Synthesis and in vitro activities of a new antiviral duplex drug linking Zidovudine (AZT) and Foscarnet (PFA) via an octadecylglycerol residue

Article abstract of DOI:10.1016/j.bmc.2008.10.081

To prepare a new antiviral duplex drug linking Zidovudine (AZT) and Foscarnet (PFA) via a lipophilic octadecylglycerol residue we condensed 1-O-4-monomethoxytrityl-3-O-octadecyl-sn-glycerol-2-hydrogenphosphonate obtained from 3-O-octadecyl-sn-glycerol wit

Full text of DOI:10.1016/j.bmc.2008.10.081

Bioorganic & Medicinal Chemistry 17 (2009) 303–310
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro activities of a new antiviral duplex drug linking
Zidovudine (AZT) and Foscarnet (PFA) via an octadecylglycerol residue
b
a
b
c
Herbert Schott a, , Klaus Hamprecht , Sarah Schott , Timm C. Schott , Reto A. Schwendener
*
a Institute for Organic Chemistry, University Tuebingen, Auf der Morgenstelle 18, D-72076 Tuebingen, Germany
b Institute of Medical Virology and Epidemiology of Viral Diseases, University Hospital of Tuebingen, D-72076 Tuebingen, Germany
c Institute of Molecular Cancer Research, University of Zuerich, CH-8057 Zuerich, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 July 2008
Revised 29 October 2008
Accepted 31 October 2008
Available online 5 November 2008
To prepare a new antiviral duplex drug linking Zidovudine (AZT) and Foscarnet (PFA) via a lipophilic octa-
decylglycerol residue we condensed 1-O-4-monomethoxytrityl-3-O-octadecyl-sn-glycerol-2-hydroge-
nphosphonate obtained from 3-O-octadecyl-sn-glycerol with AZT by the phosphonate method. The
purified condensation product was de-tritylated resulting in 30-azido-30-deoxythymidylyl-(50 ? 2-O)-3-
O-octadecyl-sn-glycerol, followed by treatment with (ethoxycarbonyl)phosphoric dichloride. The result-
ing 30-azido-30-deoxy-thymidylyl-(50 ? 2)-3-O-octadecyl-sn-glycerol-1-O-(ethoxycarbonyl)phosphonate
was purified by preparative RP-18 column chromatography. The antiviral duplex drug 30-azido-30-deoxy-
thymidylyl-(50 ? 2-O)-3-O-octadecyl-sn-glycerol-1-O-phosphonoformate trisodium salt (AZT–lipid–PFA)
was obtained after alkaline cleavage of the phosphonoformate ethylester residue. The overall yield of the
five step synthesis performed at gram scale was about 30%. According to a supposed pathway
AZT–lipid–PFA could be cleaved to yield a mixture of different antiviral compounds such as AZT, AZT-
50-monophosphate, octadecylglycerol–AZT, PFA and octadecylglycerol–PFA, possibly producing additive
and/or synergistic antiviral effects. In vitro studies showed that the duplex drug exhibits antiviral activ-
ities against HIV and especially against drug-resistant strains and clinical isolates of HSV and HCMV. The
E50 values of AZT–lipid–PFA against HIV ranged between 170 and 200 nM. The half-maximal inhibitory
doses (IC50) against highly acyclovir (ACV)-resistant HSV isolates determined by a plaque reduction assay
Keywords:
Antiviral duplex drug
Herpes simplex virus (HSV)
Human cytomegalovirus (HCMV)
Antiviral drug resistance
Hydrogenphosphonate method
ranged between 1.87 and 4.59
lM. Using ganciclovir (GCV)-sensitive, GCV resistant and drug cross-resis-
tant HCMV strains the IC50-values of AZT–lipid–PFA were between 2.78 and 1.18
l
M. With regard to PFA,
the IC50-value of AZT–lipid–PFA determined on a multi-drug-resistant HCMV strain was about 90-fold
lower than that of PFA, demonstrating the superior antiviral effect of the duplex-drug.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
patients. However, the antiviral profiles of the single drugs in the
mixtures remain unchanged.
In order to improve antiviral chemotherapy, the intense search
for new effective antiviral agents is ongoing.1,2 The successful clin-
ical application of new antiviral drugs, however, is often limited as
compounds with high in vitro activity do not meet the high expec-
tations made for drug development and licensing. An alternative to
the search for new single agents inhibiting virus replication lies in
the optimization of therapies by combining several well-known
antiviral single drugs. Such a combination therapy scheme either
aims at the simultaneous application of several single compounds,
for example, the highly active antiretroviral therapy (HAART)
against human immunodeficiency virus (HIV) or combination ther-
apies containing mixtures of two (CombivirTM , EpzicomTM, Truva-
daTM) or three (AtriplaTM, TrizivirTM) active agents in one tablet.3,4
The use of such tablets simplifies the application schedule for the
Another promising, but not yet well-investigated possibility of
antiviral combination therapy is the chemical linkage of two clin-
ically well-characterized drugs into one molecule, a so-called du-
plex-drug. After uptake of a duplex drug molecule in an infected
host cell, the drug can be cleaved into several metabolites, each
having a different virus inhibiting profile with additive or synergis-
tic properties. Antiviral single drugs are generally well justifiable
for the synthesis of corresponding duplex molecules, if the effort
for their chemical synthesis is relatively sustainable. A further
important consideration is the proven efficacy and toxic profile
of the single compounds. Synergistic or additive interactions of
the coupled drugs ought to be considered besides possible addi-
tional activities that might result from the structure of a new du-
plex molecule and therefore optimize the therapeutic effect of
such duplex compounds. The toxicity profile of two promising
single compounds should be as distinct as possible in order to
achieve reduced toxicity and optimal activity of the duplex drug
* Corresponding author. Fax: +49 7071 65782.
E-mail address: herbert.schott@uni-tuebingen.de (H. Schott).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.081

304
H. Schott et al. / Bioorg. Med. Chem. 17 (2009) 303–310
in comparison to its parent compounds. If two drugs are only effec-
tive at significantly different therapeutic concentrations, they are
not suitable for a duplex drug synthesis.
degradation of the AZT–PFA conjugate is a structurally caused disad-
vantage, which can affect the activity of AZT–PFA significantly. The
improved anti-HIV activity was predominantly evaluated in those
AZT–PFA conjugates, and was caused especially by the AZT compo-
nent. It is unknown, if these conjugates also show an anti-HSV and
anti-HCMV activity, which should based on the linked PFA
compound.
The present work described a new concept for the preparative
conjugation of AZT with PFA. According to the new synthesis both
drugs are independently linked via a glycerol lipid backbone con-
trast to the already synthesized AZT–PFA conjugates. Thereby, an
AZT–lipid–PFA duplex drug was obtained for the first time that
inhibits the viral replication of HIV, HSV as well as HCMV, as shown
in well established in vitro investigations.
Azidothymidine (AZT) used in HIV therapy as a nucleoside re-
verse transcriptase inhibitor (NRTI) as well as a chain terminator.
