Synthesis and in vitro enzymatic and antiviral evaluation of phosphoramidate d4T derivatives as chain terminators

Article abstract of DOI:10.1039/c1ob06214j

The anti-HIV activity of nucleoside analogues is highly related to their substrate specificity for cellular and viral kinase and, as triphosphate, for HIV-RT. A series of phosphoramidate d4T derivatives have been synthesized and evaluated as substrates fo

Full text of DOI:10.1039/c1ob06214j

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PAPER
Synthesis and in vitro enzymatic and antiviral evaluation of phosphoramidate
d4T derivatives as chain terminators
Shiqiong Yang,a Christophe Pannecouque,b Eveline Lescrinier,a Anne Girauta and Piet Herdewijn*a
Received 20th July 2011, Accepted 22nd September 2011
DOI: 10.1039/c1ob06214j
The anti-HIV activity of nucleoside analogues is highly related to their substrate specificity for cellular
and viral kinase and, as triphosphate, for HIV-RT. A series of phosphoramidate d4T derivatives have
been synthesized and evaluated as substrates for HIV-1 RT, and also tested for their in vitro anti-HIV
activity. Compounds 2 and 4 are able to inhibit HIV-1 replication to the same extent as d4T and
d4TMP in MT-4 cells as well as in CEM/0 cells and CEM/TK- cells. The data suggests that these
phosphoramidates are hydrolysed to d4T before exerting their antiviral activity.
In our continuous effort to search for amino acid derivatives
as PPi mimics in enzymatic DNA synthesis,14–20 3-phosphono-L-
Introduction
The 2¢,3¢-dideoxynucleoside (ddN) analogues, which are potent
inhibitors of human immunodeficiency virus (HIV), act in their
metabolic active 5¢-triphosphate forms as substrates for reverse
transcriptase (RT).1 In essence, the ddN triphosphates (ddNTPs)
compete with the natural substrates at the RT level for in-
corporation in DNA and pyrophosphate (PPi) functions as a
leaving group.1,2 In most cases, the bottleneck in the metabolic
activation of dideoxynucleosides is the enzymatic phosphorylation
of the modified nucleoside by cellular kinases. Therefore, a
number of prodrug strategies have been developed to improve
anti-HIV efficacy and reduce toxicity by delivering the modified
nucleoside monophosphate into the cell.3–11 It is worth noting that
phosphoramidates of antiviral nucleosides are especially efficient
as a nucleotide delivery system, particularly if an amino acid is
used.4 Among them, the phosphoramidate derivatives, such as a
number of alkyl and aryl phosphoramidate mono-,5,6 or di-esters7,8
of AZT, d4T, and 3¢-fluoro-dideoxythymidine (FLT), have shown
promise as potential anti-HIV agents since they show enhanced
antiviral activity and lower cytotoxicity when compared to the
parent nucleoside. In these modifications the phosphate group is
conjugated with hydrophobic amino acid esters, such as alanine,
L-phenylalanine and L-tryptophan methyl ester. These pronu-
cleotides are able to be internalized by PBMCs and CEM cells and
further transformed to their monophosphates and corresponding
triphosphates.10 Additionally, ddN triphosphate analogues were
also investigated for producing anti-ddNTP antibodies12 and
inhibiting RT-mediated drug resistance.13
alanine, L-aspartic acid, iminodiacetic acid and iminodipropionic
acid proved to be efficient leaving groups when catalyzed by HIV-
1 RT, suggesting efficient metal-chelation of these amino acid
derivatives inside the polymerase active site. 3-Phosphono-L-Ala-
dNMP (N = A, T and G) is efficiently incorporated in DNA
resulting in 77–95% conversion to a P + 1 strand at 50 mM
after 60 min polymerase reaction by HIV-1 RT.19 Therefore, we
have studied the potential of the corresponding phosphoramidate
dideoxynucleotide analogues to act as chain terminators. Hence,
in the current study, d4T monophosphate was conjugated with
3-phosphono-L-alanine mono- or di-ester and the conjugates 1
and 2 (Fig. 1) were evaluated as chain terminators of HIV-1
RT. Additionally, L-Asp-d4TMP (3) and IDA-d4TMP (4) were
synthesized and evaluated as substrates of HIV-1 RT. As well
as ester derivatives (1, 2), free acids (3, 4) have been evaluated
to investigate the structural requirements for cellular uptake of
such derivatives. Among these analogues, 3-phosphono-L-alanine
monoester phosphoramidate of d4TMP (2) proved to be efficient
in single nucleotide incorporation and resulted in 75% conversion
to P + 1 strand at 100 mM after 60 min. Full chain termination can
aLaboratory of Medicinal Chemistry, Rega Institute for Medical Research,
Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000, Leuven,
Belgium. E-mail: piet.herdewijn@rega.kuleuven.ac.be; Fax: +32 16 337340;
Tel: +32 16 337387
bDepartment of Microbiology and Immunology, Rega Institute for Medical
Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000,
Leuven, Belgium
Fig. 1 Structure of phosphoramidate derivatives of d4T.
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be observed for compound 2 at higher substrate concentration
(200 mM to 1 mM). It was found that the steady-kinetics
study of compound 2 demonstrated a typical Michaelis–Menten
kinetic profile. In addition, to increase the hydrophobicity of the
potential prodrugs, which is important for cellular uptake, the
aryl phosphoramidate analogue 5 was synthesized. The selection
of the fenol leaving group is based on the work of McGuigan
and coworkers.4,7,8 Compounds 1–5 were further tested for their
anti-HIV activity in the cells.
In addition, the hydrolysis of the phosphonate ester function of
8 was also investigated using trimethylsilyl bromide (TMSBr) as
reagent in anhydrous CH2Cl2. The reaction failed even at 0 ◦C,
and only degradation products were obtained (demonstrated by
the isolation of the nucleobase thymine). This means that we were
not able to obtain the fully deprotected congener of compound 8.
