Potential application of thymidylate kinase in nucleoside analogue activation in Plasmodium falciparum

Article abstract of DOI:10.1016/j.bmc.2010.07.006

Plasmodium falciparum thymidylate kinase (PfTMPK) shows a broad range of substrate tolerance when compared to the corresponding human enzyme. Besides 2′-deoxythymidine monophosphate (dTMP), PfTMPK can phosphorylate 3′-azido-2′,3′-dideoxythymidine monophos

Full text of DOI:10.1016/j.bmc.2010.07.006

Bioorganic & Medicinal Chemistry 18 (2010) 7302–7309
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Potential application of thymidylate kinase in nucleoside analogue activation
in Plasmodium falciparum
Huaqing Cui a,b, Luis M. Ruiz-Pérez b, Dolores González-Pacanowska b, , Ian H. Gilbert a,
⇑
⇑
a College of Life Sciences, University of Dundee, Sir James Black Centre, Dundee, DD1 5EH, UK
b Instituto de Parasitología y Biomedicina ‘López-Neyra’, Consejo Superior de Investigaciones Científicas, Avda. del Conocimiento s/n, 18100-Armilla, Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Plasmodium falciparum thymidylate kinase (PfTMPK) shows a broad range of substrate tolerance when
compared to the corresponding human enzyme. Besides 20-deoxythymidine monophosphate (dTMP),
PfTMPK can phosphorylate 30-azido-20,30-dideoxythymidine monophosphate (AZTMP) very efficiently.
In contrast, human thymidylate kinase (hTMPK) is 200 times less active towards AZTMP. We were inter-
ested to see if we could use PfTMPK to activate 30-azido-20,30-deoxythymidine (AZT) derivatives as a strat-
egy to treat malaria. P. falciparum lacks a pyrimidine nucleoside kinase which usually activates nucleoside
and nucleoside analogues to the corresponding monophosphates. Therefore, several prodrug analogues of
AZT and related nucleoside monophosphates were prepared and analysed for antiparasitic activity. The
prodrugs showed an increase in activity over the parent nucleoside analogues, which showed no inhibi-
tion of parasite growth at the concentration tested. The evidence here reported provides a strategy that
could be exploited for further anti-malarial design.
Received 16 March 2010
Revised 30 June 2010
Accepted 6 July 2010
Available online 6 August 2010
Keywords:
Malaria
Plasmodium
Thymidylate kinase
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
tion.5,6 It acts as an alternate substrate and is incorporated into the
growing DNA chain, and because it lacks a 30-hydroxyl, incorpora-
Malaria is a major health problem in many areas of the world;
due to increased resistance and failure of the current treatments
there is a need for new drugs for both treatment and prophylaxis
of malaria that are well tolerated.1 The enzyme Plasmodium falcipa-
rum thymidylate kinase (PfTMPK) is a potential drug target in
P. falciparum.2 The role of this enzyme is the phosphorylation of
deoxythymidine monophosphate to deoxythymidine diphosphate.
Kinetic data obtained with PfTMPK shows that it is able to tolerate
a broad range of substrates, which distinguishes it from other thy-
midylate kinases reported in the literature, in particular the human
enzyme.2,3 Thus, one novel property is the capacity to phosphory-
tion leads to chain termination.7,8 AZT is activated firstly to AZTMP
by the action of human thymidine kinase, secondly to AZTDP by
human thymidylate kinase and then to AZTTP by human nucleo-
side diphosphate kinase.8 The rate limiting step in the activation
of AZT is actually the phosphorylation of AZTMP to AZTDP, which
is catalysed by human thymidylate kinase, when AZT is used as
an antiviral.9 However, as AZTMP is a much better substrate for
PfTMPK than for hTMPK, it is probable that AZTMP will be acti-
vated much more rapidly in the malaria parasite compared to
the human cell. Any subsequent anti-malarial activity would rely
on AZTTP being a reasonable substrate/inhibitor of malarial DNA
polymerases.
late AZTMP 200 times more efficiently (kcat/KM 200 sꢀ1 mMꢀ1 3
)
compared to the corresponding human thymidylate kinase
(hTMPK) (kcat/KM 1 sꢀ1 mMꢀ1).4 Another feature is that PfTMPK
can phosphorylate efficiently dGMP (kcat/KM 370 sꢀ1 mMꢀ1),2,3
which compares very favourably to the value for dTMP (kcat/KM
460 sꢀ1 mMꢀ1).3
We were interested to see if we could use PfTMPK to selectively
activate AZT as a strategy to treat malaria. AZT is a prodrug used in
the treatment of HIV/AIDS; the active species is AZT triphosphate
(AZTTP), which exerts its action against the viral enzyme reverse
transcriptase, by acting as a chain terminator to DNA polymeriza-
The first step of activation of AZT, when used in the treatment of
HIV/AIDS is conversion to AZTMP by human thymidine kinase.
However, P. falciparum does not encode the enzyme pyrimidine
nucleoside kinase10 required for the formation of the nucleotide
monophosphate and so no mechanism for the activation of nucle-
oside analogues to the nucleoside monophosphate is available in
the parasite. To achieve some activity these nucleoside analogues
must be phosphorylated to the corresponding nucleotides prior
to entering the parasites. On the other hand, it is known that nucle-
otide analogues have poor bioavailability due to the presence of
the phosphate group, which is highly charged at physiological
pH, preventing the passive diffusion through cellular membranes
and there are also stability issues due to the presence of 50-nucle-
otidases.11 A way to circumvent the lack of a malarial thymidine
⇑
Corresponding authors. Tel.: +44 1382 386 240; fax: +44 1382 386 373 (I.H.G.).
E-mail addresses: dgonzalez@ipb.csic.es (D. González-Pacanowska), i.h.gilbert@
dundee.ac.uk (I.H. Gilbert).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.006

H. Cui et al. / Bioorg. Med. Chem. 18 (2010) 7302–7309
7303
kinase would be to deliver AZTMP in a masked form, or as a pro-
drug (Fig. 1). A number of phosphate masking strategies have been
developed and reported in the literature.11 One of the most suc-
cessfully applied technologies is the use of phosphoramidates
developed by McGuigan et al.12,13
The first step was to protect the amine group of guanine base.
Initially this was attempted with the BOC group, but this was
not successful, although several different conditions17 were
tested. Alternatively the use of isobutyric anhydride in the pres-
ence of an excess of trimethylchlorosilane proved to be success-
ful18 and the protected nucleoside 6 was obtained in good yield.
The inversion of the stereochemistry of C-30 was achieved in two
steps after the protection of the 50-hydroxyl with TBDPS. The 30-
OH was first oxidized to the corresponding ketone, using Dess–
Martin reagent and then reduced to alcohol with NaBH4. The
reaction produced a diastereomeric mixture of 7 and 8 with a
1:4 ratio. Stereoisomers 7 and 8 could be separated by column
chromatography. The 30 hydroxyl was then converted into the
corresponding azide 9 with inversion of configuration using di-
phenyl-2-pyridylphosphine, DIAD and NaN3 with a yield of 40%.