The non-nucleoside phosphonoformate (Foscarnet, PFA) used in
herpesvirus (HSV) and cytomegalovirus (CMV) therapy is the drug
of choice for infections with HSV that do not respond to acyclovir
(ACV) and for HCMV that do not respond to ganciclovir (GCV). Both
AZT and PFA fulfill several desirable properties that are required for
the synthesis of a potential antiviral duplex-drug. These properties
are (i) additive or synergistic antiretroviral activities, (ii) the well-
known distinct in vivo toxicity profiles, and (iii) a lack of cross-
resistance.5,6 Besides these aspects, PFA-resistant HIV mutants
show increased susceptibility to AZT whereas the hyper-suscepti-
ble phenotype is not necessarily coupled to PFA resistance.7
PFA is a potent inhibitor of HIV-1 RT acting as pyrophosphate
(PPi) analogue.8 HIV-1 RT can unblock the chain-terminating AZT
by phosphorolytic transfer of the terminal residue to an acceptor
substrate, which also interacts with the PPi binding site.9 Primer-
unblocking activity is increased in mutants of HIV-1, that are resis-
tant to AZT and this activity is inhibited by low concentrations of
PFA, which would explain the synergistic interaction between
PFA and AZT. Conformational changes occur during catalytic activ-
ity by DNA and RNA polymerases. A stable complex is formed be-
tween polymerase and primer-template (P/T) upon binding of the
next complementary dNTP on positions +1 and +2 on the template
(+1 or +2 complex, respectively), or Foscarnet (Foscarnet complex),
which might explain the inhibitory function of PFA on HIV RT.10
PFA shows a dual effect, specifically, at low concentrations it inhib-
its the removal of a terminated primer, whereas at high concentra-
tions these compounds enhance the rescue of the terminated
primer because of the PFA-dependent phosphorolysis.11
Mixtures of AZT and an excess of PFA as single entities produced
a moderate synergistic inhibitory effect in vitro against HIV-1 and
CMV replication and low toxicity in cell cultures as previously
shown by several authors.6,12–14 The suppression of CMV replica-
tion was achieved at lower drug concentrations when AZT and
PFA were combined than when the drugs were used alone.15
The first synthesized antiviral molecules linking AZT and PFA
were conjugates, in which the carboxyl or phosphonyl group of
PFA ester derivatives was directly covalently linked to the 50-hy-
droxyl group of AZT or other inhibitory dideoxynucleoside ana-
logues.16–19 The evaluated antiviral activity of these types of
conjugates neither achieved substantial gains in terms of antiviral
activity nor in therapeutic selectivity.
The more recently developed lipophilic AZT–PFA conjugates, in
which the formic terminal of PFA was esterified with a long chain al-
kyl alcohols, whereas the phosphono terminal of PFA was coupled to
the 50-hydroxyl group of AZT, have the potential to act either as PFA
prodrugs, AZT-prodrugs or both. The conjugates inhibit the in vitro
replication of wild-type HIV-1, as well as that of PFA-resistant strain
or AZT resistant clinical isolate strains, respectively. The lipophilic
AZT–PFA conjugates show a noticeably higher inhibitory activity in
comparison to the single drugs AZT and PFA, especially regarding
resistant HIV-mutants.20 The observed antiviral activities against
HIV-resistant mutants can be caused by the different inhibitory
mechanisms of viral replication of AZT and PFA as described above.
Based on the suggested mechanism the structure of the lipophilic
AZT–PFA conjugate allows either the simultaneous release of a lipo-
philic PFA derivate and of AZT or alternatively, the metabolism into
AZT-50-monophosphate PFA however, is degraded by decarboxyl-
ation during formation of AZT-50-monophosphate and is therefore
lost as the second antiviral component. For the inhibitory effect of
PFA a considerably higher drug concentration is needed in compar-
ison to AZT. Under this consideration the loss of PFA by the metabolic
2. Results and discussion
2.1. Synthetic chemistry
AZT–lipid–PFA was prepared in five steps according to the syn-
thesis scheme given in (Fig. 1) starting with 3-O-octadecyl-sn-
glyercol (1). In the first synthesis step the free terminal hydroxyl
group of 1 was blocked with the 4-monomethoxytrityl residue.
After the chromatographic purification of the reaction mixture 1-
O-4-monomethoxytrityl-3-octadecyl-sn-glycerol (2) was obtained
at 78% yield. In the second synthesis step the hydroxyl group of
the protected glycerol derivative 2 was phosphonylated at 85%
yield by treatment with salicylchlorophosphite followed by silica
gel chromatography resulting in 1-O-4-monomethoxytrityl-3-O-
octadecyl-sn-glyerol-2-hydrogenphosphonate (3). In the following
reaction 3 was condensed to the free 50-hydroxyl group of 30-azido-
30-deoxythymidine (AZT, 4) which was isolated from expired cap-
sules (ZidovudineTM, CombivirTM).2 The condensation was performed
in the presence of pivaloyl chloride according to the hydroge-
nphosphonate method. The phosphonodiester linkage of the con-
densation product was immediately oxidized with iodine to yield
the fully protected octadecylglycerol–AZT conjugate linking the
lipophilic glycerol derivative to AZT by a phosphodiester bond.
During the chromatographic fractionation of the reaction mixture
on a silica column the 4-monomethoxytrityl protecting group
was partially lost. The trityl-protected octadecylglycerol–AZT was
separated as the main product from the de-tritylated condensation
product. The isolated tritylated reaction product was treated with
acetic acid to remove the 4-monomethoxytrityl protecting group.
After de-protection the reaction mixture was re-chromatographed
yielding the expected 5 which was identical to the de-tritylated
octadecylglycerol–AZT isolated by the first chromatography of
the reaction mixture. Both de-tritylated products were combined
resulting in 75% yield of pure 5.
In the fourth synthesis step the free terminal hydroxyl group of
5
was reacted with (ethoxycarbonyl)phosphoric dichloride
(6) in trimethylphosphate resulting in the precursor (7) of
AZT–lipid–PFA, in which the formate residue was esterified with
ethanol. The reaction mixture was flash chromatographed on a
reverse
phase column (RP-18)
using
water–methanol
mixtures with increasing amounts of methanol as the eluent.
At 20% methanolic water undesired side products and trimethyl-
phosphate were eluted. By increasing the methanol amount the
desired 30-azido-30-deoxythymidylyl-(50 ? 2-O)-3-O-octadecyl-sn-
glycerol-1-O-(ethoxycarbonyl)phosphonoformate (7) was eluted,
followed by starting material 5 which had not reacted. The product
containing fractions were pooled, concentrated and lyophilized
yielding 61% of 7 as a white powder. The isolated non-reacted
educt 5 was re-used for further derivatisation. Under this aspect,
the PFA residue can be introduced at yields higher than
61%. In the last reaction step the ethylester group of 7 was

H. Schott et al. / Bioorg. Med. Chem. 17 (2009) 303–310
305
TLC. The chemical structure and the analytical purity of the prod-
uct were confirmed by NMR spectroscopy, high resolution mass
spectroscopy and elementary analysis. Elementary analysis was
within usual limits for C, H and N. In case of 5 and 8 the too low
nitrogen analysis could be caused by the incomplete conversion
of the azido group to nitrogen oxide during combustion, which
O
(CH2)17-CH3
MeOTr
O
(CH2)17-CH3
a,b
OH
OH
OH
O
2
1
was also reported before.20 The specific angles of rotation (½a 2D0
ꢀ
)
c,b
O
N
of the glycerol derivatives 2 and 3 showed the expected negative
values. The positive values (½a D20
ꢀ
) +5.69°; c 1.16, H2O) of octadecyl-
glyerol–AZT 5 was unexpected. After introduction of the PFA resi-
HN
O
O
(CH2)17-CH3
due the resulting 7 and 8 showed again negative (½a D20
ꢀ
) values.
O
O
The chemical synthesis of AZT–lipid–PFA is practicable on a pre-
parative scale and the yield of the single reaction steps, which are
not yet optimized are in the range of 70–83%. This fulfills an impor-
tant prerequisite, namely that the new antiviral duplex drug can be
synthesized at sufficiently large amounts for in vivo testing and
further development towards a possible therapeutic application.