The second synthetic route used for the synthesis of the
phosphoramidate derivatives of d4T is based on the one pot
procedure described by Ludwig and Yoshikawa et al..25,26 The
synthesis of L-Asp and IDA phosphoramidate derivatives (3 and
4) of d4T is shown in Scheme 2. 2¢,3¢-Didehydrodideoxythymidine
(d4T) was first phosphorylated and the resulting nucleoside
phosphodichloridate was then reacted with the hydrochloride
salt of the appropriate amino compound in the presence of
tributylamine. After conversion to the triethylammonium salt, the
resulted phosphoramidate compounds 9 and 10 were deprotected
in basic media and subjected to ion exchange chromatography
for purification, affording the final compounds 3 and 4 as
triethylammonium salts in good yields (total 37.2–46.7%). As the
phosphorylation of d4T requires anhydrous reaction conditions,
this one-pot method is convenient when using the commercially
available hydrochloride salt of amino acid ester.
Results and discussion
Synthesis of d4T phosphoramidate derivatives
The synthesis of the amino acid phosphoramidate derivatives of
d4T (1–5) was carried out by two synthetic routes. Compounds
1 and 2 were synthesized from d4T monophosphate (d4TMP)
and 3-phosphono-L-alanine trimethyl ester (7) by an EDAC-
mediated coupling, followed by deprotection in basic conditions.
The synthesis of compounds 1 and 2 is shown in Scheme 1.
The synthetic strategy starts by removal of the Boc protecting
group of 6 with trifluoroacetic acid in CH2Cl2.21,22 The resulting
3-phosphono-L-alanine trimethyl ester (7) can directly be used
for further amidation. The monophosphate of d4T (d4TMP)
was prepared by treatment of d4T with POCl3 as described
before.23,24 In order to avoid purification problems, this reaction
was carried out using a small amount (120 mg) of d4T. The
EDAC-mediated coupling of d4TMP with the primary amino
function of 7 yielded the phosphoramidate methyl ester precursor
(8) in 57.1%. Deprotection of both the carboxylic acid ester and
phosphonate ester is accomplished in basic condition to give
the desired products (1 and 2) in 78.6% overall yield. Column
chromatographic purification followed by HPLC was used to
purify the final products.
Scheme 2 Reagents and conditions: (a) POCl3, PO(OMe)3, 0 ◦C, 3 h; (b)
(L-Asp)Me2·HCl or (IDA)Me2·HCl, n-Bu3N, DMF dry, 0 ◦C to rt, 3 min;
(c) 1 M TEAB, rt, 30 min; (d) 0.4 M NaOH in MeOH/H2O 1 : 2, rt, 4 h.
Synthesis of phenyl phosphoramidate derivative of d4T
The synthesis of phenylphosphoramidate derivative (5) was
achieved following Scheme 3, which is similar to the method
described before for the synthesis of arylphosphoramidate deriva-
tives of d4T.27,28 In brief, phenylphosphorodichloride was con-
densed with iminodiacetate methylester hydrochloride to obtain
the monochloridate (11). Treatment of monochloridate (11) in
methylene chloride and triethylamine with d4T furnished the
desired phenylphosphoramidate derivative 5. Due to the stereo-
chemistry of the phosphorous chiral center,9,11 phenyl substituted
phosphoramidate derivatives of d4T exist as a mixture of two
possible diastereoisomers as evidenced from their 1H,13C and 31
P
NMR spectra. These isomers could not be separated by regular
column chromatography and only fractions enriched with one of
the other isomer were obtained.
Scheme 1 Reagents and conditions: (a) CF3COOH, CH2Cl2, rt, 4 h; (b)
EDAC, H2O, rt, 3 h; (c) 0.2 M NaOH in MeOH/H2O 1 : 2, rt, 4–24 h.
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product was formed. As expected, a single incorporated d4TMP
functions as a chain terminator of HIV-1 RT, Compound 2 is 10-
fold more efficient than compound 1 in the single primer-extension
assay. This suggests that the 3-phosphono-L-alanine methyl ester
can be better accommodated in the active site of the polymerase
than the diester.
Under identical HIV-1 RT concentration, L-Asp-d4TMP (3)
functioned as a suboptimal substrate: 20–25% P + 1 incorporation
product was obtained after 120 min at 500 mM concentration.
High concentration of the substrate IDA-d4TMP (4) was required
in order to incorporate d4TMP into DNA in a low yield (11–16%
P + 1 incorporation product after 120 min at 1 mM concentration).
The efficiency of compound 2 was further investigated by
30,31
determining the kinetic parameters Km and Kcat
.
Steady-state
Scheme 3 Reagents and conditions: (a) TEA, dry CH2Cl2, -78 ◦C to rt,
15 h; (b) d4T, rt, 5 d.
kinetic analysis (Table 1) indicates that the incorporation of
compound 2 by HIV-1 RT results in 26-fold increase in the Km
value, and only 1.2-fold decrease in Kcat value, compared to those
for d4TTP. Although the overall catalytic efficiency, the Kcat/Km
ratio, is decreased 31-fold, the Kcat value indicates the catalytic
efficiency of HIV-1 RT for the phosphoramidate nucleotide (2)
is only a little lower than for d4TTP. It should be mentioned
that these kinetics measure the whole process of incorporation
(including translocation and DNA release) and that the real
incorporation kinetics may be underestimated.32 Whatever, these
data indicate efficient nucleophilic displacement of 3-phosphono-
L-alanine monomethyl ester when the phosphoramidate is bound
in the active site.
Single primer extension by HIV-1 RT with d4T phosphoramidate
derivatives (1–4) as substrates
As a key enzyme to replicate the single-stranded RNA genome
and produce a double-stranded proviral DNA in HIV life-
cycle, RT represents one of the main target enzymes in treating
HIV infection. A high mutation rate with HIV-1 RT indicated
its important flexibility and high tolerance towards chemically
modified nucleotide substrates.29 In previous studies,14–19 it was
shown that HIV-1 RT was able to accept amino acid dNMPs
as substrate instead of natural dNTPs, during template-directed
DNA synthesis. The potential to use this “new leaving group”
concept for chain termination in DNA synthesis was evaluated
using d4T as example and the amino acid derivatives 1–4.