The final product AZG 3 was obtained after the two de-protec-
tions to remove the isobutyl ester and the TBDPS group under
standard conditions.
Therefore we decided to make masked phosphate derivatives of
several nucleoside analogues. Firstly we selected AZT (1) as AZTMP
is known to be a good substrate of the PfTMPK.3 Then we decided
to investigate 20,30-didehydro-20,30-dideoxythymidine (d4T) (2);
interestingly for this nucleoside analogue, the rate limiting step
in its activation as an antiviral is conversion of d4T to d4T-MP,15
which is a different step to that for AZT. This would provide an
interesting comparison to AZT. Another observation from our work
is that 20-deoxyguanosine (dGMP) is a good substrate of PfTMPK.2
Therefore we decided to investigate a masked phosphate version
of 30-azido-20,30-deoxyguanosine (AZG) (3). Finally we decided to
prepare the masked phosphate of
dine ( -AZT) (4) to see the effect of altered stereochemistry on
the activity. Therefore we prepared the phosphoramidate-based
prodrugs of AZT, d4T, AZG and -AZT (Fig. 2). Once the prodrug
a
-30-azido-20,30-dideoxythymi-
a
The synthesis of
dine (10) was prepared in four steps following literature meth-
ods.19,20 -Deoxythymidine was firstly protected in 50 position
a-AZT is shown in Scheme 2. a-Deoxythymi-
a
is inside cells, the masked nucleotide should be enzymatically
a
cleaved and the free phosphate released.
with TBDPS (attempts to use the TBDMS were not successful
due to the instability of the TBDMS group under the subsequent
Mitsunobu conditions). Mesylation of the 30-OH was then carried
out using Mitsunobu conditions with methanesulfonic acid21,22 to
give the required mesylate 12 in reasonable yield (56%). The
Mitsunobu reaction is widely used for the combination of an
alcohol and an acidic species HX (methanesulfonic acid in this
study), and results in direct replacement of the hydroxyl group
2. Results and discussion
2.1. Chemistry
30-Azido-20,30-dideoxyguanosine (AZG, 3) was synthesized fol-
lowing key conditions reported in the literature (Scheme 1).16
O
N
O
N
O
No thymidine
NH
NH
NH
kinase in
O
P
O
P
PfTMPK
O
P
O
P. falciparum
O
N
O
O
O
O
-O
O
O
-O
O
HO
O-
O-
O-
Corresponding
hTMPK much less
efficient at this step
N3
N3
N3
AZT
AZTMP
AZTDP
Chemical
synthesis
Prodrug
unmasking
PfNDK
O
N
NH
O
N
O
P
O
O
NH
O
O
N
H
O
O
P
O
P
O
P
O
O
O
-O
O
O
O
N3
O-
O-
O-
N3
AZTTP
Substrate (or inhibitor ?) of malarial DNA polymerases
Prodrug
Can penetrate cell membranes
Figure 1. The strategy used in this study. P. falciparum lacks the enzyme of thymidine kinase, which is needed to phosphorylate AZT to be AZTMP. In the present strategy, AZT
is masked as a prodrug for parasite delivery. The prodrug is released as AZTMP. AZTMP is first phosphorylated to be AZTDP by PfTMPK, a process which is 200 times more
efficient than with the corresponding human enzyme. AZTDP would be further phosphorylated by Plasmodium nucleoside diphosphate kinase14 (PfNDK) to AZTTP, which can
act as a substrate or inhibitor of Plasmodium DNA polymerases and interfere with parasite DNA replication.
O
O
N
O
N
N
N
NH
NH
NH
O
N
O
NH2
O
N
O
O
HO
O
HO
HO
HO
O
N
H
N3
O
N3
N3
1
4
2
3
Figure 2. Four selective nucleoside analogues, which are used for the study. 30-azido-20,30-dideoxythymidine AZT (1), 20,30-didehydro-20,30-dideoxythymidine d4T (2), 30-
azido-20,30-dideoxyguanosine AZG (3) and -30-azido-20,30-dideoxythymidine
-AZT (4).
a
a

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H. Cui et al. / Bioorg. Med. Chem. 18 (2010) 7302–7309
O
O
O
N
N
N
N
N
NH
NH
O
NH
O
Si
N
b
a
N
O
NH2
N
O
N
N
HO
O
N
HO
O
H
H
HO
HO
HO
7
5
6
c
O
O
O
N
N
N
N
NH
NH
O
NH
O
Si
N
e
Si
N
O
NH2
N
d
N
O
N
HO
O
N
O
N
O
H
H
N3
N3
HO
9
3
8
Scheme 1. The synthetic pathway for AZG. Reagents and conditions: (a) Trimethylchlorosilane, isobutyric anhydride, pyridine, 60%; (b) TBDMS–Cl, imidazole, pyridine, 78%;
(c) (I) Dess–Martin periodinane, DCM; (II) NaBH4, Acetone, ꢀ60 °C, 12 h, 71%; (d) PyPh2P, DIAD, NaN3, 40%; (e) (I) TBAF, THF; (II) NaOMe, DCM/MeOH, 45 °C, 72%.
O
O
O
b
a
Si
Si
HO
O
N
N
N
O
O
O
O
N
H
N
H
N
H
HO
HO
11
O
O
O
O
10
O
S
O
12
c
d
O
O
Si
N
N
HO
O
O
O
N
H
N
N3
N3
O
O
H
4
13
Scheme 2. The synthetic pathway for a-AZT. Reagents and conditions: (a) TBDPS–Cl, imidazole, pyridine, 83%; (b) MsOH, Ph3P, DIAD, THF, 24 h, 56%; (c) NaN3, DMF, 12 h,
40%; (d) TBAF, THF, 95%.
by X with strict inversion of configuration.21 This procedure
allowed us to invert the stereochemistry of the 30-C, although
there was some difficulty in the removal of the triphenylphos-
phine oxide. Conversion of 30-methanesulfonate group into azide
was performed by SN2 displacement with NaN3 in DMF, which
gave 13 with the re-inverted stereochemistry of the 30-C. The
de-protection of TBDPS by TBAF in THF produced the final prod-
2.2. Anti-malarial activity
Compounds were screened for their potency in vitro against P.
falciparum using the fluorescence-based SYBR-green assay re-
ported in the literature.25 Table 1 shows the inhibition of P. falcipa-
rum growth obtained at a compound concentration of 100 lM.
EC50 values which correspond to the concentration of compounds
that reduces P. falciparum growth by 50% were further obtained
for some of the compounds. As predicted the parent nucleosides
1–4 did not inhibit significantly P. falciparum growth at a concen-
uct
a-AZT 4.
The synthesis of phosphoramidates of AZT, d4T and
a
-AZT was
performed according to the method developed by McGuigan
et al.7,13,23 This consists in the reaction of the amino acid ester with
phenyl dichlorophosphate to form compounds 16 and 17 and sub-
sequent coupling with the 50-OH of the nucleosides in the presence
of N-methyl imidazole (Scheme 3). This procedure afforded: the
two phosphoramidates of AZT 18 and 19; the two d4T phospho-
tration of 100 lM presumably due to the lack of activation by a
thymidine kinase. By contrast, the prodrugs of the azides all
showed improved anti-malarial activities compared to the parent
nucleoside analogues with EC50 values in the micromolar range.