The successful use of the recycled AZT proves that clinically
expired drugs can be used as a cheap source of valuable starting
materials. The hydrolytic stability of dissolved 7 is very high and
the lyophilisate can be stored at room temperature for several
months without change or damage. The alkaline cleavage of the
ethylester group nearly quantitatively converts 7 into AZT–lipid–
PFA without occurrence of side products or impurities. For this
reason and because 7 can be obtained at high purity the chromato-
graphic purification of the acid labile 8 is not necessary and a
possible hydrolytic degradation during the chromatographic
procedures will be avoided. In neutral environment AZT–lipid–
PFA is very stable because a stock solution of 8 (1 mM) in water
can be stored at ꢁ20 °C more than one year without any reduction
of its antiviral activity.
P
OH
+
HO
H
O
O
MeOTr
N3
3
4
d,e,b
O
HN
O (CH2)17-CH3
O
O
Cl
O
N
O
P
O
OH
O
P
+
O
Cl
O
OH
N3
6
5
f,g,h
O
HN
O
(CH2)17-CH3
O
2.2. Antiviral activity of AZT–lipid–PFA duplex drug
N
O
O
P
O
2.2.1. Concept of AZT–PFA duplex-drug
O
ONa
We established a new structure for an AZT–lipid–PFA duplex
drug, which effectively utilizes the inhibitory advantages of a cova-
lent AZT–PFA combination, but avoids a structurally based loss of
activity through the metabolic pathway of PFA. Therefore, AZT
and PFA are, unlike previously described conjugates, not directly,
but indirectly linked via an ether lipid molecule to a new type of
AZT–lipid–PFA compound. Ether lipid conjugates may allow a ma-
jor part of the drug to enter the cell due to their lipophilic residue
and thus provide cells with a depot of the prodrug. In the course of
the metabolic pathway lipophilic octadecylglycerol–AZT as well as
octadecylglycerol–PFA prodrugs can be cleaved by enzymatic
hydrolysis in addition to the formation of PFA, AZT-50-monophos-
phate and AZT, respectively. Furthermore, the direct cytoplasmatic
release of AZT-50-monophosphate would not require initial
phosphorylation of AZT. Ether lipids are membrane interactive,
thus the lipophilic compounds could accumulate in the plasma
membrane of T-lymphocytes and monocytes or macrophages.
The released lipophilic prodrugs correspond to the previously
described, inhibitory active alkylglycerol AZT and PFA deriva-
tives.22–25 Those derivatives have several advantages in
comparison to the non-derivatized parent drug and are potent
antiviral drugs.
O
O
N3
O P
ONa
OC2H5
7
i,h
O
HN
O
O
(CH2)17-CH3
O
P
ONa
O
N
O
O
O
O
O P
ONa
N3
ONa
8
Figure 1. Synthesis of the antiviral duplex drug (30-azido-30-deoxythymidylyl-
(50 ? 2-O)-3-O-octadecyl-sn-glycerol-1-O-phosphonoformate) trisodium salt (8)
starting from 3-octadecyl-sn-glycerol (1). The reaction steps are: (a) 4-monometh-
oxytrityl chloride in pyridine; (b) purification of the reaction mixture on a silica gel
column; (c) salicylchlorophosphite in pyridine/dioxane; (d) pivaloyl chloride in
pyridine, 7 min, 0.2 M iodine in THF/water; (e) 80% acetic acid, room temperature;
(f) trimethylphosphate, 16 h, sodium carbonate until pH 6.5; (g) purification of the
reaction mixture on a LiChroprep. RP-18 column; (h) lyophilisation; (i) 1N NaOH,
cation exchange (H+-form) until pH 8.5.
2.2.2. In vitro antiviral activities of AZT–lipid–PFA
The anti-HIV activity of AZT–lipid–PFA was determined at the
National Cancer Institute (NCI, USA). The inhibitory effects against
HCMV isolates were evaluated as the half-maximal inhibitory
doses (IC50) using a modified cell-associated immediate early pla-
que reduction assay followed by statistical Probit evaluations in
the case of HCMV. A screening plaque reduction assay (PRA) fol-
lowed by Probit analysis was used for the determination of the
hydrolyzed under alkaline conditions resulting in the antiviral
active 30-azido-30-deoxythymidylyl-(50 ? 2-O)-3-O-octadecyl-sn-
glycerol-1-O-phosphonoformate trisodium salt (AZT–lipid–PFA)
(8) which was isolated by lyophilisation at 83% yield. The course
of the syntheses and the purification steps were monitored by

306
H. Schott et al. / Bioorg. Med. Chem. 17 (2009) 303–310
IC50 of HSV. The anti-HSV effects were investigated in comparison
to ACV and PFA. The evaluated antiviral potency of AZT–lipid–PFA
against HCMV was compared with GCV and PFA.
isolates. AZT–lipid–PFA (IC50 = 1.18
lM) was about 56-fold more
active than PFA (IC50 = 67.50 M) in cells infected with GCV
l
resistant HCMV-3res and about 93-fold more active in cells in-
fected with HCMV-U405cres isolates which is cross-resistant
against GCV and Cidovovir (CDV).
2.2.3. Antiviral activity of AZT–lipid–PFA in the HIV-1 infected
CEM-SS cell line
The anti-HIV-1 activity has been evaluated in vitro in the frame-
work of the In Vitro Anti-AIDS Drug Discovery Program (NCI, USA).
The standard procedure used in the test of the NCI for anti-HIV ac-
tive compounds is designed to detect agents acting at any stage of
the reproductive cycle of the virus26 The assay basically involves
killing of T4 lymphocytes by HIV. On the basis, that the values eval-
uated for 50% effective concentration (EC50) against cytopathic ef-
2.2.6. Possible causes of antiviral activity of AZT–lipid–PFA
The published AZT–PFA conjugates only report on an optimized
anti-HIV activity. The anti-HSV and anti-HCMV activity due to the
PFA component was not described. Although these conjugates link
AZT and PFA, however, they only show an antiviral effect against
HIV and therefore, such conjugates act as a monotherapeutics.
AZT–lipid–PFA seems to be at first time a duplex drug with a
broad antiviral spectrum which inhibits the replication of HIV as
well as of sensitive and resistant laboratory isolates of HSV and
HCMV. Antiviral effects already appear at IC50 ranges of 1.1–
fects ranged between 0.17 and 0.20
growth inhibitory concentration (IC50) was between 94.3 and
92.7 M and the therapeutic index TI50(IC/EC) = 454–549. Thus,
lM, the calculated 50% cell
l
AZT–lipid–PFA was judged ‘confirmed active’.
4.6 lM. The inhibitory profile of AZT–lipid–PFA against sensitive
HSV and HCMV strains correlate with that of ACV and GCV, the
drugs that are used as basic therapeutics against HSV and HCMV
infections. However, the antiviral effect of AZT–lipid–PFA against
resistant HSV and HCMV strains is markedly superior to that of
ACV and GCV. Clinical studies have proven PFA as being equivalent
to GCV for the treatment of HCMV infections27 and superior to
vidarabine for the treatment of ACV resistant HSV-strains.28 This
aspect supports mentioning, that AZT–lipid–PFA shows an up to
30-fold lower IC50 value in comparison to PFA alone against HSV
and even an up to 90-fold lower IC50 against HCMV. The antiviral
activity of the duplex drug, whose PFA component is only 14% of
the total MW, is especially in a resistance setting markly superior
to PFA alone. It can be estimated that both components (AZT and
PFA) of the duplex drug cause the antiviral effect due to additive
or synergistic cooperation, whereby the mechanism is not yet
known. A PFA therapy is accompanied by major side effects, there-
fore every effort to increase the activity of PFA is worth aiming at
which could for example be achieved by AZT–lipid–PFA.