The ability of 3-phosphono-L-alanine di- or mono-methyl ester
phosphoramidate analogues (1 and 2) to function as substrates
for HIV-1 RT was investigated by the gel-based single nucleotide
incorporation assay (Fig. 2). The natural dideoxynucleoside
triphosphate (d4TTP) was used as reference.
Antiviral evaluation of phosphoramidate analogues (1–5) of
dideoxynucleotides against HIV-1 and HIV-2
The antiviral activity of compounds 1–5 against HIV-1 and HIV-
2 were evaluated in MT-4 cells, CEM/0 and CEM/TK- cells
(Table 2). In all of these experiments, d4T and d4TMP were
used as references. Compound 1 is 14 and 11-fold less active
than compounds 2–4 in MT-4 cells and CEM/0 cells, respectively.
Compound 4 is more efficient than compound 3 in CEM/0 cells.
Compound 2 shows antiviral activity against HIV-1 and HIV-
2 at concentrations of 0.47 mM and 0.19 mM, respectively, in
MT-4 cells. Additionally, compound 2 has similar activity as
d4T in CEM/0 cells. However, all of these d4T derivatives are
much less active in CEM/TK-. These results indicate that the
d4T phosphoramidate analogues and d4TMP itself were probably
hydrolyzed to d4T in culture medium before penetration into the
CEM/TK- cells.
In general, compounds 1–4 apparently serve as a source of d4T
in cells and none of these phosphoramidate analogues possess
marked cytotoxicity. The lower activity of compound 1 may be due
to its less efficient hydrolysis to d4T than compound 2. In addition,
compound 5 has a limited activity against HIV in MT-4 cells. Three
mixtures consisting of varying ratios of the Rp and Sp isomers of
the phosphotriester were evaluated for their anti-HIV activity. As
we did not assign the absolute phosphorus stereochemistry, the
isomer showing d4.35 ppm (31P NMR) was called the upfield
isomer and the other was the downfield isomer. The isomer
mixtures denominated 5b (ratio upfield/downfield = 2 : 1) and 5c
(ratio upfield/downfield = 1 : 1) were almost equipotent, whereas
the mixture of isomers 5a (ratio upfield/downfield = 1 : 2) was
12- and 4-fold less active than 5b against HIV-1 and HIV-2,
Fig. 2 Single incorporation of primer of P1T1 (125 nM) by HIV-1 RT
with phosphoramidate substrate concentrations and time intervals (min)
as indicated, [HIV-1 RT] = 0.025 U mL-1. Panel A: incorporation of
compound 1; panel B: incorporation of compound 2; d4TTP (10 mM)
used as reference.
During a single nucleotide incorporation assay, compound 1
only resulted in 30% conversion to a P + 1 strand in 30 min
at 1 mM. However, the use of compound 2 resulted in 75%
incorporation in 60 min at 100 mM. With higher concentrations
(200 mM to 1 mM), similar results were obtained and only P+1
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Table 1 Steady-state kinetics of single nucleotide incorporation into P1T1 by HIV-1 RT
Compound
Vmax (nM min-1)
Kcat (min-1)
Km (mM)
K
cat/Km (¥10-3 min-1 mM-1)
d4TTP
Compound 2
7.92 0.39 [HIV-1 RT] = 0.0125 U mL-1 = 18.1 nM
13.22 1.08 [HIV-1 RT] = 0.025 U mL-1 = 36.2 nM
0.44 0.02
0.37 0.03
3.57 0.53
93.33 16.55
123.2
3.96
Table 2 Antiviral activity test of compounds 1–5 against HIV-1 and HIV-2
MT-4 cellsa
CEM/0b
CEM/TK-c
CEM/0b
Compound
IC50 (mM) HIV-1
IC50 (mM) HIV-2
CC50 (mM)
IC50 (mM) HIV-2
IC50 (mM) HIV-2
CC50 (mM)
1
2
3
4
4.26 0.58
0.47 0.26
0.36 0.12
0.31 0.02
183.66 28.92
14.90 1.01
17.11 0.63
0.30 0.03
0.39 0.10
1.18 0.29
0.19 0.03
0.24 0.05
0.21 0.09
81.39 13.51
22.54 0.80
19.04 1.43
0.19 0.07
0.33 0.10
>259
16.52 1.11
1.44 0.11
6.55 4.88
2.13 0.46
>239
>239
>239
258.64 0.00
176.52 28.65
183.66 28.92
155.14 19.99
>239
>239
>239
259
266
298
298
>239
>239
>239
268
145.76 4.73
169.70 2.86
172.68 3.72
>239
>239
>239
5a
5b
5c
d4T
d4TMP
145.76 4.73
187.05 2.33
3.02 1.52
1.64 0.48
217.42 11.07
170.11 21.34
411
a HIV-1 (IIIB), HIV-2 (ROD). b CEM/0 cells (ROD). c CEM thymidine kinase deficient cells (ROD).
respectively. The diastereomeric excess was determined by 31P
NMR after high pressure liquid chromatography (HPLC). The
observed differences in anti-HIV activity result from differences
in hydrolytic stability of the different isomers. Most probably the
poor anti-HIV activity of compound 5a–c is due to the fact that
both carboxylic acids are present as their methyl esters.
5 lacks anti-HIV activity. This fully protected phosphoramidate
seems to be resistant to enzymatic/chemical degradation to d4T
in the given reaction circumstances. Preliminary results suggest
a difference in hydrolytic stability of the different diastereomers
of the phosphotriester in cell culture. The synthesis of new ester
derivatives of the compounds mentioned in this article will be
necessary to find a good balance between enzymatic stability,
cellular uptake and intracellular processing.
Conclusion
In summary, the investigation of the synthesis and evaluation of
the phosphoramidate analogues of d4TMP as chain terminators
for HIV-1 RT has shown that compound 2 is a good substrate
of HIV-1 RT resulting in 75% incorporation in 60 min at 100
mM. Compounds 1, 3 and 4 are suboptimal substrates of HIV-
1 RT and higher concentrations are needed to reach 11%–38%
incorporation after 120 min. The capability to function as leaving
groups in the enzymatic synthesis of DNA catalyzed by HIV-1 RT
increase in the order of iminodiacetic acid < L-aspartic acid <
3-phosphono-L-alanine mono methylester, when conjugated with
d4TMP.
