t, 6H, H-arom.), 7.41 (d, 6H, H-
arom.), 9.18 (s, 1H, H-6), 10.90 (bs, 1H, NH). 13C NMR (CDCl3, ppm) δ: 13.80 (CH3), 22.37 (CH2),
23.54 (d, JP,C = 4.4, CH3), 23.72 (d, JP,C = 3.4, CH3), 23.76 (d, JP,C = 4.4, CH3), 23.92 (d, JP,C = 3.4,
CH3), 24.50, 28.92, 29.03, 29.04, 29.19, 29.30 (2H), 29.38 (10H), 31.60 and 36.80 (CH2), 41.31 (C-
1´), 65.60 (JP,C = 165.8, P-C), 66.14 (C-3´), 70.41 (JP,C = 6.4, P-O-C), 71.41 (JP,C = 6.4, P-O-C),
79.08 (d, JP,C = 9.8, C-2´), 86.97 (Tr), 126.68 (3C, Tr), 127.71 (6C, Tr), 128.37 (6C, Tr), 143.51 (3C,
Tr), 159.14 (C-4), 162.76 (C-2), 163.30 (C-6), 176.87 (C=O). 31P{1H} NMR (CDCl3, ppm) δ: 19.57.
ESI MS, m/z: 969.6 [M.H2O+Na]+ (100), 951.7 [M+Na]+ (16), 947.2 [M.H2O+H]+ (7), 929.3 [M+H]+
(4). Anal. Calcd. for C54H81N4O7P. H2O: C, 68.47; H, 8.83; N, 5.91; P, 3.27. Found: C, 68.51; H,
9.02; N, 5.81; P, 3.56.
5.3. 1-{(2S)-2-[(Diisopropoxyphosphoryl)methoxy-3-(triphenylmethoxy)]propyl}-N4-octadeca-
noyl-5-azacytosine (3). The starting compound 1 (1.5 g, 2.5 mmol) was treated with stearoyl
chloride (1.2 mL, 3.5 mmol) in pyridine (5 mL) and the mixture worked up analogously as described
for 2. Chromatography of the crude product in system toluene – ethyl acetate (2:1) gave 1.82 g (83
_
1
%) of 3 as a yellowish syrup. [α]D 11.1 (c 0.225, CHCl3). H NMR (DMSO-d6, ppm) δ: 084 (t, 3H,
JCH3,CH2 = 7.0, CH3), 1.21 (overlay, 12H, CH3), 1.21 (m, 12 H, CH2), 1.54 (m, 2H, CH2), 2.38 (t, 2H,
JCH2,CH2 = 7.6, CH2), 2.99 (dd, 1H, J3´a,2´ = 5.6, Jgem = 10.8, H-3´a), 3.05 (dd, 1H, J3´b,2´ = 2.8, Jgem
=
10.8, H-3´b), 3.57 (dd, 1H, J1´a,2´ = 5.7, Jgem = 12.6, H-1´a), 3.78 (dd, 1H, J1´b,2´ = 6.2, Jgem = 12.6, H-
1´b), 3.80 (d, 2H, JP,CH = 9.0, PCH2), 4.56 (m, 1H, H-2´), 4.60 (m, 2H, P-OCH), 7.24 (t, 3H, H-
arom.), 7.29 (t, 6H, H-arom.), 7.39 (d, 6H, H-arom.), 9.45 (s, 1H, H-6), 10.71 (s, 1H, NH). 13C NMR
(DMSO-d6, ppm) δ: 14.07 (CH3), 22.23 (CH2), 23.78 (d, JP,C = 4.6, CH3), 23.82 (d, JP,C = 4.6, CH3),
23.93 (d, 2C, JP,C = 3.6, CH3), 24.38, 28.79, 29.13 (2C), 29.15 (9C), 31.41 and 36.29 (CH2), 46.20
(C-1´), 64.12 (d, JP,C = 164.6, P-C), 64.36 (C-3´), 70.33 (d, 2C, JP,C = 6.4, P-O-C), 78.56 (d, JP,C
=
11.4, C-2´), 86.32 (Tr), 127.11 (3C, Tr), 127.93 (6C, Tr), 128.37 (6C, Tr), 143.87 (3C, Tr), 158.19
15

31
(C-4), 161.10 (C-2), 161.78 (C-6), 176.79 (C=O). P{1H} NMR (CDCl3, ppm) δ: 19.62. ESI MS,
m/z: 1802.9 [2(M.H2O)+Na]+ (56), 913.4 [M.H2O+Na]+ (100), 895.4 [M+Na]+ (10), 890.9
[M.H2O+H]+ (3). HRMS (ESI): For C50H73N4O7PNa (MNa)+ calculated: 895.511460; found:
895.512663. Anal. Calcd. for C50H73N4O7P: C, 68.78; H, 8.43; N, 6.42; P, 3.55. Found: C, 68.71; H,
8.57; N, 6.20; P, 3.63.
(S,E)-(8-Amino-3-(hydroxymethyl)-6,10-dioxo-2-oxa-5,7,9-triazahentriacont-7-en-1-
yl)phosphonic acid (4). A solution of 2 (204 mg, 0.22 mmol) in acetonitrile (10 mL) was kept in
dark with bromotrimethylsilane (0.4 mL, 3 mmol) for 24 at room temperature and then evaporated.