The strong antiviral effect of AZT–lipid–PFA is most likely based
on the action of several different mechanisms. The duplex drug
optimizes an AZT + PFA combination therapy and also acts as a de-
pot for several different inhibitory prodrugs and drug metabolites.
The proposed pathway in Fig. 2 suggests that AZT–lipid–PFA can be
enzymatically cleaved in several steps to a mixture of lipophilic
prodrugs of PFA and AZT (metabolites A and B in Fig. 2), unmodi-
fied PFA and AZT besides AZT-50-monophosphate, with all com-
pounds acting additively or synergistically. For example
metabolite A in combination with AZT is strongly synergistically
in HT4-6C cells infected with HIV-1.25 As the exact metabolism
of AZT–lipid–PFA is not yet determined, the supposed pathway
shown in Fig. 2 is hypothetical. A major issue in terms of the anti-
viral effect of AZT–lipid–PFA is the question if metabolic cleavage
takes place in- or out-side of the target cell and at what time, in
which succession and amounts the different metabolites are
formed. These aspects are responsible for gradual differences in
the antiviral effects of AZT–lipid–PFA when the IC50-values are
determined in different culture media or cell lines. On the other
hand, it may be postulated that due to the high variety of the dif-
ferent inhibitory metabolites formed by the AZT–lipid–PFA, the du-
plex drug would be much less susceptible to resistance than the
single drugs.
2.2.4. Antiviral activity of AZT–lipid–PFA against HSV type 1
isolates
The inhibitory potencies of AZT–lipid–PFA, ACV and PFA against
HSV-1 isolates are summarised in Table 1. In comparison to ACV
the antiviral capacity of AZT–lipid–PFA against the HSVsens KOS
multiple passage laboratory strain corresponds nearly to that of
ACV but is about 9-fold higher against the highly ACV resistant
UL23 KOS-M strain. The replication of clinical HSV cell isolates
was inhibited 2 times more effectively than the replication of the
HSV laboratory strains (KOS and KOS-M). Surprisingly,
AZT–lipid–PFA inhibited the replication of the ACV sensitive pre-
therapeutic clinical isolates of two recipients of stem cell trans-
plants (HSV-K161, HSV-L177) 4- to 6-fold less effectively as ACV.
However, the inhibition of the corresponding sequential ACV resis-
tant isolates (HSV-K143) was 16 times more potent than ACV.
Remarkably, the antiviral effect of PFA in comparison to AZT–li-
pid–PFA was about 25- to 30-fold lower against the HSVsens and
HSVres isolates. Due to this very low inhibitory effect the IC50-val-
ues of PFA were not further confirmed.
2.2.5. Antiviral activity of AZT–lipid–PFA against clinical HCMV
isolates
Details of the inhibitory effects of AZT-lipid-PFA, GCV and PFA
against sensitive and resistant clinical HCMV isolates were pre-
sented at 10th International CMV Workshop in Williamsburg,
2005. The small difference of IC50-values of GCV (IC50 = 3.2
lM)
and of AZT–lipid–PFA (IC50 = 2.8 M) show that both drugs have
l
a comparable antiviral potency against the HCMV-1 sensitive
Table 1
Antiviral activity of acyclovir (ACV), ganciclovir (GCV), Foscarnet (PFA) on replication
of different HSV virus strains and clinical HCMV isolates in comparison to AZT–lipid–
PFA, expressed by the 50% inhibitory concentration (IC50) on viral plaque formation
Virus strain
IC50
GCV
(l
M) of antiviral drugsa
PFA
ACV
AZT–lipid–PFA
HSV-1 isolates
KOS
KOS-M
HSV-K161 sens
HSV-K143 res
HSV-L177 sens
HSV-L182 res
3.04 1.65
31.20 1.5
0.58 0.11
75.07 8.15
0.32 0.11
9.51 0.41
—
—
ND
ND
ND
ND
125
112
ND
ND
64
4.02 0.81
3.39 0.93
2.27 1.01
4.59 1.07
1.92 0.15
1.87 0.66
The structure of AZT–lipid–PFA allows as first ever described
complex, especially in contrast to all other previously reported
conjugates, an enzymatic pathway that can release AZT-50-mono-
phosphate without decarboxylation of PFA. In the case that AZT-
50-monophosphate is released from AZT–lipid–PFA after cell-up-
take, an AZT-resistance based upon a kinase deficiency could be
circumvented. If AZT-50-monophosphate is cleaved outside the cell,
the amphipatic AZT–lipid–PFA is transformed into a lipophilic
46
HCMV isolates
HCMV-1sens
HCMV-3res
ND
ND
3.22 0.47
16.40 5.90
ND
ND
67.50 8.71
400.12 69.41 4.29 2.07
2.78 0.23
1.18 0.55
HCMV-U405cres ND
a
Results of three individual experiments; ND, not determined.

H. Schott et al. / Bioorg. Med. Chem. 17 (2009) 303–310
307
Octadecanol
+
+
+
OH
O
N
OH
CH3
O
N
HN
CH3
OH
OH
O
HN
O
O P
O
O
O
O
OH
O P
O
O
OH
O
N3
O
O
O
N3
O P
P
O
OH
OH
OH
OH
OH
Glycerol-AZT
h
AZT-glycerol-PFA
g
Glycerol-PFA
f
O-(CH2)17-CH3
HN
O-(CH2)17-CH3
HN
O
N
O
N
CH3
O-(CH2)17-CH3
OH
CH3
O
P
OH
O
a
b
AZT-5'-mono-
phosphate
O
O
+
O
O
O P
O
+
PFA
O
O
OH
O
O
P
e
O
O
N3
O
N3
OH
5
OH
O P
OH
OH
8
OH
AZT-lipid-PFA
AZT
Octadecylglycerol-AZT
(metabolite B)
AZT
e
Octadecylglycerol-PFA
(metabolite A)
O-(CH2)17-CH3
OH
c
d
AZT-5'-mono-
phosphate
PFA
OH
Octadecylglycerol
Figure 2. Possible enzymatic pathway of the protonated AZT–lipid–PFA duplex drug resulting in a mixture of lipophilic prodrugs of PFA, AZT and unmodified PFA, AZT-50-
monophosphate and AZT. Depending on the medium the duplex drug and its metabolites either exits in a salt or protonated form.
octadecylglyerol–PFA prodrug (metabolite A, Fig. 2). Due to their
increased lipophilicity the PFA prodrugs should permeate the cell
membrane even more efficiently than the unaltered duplex drug.