In addition, compound 3 is 3-fold less efficient than compound
4 in CEM/0 cells. Compounds 2 and 4 exhibit similar anti-HIV
activity as d4T and d4TMP in MT-4 cells and is 1.5–2-fold more
efficient than d4T in CEM/0 cells. However, these compounds
are less potent in CEM/TK- cells, as also observed for d4T and
d4TMP. This suggests that d4TMP and the phosphoramidate
analogues (1–4) are hydrolyzed to d4T in the extracellular
medium or in an intracellular compartment. Indeed, following
the McGuigan approach, the phosphoramidate diesters are taken
up by cells and release the nucleoside monophosphate into the
cell.7 The negatively charged phosphoramidate monoesters, as
produced by Wagner and coworkers,5 can also be taken up by cells.
The outcome of the phosphoramidate prodrug approach seems to
be cell type and compound dependent. This, together with the
different stability of the prodrugs in the extracellular medium,
makes this approach difficult to predict.33 In addition, compound
Experimental section
General for chemical synthesis
For all reactions, analytical grade solvents were used. All moisture
sensitive reactions were carried out in oven-dried glassware
(135 ◦C). A Bruker Avance II 600 MHz or 500 MHz spectrometer
and a Bruker Avance 300 MHz apparatus were used for 1H
NMR, 13C NMR and 31P NMR. All chemical shifts are listed
in ppm. For sake of clarity of the NMR signal assignment,
sugar protons and carbons are numbered with a prime. 31P
NMR chemical shifts are referenced to an external 85% H3PO4
standard (d = 0.000 ppm). UV spectra were recorded on a Varian
Cary-300-Bio UV/Vis Spectrophotometer. Mass spectrometry
was performed on a quadrupole orthogonal acceleration time-of-
flight mass spectrometer (Q-Tof-2, Micromass, Manchester, UK)
equipped with a standard electrospray probe (Z-spray, Micromass,
Manchester, UK). Samples were dissolved in acetonitrile/water
(1 : 1 v/v) and infused with a flow rate of 5 mL min-1. Electrospray
capillary voltage was set to 3000 V and cone voltage to 30 V.
Nitrogen gas was used for nebulization and desolvation. Accurate
masses were determined by coinfusion of the samples with leucine
enkephalin (YGGFL) and recalibration of the spectrum using the
peak at m/z 556.2771 as lock mass. Precoated aluminum sheets
(MN ALUGRAM SIL G/UV254 20 ¥ 20 cm) were used for TLC;
the spots were examined with UV light. Column chromatography
˚
was performed on ICN silica gel 63-200, 60 A.
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Experimental procedure for the synthesis of intermediate
8 and compounds 1, 2: synthesis of 3-phosphono-L-alanine
trimethyl ester (7). (S)-methyl N-Boc-2-amino-3-(dimethoxyl-
phosphoryl)propanoate (6) (0.84 g, 2.70 mmol) was synthesized
according to the procedure described before by E. C. R. Smith
et al.34 It was dissolved in CH2Cl2 (12 mL) and trifluoroacetic
acid (3 mL, 40.39 mmol) was added in two portions in 1 h. The
reaction mixture was stirred at room temperature for 4 h. The
solvents were evaporated in vacuo and the obtained TFA salt was
6.1, J2 = 1.8 Hz, 1H, H-2¢), 5.92 (dd, J1 = 6.7, J2 = 1.8 Hz, 1H,
H-3¢), 5.08 (m, 1H, H-4¢), 3.94 (m, 2H, H-5¢ and H-5¢¢), 3.68–3.80
(m, 9H, 3 OMe), 3.54 (m, 1H, -CHCOO), 2.28–2.36 (m, 2H, -
CH2PO3), 1.90 (q, 3H, -CH3); 13C NMR (75 MHz, D2O): d 174.15
(d, 3J(C, P) = 5.1 Hz, -COOH), 166.33 (C-4), 151.88 (C-2), 137.96
3
(C-6), 134.19 (C-2¢), 124.80 (C-2¢), 110.97 (C-5), 89.61 (d, J(C,
P) = 12.8 Hz, C-4¢), 85.68 (C-1¢), 64.68 (d, 2J (C, P) = 5.1 Hz, C-5¢),
2
2
52.59 (d, J(C, P) = 3.0 Hz, -CHCOOH), 52.49 (d, J (C, P) =
5.7 Hz, POCH3), 49.60 (COOCH3), 28.27 (d, 1J(C, P) = 140.1 Hz,
-CH2PO3), 11.10 (CH3); 31P NMR (121 MHz, D2O): d 31.86 (-
CH2PO3), 4.79 (N-PO3); MS for C16H25N3O11P2 (M–H)- calcd:
496.1, found: 495.8.
◦
dissolved in MeOH (8 ml) and cooled to 0 C. Then, the pH of
solution was adjusted to 8 by adding triethylamine (2.2 mL). The
solvents were removed in vacuo and the residue was purified by
silica column chromatography (n-hexane/EtOAc/MeOH, 6 : 5 : 1
to 4 : 4 : 1). Then, the N-deprotected product 7 (0.43 g, 75.5%)
was obtained as yellowish syrup and it can directly be used for
the subsequent amidation reaction. TLC (CH2Cl2/CH3OH 9 : 1,
v/v): Rf 0.36; 1H NMR (300 MHz, CDCl3): d 12.28 (br, 2H), 4.12
(m, 1H), 3.74–3.79 (m, 9H), 2.35–2.62 (m, 2H); 31P NMR (121
MHz, CDCl3): d 29.56; MS for C6H14N1O5P1 (M + H)+ calcd:
212.1 found: 211.9.