The residue was coevaporated with toluene (2x 20 mL), washed with diethylether (to remove TrOH)
and crystallized from 96% ethanol. The crystalline product was filtered, washed with ethanol and
ether and dried in vacuo. Yield: 90 mg (68%) of a mixture of 4, octadecanoyl amide and final
1
13
degradation product of HPMP-5-azaC (according H and C NMR) as a white solid. -ESI MS, m/z:
1183.3 [2M-H]- (100), 591.3 [M-H]- (56). HRMS (FAB): For C28H58N4O7P (MH)+ calculated:
593.404314; found: 593.406317.
(S,E)-Diisopropyl {8-amino-6,10-dioxo-3-[(trityloxy)methyl]-2-oxa-5,7,9-triazaheptacos-7-en-1-
yl}phosphonate (5). 80% Trifluoroacetic acid (15 mL) was added to 3 (1.7 g, 2.0 mmol), the
mixture shaken shortly (3-5 min) in stoppered flask and evaporated. The residue was coevaporated
with toluene (50 mL) and then partitioned between ethyl acetate and saturated solution of sodium
bicarbonate (150 mL each). An organic layer was dried over sodium sulfate and evaporated. The
residue was chromatographed on a column of silica gel in ethyl acetate to afford 5 as a colorless oil
1
(RF 0.4). Yield: 590 mg (53%). [α]D _10.6 (c 0.324, CHCl3). H NMR (CDCl3, ppm) δ: 0.88 (t, 3H,
JCH3,CH2 = 7.0, CH3), 1.26 (m, 12H, CH2), 1.315 (d, 3H, JCH3,CH = 6.1, CH3), 1.32 (d, 3H, JCH3,CH
=
6.1, CH3), 1.33 (d, 3H, JCH3,CH = 6.1, CH3), 1.34 (d, 3H, JCH3,CH = 6.1, CH3), 1.65 (m, 2H, CH2), 2.33
(m, 2H, CH2), 3.17 (dd, 1H, J3´a,2´ = 5.4, Jgem = 10.2, H-3´a), 3.21 (dd, 1H, J3´b,2´ = 4.3, Jgem = 10.2,
H-3´b), 3.24 (ddd, 1H, J1´a,2´ = 4.4, J1´a,NH = 7.9, Jgem = 14.0, H-1´a), 3.50 (ddd, 1H, J1´b,2´ = 3.40,
16

J1´b,NH = 6.90, Jgem = 14.0, H-1´b), 3.67 (m, 1H, H-2´), 3.76 (dd, 1H, JP,CHa = 8.6, Jgem = 13.8, PCHa),
3.96 (dd, 1H, JP,CHb= 8.1, Jgem = 13.8, PCHb), 4.75 (m, 2H, P-OCH), 6.10 (br, 1H, NH), 7.24 (t, 3H,
H-arom.), 7.29 (t, 6H, H-arom.), 7.44 (d, 6H, H-arom.), 8.50 (br, 2H, NH2), 13.00 (br, 1H, NH). 13C
NMR (CDCl3, ppm) δ: 14.11 (CH3), 22.66 (CH2), 23.95 (d, JP,C = 4.6, CH3), 24.03 (d, JP,C = 4.9,
CH3), 24.035 (d, JP,C = 3.6, CH3), 24.10 (d, JP,C = 3.9, CH3), 24.83, 29.04, 29.24, 29.34, 29.41,
29.56, 29.61, 29.64 (2C), 29.67 (4C), 31.90 and 37.98 (CH2), 41.45 (C-1´), 63.85 (C-3´), 65.37 (d,
JP,C = 167.7, P-C), 71.02 (d, 2C, JP,C = 6.7, P-O-C), 71.33 (d, JP,C = 6.5, C-2´), 86.84 (Tr), 127.05
(3C, Tr), 127.83 (6C, Tr), 128.61 (6C, Tr), 143.64 (3C, Tr). FAB MS, m/z: 886 [M+Na]+ (20), 864
[M+H]+ (1), 243 [(C6H5)3C]+ (100). Anal. Calcd. for C49H75N4O7P.1½ H2O: C, 66.12; H, 8.83; N,
6.29; P, 3.48. Found: C, 66.09; H, 8.77; N, 6.43; P, 3.78.
(S,E)-Diisopropyl [8-amino-3-(hydroxymethyl)-6,10-dioxo-2-oxa-5,7,9-triazaheptacos-7-en-1-
yl]phosphonate (6). The compound was eluted from the column as a more polar by-product (RF
0.25) after isolation of 5. Yield: 85 mg (7 %) as a white solid. 1H NMR (CDCl3, ppm) δ: unsufficient
quality of the spectrum for analysis. 13C NMR (CDCl3, ppm) δ: 14.10 (CH3), 24.00 (d, 4C, JP,C = 4.0,
4xCH3), 29.12, 29.27, 29.33, 29.42, 29.57, 29.67 (6C), 31.89 and 37.81 (CH2), 39.41 (C-1´), 60.81
(C-3´), 64.43 (d, JP,C = 169.15, P-C), 71.46 (d, JP,C) = 6.7, P-O-C), 71.48 (d, JP,C) = 6.7, P-O-C),
81.85 (d, JP,C = 7.7, C-2´). FAB MS, m/z: 643 [M+Na]+ (20), 621 [M+H]+ (25).