In case that the PFA residue is cleaved enzymatically outside the
cell, metabolite B, which is a lipophilic prodrug of AZT-50-mono-
phosphate is formed. This metabolite can presumably easily be ta-
ken up by the cell. The lipid–AZT prodrug can target the virus
replication cycle in two ways, firstly by inhibiting viral RT by AZT
and secondly by inducing the production of defective virus
particles by the lipid.29 As the inhibitory effect of PFA is mostly
depending on the intracellular drug concentration an increased
cell-uptake raises the antiviral activity of PFA. In addition, due to
the high IC50-values of PFA in comparison to those of AZT, the
intracellular PFA concentration seems to be responsible for the
occurrence of additive or synergistic effects of PFA and AZT. In case
that PFA and AZT occur simultaneously at sufficient concentrations
within the cell, AZT resistant mutations can be suppressed by PFA-
resistant mutations and vice versa.10
(Fig. 2) corresponds to the synthetic octadecylglyerol–PFA prodrug
as described previously.23–25 The evaluated inhibitory activity of
these synthetic PFA prodrugs is 40- to 140-fold greater than that
of free PFA in cells infected with HIV-1, HSV-1 and HCMV and they
are also potent inhibitors of both wild-type HIV-1LAI and most NRTI
resistant HIV-1 variants. However, the PFA prodrug was not active
against PFA resistance. The antiviral activity of AZT–lipid–PFA
against HIV-1, which was evaluated by the NCI, correlated with
the described IC50-values of the synthetic octadecylglycerol–PFA
prodrugs. As octadecylglycerol–PFA can be released by metabolism
of AZT–lipid–PFA, it can be expected that AZT–lipid–PFA is also
effective against HIV variants, which are susceptible to the syn-
thetic PFA prodrug. The inhibitory effect of the duplex drug against
those HIV variants should be as good as or even better than the
synthesized PFA prodrug, because metabolized AZT–lipid–PFA
yields several additional AZT metabolites besides octadecylglycer-
ol–PFA. Those metabolites enhance the anti-HIV activity
synergistically.25
A preferred site of enzymatic cleavage of AZT–lipid–PFA is the
natural phosphodiester bond, which links AZT to the octadecyl-
glycerol residue. The removal of terminal nucleotides, in this case
AZT-50-monophosphate, by exonucleases and/or the primer
unblocking reaction by pyrophosphorolysis are well-known mech-
anisms for the degradation of nucleotide derivatives. Therefore,
AZT-50-monophosphate should easily be cleaved enzymatically
from the AZT–lipid–PFA molecule. The remaining metabolite A
If the enzymatic metabolism of the duplex drug does not initi-
ate with the cleavage of AZT-50-monophosphate, but with the
cleavage of the octadecyl residue (see metabolism f, g, h of Fig. 2)
hydrophilic molecules appear. The resulting metabolites are pre-
sumably cleaved into PFA + AZT outside of the cells. However,
based on the high inhibitory activity of AZT–lipid–PFA the possible
breakdown of the octadecyl residue is probably not the first step of
the metabolism.

308
H. Schott et al. / Bioorg. Med. Chem. 17 (2009) 303–310
A comparison of the inhibitory effect of each drug on the basis
drugs.21 The synthesized compounds 1, 2, 3, 4, and AZT were rigor-
ously dried by repetitive addition of dry pyridine and rotary evap-
oration before they were further derivatized in pyridine or
pyridine containing solutions. All reactions were performed at
room temperature if not stated differently. The concentrations of
the reaction mixtures, solutions, organic layers and eluted fractions
were done in vacuo at a bath temperature of about 45 °C. Prepara-
tive column chromatography was carried out at room temperature
by flash chromatography on self-packed dry silica gel or equili-
of the IC50-values, which had been evaluated in the different sys-
tems, only provides preliminary data. Based upon this consider-
ation we have compared our IC50-values with those described in
the literature. Additive or synergistic effects of AZT–lipid–PFA
can indirectly be postulated by comparison of IC50-values of ACV
and octadecylglycerol–PFA, which had been previously evaluated
for HSV-1.23 The published values show that ACV (IC50 = 0.03
is 30-fold more active in the used test than the synthetic octadecyl-
glycerol–PFA (IC50 = 1.1 M). The IC50-values of ACV in different
sensitive HSV-1 isolates, which we have evaluated (see Table 1)
are between 0.32 and 3.04 M and therefore only maximally 6-fold
lower than AZT–lipid–PFA (IC50 = 1.92–4.02 M). The improved
lM)
l
brated RP-18 reversed phase (LiChroprep, 40–60 lm) columns,
using mixtures of eluents with increasing amounts of methanol.
The eluent was collected in 20 ml fractions and the eluted com-
pounds were identified by TLC.
l
l
HSV activity of AZT–lipid–PFA in comparison to the published
octadecylglycerol–PFA indicate that the AZT component contrib-
utes to the anti-HSV activity. The contributional effect of AZT is
shown even more significantly in the case of resistant HSV-1 iso-
lates, respectively, which had not been evaluated by Hostetler et
3.2. Synthesis of AZT–Lipid–PFA
3.2.1. Synthesis of 1-O-4-monomethoxytrityl-3-O-octadecyl-sn-
glycerol (2)
al.23 IC50 = 1.87–4.59
lower than ACV (IC50 = 9.51–75.07
l
M of AZT–lipid–PFA is maximally 16 times
M). We conclude that the
3-O-Octadecyl-sn-glyercol 1 (75 g, 218.6 mmol) was dissolved
in dry pyridine (380 g) and 4-monomethoxytritylchloride (75 g,
240 mmol) was added. The resulting mixture was stirred under
exclusion of moisture for 3 days. After cooling to 5 °C the precipi-
tate was removed by filtration and washed with cold toluene. The
filtrate was combined with the wash solution and concentrated to
a sirup which was coevaporated with toluene (2ꢂ 100 ml) and di-
luted with petroleum ether (300 ml). The obtained solution was
purified on a silica gel column using a petroleum ether/chloroform
gradient with increasing parts of chloroform. The pooled fractions
of the desired product 2 (UV-, trityl- and glycerol derivative-posi-
tive) were concentrated in order to yield 105 g of 2 (78%) as color-
less sirup, which crystallized by standing openly at room
temperature for several days; mp: 48 °C; RF = 0.40 (chloroform/
l
simultaneous release of AZT and octadecylglycerol–PFA from
AZT–lipid–PFA supports the PFA-activity additively or synergisti-
cally and increases the inhibitory effect. Our assumption is sup-
ported by the work of Snoeck et al.15 which described additive
effects of AZT and PFA against HCMV. The significantly lower
IC50-values of AZT–lipid–PFA and its superior antiviral effects in
sensitive and resistant virus isolates in comparison to those estab-
lished show that the duplex drug could have the potential for a
new, effective therapeutic alternative to the monotherapy with
AZT, ACV, GCA or PFA.
The metabolite B, which is released from AZT–lipid–PFA, is the
position-isomer compound of the synthetic octadecylglycerol–AZT
prodrug previously described22 which is 48 times more active than
unmodified AZT in reducing HBV replication in 2.2.15 cells. Struc-
ture activity relationships of phospholipid AZT conjugated have
shown, that the antiviral activity remains if AZT is linked on posi-
tion 2 or 3 to the glycerol backbone.29 This allows the presumption
that AZT–lipid–PFA will also have an antiviral activity against HBV,
which might expand the activity spectrum of duplex this drug.
petroleum ether 1:1, v/v); MS (FAB+), 615.3 [MꢁH+]; ½a 2D0
ꢁ3.47°
ꢀ
(c 3.57, benzene). Anal. calcd (C41H60O4): C, H. 1H NMR (CDCl3,
250 MHz); d = 0.87 (t, 3H, J = 6.7 Hz, –CH3), 1.2–1.3 (m, 30H, –
(CH2)15–), 2.46, (d, 1H, J = 4.5 Hz, OH), 3.19 (dd, 2H J1 = 5.5 Hz,
J2 = 1.8 Hz, (C300)H2), 3.45 (t, 2H, J = 7 Hz, O–CH2–(CH2)16–CH3),
3.49 (m, 2H, (C100)H2) 3.77 (s, 3H, aryl-O–CH3), 3.94 (m, 1H,
(C200)H), 6.79–7.46 (m, 14H, aryl-H). 13C NMR (CDCl3, 62 MHz):
d = 14.1 (–CH3), 22.7 (–CH2–CH3), 26.1 (–CH2–), 29.4–29.7 (–
CH2)15–), 31.9 (O–CH2–CH2–(CH2)14–), 55.2 (phenyl-O–CH3), 64.6
(C300), 69.9 (C200), 71.6 (–O–CH2–(CH2)16–), 72.1 (C100), 86.4 (O–C-
phenyl3), 113.1 (aryl-C), 126.9–130.4 (aryl-C), 135.6 (aryl-C),
144.4 (aryl-C), 158.6 (aryl-C–OMe).