Deprotection reaction
To the obtained ester intermediate 8 (39 mg, 0.08 mmol) was added
6 mL 0.2 M NaOH in MeOH/H2O 1 : 2 and the mixture were
stirred at room temperature. The progress of the reaction was mon-
itored by TLC (i-PrOH/NH3/H2O 7 : 1 : 2) until the disappearance
of ester. The hydrolysis is stopped after 4 h and 24 h, respectively,
to give the desired products as 1 and 2. The reaction mixture
was neutralized by addition of 1 M triethylammonium acetate
(TEAA). The solvent was evaporated to dryness in vacuum. The
residue was purified by silica column chromatography eluting with
CHCl3, CHCl3/MeOH = 5 : 1, CHCl3/MeOH/H2O = 5 : 2.5 : 0.4,
yielding compound 1 (18 mg, 46.6%), then i-PrOH/NH3/H2O
(7 : 1 : 1 to 7 : 1 : 1.5) and compound 2 (12 mg, 32.0%) as white
solid. An analytical sample was obtained by purification with
preparative HPLC (waters 1525-2487 system) with a gradient of
CH3CN in 50 mM TEAB buffer (pH = 7.4) on using Prep C18 5
mm column 19 ¥ 150 mm at the flow rate of 3 mL min-1.
Synthesis of d4TMP
To a solution of 3¢-deoxy-2¢,3¢-didehydrothymidine (100 mg,
0.45 mmol) in trimethylphosphate (1.25 mL) was added phos-
phoryl chloride (0.11 mL, 1.18 mmol) at 0 ◦C. The reaction
mixture was stirred at room temperature for 1.5 h and another
0.11 mL of phosphoryl chloride was added. The reaction was
continued to stir for 15 h and quenched by adding 1 M TEAB
(pH 7.4) 15 mL. After the solvents were evaporated in vacuo,
the residue was purified by silica column chromatography eluting
with CHCl3, CHCl3/MeOH 5 : 1 to i-PrOH/NH3/H2O 7 : 1 : 1 to
yield the desired d4TMP ammonium salt (55 mg, 36.5%) as white
2¢,3¢-Dideoxy-2¢,3¢-didehydrothymidine-5¢-[3-(dimethoxyphos-
phoryl)-L-alanine] phosphoramidate (1). TLC (i-PrOH/NH3/
H2O 7 : 1 : 2, v/v): Rf 0.53; UV: (H2O) lmax = 266 nm (log e =
3.97); 1H NMR (300 MHz, D2O, 5 ◦C): d 7.70 (s, 1H, H-6), 6.97
(m, 1H, H-1¢), 6.48 (ddd, J1 = 6.1 Hz, J2 = 1.8 Hz, J3 = 1.8 Hz,
1H, H-2¢), 5.92 (dd, J1 = 6.0 Hz, J2 = 1.8 Hz, J3 = 1.8 Hz, 1H,
H-3¢), 5.07 (m, 1H, H-4¢), 3.97 (m, 2H, H-5¢ and H-5¢¢), 3.70–
3.75 (m, 6H, 2 OMe), 3.57 (m, 1H, -CHCOO), 2.23-2.35 (m, 2H,
-CH2PO3), 1.92 (q, 3H, -CH3); 13C NMR (75 MHz, D2O, 5 ◦C): d
178.14 (d, 3J(C, P) = 9.0 Hz, -COOH), 166.42 (C-4), 151.89 (C-2),
137.92 (C-6), 134.10 (C-3¢), 124.86 (C-2¢), 111.24 (C-5), 89.61 (C-
1¢), 85.85 (C-4¢), 65.10 (d, 2J (C, P) = 5.3 Hz, C-5¢), 52.64 (dd, 2J1(C,
P) = 2.0 Hz, 2J2(C, P) = 6.8 Hz, -CHCOOH), 51.87 (d, 2J (C, P) =
3.4 Hz, POCH3), 29.15 (d, 1J(C, P) = 135.6 Hz, -CH2PO3), 11.10
(CH3); 31P NMR (121 MHz, D2O): d 33.90 (-CH2PO3), 5.66 (N-
PO3); HRMS for C15H23N3O11P2 (M-H)- calcd: 482.0735, found:
482.0738.
1
solid. H NMR (300 MHz, D2O): d 7.63 (s, 1H, H-6), 6.96 (m,
1H, H-1¢), 6.51 (dd, J1 = 1.6 Hz, J2 = 6.0 Hz, 1H, H-2¢), 5.91 (d,
J1 = 6.2, J2 = 1.5 Hz, 1H, H-3¢), 5.07 (m, 1H, H-4¢) 3.92 (m, 2H,
H-5¢ and H-5¢¢), 1.90 (q, 3H, -CH3); 13C NMR (75 MHz, D2O):
d 166.34 (C-4), 151.89 (C-2), 137.76 (C-6), 134.16 (C-3¢), 124.60
3
(C-2¢), 111.10 (C-5), 89.70 (C-1¢), 85.99 (d, J(C, P) = 8.3 Hz, C-
4¢), 65.06 (d, 2J (C, P) = 4.6 Hz, C-5¢), 11.08 (CH3); 31P NMR (121
MHz, D2O): d 1.57 (ammonium salt) or 3.36 (triethylamine salt);
MS for C10H13N2O7P1 (M-H)- calcd: 303.0, found: 302.6.
Coupling reaction
2¢,3¢-Dideoxy-2¢,3¢-didehydrothymidine-5¢-monophosphate am-
monium salt (50 mg, 0.15 mmol) and the crude 3-phosphono-
L-alanine trimethyl ester (103 mg, 0.49 mmol) were suspended in
1 mL water under Argon. Then, N-ethyl-N¢-(3-dimethylamino-
propyl)-carbodiimide hydrochloride (EDAC, 94 mg, 0.49 mmol)
was added to the suspension, and the reaction was stirred for 2 h
at room temperature. The progress of the reaction was monitored
by TLC (CHCl3/MeOH/H2O 5 : 3 : 0.5). The reaction mixture
was evaporated in vacuum at 30 ◦C and further lyophilized to
dryness. The residue was purified by silica column chromatography
eluting with CHCl3, CHCl3/MeOH = 5 : 1, CHCl3/MeOH/H2O =
5 : 2 : 0.25 to yield compound 8 (42 mg, 57.1%) as yellowish solid.