N-(5-{[(5S)-2-(2-(Hexadecyloxy)ethoxy)-2-oxido-1,4,2-dioxaphosphinan-5-yl]methyl}-4-oxo-4,5-
dihydro-1,3,5-triazin-2-yl)docosanamide (7). A solution of HDE ester of cHPMP-5-azaC17 (trans-
isomer, 200 mg, 0.38 mmol) in pyridine (4 mL) was stirred with behenoyl chloride (500 mg, 1.39
mmol) at room temperature for 24 h, followed by 2 h at 40°C. The mixture was evaporated, the
residue chromatographed on a column of silica gel (30 mL) in chloroform, followed by a gradient of
MeOH in chloroform (1-4 %). Yield: 120 mg (37 %) as a white solid. [α]D _5.9 (c 0.249, CHCl3).
17

1
The isolated compound was a single pure diastereoisomer (Fig. 3). H NMR (CDCl3, ppm) δ: 0.88
(m, 6H, CH3(CH2)14CH2, CH3(CH2)19CH2), 1.21-1.40 (m, 62H, CH3(CH2)13CH2CH2,
CH3(CH2)18CH2CH2), 1.59 (m, 2H, CH3(CH2)13CH2CH2), 1.62 (m, 2H, CH3(CH2)18CH2CH2), 2.42 (t,
2H, Jvic = 7.4, CH3(CH2)18CH2CH2), 3.49 (m, 2H, CH3(CH2)13CH2CH2), 3.64 (m, 1H, H-1´a), 3.68
(m, 2H, POCH2CH2), 3.87-3.95 (m, 3H, PCHa, H-1´b,2´), 4.16-4.30 (m, 4H, PCHb, H-3´a,
POCH2CH2), 4.37 (ddd, 1H, Jgem = 11.7, JP,3´b = 10.0, J3´b,2´ = 1.7, H-3´b), 9.13 (bs, 1H, NH), 9.59 (s,
1H, H-6). 13C NMR (CDCl3, ppm) δ: 14.11 (CH3(CH2)14CH2, CH3(CH2)19CH2), 22.68, 24.60, 26.06,
28.99, 29.23, 29.35, 29.40, 29.50, 29.57, 29.60, 29.65, 29.69, 31.92 (CH3(CH2)14CH2,
CH3(CH2)19CH2), 37.72 (CH3(CH2)19CH2), 39.16 (C-1´), 63.75 (d, JP,C = 145.1, P-C), 64.76 (d, JP,C
=
6.7, POCH2CH2), 69.56 (d, JP,C = 5.4, POCH2CH2), 71.54 (CH3(CH2)14CH2), 73.04 (d, JP,C = 8.3, C-
3´), 74.13 (d, JP,C = 5.2, C-2´), 158.13 (C-4), 162.31 (C-2), 164.42 (C-6), 175.53 (C=O). -ESI MS,
m/z: 869.5 [M.H2O-H]- (100). HRMS (FAB): For C47H90N4O7P (MH)+ calculated: 853.654716;
found: 853.657447. HRMS (FAB): For C47H89N4O7PNa (M+Na)+ calculated: 875.636660; found:
875.635124. Anal. Calcd. for C47H89N4O7P: C, 66.16; H, 10.51; N, 6.57; P, 3.63. Found: C, 66.64; H,
10.23; N, 6.68; P, 3.68.
N-(5-{[(5S)-2-Hydroxy-2-oxido-1,4,2-dioxaphosphinan-5-yl]methyl}-4-oxo-4,5-dihydro-1,3,5-
triazin-2-yl)stearamide (8). A mixture of cHPMP-5-azaC17(100 mg, 0.38 mmol), 1M methanolic
tetrabutylammonium hydroxide (0.38 mL) and methanol (50 mL) was evaporated to dryness and the
residue coevaporated with pyridine. Pyridine (5 mL) was added, followed by stearoyl chloride (0.7
mL, 2 mmol) and the mixture stirred for 4 days at room temperature. Hexane (200 mL) was added,
the mixture stirred for 10 min and let to stand. Hexane layer was removed after its complete
separation. The operation with hexane was repeated still twice. The remaining product was purified
by a reverse phase HPLC in gradient of water - acetonitrile. Approprite UV absorbing fractions were
evaporated to give 8 as a white solid in yield 60 mg (30%). FAB MS, m/z: 551 [M+Na]+ (10). HRMS
(FAB): For C25H46N4O6P (MH)+ calculated: 529.315499; found: 529.316864. HRMS (FAB): For
18

C25H45N4O6PNa (M+Na)+ calculated: 551.297444; found: 551.295808. For records of 1H, 13C and 31P
NMR spectra see: Supporting information.