It is reasonable to assume that the prodrugs are internalised and
inside the parasites, are metabolized to yield the nucleoside mono-
phosphates. These are then converted to the diphosphate form by
PfTMPK and subsequently to the triphosphates by Plasmodium
nucleoside diphosphate kinase.14 We hypothesise that the com-
pounds act at the level of DNA replication, where they are chain
terminators of Plasmodium DNA polymerisation.
ramidates 20 and 21; and the phosphoramidate of
a-AZT 23. For
the synthesis of the AZG phosphoramidate 22, the phosphochlori-
date 17 was coupled to the nucleoside following the Uchiyama
procedure24 in the presence of tert-butyl magnesium chloride in
THF. Again also this method worked well and the desired prodrug
22 was obtained in good yield. The masking group derived from
valine was slightly more active against P. falciparum than that
from the alanine derivative for both AZT and d4T (see later), there-
Interestingly the d4T prodrugs had significantly lower activity—
it may be that d4T monophosphate is a poor substrate of the
PfTMPK. It appeared to be possible to change the stereochemistry
fore AZG and
a-AZT were only coupled with valine derived
phosphoramidate.
from b to
a with little effect on activity or to replace the thymine

H. Cui et al. / Bioorg. Med. Chem. 18 (2010) 7302–7309
7305
R2
O
O
P
Cl
O
P
Cl
R1
H
N
HCl.H2N
a
Cl
O
O
O
O
R1
14
16. R1 = CH3; R2 = CH3
O
R2
15
17. R1 = CH(CH3)2; R2 = CH2CH3
b
b
c
b
O
O
N
O
N
N
N
NH
NH
NH
R1
O
R1
O
O
H
O
P
O
O
O
P
O
O
P
O
N
H
N
O
NH2
or
O
O
O
N
N
H
N
P
O
O
N
H
O
O
O
O
O
or
O
O
O
O
N
H
N3
O
O
O
N3
R2
N3
R2
22
20. R1 = CH3; R2 = CH3
21. R1 = CH(CH3)2; R2 = CH2CH3
23
18. R1 = CH3; R2 = CH3
19. R1 = CH(CH3)2; R2 = CH2CH3
Scheme 3. Synthesis of phosphoramidates of AZT, d4T, AZG and
THF, 12 h, 46%.
a
-AZT. Reagents and conditions: (a) TEA, DCM, ꢀ78 °C, 70%; (b) NMI, THF, ꢀ78 °C to rt, 70–80%; (c) tBuMgCl,
3. Conclusion
Table 1
Inhibition of growth of P. falciparum cultured in vitro
In this paper, we have taken advantage of the unique substrate
Compound
Nucleoside
(%) Growth inhibition at 100
lM
EC50 (lM)
specificity of PfTMPK compared to that of the human enzyme for
designing compounds that would be toxic to Plasmodium parasites.
In particular we have exploited the observations that both AZTMP
and dGMP are good substrates for the PfTMPK but not for human
TMPK. Since malaria parasites lack a thymidine kinase activity that
would be required for the phosphorylation of nucleoside ana-
logues, a prodrug strategy was devised in order to efficiently deli-
ver the nucleoside monophosphates into cells. Three nucleoside
1 (AZT)
18
19
2 (d4T)
20
21
AZT
AZT
AZT
d4T
d4T
d4T
AZG
AZG
0
>100
120
80
>100
>100
>100
>100
66
35 2%
69 1%
0
9
1%
18 1%
0
80 1%
0
3 (AZG)
22
4 (a-AZT)
a-AZT
-AZT
>100
67
23
a
78 2%
derivatives of AZT, AZG and a-AZT exhibit significant growth inhi-
bition of Plasmodium with EC50 values in micromolar range, while
d4T derivatives were less active. Further optimization of the nucle-
oside moiety to provide improved cellular uptake, activation and
binding to Plasmodium DNA polymerases could yield more potent
inhibitors of Plasmodium growth.
with a guanine. We can speculate that a-AZTMP and AZG-MP are
both substrates for PfTMPK in a similar way to AZTMP. The valine
derivative 19 was marginally more active than the alanine deriva-
tive 18 for the AZT derivatives and hence we used the valinyl deriv-
4. Experimental
atives for the a-AZTMP and AZG-MP prodrugs.
Although the present compounds have insufficient activity for
the progressing into antimalarials, all the prodrugs clearly showed
improved activity against the parasite compared to the parent
nucleoside analogues. It has been described that apart from inhib-
iting viral reverse transcriptase and incorporating into viral DNA,26
these kinds of nucleoside analogues also exhibit some affinity for
cellular polymerases and can be incorporated into mitochondrial
DNA where they arrest DNA chain extension and are able to affect
an exonucleolytic repair mechanism.27 It is therefore possible that
the antiplasmodial activity observed is due to direct incorporation
into DNA or inhibition of parasite replication.
4.1. In vitro assays
In vitro activity against the erythrocytic stages of P. falciparum
was determined by using a SYBR-green assay.25 The parasite P. fal-
ciparum 3D7 was cultured using standard methods, and synchro-
nized using 5% sorbitol as previously described.28 Compounds
were dissolved in DMSO at 100 mM and added to 48 h post-syn-
chronization parasite cultures incubated in RPMI 1640 medium
with hypoxanthine (150
lM), NaHCO3 (0.2%), gentamycin
(12.5 g/mL), Albumax (0.5%), human serum (2%) and washed hu-
l
man red cells O+ at 5% haematocrit (0.3% parasitaemia). Chloro-
quine was used as standard drug. Experiments were carried out
at least twice independently and the different concentrations were
The lack of potent activity of these compounds may be due to
several reasons. First, reduced cell permeability to P. falciparum
may be an issue. Second, we ignore how efficiently the prodrug
is converted to the active species. Finally, the nucleoside triphos-
phates may be relatively poor substrates or inhibitors of Plasmo-
dium DNA polymerase. Further information regarding the uptake
and intracellular metabolism of these compounds would aid in
the design of compounds with increased antiplasmodial activity.
tested in duplicate. After 48 h of growth, 100
(Molecular Probes) in lysis buffer (Tris 20 mM, pH 7.5, EDTA
5 mM, saponin 0.008%, triton X-100 0.08%, 0.2 l of SYBR-green/
ll of SYBR-green I
l
ml of lysis buffer) was added to each well, and mixed and after
1 h of incubation in the dark at rt, fluorescence was measured with

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H. Cui et al. / Bioorg. Med. Chem. 18 (2010) 7302–7309
excitation and emission wavelength bands centred at 485 and
530 nm. The percent inhibition of each compound at each concen-
tration was determined. EC50 values were calculated from hyper-
bolic or sigmoidal dose–response curves using Sigmaplot 10.0.