2¢,3¢-Dideoxy-2¢,3¢-didehydrothymidine-5¢-[3-(methoxyphos-
phoryl)-L-alanine] phosphoramidate (2). TLC (i-PrOH/NH3/
H2O 7 : 1 : 2, v/v): Rf 0.40; UV: (H2O) lmax = 266 nm (log e =
3.97); 1H NMR (600 MHz, D2O, 5 ◦C): d 7.62 (s, 1H, H-6), 6.90
(m, 1H, H-1¢), 6.45 (d, J = 6.0 Hz, 1H, H-2¢), 5.89 (m, 1H, H-3¢),
5.12 (m, 1H, H-4¢), 3.92 (m, 2H, H-5¢ and H-5¢¢), 3.61 (m, 1H,
-CHCOOH), 3.45 (q, 3H, OMe), 1.86-1.99 (m, 2H, -CH2PO3),
1.84 (q, 3H, -CH3); 13C NMR (150 MHz, D2O, 5 ◦C): d 181.9
(-COOH), 169.63 (C-4), 155.02 (C-2), 141.05 (C-6), 137.28 (C-3¢),
127.53 (C-2¢), 114.25 (C-5), 92.54 (C-1¢), 88.92 (d, 3J(C, P) = 9.1 Hz,
1
TLC (i-PrOH/NH3/H2O 7 : 1 : 2, v/v): Rf 0.62; H NMR (300
MHz, D2O): d 7.64 (s, 1H, H-6), 6.99 (m, 1H, H-1¢), 6.47 (dd, J1 =
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C-4¢), 67.60 (d, 2J (C, P) = 4.8 Hz, C-5¢), 55.73 (d, 2J(C, P) = 18.1 Hz,
-CHCOOH), 53.98 (d, 2J (C, P) = 4.8 Hz, POCH3), 33.87 (d, 1J(C,
151,85 (C-2T), 137.87 (C-6), 134.05 (C-2¢), 124.81 (C-3¢), 111.10
(C-5), 89.50 (C-1¢), 85.70 (C-4¢, J = 9.65 Hz), 64.52 (C-5¢), 46.30
3
P) = 130.5 Hz, J(C, P) = 7.5 Hz, -CH2PO3), 14.21 (CH3); 31P
(Et3N+), 39.70 (CHa), 34.75 (CH2b), 11.13, 7.86 (Et3N+) ppm. 31
P
NMR (202 MHz, D2O): d 22.0 (-CH2PO3), 6.10 (N-PO3); HRMS
NMR (D2O, 121 MHz): d 6.34 ppm. HRMS (negative ionization):
for C14H21N3O11P2 (M-H)- calcd: 468.0578, found: 468.0600.
calculated for C14H17N3O10P-: 418.0657, found: 418.0659.
2¢,3¢-Dideoxy-2¢,3¢-didehydrothymidine-5¢-(dimethyliminodiace-
tate)phosphoramidate triethylammonium salt (10, intermediate for
compound 4. The protocol for the synthesis of 10 is identical
to the previously described method for the synthesis of the
phosphoramidate (9) using a solution of dimethyl iminodiacetic
acid hydrochloride (434 mg, 2.2 mmol, 10 eq.) and n-tributylamine
(500 mL, 30 eq.) in dried DMF (2.2 mL) in the second step. The
corresponding compound 10 (74 mg, colourless solid, yield 76%)
was obtained and stored at -20 ◦C.
General procedure for synthesis of intermediates 9, 10 and
compounds 3, 4: 2¢,3¢-dideoxy-2¢,3¢-didehydrothymidine-5¢-
(dimethyl-L-aspartate)phosphoramidate triethylammonium salt
(9, intermediate for compound 3)
All solids were dried in a desiccator under high vacuum one day
prior to reaction. The nucleoside (50 mg, 0.22 mmol) was placed
under argon atmosphere and dissolved in trimethylphosphate (500
mL, 50 mL/10 mg nucleoside). Upon cooling down to 0 ◦C,
phosphorous trichloride (42 mL, 0.44 mmol, 2 eq.) was added
slowly via syringe. The resulting solution was stirred for 3 h at
0 ◦C and the reaction was monitored by TLC (i-PrOH/NH3/H2O
6 : 3 : 1, v/v). Upon total conversion of the starting nucleoside,
a solution of dimethyl L-aspartic acid hydrochloride (434 mg,
2.2 mmol, 10 eq.) and n-tributylamine (500 mL, 30 eq.) in dried
DMF (2.2 mL) was added via syringe. The resulting mixture was
stirred vigorously for 3 min and pipetted into a cold TEAB 1 M
solution (5 mL). The resulting clear mixture was further stirred
at room temperature for 30 min. Upon completion, all volatiles
were evaporated under high vacuum. The product was purified
by flash chromatography using a gradient of methanol and water
in chloroform (5 : 1 : 0, 5 : 2 : 0.25, 5 : 3 : 0.5). The corresponding
fractions were evaporated, lyophilized and the final compound
(70 mg, colourless solid, yield 73%) was stored at -20 ◦C.
TLC (i-PrOH/NH3/H2O 6 : 3 : 1, v/v) Rf 0.50; 1H NMR (D2O,
300 MHz): d 7.52 (s, 1H, H-6), 6.85 (s, 1H, H-1¢), 6.39 (s, 1H,
J = 5.97 Hz, H-2¢), 5.83 (s, 1H, J = 5.58 Hz, H-3¢), 4.95 (bs,
1H, H-4¢), 3.80–3.60 (m, 5H, H-5¢ and 2 ¥ CH2), 3.57 (bs, 6H,
CH3-O), 3.11 (q, 6H, N-CH2-CH3, Et3N+), 1.82 (s, 3H, CH3) 1.19
(t, 9H, N-CH2-CH3, Et3N+) ppm. 13C NMR (D2O, 126 MHz): d
173.83 (2 ¥ CO Ac), 166.62 (C-4T), 152.144 (C-2T), 138.44 (C-
6), 134.62 (C-2¢), 124.73 (C-3¢), 111.06 (C-5¢), 89.70 (C-1¢), 85.85
(C-4¢, J = 10.5 HZ), 64.85 (C-5¢), 52.13 (CH2 IDA), 48.69 (CH3-
O), 46.58 (Et3N+), 11.37, 8.16 (Et3N+) ppm. 31P NMR (D2O, 121
MHz): d 6.45 ppm. HRMS (negative ionization): calculated for
C16H21N3O10P-: 446.0970, found: 446.0976.