1-{(2S)-2-[(Diisopropoxyphosphoryl)methoxy-3-(triphenylmethoxy)]propyl}-N4-
docosanoylcytosine (10). Method A. Behenoyl chloride (1.13 g, 3.16 mmol) was added to a solution
of 9 (ref. 19, 1.33 g, 2.2 mmol) in pyridine (25 mL) and the mixture was stirred at room temperature for
2 days. Methanol (2 mL) was added to quench the reaction. The solution was evaporated, the residue
coevaporated with toluene and chromatographed on a column of silica gel (400 mL) in system ethyl
_
acetate – acetone – ethanol – water (36:6:1:1). Yield: 1.64g (80%) of 10 as a white foam. [α]D 18.0 (c
0.286, CHCl3). 1H NMR (CDCl3, ppm) δ: 0.88 (t, 3H, JCH3,CH2 = 7.0, CH3), 1.30 (m, 28 H, CH2), 1.68
(m, 2H, CH2), 2.49 (t, 2H, JCH2,CH2 = 7.6, CH2), 3.06 (dd, 1H, J3´a,2´ = 3.4, Jgem = 10.6, H-3´a), 3.43
(dd, 1H, J3´b,2´ = 3.2, Jgem = 10.6, H-3´b), 3.78 (dd, 1H, J1´a,2´ = 8.6, Jgem = 13.6, H-1´a), 3.84 (d, 2H,
JP,CH = 8.4, PCH2), 3.98 (m, 1H, H-2´), 4.33 (dd, 1H, J1´b,2´ = 3.2, Jgem = 13.6, H-1´b), 4.67 (m, 1H, P-
OCH), 4.74 (m, 1H, P-OCH), 7.23 (t, 3H, Tr), 7.30 (t, 6H, Tr), 7.44 (d, 6H, Tr), 7.65 (d, 1H, J5,6
=
7.4, H-5), 7.84 (d, 1H, J6,5 = 7.4, H-6), 10.2 (bs, 1H, NH). 13C NMR (CDCl3, ppm) δ: 14.03 (CH3),
22.58 (CH2), 23.92 (d, 2C, JP,C = 4.9, CH3), 23.96 (d, 2C, JP,C = 3.9, CH3), 24.91, 29.10, 29.26, 29.29,
29.43, 29.55 (2C), 29.60 (10C), 31.82 and 37.405 (CH2), 51.76 (C-1´), 61.79 (C-3´), 64.39 (d, JP,C
=
169.2, P-C), 70.77 (d, P-O-C), 71.06 (d, P-O-C), 78.48 (d, JP,C = 13.6, C-2´), 86.63 (Tr), 96.03 (C-5),
127.08 (3C, Tr), 127.84 (6C, Tr), 128.46 (6C, Tr), 143.38 (3C, Tr), 150.31 (C-6), 155.79 (C-2),
162.69 (C-4). ESI MS, m/z: 950.5 [M+Na]+ (29). Anal. Calcd. for C55H82N3O7P: C, 71.17; H, 8.90; N,
4.53; P, 3.34. Found: C, 71.03; H, 9.13; N, 4.68; P, 3.52.
Method B. A suspension of behenic acid (425 mg, 1.25 mmol) and 1,1´-carbonyldiimidazole (162
mg, 1 mmol) in acetonitrile (5 mL) was stirred for 30 min at room temperature. Compound 9 (200 mg,
0.33 mmol) was added. The mixture was refluxed for 4 h and evaporated. The residue was
chromatographed on a column of silica gel in system ethyl acetate – acetone – ethanol – water
(36:6:1:1) to give 10 as a white foam. Yield: 190 mg (62%).
19

1-{(2S)-2-[(Diisopropoxyphosphoryl)methoxy]-3-hydroxypropyl}-N4-docosanoylcytosine (11). A
mixture of 10 (1.3 g, 1.4 mmol) and 80% trifluoroacetic acid (10 mL) was stirred vigorously in a
stoppered flask for 5 min and evaporated. The residue was coevaporated with water (10 mL),
-
dissolved in another portion of water (50 mL) and neutralized with Dowex 1 in HCO3 form to pH 7.
Ethyl acetate was (150 mL) added, the mixture shaken well and Dowex 1 removed by suction. An
organic layer was dried over magnesium sulfate and evaporated. The residue was chromatographed on
a column of silica gel (100 mL) in system chloroform – methanol (9:1) to give 11 in yield 870 mg
_
(91%) as a white solid. [α]D 6.4 (c 0.234, CHCl3). 1H NMR (CDCl3, ppm) δ: 0.88 (t, 3H, JCH3,CH2
=
7.0, CH3), 1.21-1.35 (m, 48H, CH2, CH3), 1.66 (m, 2H,CH2), 2.46 (t, 2H, JCH2,CH3 = 7.6, CH2), 3.59
(dd, 1H, J3´a,2´ = 3.4, Jgem = 12.6, H-3´a), 3.67 (dd, 1H, J3´b,2´ = 5.5, Jgem = 12.6, H-3´b), 3.80 (dd, 1H,
JP,CHa = 8.8, Jgem = 12.6, PCHa), 3.85 (dd, 1H, JP,CHb = 8.8, Jgem = 12.6, PCHb), 3.87 (m, 1H, H-2´),
4.05 (dd, 1H, J1´a,2´ = 6.4, Jgem = 13.8, H-1´a), 4.17 (dd, 1H, J1´b,2´ = 3.6, Jgem = 13.8, H-1´b), 4.70 (m,
1H, P-OCH), 4.75 (m, 1H, P-OCH), 7.43 (d, 1H, J5,6 = 7.3, H-5), 7.77 (d, 1H, J6,5 = 7.3, H-6), 9.34
(bs, 1H, NH). 13C NMR (CDCl3, ppm) δ: 13.78 (CH3), 22.35 (CH2), 23.67 (d, 2C, JP,C = 4.9, CH3),
23.71 (d, 2C, JP,C = 3.9, CH3), 24.53, 29.02, 29.13, 29.20, 29.29, 29.32, 29.36 (2C), 30.15 (10C),
31.58 and 37.29 (CH2), 50.56 (C-1´), 59.57 (C-3´), 64.44 (d, JP,C = 169.6, P-C), 71.19 (d, JP,C = 6.4, P-
O-C), 71.29 (d, JP,C = 6.4, P-O-C), 79.88 (d, JP,C = 10.6, C-2´), 96.18 (C-5), 150.33 (C-6), 156.45 (C-
2), 162.40 (C-4), 173.36 (C=O). 31P{1H} NMR (CDCl3, ppm) δ: 20.17. ESI MS, m/z: 708.4 [M+Na]+
(53), 686.3 [MH]+ (100). Anal. Calcd. for C36H68N3O7P. H2O : C, 61.43; H, 10.02; N, 5.97; P, 4.40.