3. Experimental
3.1. General chemistry
1H and 13C NMR spectra were obtained on a Bruker AC 250 spec-
trometer at 250 and 62.9 MHz or on a Bruker Avance 400 spectrom-
eter at 400 and 100 MHz, respectively. CDCl3, DMSO-d6 and D2O
were used as solvents and Me4Si as internal standard. 31P NMR data
were obtained on a Bruker Avance 400 spectrometer at 161 MHz,
using H3PO4 as the external standard. Mass spectra were measured
on a Finnigan TSQ 70 or on a Finnigan MAT 95 instrument. For FAB
Mass spectra, all compounds were measured in a NBA-or glycerine
matrix. HRMS were measured on a Bruker Apex II FT-ICR instru-
ment. Optical rotations were recorded on a Perkin Elmer 341 polar-
imeter, using a 10 cm quartz cell at k = 589 nm (Na-D-line). Melting
points (mp) were measured on a Stuart Scientific SMP3 apparatus
and are uncorrected. TLC was performed on pre-coated silica gel
plates 60 F254 (0.25 mm, Merck). The nucleotides-glycerol deriva-
tives and impurities were detected as previously described30 using
UV-light for visualization and spray reagents as developing agents.
The following compounds were synthesized in our laboratory in
analogy to previously published methods: salicylchlorophosph-
ite,31 3-octadecyl-sn-glyerol (compound 1 in Fig. 1)32 (ethoxycar-
bonyl)phosphoric dichloride,33 4-monomethoxytritylchloride.34
30-Azido-30-deoxythymidine (AZT) was extracted from expired
3.2.2. Synthesis of 1-O-4-monomethoxytrityl-3-O-octadecyl-sn-
glycerol-2-hydrogenphosphonate (3)
A solution of 2 (90 g, 146 mmol) in dry pyridine (140 g) was di-
luted by adding dry dioxane (340 ml). Salicylchlorophosphite (30 g,
148 mmol) dissolved in dry dioxane (140 ml) was added and the
reaction mixture shaked under exclusion of moisture for 2 h. The
reaction was stopped by adding saturated Na2CO3 (100 ml) and
subsequently concentrated to a sirup which was diluted with chlo-
roform (800 ml) and extracted with saturated Na2CO3 (400 ml).
The chloroform phase was concentrated to a sirup which was
mixed with ether (600 ml) containing triethylamine (20 ml). This
solution was chromatographed on a silica gel column using ether
as the first and a chloroform/methanol mixture with increasing
parts of methanol as the second eluent. Product containing frac-
tions (UV-, trityl-, and glycerol derivative-positive) were pooled
and concentrated to yield 84 g (85.0%) of 3 as sirup. RF = 0.57 (chlo-
roform/methanol 8:2 v/v); MS (FABꢁ) 679.3 [MꢁHꢁ]; ½a 2D0
ꢁ2.76°
ꢀ
(c 1.42, THF): Anal. calcd (C41H61O6PꢃH2O) C, H. 1H NMR (CDCl3,
250 MHz): d = 0.87 (t, 3H, J = 6.7 Hz, –CH3), 1.2–1.3 (m, 30H, –
(CH2)15–), 1.46 (m, 2H, –CH2–), 3.38 (m, 2H, O–CH2–(CH)16–),
3.61 (d, 2H, J = 5.4 Hz, (C100)H2) 3.75 (s, 3H, phenyl-O–CH3), 4.55

H. Schott et al. / Bioorg. Med. Chem. 17 (2009) 303–310
309
(m, 1H, (C200)H), 5.73 (s, 0.5H, OPHO(OH)) 6.79–7.46 (m, 14H, aryl-
H), 12.66 (br, s, 1H, PHO(OH)), 8.28 (s 0.5H, OP(OH)(OH)). 13C NMR
(CDCl3, 62.9 MHz: d = 14.1 (–CH3), 22.7 (–CH2–CH3), 26.1 (–CH2–),
29.4–29.7 (–CH2)15–), 31.9 (O–CH2-CH2–(CH2)14–), 55.1 (phenyl–
O–CH3), 64.6 (C300), 71.2 (C200), 71.5 (–O–CH2–(CH2)16–), 72.7
(C100), 86.2 (O–C-phenyl3), 113.1 (aryl-C), 126.9–130.4 (aryl-C),
135.6 (aryl-C), 144.4 (aryl-C), 158.5 (aryl-C–OMe). 31P NMR (CDCl3,
161 MHz): d = 4, 1.
dient 50% methanol (600 ml) in the mixing vessel/80% methanol
(600 ml) in the reservoir, and (3) 80 % methanol (1 l). The eluted
compounds were identified by TLC (chloroform/methanol/25%
ammonium hydroxide; 20/10/3, v/v/v). The condensation product
7 (RF = 0.55) left the column prior to the un-reacted starting mate-
rial 5 (RF = 0.75). The additionally isolated un-reacted starting
product (6.1 g, 24%) was identical to compound 5 and could be re-
used for a new condensation reaction. Fractions containing the de-
sired compounds were pooled, concentrated and then freeze dried
to a colorless solid. The column chromatography purification of the
whole reaction mixture yielded 20 g (61%) of 7 mp 136–140 °C
3.2.3. Synthesis of 30-azido-30-deoxythymidylyl-(50 ? 2-O)-3-O-
octadecyl-sn-glycerol (5)
Compound 3 (84 g, 123 mmol) and 30-azido-30-deoxythymidine
4 (33 g, 124 mmol) were dissolved in dry pyridine (260 g). Pivaloyl
chloride (76 ml, 617 mmol) was added and the reaction mixture
was stirred under exclusion of moisture for 7 min before the con-
densation reaction was stopped by addition of water (100 ml) un-
der cooling. The stirred reaction mixture was oxidized within 2 h
using a solution (500 ml) of iodine (0.2 M) dissolved in THF/pyri-
dine (18:1, v/v). Then, the solution was decolorized by adding a
small amount of solid NaHSO3 and concentrated to a sirup after
addition of saturated Na2CO3 (60 ml), which was coevaporated with
toluene (3ꢂ 50 ml), diluted with chloroform (600 ml) and then ex-
tracted with 50% aqueous NaHCO3 (200 ml). The separated chloro-
form phase was fractionated on a silica gel column using a
chloroform/methanol gradient as eluent. The condensation product
protected with the 1-O-4-monomethoxytrityl group was eluted as
main product, followed by a small amount of the desired de-trity-
lated compounds (5), which were collected when the eluent con-
tained more than 50% methanol. The pooled and concentrated
fractions (62 g) containing the tritylated condensation product
were diluted with a mixture (150 ml) of chloroform/methanol
(8:1, v/v), and then de-tritylated using 80% aqueous acetic acid
(60 ml) for 12 h, concentrated to a sirup which was mixed with
ether (300 ml) and then re-chromatographed on a silica gel column
using a chloroform/methanol gradient. The pooled fraction of the
desired product 5 (UV, sugar moiety positive and trityl negative)
were concentrated to a foam which was combined with the foam
obtained from the first chromatography of the reaction mixture.
The total yield of the 5 was 63 g (75.8%) which was dried in vacuo
over P4O10. RF = 0.75 (chloroform/methanol/25% ammoniumhy-
RF = 0.55
(chloroform/methanol/25%
ammoniumhydroxide
20:10:3, v/v/v); HRMS (ESIꢁ) obsd 808.36580, calcd for
C34H60N5O13P2 808.36574 [MꢁHꢁ]; ½a D20
ꢁ1.97° (c 1.06, water).