2¢,3¢-Dideoxy-2¢,3¢-didehydrothymidine-5¢-iminodiacetate phos-
phoramidate triethylammonium salt (4). The deprotection of
compound 10 (74 mg) is carried out in the same way as the
deprotection of 9. The final compound 4 was obtained as a white
solid (yield 51–60%) and stored at -20 ◦C.
TLC (i-PrOH/NH3/H2O 6 : 3 : 1, v/v) Rf 0.77; 1H NMR (D2O,
300 MHz): d 7.56 (s, 1H, H-6), 6.89 (s, 1H, H-1¢), 6.41 (s, 1H, J =
5.97 Hz, H-2¢), 5.86 (s, 1H, J = 5.37 Hz, H-3¢), 5.00 (bs, 1H, H-4¢),
3.97 (m, 1H, Ha), 3.88 (bs, 2H, H-5¢), 3.70, 3.65 (2 s, 2 ¥ 3H, CH3-
O), 3.13 (q, 6H, N-CH2-CH3, Et3N+), 2.72–2.70 (m, 2H, Hb), 1.83
(s, 3H, CH3), 1.21 (t, 9H, N-CH2-CH3, Et3N+) ppm. 13C NMR
(D2O, 75 MHz): d 173.04 (CO Asp 2x), 166.57 (C-4T), 152.16
(C-2T), 138.19 (C-6), 134.05 (C-2¢), 125.24 (C-3¢), 111.25 (C-5¢),
89.75 (C-1¢), 85.58 (C-4¢, J = 8.71 Hz), 64.71 (C-5¢), 52.80 (CH3-
O), 50.93 (CH3-O), 43.21 (Et3N+), 38.20 (CHa), 35.48 (CHb), 11.27
(CH3-T), 8.14 (Et3N+) ppm. 31P NMR (D2O, 121 MHz): d 5.59
ppm. HRMS (negative ionization): calculated for C16H21N3O10P-:
446.0970, found: 446.0966.
1H NMR (D2O, 300 MHz): d 7.51 (s, 1H, H-6), 6.85 (s, 1H,
H-1¢), 6.38 (s, 1H, J = 6.18 Hz, H-2¢), 5.84 (s, 1H, J = 5.58 Hz,
H-3¢), 4.97 (bs, 1H, H-4¢), 3.91–3.88 (m, 5H, H-5¢), 3.67, 3.64 (2 s,
4H, 2 ¥ CH2), 3.11 (q, 6H, N-CH2-CH3, Et3N+), 1.81 (s, 3H, CH3),
1.19 (t, 9H, N-CH2-CH3, Et3N+) ppm. 13C NMR (D2O, 151 MHz):
d 178.91 (2 ¥ CO Ac), 166.42 (C-4), 151.94 (C-2), 137.88 (C-6),
134.00 (C-2¢), 124.81 (C-3¢), 111.14 (C-5¢), 89.62 (C-1¢), 85.60 (C-
4¢, J = 9.50 Hz), 64.92 (C-5¢), 52.96 (CH2 IDA), 46.37 (Et3N+),
11.16, 8.10 (Et3N+) ppm. 31P NMR (D2O, 121 MHz): d 6.97
ppm. HRMS (negative ionization): calculated for C14H17N3O10P-:
418.0657, found: 418.0663.
2¢,3¢-Dideoxy-2¢,3¢-didehydrothymidine-5¢-(L-aspartyl)phospho-
ramidate triethylammonium salt (3). The previously prepared
compound (70 mg) was dissolved in 0.4 M sodium hydroxide
solution in water : methanol 2 : 1 (1 mL) and stirred at room
temperature until complete deprotection was achieved. Solvents
were evaporated and the residue was submitted to preparative
ion exchange chromatography (Source 15Q, Pharmacia) running
a gradient of TEAB in water (0 to 30% in 15 min). The final
compound was obtained as a white solid (yield 51–64%) and stored
at -20 ◦C.
2¢,3¢-Dideoxy-2¢,3¢-didehydrothymidine-5¢-(phenyl dimethyl-imi-
nodiacetate phosphate) (5). In a dry flask, iminodiacetate
methylester hydrochloride (0.99 g, 5 mmol) and dry CH2Cl2
(37.5 mL) were added and cooled to - 78 ◦C. Then phenyl
dichlorophosphate (1.05 g, 0.74 mL, 5 mmol) was added into
◦
the flask. The reaction mixture were stirred and kept at -78 C.
Anhydrous triethylamine (2.1 mL, 15 mmol) was added dropwise
◦
at - 78 C. After 15 min, the reaction mixture was left to warm
up to room temperature and stirred overnight. The formation
of the phosphonochloridate was monitored by 31P NMR and an
additional portion of triethylamine (2.1 mL, 15 mmol) was added
to the reaction mixture and the contents were allowed to stir.
After stirring at room temperature for 2 h, d4T (0.45 g, 2.0 mmol)
was added to the above reaction flask and the reaction mixture was
stirred at room temperature for 5 days. The suspension was filtered
1H NMR (D2O, 300 MHz): d 7.58 (s, 1H, H-6), 6.88 (s, 1H, H-
1¢), 6.39 (s, 1H, J = 6.09 Hz, H-2¢), 5.84 (s, 1H, J = 6.06 Hz, H-3¢),
5.00 (bs, 1H, H-4¢), 3.90 (bs, 2H, H-5¢), 3.70–3.67 (m, 1H, Ha),
3.14 (q, 6H, N-CH2-CH3, Et3N+), 2.61–2.58 (m, 2H, Hb), 1.83 (s,
3H, CH3), 1.21 (t, 9H, N-CH2-CH3, Et3N+) ppm. 13C NMR (D2O,
125 MHz): d 178.24 (CO Asp), 176.08 (CO Asp), 166.32 (C-4T),
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and the filtrate was washed with anhydrous CH2Cl2 2 ¥ 25 mL.