Found: C, 61.62; H, 10.09; N, 5.78; P, 4.70.
1-{(S)-N4-Docosanoyl-[3-hydroxy-2-(phosphonomethoxy)propyl]}cytosine (12). Method A.
Bromotrimethylsilane (1.3 mL, 10 mmol) was added at room temperature to a suspension of 11 (997
mg, 1.45 mmol) in acetonitrile (50 mL), the mixture stored in dark for three days and evaporated. The
20

residue was coevaporated with toluene (3x 30 mL). Methanol (50 mL) was added, the mixture
neutralized with ammonia to pH 7 and evaporated. The solid residue was crystallized from water,
washed with acetone, followed by diethyl ether and dried in vacuo. Yield: 380 mg (39 %) of 12 as a
white solid.
Method B. A suspension of monoester 13 (120 mg, 0.2 mmol) in acetonitril (3 mL) was treated with
bromotrimethylsilane (0.08 mL, 0.6 mmol) at room temperature for three days in dark. The further
work up was the same as in Method A. Yield: 40 mg (35 %) of 12 as a white solid. Mp. 180-185 °C
1
(methanol). H NMR (CD3OD, ppm) δ: 0.90 (t, 3H, JCH3,CH2 = 7.0, CH3), 1.29 (m, 36H, CH2), 1.60
(m, 2H, CH2), 2.25 (t, 2H, JCH2CH2 = 7.6, CH2), 3.54 (dd, 1H, JP,CHa = 9.4, Jgem = 12.6, PCHa), 3.66
(dd, 1H, JP,CHb = 10.6, Jgem = 12.6, PCHb), 3.68 (dd, 1H, J3´a,2´ = 3.8, Jgem = 12.4, H-3´a), 3.69 (m, 1H,
H-2´), 3.77 (dd, 1H, J3´b,2´ = 3.7, Jgem = 12.4, H-3´b), 3.92 (dd, 1H, J1´a,2´ = 7.8, Jgem = 13.8, H-1´a),
4.23 (dd, 1H, J1´b,2´ = 3.6, Jgem = 13.8, H-1´b), 7.41 (d, 1H, J5,6 = 7.3, H-5), 8.10 (d, 1H, J6,5 = 7.3, H-
6). 13C NMR (CD3OD, ppm) δ: 12.67 (CH3), 21.98, 24.33, 24.58, 28.44, 28.57, 28.68 (2C), 28.83,
29.08 (10C), 31.32 and 36.40 (CH2), 50.56 (C-1´), 60.15 (C-3´), 66.68 (d, JP,C = 157.2, P-C), 79.79 (d,
JP,C = 12.6, C-2´), 96.13 (C-5), 151.20 (C-6), 157.40 (C-2), 162.71 (C-4), 174.20 (C=O). 31P{1H}
NMR (CD3OD, t = 50°C, ppm) δ: 15.59. -ESI MS, m/z: 1201.4 [2M-H]- (16), 600.4 [M-H]- (100).
HRMS (-ESI): For C30H55N3O7P (M-H)- calculated: 600.3778; found: 600.3788. Anal. Calcd. for
C30H56N3O7P: C, 59.88; H, 9.38; N, 6.98; P, 5.15. Found: C, 61.23; H, 9.59; N, 6.71; P, 4.91.
N4-Docosanoyl-1-{(2S)-2-[(isopropoxyphosphoryl)methoxy]-3-hydroxypropyl}cytosine (13).
Bromotrimethylsilane (0.4 mL, 3 mmol) was added at 0 °C dropwise to a suspension of 11 (422 mg,
0.62 mmol) in acetonitrile (5 mL), the mixture kept in refrigerator overnight and evaporated. 0.5 %
NH4OH was added to a solid residue to neutralize the mixture to pH 7. The solid was filtered off,
washed with water, followed by acetone and diethyl ether and dried in vacuo. Yield: 210 mg (53 %)
_
of a white solid. Mp. 120 °C (CH3OH). [α]D 23.2 (c 0.241, CH3OH). 1H NMR (CD3OD, t = 40°C,
ppm) δ: 0.90 (m, 3H, CH3(CH2)19CH2), 1.18, 1.21 (2 × d, 2 × 3H, Jvic = 6.2, (CH3)2CH), 1.25-1.40 (m,
21

36H, CH3(CH2)18CH2CH2), 1.66 (m, 2H, CH3(CH2)18CH2CH2), 2.43 (t, 2H, JCH2,CH2 = 7.4,
CH3(CH2)19CH2), 3.53 (dd, 1H, J3´a,2´ = 3.9, Jgem = 12.2, H-3´a), 3.54 (dd, 1H, JP,CHa = 9.2, Jgem = 12.8,
PCHa), 3.67 (dd, 1H, JP,CHb = 9.8, Jgem = 12.8, PCHb), 3.73 (m, 1H, H-2´), 3.77 (dd, 1H, J3´b,2´ = 4.1,
Jgem = 12.2, H-3´b), 3.94 (dd, 1H, J1´a,2´ = 7.6, Jgem = 13.7, H-1´a), 4.21 (dd, 1H, J1´b,2´ = 3.4, Jgem
=
13.7, H-1´b), 4.45 (dsep, 1H, Jvic = 6.2, JH,P = 8.1, P-OCH), 7.40 (d, 1H, J5,6 = 7.3, H-5), 8.06 (d, 1H,
J6,5 = 7.3, H-6). 13C NMR (CD3OD, t = 40°C, ppm) δ: 14.40 (CH3(CH2)19CH2), 23.69
(CH3(CH2)18CH2CH2), 24.81 (d, JP,C = 3.8, (CH3)2CH), 24.90 (d, JP,C = 4.1, (CH3)2CH), 26.08
(CH3(CH2)18CH2CH2), 30.16, 30.40, 30.42, 30.56, 30.66, 30.71, 33.03 (CH3(CH2)18CH2CH2), 38.18
(CH3(CH2)18CH2CH2), 52.49 (C-1´), 61.62 (C-3´), 67.50 (d, JP,C = 160.4, P-C), 69.36 (d, JP,C = 6.1,
(CH3)2CH), 81.15 (d, JP,C = 11.8, C-2´), 97.89 (C-5), 152.75 (C-6), 159.02 (C-2), 164.35 (C-4),
175.80 (C=O).