CHCH3, i-Bu), 2.52–2.57 (m, 1H, H20), 2.27–2.30 (m, 1H, H20), 1.09
(d, J = 6.5 Hz, 6H, CHCH3, i-Bu), 0.82 (s, 9H, SiCCH3), 0.00 (s, 6H,
SiCH3); 13C NMR (125 MHz, DMSO-d6) d 180.1 (COCH), 154.8
(C6), 148.3, 148.1 (C2 and C4), 137.1 (C8), 120.2 (C5), 87.1 (C10),
82.9 (C40), 70.0 (C30), 63.1 (C50), 39.3 (C20), 34.7 (CHCH3, i-Bu),
25.8 (SiCCH3), 18.9, 18.8 (CHCH3, i-Bu), 18.0 (SiCCH3), ꢀ5.5 (SiCH3);
LC–MS (ES+): m/z (%) 452 (100) [M+H]+; HRMS (ES+): calcd for
4.2. Chemistry
General: Chemical and solvents were purchased from the Sig-
ma-Aldrich Chemical Company, Fluka, VWR, Acros, Fisher Chemi-
cals and Alfa Aesar. 1H NMR, 13C NMR, 31P NMR were recorded
on a Bruker Avance DPX 500 spectrometer (1H at 500.1 MHz, 13C
at 125.8 MHz and 31P at 202 MHz). Chemical shift (d) are expressed
in ppm. Signal splitting patters are described as singlet (s), broad
singlet (br s), double (d), triplet (t), quarter (q), multiplet (m).
Low resolution electrospray (ES) mass spectra were recorded either
on a Applied Biosystem Mariner API-TOF biospectrometry Work-
station spectrometer or on a Bruker MicroTof mass spectrometer,
run in a positive ion mode, using either methanol, methanol/water
(95:5), or water/acetonitrile (1:1) + 0.2% formic acid as the mobile
phase. High resolution electrospray measurements were per-
formed on a Bruker Daltonics MicroTOF mass spectrometer. Col-
umn chromatography was carried out using Silica Gel 60 from
Fluka. Thin layer chromatography (TLC) was carried out on Merck
Silica Gel 60 F254 plates using UV light or PMA for visualization.
TLC data are given as the Rf value with the corresponding eluent
system specified in brackets.
C
20H34N5O5Si1 [M+H]+ 452.2324 m/z, found 452.2324 m/z.
4.2.3. 50-O-tert-Butyldimethylsilyl-N2-isobutyryl-20-30-alpha-
deoxyguanosine16 (8)
To a chilled solution (0 °C) of Dess–Martin periodinane (424 mg,
1 mmol) in DCM (3 ml) was added 7 (320 mg, 0.7 mmol) and the
result solution stirred at 0 °C. After 60 min, the cooling bath was
removed and stirred continued at rt. After 3 h, the reaction mixture
was cooled to ꢀ60 °C and anhydrous 2-propanol (3 ml) was added.
Stirring was continued for a further 12 h at ꢀ60 °C. Acetone (3 ml)
was added and the slurry was allowed to come to rt. The resulting
solution was washed with NaHCO3, H2O and Brine. After being dried
with MgSO4 and evaporated the solvent, the product was purified by
chromatography on silica to give 8 (224 mg, 71%) as a white solid.
TLC (5% MeOH/DCM) Rf = 0.20; 1H NMR (500 MHz, DMSO-d6) d
12.05 (s, 1H, NH), 11.70 (s, 1H, NH), 8.14 (s, 1H, H8), 6.11 (d, 1H,
J = 4.1 Hz, H10), 5.36 (d, J = 3.3 Hz, 1H, 30-OH), 4.34 (d, J = 3.7 Hz,
1H, H30), 3.91–3.96 (m, 2H, H40 and H50), 3.73–3.76 (m, 1H, H50),
2.67–2.75 (m, 2H, H20 and CHCH3, i-Bu), 2.21 (d, J = 14.5 Hz, 1H,
H20), 1.09 (d, J = 6.8 Hz, 6H, CCH3, i-Bu), 0.82 (s, 9H, SiCCH3), 0.00
(s, 6H, SiCH3); 13C NMR (125 MHz, DMSO-d6) d 180.1 (COCH3),
154.8 (C6), 148.2 (C4 and C2), 138.0 (C8), 119.8 (C5), 85.4 (C10),
82.5 (C40), 68.8 (C30), 62.1 (C50), 41.0 (C20), 34.7 (CCH3, i-Bu), 25.8
(SiCCH3), 18.9, 18.8 (CCH3, i-Bu), 18.0 (SiCCH3), ꢀ5.3, ꢀ5.4 (SiCH3);
LC–MS (ES+): m/z (%) 452 (100) [M+H]+; HRMS (ES+): calcd for
4.2.1. N2-Isobutyryl-20-deoxyguanosine18 (6)
20-Deoxyguanosine (1 g, 3.744 mmol) was co-evaporated with
pyridine (20 ml) three times and dried overnight. It was then sus-
pended in dry pyridine (20 ml). Trimethylchlorosilane (2.37 ml,
18.73 mmol) was added. The resulting solution was stirred at rt
for 2 h. Isobutyric anhydride (3.11 ml, 18.73 mmol) was added
and the mixture was stirred at rt under Ar for 4 h. The reaction
was cooled in an ice bath, and H2O (6 ml) was added. After 15 min
33% aqueous ammonia (6 ml) was added and the reaction was stir-
red for 15 min. The solution was then evaporated to near dryness
and the residue was dissolved in H2O (60 ml). The aqueous layer
was washed with DCM (50 ml) and crystallization occurred quickly
in water. The compound was then filtered and dried. The product
could further purified by chromatography (MeOH/DCM = 20:80) to
yield the product 6 (763 mg, 60%) as a white solid. TLC (10%
MeOH/DCM) Rf = 0.09; 1H NMR (500 MHz, DMSO-d6) d 12.05 (br s,
1H, NH), 11.72 (br s, 1H, NH), 8.24 (s, 1H, H8), 6.21 (q, J = 6.5 Hz,
1H, H10), 5.32 (d, J = 3.8 Hz, 1H, 30OH), 4.97 (t, J = 5.4 Hz, 1H, 50OH),
4.37 (m, 1H, H30), 3.83 (m, 1H, H40), 3.51–3.56 (m, 2H, H50 and
H50), 2.76–2.78 (m, 1H, CHCH3, i-Bu), 2.54–2.56 (m, 1H, H20), 2.27–
2.29 (m, 1H, H20), 1.12 (dd, 6H, CHCH3 i-Bu); 13C NMR (125 MHz,
DMSO-d6) d 180.1 (COCH), 154.8 (C6), 148.4, 148.1 (C2 and C4),
137.4 (C8), 120.1 (C5), 87.7 (C10), 82.9 (C40), 70.4 (C30), 61.4 (C50),
39.3 (C20), 34.7 (CHCH3), 18.8 (CHCH3); LC–MS (ES+): m/z (%) 222
(71) [C9H12N5O2]+, 338 (100) [M+H]+; HRMS (ES+): calcd for
C
20H34N5O5Si1 [M+H]+ 452.2324 m/z, found 452.2321 m/z.