ꢀ
Anal. calcd (C34H59N5O13Na2P2ꢃ2H2O): C, H, N. 1H NMR (250 MHz,
D2O): d = 0.83 (t, 3H, J = 6.6 Hz, –CH3), 1.16–1.31 (m, 35H, –
(CH2)16–CH3), 1.54 (m, 2H, –CH2–), 1.89 (s, 3H, –CH3), 2.43 (m,
2H, H-20), 3.37–3.72 (m, 4H, Hglycerol), 4.03–4.41 (m, 5H, H-50, H-
500, Hglycerol, –O–CH2–), 4.41–4.56 (m, 2H, H-30, H-40), 6.16 (m, 1H,
H-10), 7.80 (s, 1H, H-6). 13C NMR (62.9 MHz, D2O): d = 14.5 (–
CH3), 16.3 (–CH3), 16.5 (–CH3), 25.3, 28.7, 32.0–32.4 (–(CH2)15–),
34.6 (C20), 39.9, 63.8, 63.9, 64.1 (C5’), 67.9, 68.7 (CH2glycerol), 73.1
(CHglycerol), 74.3 (O–CH2–), 76.9 (CHglycerol), 86.2 (C40), 88.1 (C10),
113.4 (C5), 139.8 (C6), 153.5 (C2), 168.6 (C4), 173.3 (PCO), 31P
NMR (161 MHz, D2O): d = ꢁ1.02, ꢁ5.02.
3.2.5. Synthesis of 30-azido-30-deoxythymidylyl-(50 ? 2-O)-3-O-
octadecyl-sn-glycerol-1-O-phosphonoformate trisodium salt (8)
A solution of 7 (9 g, 10 mmol) in 1 M NaOH (135 ml) was stirred
for 20 min. Then, the pH value of the solution was reduced to 8.5
by adding a cation-exchanger resin. The exchanger was removed
by filtration and the obtained filtrate freeze dried to yield the col-
orless product 8 (9 g, 98%). mp: 218–220 °C dec RF = 0.14 (chloro-
form/methanol/25% ammoniumhydroxide; 20:10:3, v/v/v); HRMS
(ESIꢁ) obsd 780.33606, calcd for C32H56N5O13P2 780.33533
[MꢁHꢁ]; ½a 2D0
ꢁ2.03° (c 1.08, water); Anal. calcd (C32H56N5O13-
ꢀ
Na3P2ꢃ4H2O): C, H. 1H NMR (250 MHz, D2O): d = 0.91 (t, 3H,
J = 6.6 Hz, –CH3), 1.22–1.38 (m, 32H, –(CH2)16–), 1.63 (m, 2 H, –
CH2–), 1.97 (s, 3H, –CH3), 2.52 (m, 2H, H-20), 3.51–3.81 (m, 5H,
Hglycerol), 4.03–4.21 (m, 3H, H-40 H-50,H-500), 4.41–4.63 (m, 1H, H-
30), 6.15 (m, 1H, H-10), 7.82 (s, 1H, H-6). 13C NMR (62.9 MHz,
D2O): d = 14.6 (–CH3), 16.5 (–CH3), 25.3, 28.7, 32.1–32.5 (–
(CH2)14–), 34.6 (C20), 40.2, 64.3 (C50), 67.7 (CH2glycerol), 73.4 (CHglyc-
erol), 74.4 (O–CH2–), 74.8, 77.5 (CHglycerol), 88.7 (C10), 88.9 (C40),
113.4 (C5), 139.9 (C6), 154.5 (C2), 168.9 (C4), 178.4 (PCO), 182.0.
31P NMR (161 MHz, D2O): d = 1.33, ꢁ3.43.
droxide 20:10:3 v/v/v); MS (FABꢁ) 672.2 [MꢁHꢁ]; ½a 2D0
ꢀ
5.69°
(c 1.16, H2O); ½a D20
ꢀ
+7.57° (c 1.17, THF). Anal. calcd (C31H55O9N5-
NaPꢃH2O)C, H. 1H NMR (D2O, 400 MHz): d = 0.88 (m, 3H, –CH3),
1.21–1.38 (m, 32H, –(CH2)16–), 1.61 (m, 2H, O–CH2–), 1.94 (m, 3H,
–CH3), 2.48 (m, 2H, H-20), 3.45–3.81 (m, 5H, Hglycerol), 3.97–4.41
(m, 4H, H-30, H-40, H-50, H-500), 4.80 (s, br, –OH + HDO), 6.23 (s, br,
1H, H-10), 7.76–7.87 (m, 1H, H-6). 13C NMR (D2O, 100 MHz):
d = 11.9 (CH3), 13.9 (–CH2–CH3), 22.7, 26.2, 29.3–30.4 (–CH2)15–),
32.0 (O–CH2–CH2–(CH2)14–), 61.5 (C30) 62.4 (C50), 66.5, 71.0 (CHglycerol),
71.7 (O–CH2–), 74.4 (CHglycerol) 76.1 (CHglycerol), 83.7 (C10), 85.5
(C40), 110.8 (C5), 119.2, 137.0 (C6), 150.9 (C2), 166.0 (C4). 31P
NMR (D2O, 161 MHz): d = 0.36 (O–PO(OH)–O), 18.4 (O–PO(H)–O).
3.3. Virological methods
3.3.1. Antiviral drugs
The stock solution (196 mM) of ganciclovir (GCV, CymeveneÒ)
contained 543 mg sodium-GCV corresponding to 50 mg GCV/ml
phosphate buffered saline (PBS). The original stock solution
(80 mM) of Foscarnet (PFA, FoscavirÒ) contained 24.0 mg triso-
dium-Foscarnet ꢂ 6H2O/ml. The stock solution (111 mM) of acy-
clovir (ACV) contained 27.44 mg sodium-ACV/ml PBS. The stock
solution (100 mM) of AZT–lipid–PFA contained 8.4 mg/ml in PBS.
Further solutions were prepared in Eagles minimal essential
medium (MEM) supplemented with 10% fetal calf serum (FCS),
10,000 U/ml penicillin G, 10 mg/ml streptomycin sulfate (MEM–
10% FCS) starting from 1 mM GCV, 80 mM PFA, 100 mM ACV and
100 mM AZT–lipid–PFA in MEM ꢁ10% FCS, respectively.
3.2.4. Synthesis of 30-azido-30-deoxythymidylyl-(50 ? 2-O)-3-O-
octadecyl-sn-glycerol-1-O-(ethoxycarbonyl)phosphonate (7)
(Ethoxycarbonyl)phosphoric dichloride 6 (25 g, 130 mmol) was
dissolved in trimethylphosphate (75 ml) in a 250 ml round-bot-
tomed flask equipped with a drying tube. After addition of 5
(25 g, 37 mmol) the reaction mixture was stirred for 16 h. The reac-
tion was stopped by pouring the solution slowly into a stirred and
cooled solution of saturated aqueous Na2CO3 (about 225 ml). The
pH value of the resulting mixture (about 370 ml) should be kept
between 6.5 and 7.0. The obtained solution was halved and puri-
fied within two runs by flash chromatography on a RP-18 (40–
3.3.2. Cell culture
60
l
M) column (25 ꢂ 5 cm) using a three step water/methanol elu-
HCMV strains were propagated using human foreskin fibroblast
(HFF) monolayers in MEM ꢁ10% FCS, which were used between
tion gradient (1) 50% methanol (100 ml), (2) linear increasing gra-

310
H. Schott et al. / Bioorg. Med. Chem. 17 (2009) 303–310
6. Kong, X. B.; Zhu, Q. Y.; Ruprecht, R. M.; Watanabe, K. A.; Zeidler, J.; Gold, J. W.
M. W.; Polsky, B.; Armstrong, D.; Chou, T. C. Antimicrob. Agents Chemother. 1991,
35, 2003.