ESI MS, m/z: 1285.4 [2M-H]- (20), 642.4 [M-H]- (100). HRMS (-ESI): For C33H61N3O7P (M-H)-
calculated: 642.4247; found: 642.4256. Anal. Calcd. for C33H62N3O7P: C, 61.56; H, 9.71; N, 6.53; P,
4.81. Found: C, 61.67; H, 9.67; N, 6.68; P, 4.51.
Pivaloyloxymethylation of cyclic cidofovir (cHPMPC). A suspension of cHPMPC (400 mg, 1.53
mmol) in methanol (50 mL) was stirred with 1M methanolic tetrabutylammonium hydroxide (1.6 mL)
till dissolution and evaporated. The residue was coevaporated with dioxane (2 x 20 mL), then
dissolved in dioxane (15 mL) and stirred with chloromethyl pivalate (1.5 mL, 10.4 mmol) at 90°C for
1h. After cooling to room temperature, the reaction was quenched with methanol (5 mL) and the
mixture evaporated. The residue was chromatographed on a column of silica gel (150 mL) in system
chloroform – methanol (9:1) to give products 14 (RF 0.65) and 15 (RF 0.30).
({(5S)-2-Oxido-5-[(N4-pivaloylcytosin-1-yl)methyl]-1,4,2-dioxaphosphinan-2-yl}oxy)methyl
pivalate (14). Yield: 281 mg (40 %) as a white foam. According to NMR spectrum the compound is
a mixture of 2 diastereoisomers (A:B ~ 5:3). 1H NMR (CDCl3, ppm) δ: 1.20 (s, 9H, (CH3)3CCOO-B),
1.23 (s, 9H, (CH3)3CCOO-A), 1.27 (s, 9H, (CH3)3CCON-A,B), 3.55 (dd, 1H, J1´a,2´ = 7.7, Jgem = 14.0,
22

H-1´a-A), 3.64 (dd, 1H, J1´a,2´ = 8.2, Jgem = 13.9, H-1´a-B), 3.83 (dd, 1H, JP,CHa = 1.8, Jgem = 14.2,
PCHa-B), 3.92 (d, 1H, Jgem = 14.7, PCHa-A), 4.08-4.23 (m, 5H, H-2´-A,B, H-3´a-A, PCHb-A,B),
4.38-4.46 (m, 3H, H-3´a-A, H-3´-B), 5.58 (dd, 1H, Jgem = 5.2, JP,CHa = 12.1, OCHaHbO-B), 5.69 (dd,
1H, Jgem = 5.2, JP,CHa = 11.9, OCHaHbO-B), 5.73 (dd, 1H, Jgem = 5.2, JP,CHb = 13.9, OCHaHbO-A),
5.75 (dd, 1H, Jgem = 5.2, JP,CHb = 13.9, OCHaHbO-B), 7.39 (d, 1H, J5,6 = 7.3, H-5-A), 7.40 (d, 1H, J5,6
= 7.3, H-5-B), 7.54 (d, 1H, J6,5 = 7.3, H-6-A), 7.59 (d, 1H, J6,5 = 7.3, H-6-B), 8.15 (bs, 2H, NH-A,B).
13C NMR (CDCl3, ppm) δ: 26.74 ((CH3)3CCOO-B), 26.80 ((CH3)3CCOO-A), 27.04 ((CH3)3CCON-
A,B), 38.65 ((CH3)3CCOO-B), 38.72 ((CH3)3CCOO-A), 40.31 ((CH3)3CCON-A,B), 49.81 (C-1´-A),
49.91 (C-1´-B), 64.11 (d, JP,C = 144.3, P-C-A), 64.57 (d, JP,C = 147.2, P-C-B), 70.79 (d, JP,C = 6.9, C-
3´-B), 72.23 (d, JP,C = 9.1, C-3´-A), 73.19 (d, JP,C = 4.8, C-2´-B), 73.54 (d, JP,C = 5.3, C-2´-A), 81.42
(d, JP,C = 6.2, OCH2O-A), 81.77 (d, JP,C = 5.4, OCH2O-B), 96.26 (C-5-A), 96.32 (C-5-B), 149.80 (C-
6-A), 149.96 (C-6-B), 155.72 (C-2-A), 155.77 (C-2-B), 162.74 (C-4-A,B), 176.91 ((CH3)3CCOO-A),
176.93 ((CH3)3CCOO-B), 178.04 ((CH3)3CCON-B), 178.08 ((CH3)3CCON-A). 31P{1H} NMR
(CDCl3, ppm) δ: 10.71 (A), 12.89 (B). ESI MS, m/z: 940.7 [2M+Na]+ (20), 482.1 [M+Na]+ (82), 460.1
[MH]+ (100). HRMS (-ESI): For C19H31N3O8P (MH)+ calculated: 460.1849; found: 460.1844. Anal.