4.2.4. 30-Azido-50-O-tert-butyldimethylsilyl-N2-isobutyryl-20,30-
dideoxyguanosine16 (9)
To a stirred slurry of 8 (210 mg, 0.47 mmol) and NaN3 (91 mg,
1.4 mmol) in DMF (2 ml) was added a solution of diphenyl-2-pyr-
idylphosphine (184 mg, 0.7 mmol) and DIAD (137 ll, 0.7 mmol) in
DMF (2 ml). After 8 h, H2O was added to stop the reaction, and DMF
was removed by evaporation. The reaction mixture was purified by
column chromatography using ethyl acetate/hexane (9:1) to give 9
as a solid (89 mg, 40%). TLC (10% MeOH/DCM) Rf = 0.49; 1H NMR
(500 MHz, CHCl3-d1) d 11.90 (s, 1H, N–H), 8.40 (br s, 1H, NH),
7.87 (s, 1H, H8), 6.05 (t, J = 6.1 Hz, 1H, H10), 4.30 (q, J = 5.3 Hz, 1H,
H30), 3.94–3.95 (m, 1H, H40), 3.70–3.78 (m, 2H, H50 and H50),
2.50–2.56 (m, 2H, H20 and CHCH3), 2.37–2.41 (m, 1H, H20), 1.14–
1.22 (m, 6H, CHCH3, i-Bu), 0.80 (s, 9H, SiCCH3), 0.00 (s, 3H, SiCH3),
0.00 (s, 3H, SiCH3); 13C NMR (125 MHz, CHCl3-d1) d 178.2 (CO),
155.4 (C6), 147.5 (C2 and C4), 136.5 (C8), 121.6 (C5), 85.0 (C10),
83.5 (C40), 62.9 (C30), 60.6 (C50), 38.4 (C20), 36.6 (CCH3, i-Bu), 25.9
(SiCCH3), 19.0, 18.9 (CCH3), 18.4 (SiCCH3), ꢀ5.3, ꢀ5.5 (SiCH3); LC–
MS (ES+): m/z (%) 477 (100) [M+H]+.
C
14H20N5O5 [M+H]+ 338.1459 m/z, found 338.1457 m/z.
4.2.2. 50-O-tert-Butyldimethylsilyl-N2-isobutyryl-20-deoxy-30-
beta-guanosine16 (7)
4.2.5. 30-Azido-20,30-dideoxyguanosine29 (3)
Compound 9 (210 mg, 0.44 mmol) was dissolved in 6 ml THF.
A stirred solution of 6 (750 mg, 2.22 mmol) in 5 ml pyridine was
treated with TBDMS–Cl (361 mg, 2.4 mmol). After 16 h, the reac-
tion mixture was transferred into ethyl acetate and the solution
was washed with H2O, brine and dried over MgSO4. The reaction
mixture was evaporated and purified by chromatography on silica
to yield 7 as a white solid (780 mg, 78%). TLC (5% MeOH/DCM)
Rf = 0.18; 1H NMR (500 MHz, DMSO-d6) d 12.05 (s, 1H, NH), 11.68
(s, 1H, NH), 8.13 (s, 1H, H8), 6.17–6.19 (m, 1H, H10), 5.34 (d,
J = 3.7 Hz, 1H, 30-OH), 4.34 (s, 1H, H30), 3.83 (d, J = 3.2 Hz, H40),
3.72–3.75 (m, 1H, H50), 3.65–3.68 (m, 1H, H50), 2.71–2.76 (m, 1H,
TBAF (440 ll, 0.44 mmol) was added into the solution. The reaction
was stirred at rt for 3 h and TLC was used to observe the disappear-
ance of the starting compound 9. The solvent of THF was evapo-
rated to remove. Without purification, we further de-protected
the isobutyric group by 0.5 M NaOMe in the solution of 3 ml
DCM/MeOH (1:1) in 45 °C overnight. After purification by column
chromatography using 20% MeOH in DCM as eluent to yield
required compound 3 as a white solid (90 mg, 72%). TLC (10%
MeOH/DCM) Rf = 0.12; 1H NMR (500 MHz, DMSO-d6) d 10.78 (br
s, 1H, NH), 7.93 (s, 1H, H8), 6.60 (s, 2H, NH2), 6.07 (t, J = 6.4 Hz,

H. Cui et al. / Bioorg. Med. Chem. 18 (2010) 7302–7309
7307
1H, H10), 5.16 (t, J = 5.4 Hz, 1H, OH), 4.57 (m, 1H, H30), 3.87 (q,
J = 4.6 Hz, 1H, H40), 3.56 (m, 1H, H50), 2.77 (m, 1H, H20), 2.43 (m,
1H, H20); 13C NMR (125 MHz, DMSO-d6) d 156.7 (C6), 153.8 (C2
and C4), 150.8 (C5), 135.3 (C8), 84.5 (C10), 82.1 (C40), 61.3 (C30),
60.9 (C50), 36.2 (C20); LC–MS (ES+): m/z (%) 152 (100) [C5H6N5O]+,
293 (76) [M+H]+; HRMS (ES+): calcd for C10H13N8O3 [M+H]+
293.1105 m/z, found 293.1094 m/z.
(d, J = 3.8 Hz, 2H, H50 and H50), 2.75 (m, 1H, H20), 2.08 (m, 1H,
H20), 1.88 (s, 3H, CH3), 1.00 (s, 9H, SiCCH3); 13C NMR (125 MHz,
CHCl3-d1) d 163.9 (C4), 150.4 (C2), 135.6 (C6), 135.5, 135.3, 132.6,
132.4, 130.14, 130.11, 128.0 (C-Ph), 110.6 (C5), 86.6, 86.5 (C10
and C40), 64.2 (C50), 61.6 (C30), 38.5 (C20), 26.9 (SiCCH3), 19.2
(CH3), 12.8 (SiCCH3); LC–MS (ES+): m/z (%) 506 (100) [M+H]+.
4.2.9. 30-Azido-30-deoxy-
Compound 13 (60 mg, 0.12 mmol) was de-protected the TBDPS
group by TBAF (120 l, 0.12 mmol) in 3 ml THF using the proce-
a
-thymidine30 (4)
4.2.6. 50-O-tert-Butyldiphenylsilyl-
-Deoxythymidine (726 mg, 3 mmol) in dry DMF (6 ml) was
added to a stirred solution of TBDPS–Cl (780 l, 3 mmol) and imid-
a-thymidine (11)
a
l
l
dure of compound 3, the reaction mixture was then purified by col-
umn chromatography using 5% MeOH/DCM as eluent to give the
product 4 as a white solid (30 mg, 95%). TLC (10% MeOH/DCM)
Rf = 0.17; 1H NMR (500 MHz, acetone-d6 ) d 9.87 (br s, 1H, NH),
7.43 (s, 1H, H6), 6.06 (q, J = 3.8 Hz, 1H, H10), 4.31–4.34 (m, 1H,
H30), 4.21 (q, J = 4.0 Hz, 1H, H40), 3.52–3.58 (m, 2H, H50 and H50),
2.74–2.75 (m, 1H, H20), 2.05–2.10 (m, 1H, H20), 1.72 (s, 3H, CH3);
13C NMR (125 MHz, acetone-d6 ) d 164.4 (C4), 151.3 (C2), 136.6
(C6), 110.5 (C5), 86.9, 86.6 (C10 and C40), 63.0 (C50), 62.1 (C30),
38.4 (C20), 12.6 (CH3); LC–MS (ES+): m/z (%) 127 (100)
[C5H7N2O2]+, 268 (38) [M+H]+; HRMS (ES+): calcd for C10H14N5O4
[M+H]+ 268.1040 m/z, found 293.1039 m/z.