7. Smith, R. A.; Anderson, D. J.; Preston, B. D. J. Virol. 2006, 80, 7169.
8. Marchand, B.; Tchesnokov, E. P.; Gotte, M. J. Biol. Chem. 2007, 282, 3337.
9. Goldschmidt, V.; Marquet, R. Int. J. Biochem. Cell Biol. 2004, 36, 1687.
10. Meyer, P. R.; Matsuura, S. E.; Zonarich, D.; Chopra, R. R.; Pendarvis, E.; Bazmi, H.
Z.; Mellors, J. W.; Scott, W. A. J. Virol. 2003, 77, 6127.
11. Cruchaga, C.; Ansó, E.; Rouzaut, A.; Martinez-Irujo, J. J. J. Biol. Chem. 2006, 281,
27744.
12. Eriksson, B. F.; Schinazi, R. F. Antimicrob. Agents Chemother. 1989, 33, 663.
13. Koshida, R.; Vrang, L.; Gilljam, G.; Harmenberg, J.; Oberg, B.; Wahren, B.
Antimicrob. Agents Chemother. 1989, 33, 778.
passage 15 and 30 for resistance testing and virus propagation.
HSV strains were propagated in monolayers of Vero cells in MEM
ꢁ10% FCS. Fibroblast and Vero cells were tested mycoplasma free.
3.3.3. Viral strains
Three clinical HCMV strains were used. Strain HCMV-1sens was
phenotypically GCV sensitive, while strain HCMV-3res with the
UL97 mutation C603W showed GCV resistance. Both strains were
derived from a chemosusceptibility trial of the German Society
for Virology in 2000. The GCV/CDV cross-resistant HCMV strain
U405cres was isolated from a pediatric stem cell transplant recipi-
ent.35 The ACV sensitive HSV-1 strain KOS was propagated for 3
14. Palmer, S.; Rasmussen, H.; Harmenberg, J.; Cox, S. Antiviral Chem. Chemother.
1996, 7, 14.
15. Snoeck, R.; Andrei, G.; Schols, D.; Balzarini, J.; De Clercq, E. Eur. J. Clin. Microbiol.
Infect Dis. 1992, 11, 1144.
weeks in the presence of 13 lM ACV resulting in the highly ACV
16. Rosowsky, A.; Saha, J.; Fazely, F.; Koch, J.; Ruprecht, R. M. Biochem. Biophys. Res.
resistant KOS-M strain. Sequential pairs of HSV-K and HSV-L
strains were isolated from two adult stem cell transplant recipients
K and L. The HSV-K161sens and HSV L177 isolate were both with
ACV sensitivity and represent the first pre-treatment viral isolates,
whereas the resistant strains (HSV-K161sens and HSV-L177) are
longitudinal viral isolates from patients K and L with clinically sus-
pected ACV resistance.
Commun. 1990, 172, 288.
17. Saha, J.; Ruprecht, R. M.; Rosowsky, A. Nucleosides Nucleotides 1991, 10, 1465.
18. Charvet, A. S.; Camplo, M.; Faury, P.; Graciet, J. C.; Mourier, N.; Chermann, J. C.;
Kraus, J. L. J. Med. Chem. 1994, 37, 2216.
19. Meier, C.; Aubertin, A. M.; de Monte, M.; Faraj, A.; Sommadossi, J. P.; Perigaud,
C.; Imbach, J. L.; Gosselin, G. Antiviral Chem. Chemother. 1998, 9, 41.
20. Rosowsky, A.; Fu, H.; Pai, N.; Mellors, J.; Richman, D. D.; Hostetler, K. Y. J. Med.
Chem. 1997, 40, 2482.
21. Schott, S. Pharm. Ztg 146 2001, 42, 24.
22. Hostetler, K. Y.; Beadle, J. R.; Kini, G. D.; Gardner, M. F.; Wright, K. N.; Wu, T. H.;
Korba, B. A. Biochem. Pharmacol. 1997, 53, 1815.
23. Hostetler, K. Y.; Kini, G. D.; Beadle, J. R.; Aldern, K. A.; Gardner, M. F.; Border, R.;
Kumar, R.; Barshak, L.; Sridhar, C. N.; Wheeler, C. J.; Richman, D. D. Antiviral Res.
1996, 31, 59.
24. Hammond, J. L.; Koontz, D. L.; Bazmi, H. Z.; Beadle, J. R.; Hostetler, S. E.; Kini, G.
D.; Aldern, K. A.; Richman, D. D.; Hostetler, K. Y.; Mellors, J. W. Antimicrob.
Agents Chemother. 2001, 45, 1621.
25. Hostetler, K. Y.; Hammond, J. L.; Kini, G. D.; Hostetler, S. E.; Beadle, J. R.; Aldern,
K. A.; Chou, T. C.; Richman, D. D.; Mellors, J. W. Antiviral Chem. Chemother. 2000,
11(31), 59.
3.3.4. Assays for the determination of the in vitro antiviral
activity against HCMV and HSV
Phenotypic HCMV susceptibility assays were performed using a
simplified micro-culture-based plaque reduction assay with cell-
associated virus strains, as reported previously.35,36 The HSV pla-
que reduction assay was performed in analogy to published
methods.28
26. Weislow, O. S.; Kiser, R.; Fine, D. L.; Bader, J.; Shoemaker, R. H.; Boyd, M. R. J.
Natl. Cancer Inst. 1989, 81, 577.
Acknowledgments
27. Balfour, H. H., Jr. N. Engl. J. Med. 1999, 340, 1255.
28. Safrin, S.; Crumpacker, C.; Chatis, P.; Davis, R.; Hafner, R.; Rush, J.; Kessler, H.
A.; Landry, B.; Mills, J. N. Engl. J. Med. 1991, 325, 551.
29. Morris-Natschke, S. L.; Ishaq, K. S.; Kucera, L. S. Curr. Pharm. Des. 2003, 9, 1441.
30. Schott, H.; Häussler, M. P.; Gowland, P.; Horber, D. H.; Schwendener, R. A.
Antiviral Chem. Chemother. 1994, 5, 387.
For the performance of the NMR analysis we thank Dr. Goltz.
The excellent technical support of E. Mikeler gratefully acknowl-
edged. The authors thank the National Cancer Institute (Bethesda,
USA) for the in vitro HIV testing of AZT–lipid–PFA.
31. Anschütz, R.; Emery, W. O. Liebigs Ann. Chem. 1887, 239, 301.
32. Hirth, G.; Barner, R. Helv. Chim. Acta 1982, 65, 1059.
33. Vaghefi, M. M.; McKernan, P. A.; Robins, R. K. J. Med. Chem. 1986, 29, 1389.
34. Gomberg, M.; Buchler, C. C. J. Am. Chem. Soc. 1923, 45, 207.
35. Eckle, T.; Lang, P.; Prix, L.; Jahn, G.; Klingebiel, T.; Handgretinger, R.; Selle, B.;
Niethammer, D.; Hamprecht, K. Bone Marrow Transplant 2002, 30, 433.
36. Prix, L.; Maierl, J.; Jahn, G.; Hamprecht, K. J. Clin. Virol. 1998, 11, 29.
References and notes
1. Pauwels, R. Antiviral Res. 2006, 71, 77.
2. Simons, C.; Wu, Q.; Htar, T. T. Curr. Top. Med. Chem. 2005, 5, 1191.
3. De Clercq, E. Biochem. Pharmacol. 2007, 73, 911.
4. Oversteegen, L.; Shah, M.; Rovini, H. Nat. Rev. Drug Discovery 2007, 6, 951.
5. Tachedjian, G.; Mellors, J.; Bazmi, H.; Birch, C.; Mills, J. J. Virol. 1996, 70, 7171.