Calcd. for C19H30N3O8P: C, 49.67; H, 6.58; N, 9.15; P, 6.74. Found: C, 49.99; H, 6.72; N, 8.88; P,
6.61
({(5S)-5-[(Cytosin-1-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl}oxy)methyl pivalate (15).
Yield: 109 mg (19 %) as a white foam. According to NMR the compound is a mixture of 2
diastereoisomers 3:1 (Fig.3). HPLC separation was performed with a linear gradient MeOH – water
(0-100 %, flow rate 5 mL/min), diastereoisomer A was eluted at concentration 45 % MeOH, isomer B
at 50 % MeOH. Major diastereoisomer A: 1H NMR (CDCl3, ppm) δ: 1.25 (s, 9H, (CH3)3C), 4.05 (dd,
1H, J1´a,2´ = 7.7, Jgem = 14.4, H-1´a), 3.94 (d, 1H, Jgem = 14.7, PCHa), 4.04-4.12 (m, 2H, H-1´b,2´),
4.15 (dd, 1H, JP,CHb = 11.2, Jgem = 14.7, PCHb), 4.25 (t, 1H, J3´a,2´ = Jgem = 11.5, H-3´a), 4.42 (ddd, 1H,
JP,3´b = 17.0, Jgem = 11.5, J3´b,2´ = 2.0, H-3´b), 5.71 (dd, 1H, Jgem = 5.2, JP,CHa = 11.8, OCHaHbO), 5.74
23

(dd, 1H, Jgem = 5.2, JP,CHb = 14.1, OCHaHbO), 5.75 (d, 1H, J5,6 = 7.3, H-5), 7.22 (d, 1H, J6,5 = 7.3, H-
6). 13C NMR (CDCl3, ppm) δ: 26.81 ((CH3)3C), 38.71 ((CH3)3C), 49.21 (C-1´), 64.05 (d, JP,C = 144.9,
P-C), 72.55 (d, JP,C = 9.1, C-3´), 74.30 (d, JP,C = 5.3, C-2´), 81.39 (d, JP,C = 6.2, OCH2O), 94.31 (C-
5), 146.45 (C-6), 156.42 (C-2), 166.19 (C-4), 176.98 (C=O). 31P{1H} NMR (CDCl3, ppm) δ: 10.94.
Minor diastereoisomer B: 1H NMR (CDCl3, ppm) δ: 1.22 (s, 9H, (CH3)3C), 3.56 (dd, 1H, J1´a,2´ = 8.3,
Jgem = 14.6, H-1´a), 3.86 (dd, 1H, JP,CHa = 1.7, Jgem = 14.2, PCHa), 4.09-4.12 (m, 2H, H-1´b,2´), 4.23
(dd, 1H, JP,CHb = 9.8, Jgem = 14.2, PCHb), 4.40-4.49 (m, 2H, H-3´), 5.59 (dd, 1H, Jgem = 5.2, JP,CHa
=
12.0, OCHaHbO), 5.68 (d, 1H, J5,6 = 7.2, H-5), 5.77 (dd, 1H, Jgem = 5.2, JP,CHb = 14.0, OCHaHbO), 7.28
(d, 1H, J6,5 = 7.2, H-6). 13C NMR (CDCl3, ppm) δ: 26.78 ((CH3)3C), 38.69 ((CH3)3C), 49.41 (C-1´),
64.60 (d, JP,C = 147.1, P-C), 71.03 (d, JP,C = 6.9, C-3´), 73.84 (d, JP,C = 4.9, C-2´), 81.78 (d, JP,C = 5.6,
OCH2O), 93.72 (C-5), 147.04 (C-6), 156.31 (C-2), 165.84 (C-4), 176.95 (C=O). 31P{1H} NMR (CDCl3,
ppm) δ: 12.86. ESI MS, m/z: 772.7 [2M+Na]+ (15), 750.9 [2M+H]+ (20), 398.0 [M+Na]+ (22), 376.0
[MH]+ (28). HRMS (ESI): For C14H22N3O7PNa (M+Na)+ calculated: 398.10876; found: 398.10888.
Anal. Calcd. for C14H22N3O7P: C, 44.80; H, 5.91; N, 11.20; P, 8.25. Found: C, 44.99; H, 6.14; N, 10.96;
P, 8.37.