azole (450 mg, 6.6 mmol) in dry DMF (6 ml) at rt. After 4 h, water
(30 ml) was added and the mixture was extracted with diethyl
ether (50 ml ꢁ 2). The combined extracts were washed with satu-
rated NaHCO3 (50 ml) and H2O (50 ml), dried with MgSO4 and re-
duced in vacuo. Column chromatography with 5% MeOH/DCM as
eluent gave the product 11 as a white solid (1.19 g, 83%). TLC
(10% MeOH/DCM) Rf = 0.33; 1H NMR (500 MHz, DMSO-d6)
d
11.28 (s, 1H, NH), 7.76 (s, 1H, H6), 7.44–7.65 (m, 10H, H-Ph), 6.20
(q, J = 3.5 Hz, 1H, H10), 5.44 (d, J = 2.9 Hz, 1H, 30-OH), 4.36 (s, 1H,
H30), 4.28 (s, 1H, H40), 3.62–3.65 (m, 2H, H50 and H50), 2.58–2.63
(m, 1H, H20), 1.93–1.96 (m, 1H, H20), 1.78 (s, 3H, CH3), 1.01 (s,
9H, SiCCH3); 13C NMR (125 MHz, DMSO-d6) d 163.8 (C2), 150.5
(C4), 136.9 (C6), 135.1, 132.7, 132.6, 130.0, 128.0 (C-Ph), 108.7
(C5), 88.2 (C10), 85.4 (C40), 70.5 (C30), 64.4 (C50), 39.0 (C20), 26.6
(C(CH3)3), 18.7 (CH3), 12.3 (CH3); LC–MS (ES+): m/z (%) 481 (100)
[M+H]+; HRMS (ES+): calcd for C26H33N2O5Si1 [M+H]+ 481.2153
m/z, found 481.2150 m/z.
4.2.10. Phenyl methoxyalaninyl phosphochloridate (16) and
phenyl ethyloxyvalinyl phosphochloridate (17)
The procedure for the preparation of 16 and 17 was carried out
according to the literature methods.23,31
4.2.11. General procedure: Preparation of 30-azido-30-deoxy-
nucleoside phosphoramidites
4.2.7. 30-O-Methanesulfonyl-50-O-tert-butyldiphenylsilyl-
a-
thymidine (12)
30-Azido-30-deoxy-nucleoside (0.3 mmol, 1 equiv) was dis-
solved in THF (2 ml), and phosphorochloridate (0.9 mmol, 3 equiv)
and N-methylimidazole (1.8 mmol, 6 equiv) were added dropwise
with vigorous stirring in ꢀ78 °C. After 12 h, the solvent was re-
moved under vacuum. The residue was dissolved in chloroform
(15 ml) and washed with 1 M hydrochloric acid solution
(2 ꢁ 15 ml), then water (3 ꢁ 15 ml). The organic phase was dried
with MgSO4 and evaporated under vacuum, and the residue was
purified by chromatography on silica by elution with 5% MeOH
in DCM to give the final product.
Methanesulfonic acid (35 ll, 0.53 mmol) was added to a solu-
tion of 11 (120 mg, 0.25 mmol) and triphenylphosphine (196 mg,
0.75 mmol) in dry THF (0.8 ml) under Ar. The temperature was
raised to 40 °C and DIAD (147 ll, 0.75 mmol) was added dropwise
over a period of 12 min with vigorous stirring, and with careful
exclusion of moisture. The reaction mixture became a yellow col-
our, but changed to a white and viscous after 15 min. The mixture
was stirred vigorously for 24 h at 40 °C under Ar. The volatiles were
then removed under reduced pressure and the residue was re-dis-
solved in DCM and purified by column chromatography using 5%
MeOH in DCM as eluent to give product 12 as a white solid
(78 mg, 56%). TLC (10% MeOH/DCM) Rf = 0.22; 1H NMR (500 MHz,
DMSO-d6) d 11.38 (s, 1H, NH), 7.60 (s, 1H, H6), 7.42–7.67 (m,
10H, H-Ph), 6.21 (t, J = 6.9 Hz, 1H, H10), 5.50 (q, J = 3.8 Hz, 1H,
H30), 4.68 (q, J = 4.9 Hz, 1H, H40), 3.82–3.86 (m, 1H, H50), 3.73–
3.76 (m, 1H, H50), 3.36 (s, 3H, SCH3), 2.64–2.67 (m, 2H, H20 and
H20), 1.81 (s, 3H, CH3), 1.00 (s, 9H, SiCCH3); 13C NMR (125 MHz,
DMSO-d6) d 163.8 (C2), 150.4 (C4), 136.7 (C6), 135.11, 135.07,
132.51, 132.45, 131.5, 129.95, 127.93, 127.89 (C-Ph), 109.7 (C5),
84.8 (C10), 81.5 (C40), 80.2 (C30), 62.0 (C50), 37.7, 37.5 (C20 and
SCH3), 26.5 (SiC), 18.7 (SiCCH3), 12.1 (CH3); LC–MS (ES+): m/z (%)
559 (100) [M+H]+; HRMS (ES+): calcd for C27H34N2O7S1Si1 [M+H]+
559.1915 m/z, found 559.1920 m/z.
4.2.12. 30-Azido-50-phenyl methoxyalaninyl phosphate-30-
deoxy-thymidine (18)
30-Azido-30-deoxy-thymidine (80 mg, 0.3 mmol) was reacted
with phenyl methoxyalaninyl phosphochloridate 16 (249 mg,
0.9 mmol) to give the final product 18 as a white solid (120 mg,
80%). TLC (10% MeOH/DCM) Rf = 0.52; 1H NMR (500 MHz, CHCl3-
d1) d 8.73 (d, J = 9.1 Hz, 1H, NH), 7.09–7.28 (m, 6H, H-Ph and H6),
6.06–6.13 (m, 1H, H10), 4.17–4.32 (m, 3H, H50, H50 and H40), 3.90–
4.03 (m, 2H, H30 and NH), 3.75–3.79 (m, 1H, CHNH), 3.63–3.70
(m, 3H, CH3), 2.26–2.36, 2.04–2.16 (m, 2H, H20), 1.78–1.83 (m,
3H, H7), 1.28–1.32 (m, 3H, CH3); 13C NMR (125 MHz, CHCl3-d1) d
173.8 (CO), 163.5 (C4), 150.4, 150.13 (C2), 150.10 (C-Ph), 135.32,
135.28 (C6), 129.9, 125.4, 125.3, 120.10, 120.06, 120.0 (C-Ph),
111.44, 111.42 (C5), 85.2, 84.9 (C10), 82.43, 82.37, 82.3, 82.2 (C40),
65.8, 65.72, 65.67 (C50), 60.4, 60.3 (C30), 52.6 (OCH3), 50.4, 50.2
(CHNH), 37.3 (C20), 21.0, 20.94, 20.89 (NHCHCH3), 12.5, 12.4 (C7);
31P NMR (202 MHz, CHCl3-d1) d 2.50, 2.80; LC–MS (ES+): m/z (%)
509 (100) [M+H]+; HRMS (ES+): calcd for C20H26N6O8P1 [M+H]+
509.1544 m/z, found 509.1563 m/z.
4.2.8. 30-Azido-50-O-tert-butyldiphenylsilyl-30-deoxy-
a-
thymidine (13)
A
solution of 12 (151 mg, 0.3 mmol) and NaN3 (39 mg,
0.6 mmol) in DMF (5 ml) was heated to 60 °C overnight. The reac-
tion mixture was evaporated in vacuo with toluene. The solution
was cooled and filtered. The filtrate was co-evaporated with tolu-
ene to remove DMF. The final azide was purified by column chro-
matography using 5% MeOH/DCM as eluent to give 13 as a white
solid (61 mg, 40%). TLC (10% MeOH/DCM) Rf = 0.28; 1H NMR
(500 MHz, CHCl3-d1) d 8.94 (s, 1H, NH), 7.32–7.58 (m, 10H, H-Ph),
7.23 (s, 1H, H6), 6.16 (m, 1H, H10), 4.25 (m, 2H, H30 and H40), 3.64
4.2.13. 30-Azido-50-phenyl ethyloxyvalinyl phosphate-30-deoxy-
thymidine (19)
30-Azido-30-deoxy-thymidine (80 mg, 0.3 mmol) was reacted
with phenyl ethyloxyvalinyl phosphochloridate 17 (287 mg,
0.9 mmol) to give the final product 19 as a white solid (103 mg,

7308
H. Cui et al. / Bioorg. Med. Chem. 18 (2010) 7302–7309
70%). TLC (10% MeOH/DCM) Rf = 0.50; 1H NMR (500 MHz, DMSO-d6)
d 11.34 (s, 1H, NH), 7.49 (s, 1H, H6), 7.15–7.21, 7.31–7.37 (m, 5H, H-
Ph), 6.11–6.15 (m, 1H, H10), 5.91–5.97 (m, 1H, NH), 4.45 (q,
J = 1.9 Hz, 1H, H30), 4.20–4.28 (m, 2H, H50), 4.14–4.18 (m, 1H, H40),
3.99–4.15 (m, CH2), 3.50–3.56 (m, 1H, CHNH), 2.29–2.42 (m, 2H,
H20and H20), 1.90–1.96 (m, 1H, CH), 1.75, 1.77 (s, 3H, H7), 1.13–
1.18 (m, 3H, CH3), 0.75–0.88 (m, 6H, CH3); 13C NMR (125 MHz,
DMSO-d6) d 172.4, 172.2 (CO), 163.6 (C4), 150.7, 150.6, 150.3 (C2
and C-Ph), 135.8, 135.7 (C6), 129.52, 129.49, 124.59, 124.5, 120.2,
120.1, 120.02, 120.0 (C-Ph), 109.91, 109.87 (C5), 83.7 (C10), 81.51,
81.45, 81.35 (C40), 65.6, 65.4 (C50), 60.33, 60.29, 60.2, 60.1, 60.0
(CHNH and OCH2CH3), 35.7, 35.6 (C20), 31.4, 31.3, 31.2, 31.1
(CH3CHCH3), 18.83, 18.75, 18.03, 17.79 (CH3CHCH3), 14.0
(OCH2CH3), 12.03, 11.99 (C7); 31P NMR (202 MHz, DMSO-d6) d
4.60, 4.70; LC–MS (ES+): m/z (%) 551 (100) [M+H]+; HRMS (ES+):
calcd for C23H32N6O8P1 [M+H]+ 551.2014 m/z, found 551.2002 m/z.
for 15 min. Then phenyl ethyloxyvalinyl phosphochloridate 17
(95 mg, 0.3 mmol) in dry THF was added dropwise and stirred
overnight. A saturated solution of NH4Cl was added, and the sol-
vent was removed under reduced pressure to give the final 22 as
a white solid (40 mg, 46%). TLC (10% MeOH/DCM) Rf = 0.31; 1H
NMR (500 MHz, acetone-d6) d 11.20 (s, 1H, NH); 7.79 (s, 1H, H8),
6.97–7.23 (m, 5H, H-Ph), 6.54, 6.61 (s, 2H, NH2), 6.12–6.15 (m,
1H, H10), 4.81–4.86 (m, 1H, NH), 4.66–4.69 (m, 1H, H30), 4.41–
4.45, 4.53–4.58 (m, 1H, H30), 4.13–4.17, 4.20–4.27 (m, 1H, H50),
4.03–4.06, 4.08–4.11 (m, 1H, H40), 3.90–4.02 (m, 2H, CH2CH3),
3.57–3.65 (m, 1H, CHNH), 2.81–2.88, 2.91–2.97 (m, 1H, H20),
2.39–2.46 (m, 1H, H20), 1.84–1.90 (m, 1H, CH3CHCH3), 1.02–1.07
(m, 3H, CH2CH3), 0.72–0.77 (m, 6H, CH3CHCH3); 13C NMR
(125 MHz, acetone-d6 ) d 172.48, 172.45, 172.3 (CO), 157.7 (C6),
153.91, 153.87 (C2 and C4), 151.22, 151.17, 151.1, 150.9, 150.8
(C5 and C-Ph), 136.7, 136.4 (C8), 129.52, 129.45, 124.6, 120.5,
120.44, 120.39, 120.35, 117.8, 117.7 (C-Ph), 84.4, 84.2 (C10),
82.82, 82.81, 82.7 (C40), 66.0, 65.92, 65.89, 65.85 (C50), 61.7 (C30),
60.59, 60.57, 60.40, 60.36 (CHNH and OCH2CH3), 36.4, 36.2 (C20),
32.0, 31.9, 31.8, 31.7 (CH3CHCH3), 18.5, 17.3, 17.1 (CH3CHCH3),
13.64, 13.62 (CH2CH3); 31P NMR (202 MHz, acetone-d6 ) d 4.38,
4.63, 4.87; LC–MS (ES+): m/z (%) 576 (100) [M+H]+; HRMS (ES+):
calcd for C23H31N9O7P1 [M+H]+ 576.2079 m/z, found 576.2070 m/z.