Antiviral Activity Assays
The compounds were evaluated against different herpesviruses, including 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) strain G, varicella-zoster virus (VZV) strains Oka and YS, TK-
VZV strains 07-1 and YS-R, human cytomegalovirus (HCMV) strains AD-169 and Davis as well as
poxviruses, including vaccinia virus Lederle and Western Reserve (WR) strains, cowpox virus strain
Brighton, camelpox virus strains CML-1 and CML-14 and orf virus strain NZ2. The antiviral assays
were based on inhibition of virus-induced cytopathicity or plaque formation in human embryonic lung
(HEL) fibroblasts. 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
24

forming units (PFU). 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 compound concentration required to reduce virus-induced cytopathogenicity
or viral plaque formation by 50%.
Cytostatic and Cytotoxic Assays.
Cytotoxicity measurements were based on the inhibition of cell growth. HEL cells were seeded at a
rate of 5 x 103 cells/well into 96-well microtiter plates and allowed to adhere and proliferate for 24 h.
Then, medium containing different concentrations of the test compounds was added. After 3 days of
further incubation 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 of the test compounds was expressed
as the minimum cytotoxic concentration (MCC) or the compound concentration that caused a
microscopically detectable alteration of cell morphology. Selectivity indexes were calculated as the
ratio of CC50 to EC50.
Acknowledgements
This work was supported by the Subvention for development of research organization RVO
61388963 and by the grant FR-TI4/625 by the Ministry of Industry and Trade of the Czech Republic
and the “Geconcerteerde Onderzoeksacties” (GOA), Krediet nr. 10/014” of the KU Leuven.
25

The authors´ thanks are due to the staff of the mass spectrometry (Dr. J. Cvačka, Head) and analytical
departments of the Institute (Dr S. Matějková, Head) and to Anita Camps, Steven Carmans, Frieda De
Meyer, Leentje Persoons, and Lies Van den Heurck for excellent technical assistance with the
antiviral assays.
References and notes
1. De Clercq, E. Clin. Microbiol. Rev. 2003, 16, 569.
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1998, 17, 339.
3. Field, H.J.; De Clercq, E. Antivir. Chem. Chemother. 2008, 19, 51.
4. Hostetler, K.Y. Antiviral Res. 2009, 82, A84.
5. Painter, W.; Robertson, A.; Trost, L.C.; Godkin, S.; Lampert, B.; painter, G. Antimicrob.
Agents Chemother. 2012, 56, 2726.
6. Krečmerová, M.; Masojídková, M.; Holý, A. Collect. Czech. Chem. Commun. 2006, 71, 579.
7. Krečmerová, M.; Holý, A.; Masojidková, M. Collect. Czech. Chem. Commun. 2007, 72, 927.
8. Krečmerová, M.; Holý, A.; Pískala, A.; Masojídková, M.; Andrei, G.; Naesens, L.; Neyts, J.;
Balzarini, J.; De Clercq, E.; Snoeck, R. J. Med. Chem. 2007, 50, 1069.
9. Holý, A.; De Clercq, E.; Votruba, I. In Nucleotide Analogues as Antiviral Agents; Martin, J. C.
Ed.; American Chemical Society, ACS Symposium Series #401, Washington D.C., 1989; pp
51-71.
10. Krečmerová, M.; Masojídková, M.; Holý, A. Bioorg. & Med. Chem. 2010, 18, 387-395.
11. Dračínský, M.; Krečmerová, M.; Holý, A. Bioorg. & Med. Chem. 2008, 16, 6778-6782.
12. Naesens, L.; Andrei, G.; Votruba, I.; Krečmerová, M.; Holý, A.; Neyts, J.; De Clercq, E.;
Snoeck, R. Biochem. Pharmacol. 2008, 76, 997-1005.
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13. Yoshida, M.; Suda, K.; Tsuboyama, A.; Sasaki, R.; Kitagawa, S.; Kano, Y.; Muto, Y.; Takaku,
F.; Sakamoto, S.; Miura, Y. Eur. J. Hematol. 1989, 43, 1.
14. Krečmerová, M.; Otmar, M. Future Med. Chem. 2012, 4, 991.
15. Pískala, A., Masojídková, M., Šaman, D. Collect. Czech. Chem. Commun. 1996, 61, S23.
16. Phiasivongsa, P., Redkar, S. Patent US 2007/0072796 A1, 2007.
17. Krečmerová, M.; Holý, A.; Pohl, R.; Masojídková, M.; Andrei, G.; Naesens, L.; Neyts, J.;
Balzarini, J.; De Clercq, E.; Snoeck, R. J. Med. Chem. 2007, 50, 5765.
18. Bischofberger, N.W.; Jones, R.J.; Arimilli, M.N.; Lin, K.-Y. et al. US patent 6225460. Issued on
May 01/2001.
19. Holý, A. Collect. Czech. Chem. Commun. 1993, 58, 649.
Figure 1. Chemical structures of cidofovir and related 5-azacytosine derivatives
Figure 2. An example of the mass spectrum (electrospray ionization, ESI) of an N4-acyl derivative of
HPMP-5-azaC.
Figure 3. Structure of diastereoisomers of cyclic esters 15 and 7.
Scheme 1. Synthesis and ring-opening reactions of N4-acyl derivatives of HPMP-5-azaC esters.
Scheme 2. The course of hydrolytic decomposition of HPMP-5-azaC.
Scheme 3. Acylation of cyclic derivatives of HPMP-5-azaC.
Scheme 4. Preparation of N4-docosanoyl (behenoyl) derivatives of cidofovir.
Scheme 5. Pivaloyloxymethylation of cyclic cidofovir
27

Figure 1.
Figure 2.
28

Figure 3.
29

Scheme 1.
Scheme 2.
30