4.2.14. 50-Phenyl methoxyalaninyl phosphate-20,30-didehydro-
30-deoxythymidine (20)
20,30-Didehydro-30-deoxythymidine (40 mg, 0.18 mmol) was re-
acted with phenyl methoxyalaninyl phosphochloridate 16
(149 mg, 0.54 mmol) to give the final product 20 as white solid
(63 mg, 75%). TLC (10% MeOH/DCM) Rf = 0.45; 1H NMR (500 MHz,
CHCl3-d1) d 8.29 (d, J = 16.1 Hz, 1H, NH), 7.18–7.37 (m, 6H, H-Ph
and H6), 7.03–7.07 (m, 1H, H30), 6.30–6.31 (m, 1H, H10), 5.92 (q,
J = 6.45 Hz, 1H, H20), 5.04 (q, J = 5.81 Hz, 1H, H40), 4.26–4.35,
4.39–4.43 (m, 2H, H50), 3.96–4.05 (m, 1H, CHNH), 3.73 (d,
J = 5.85 Hz, 1H, OCH3), 3.57–3.69 (m, 1H, NH), 1.84, 1.89 (d,
J = 0.8 Hz, 3H, H7), 1.36 (q, J = 10.0 Hz, 3H, CHCH3); 13C NMR
(125 MHz, CHCl3-d1) d 173.91, 173.85 (CO), 163.40, 163.35 (C4),
150.62, 150.57 (C2 and C-Ph), 135.9, 135.6 (C6), 133.4, 133.1
(C30), 129.82, 129.77 (C20), 127.5, 127.3, 125.28, 125.22, 120.19,
120.15, 120.07, 120.03 (C-Ph), 111.4, 111.3 (C5), 89.9, 89.6 (C10),
84.72, 84.65, 84.57 (C40), 67.2, 67.1, 66.6, 66.5 (C50), 52.62, 52.60
(CHNH), 50.2, 50.1 (OCH3), 21.05, 21.01, 20.98, 20.94 (NHCHCH3),
12.4, 12.3 (C7); 31P NMR (202 MHz, CHCl3-d1) d 2.46, 3.10; LC–
MS (ES+): m/z (%) 466 (100) [M+H]+; HRMS (ES+): calcd for
4.3.17. 30-Azido-50- phenyl ethyloxyvalinyl phosphate-30-deoxy-
a
-thymidine (23)
30-Azido-30-deoxy-
a-thymidine (38 mg, 0.14 mmol) was re-
acted with phenyl ethyloxyvalinyl phosphochloridate 17
(134 mg, 0.42 mmol) to give the final product 23 as a white solid
(40 mg, 52%). TLC (10% MeOH/DCM) Rf = 0.34; 1H NMR (500 MHz,
CHCl3-d1) d 9.21, 9.18 (s, 1H, NH), 7.09–7.30 (m, 6H, H-Ph and
H6), 6.02–6.06 (m, 1H, H10), 4.30 (d, J = 2.5 Hz, 1H, H40), 4.04–
4.18 (m, 5H, H30, H50, H50 CH2CH3), 3.66–3.79 (m, 2H, CHNH, NH),
2.64–2.70, 2.38–2.44 (m, 1H, H20), 1.91–2.09 (m, 2H, CH3CHCH3,
H20), 1.86, 1.87 (d, J = 1.1 Hz, 3H, CH3), 1.16–1.24 (m, 3H, CH3),
0.80–0.87 (m, 6H, CH3); 13C NMR (125 MHz, CHCl3-d1) d 172.8,
172.7 (CO), 163.89, 163.86 (C4), 150.7, 150.6 (C2), 150.4, 150.3
(C-Ph), 135.2, 135.1 (C6), 129.84, 129.80, 125.3, 125.2, 120.34,
120.30, 120.04, 120.00 (C-Ph), 111.0, 110.8 (C5), 86.5, 86.4 (C10),
84.2, 84.1 (C40), 66.1, 66.0 (C50), 61.5, 61.4 (C30), 61.0 (CH2CH3),
60.2, 60.0 (CHNH), 37.9 (C20), 32.1, 32.03, 31.98 (CH3CHCH3),
19.0, 17.3 (CH3CHCH3), 14.3, 14.2 (CH2CH3), 12.7 (C70); 31P NMR
(202 MHz, CHCl3-d1) d 3.66, 4.00; LC–MS (ES+): m/z (%) 568 (100)
[M+NH3]+, 551 (73) [M+H]+; HRMS (ES+): calcd for C23H32N6O8P1
[M+H]+ 551.2014 m/z, found 551.2026 m/z.
C
20H24N3O8P1 [M+H]+ 466.1374 m/z, found 466.1356 m/z.
4.3.15. 50-Phenyl ethyloxyvalinyl phosphate-20,30-didehydro-30-
deoxythymidine (21)
20,30-Didehydro-30-deoxythymidine (40 mg, 0.18 mmol) was re-
acted with phenyl ethyloxyvalinyl phosphochloridate 17 (0.54
mmol) to give the final product 21 as a solid (50 mg, 60%). TLC
(10% MeOH/DCM) Rf = 0.45; 1H NMR (500 MHz, DMSO-d6)
d
11.37 (s, 1H, NH), 7.30–7.36, 7.15–7.18 (m, 6H, H-Ph and H6), 6.84
(d, J = 1.9 Hz, 1H, H30), 6.38–6.44 (m, 1H, H20), 6.01 (d, J = 1.9 Hz,
1H, H10), 5.87–5.93 (m, 1H, NH), 4.97, 5.01 (s, 1H, H40), 3.95–4.27
(m, 4H, H50 and OCH2CH3), 3.42–3.49 (m, 1H, CHNH), 1.84–1.92
(m, 1H, CH3CH2CH3), 1.66, 1.70 (d, J = 0.6 Hz, 3H, H7), 1.11–1.17
(m, 3H, OCH2CH3), 0.72–0.82 (m, 6H, CH3CHCH3); 13C NMR
(125 MHz, DMSO-d6) d 172.5, 172.2 (CO), 163.77, 163.75 (C4),
150.8, 150.54, 150.50 (C2 and OCPh), 136.0, 135.9 (C6), 133.5,
133.4 (C30), 129.50, 129.48 (C20), 126.93, 126.90, 124.6, 124.5,
120.19, 120.15, 120.0, 119.9 (C-Ph), 109.69, 109.66 (C5), 89.0, 88.9
(C10), 84.4, 84.34, 84.27 (C40), 66.8, 66.2 (C50), 60.4, 60.24, 60.17,
60.0 (CHNH), 54.9 (OCH2CH3), 31.4, 31.3, 31.23, 32.17 (CH3CHCH3),
18.8, 18.7, 18.1, 17.8 (CH3CHCH3), 14.03, 13.98 (OCH2CH3), 11.94,
11.91 (C7); 31P NMR (202 MHz, DMSO-d6) d 4.11, 4.65; LC–MS
(ES+): m/z (%) 508 (100) [M+H]+; HRMS (ES+): calcd for C23H31N3O8P1
[M+H]+ 508.1843 m/z, found 508.1838 m/z.
Acknowledgments
The research leading to these results has received funding from
AntiMal, an FP6-funded integrated project under contract number
LSHP-CT-2005-0188. We would like to thank Gina MacKay for car-
rying out the HRMS and Dr. Gian Filippo Ruda for the help with
chemistry.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.07.006.
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