Solvent was removed under reduced pressure. The crude reaction
product was purified by column chromatography (100% CHCl3 to
5% MeOH in CHCl3) to yield compound 5 as yellowish syrup (0.9
g, 86.0%). Preparative HPLC (waters 1525–2487 system) with a
gradient of CH3CN in H2O on C18 5 mm column 19 ¥ 150 mm
was used to purify the syrup. Sample 5a, 5b and 5c were obtained
as (not assigned) fractions of Rp and Sp with the ratio of 1 : 2, 2 : 1
8.3. Products were visualized by phosphor imaging. The amount
of radioactivity in the bands corresponding to the products of
enzymatic reactions was determined with the imaging device
Cyclone and the associated Optiquant image analysis software
(Perkin-Elmer).
Steady-state kinetics of single nucleotide incorporation. To de-
termine the kinetic parameters for the incorporation of compound
2 and a natural nucleoside d4TTP, a steady-state kinetics assay
was carried out. The reaction was started by adding HIV-1 RT
to P1-T1 complex, RT buffer, compound 2 and d4TTP. The final
mixture (20 mL) contained 125 nM primer-template complex, and
various concentrations of compound 2 or d4TTP, buffer, 0.025 U
mL-1 HIV-1 RT for 5 and 0.0125 U mL-1 HIV-1 RT for d4TTP,
respectively. The range of concentrations for phosphoramidates
was optimized according to a Km value for the incorporation
of an individual nucleotide. Enzymatic reactions were carried
out to attain 5–25% incorporation with appropriate substrate
concentrations (5–200 mM used for compound 2 and 1–20 mM
used for d4TTP) incubated at 37 ◦C and run for 8–10 different time
intervals (1–10 min). The amount of primer template (125 nM) was
3.5-fold excess of HIV-1 RT (36.2 nM). Products were separated
on a 20% polyacrylamide gel and quantitated by phosphor
imaging. The incorporation velocities were calculated based on the
percentage of single-nucleotide extension product (P + 1 band).
The kinetic parameters (Vmax and Km) were determined by plotting
V (nM min-1) versus substrate concentration (mM) and fitting
the data point to a nonlinear Michaelis–Menten regression using
GraphPad Prism software. The Kcat was calculated according to
the used enzyme concentration.
and 1 : 1, determined by the relative intensity of two peaks of 31
P
NMR (d 4.35, 5.11 ppm).
TLC (CHCl3/CH3OH 18 : 1, v/v): Rf 0.35–0.40; 1H NMR (500
MHz, CDCl3, 5 ◦C): d 8.20 (s, 1H, NH), 7.34–7.36 (m, 2H, phenyl),
7.32 (s, 1H, H-6), 7.17–7.21 (m, 3H, phenyl), 7.03 (m, 1H, H-1¢),
6.34 (m, 1H, H-2¢), 5.88 (m, 1H, H-3¢), 5.04 (m, 1H, H-4¢), 4.37
(m, 2H, H-5¢ and H-5¢¢), 3.88–3.98 (m, 4H, 2-CH2), 3.67 (s, 6H,
2-OCH3), 1.78 (q, 3H, -CH3); 13C NMR (126 MHz, CDCl3, 5 ◦C):
d 170.20 (-COOCH3), 163.41 (C-4), 150.52 (C-2), 135.67 (C-6),
133.19 (C-2¢), 129.68 (C-phenyl), 126.93 (C-3¢), 125.22 (C-phenyl),
120.15 (C-phenyl), 111.27 (C-5), 89.29 (C-1¢), 84.48 (d, 3J(C, P) =
2
8.8 Hz, C-4¢), 65.98 (d, J (C, P) = 3.1 Hz, C-5¢), 52.24 (-OCH3),
47.44 (-CH2), 47.41 (-CH2), 12.35 (CH3); 31P NMR (202 MHz,
CDCl3): d 4.35, 5.11; HRMS for C22H26N3O10P (M-H)- calcd:
522.1288, found: 522.1281.
Enzymatic synthesis
Oligodeoxyribonucleotides. DNA oligonucleotides P1 and T1
were purchased from Sigma Genosys and Eurogentec. The
concentrations were determined with a Varian Cary-300-Bio
UV Spectrophotometer. The lyophilized oligonucleotides were
dissolved in diethylpyrocarbonate (DEPC)-treated water and
stored at -20 ◦C. The primer oligonucleotides were 5¢-labeled with
[g-33P] ATP (Perkin Elmer) using T4 polynucleotide kinase (New
England Biolabs) according to the standard procedures. Labeled
oligonucleotides were further purified with IllustraTM MicrospinTM
G-25 columns (GE Healthcare).
Biological evaluation of phosphoramidate analogues of
dideoxynucleotides against HIV-1 and HIV-2
The biological evaluation of phosphoramidate analogues of d4T
against HIV-1 and HIV-2 was carried out including d4T and its
5¢-O-monophosphate as references.
The methodology of the anti-HIV assays was as follows.
The anti-HIV activity and cytotoxicity of the compounds were
evaluated against wild-type HIV-1 strain IIIB and HIV-2 strain
ROD in MT-4 cell cultures using the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) method.35 Briefly, virus
stocks were titrated in MT-4 cells and expressed as the 50% cell
culture infective dose (CCID50). MT-4 cells were suspended in
culture medium at 1 ¥ 105 cells mL-1 and infected with HIV
at a multiplicity of infection of 0.02. Immediately after viral
infection, 100 mL of the cell suspension was placed in each well of
a flat-bottomed microtiter tray containing various concentrations
of the test compounds. The test compounds were dissolved in
DMSO at 50 mM or higher. After 4 days of incubation at
37 ◦C, the number of viable cells was determined using the MTT
method. Compounds were tested in parallel for cytotoxic effects
in uninfected MT-4 cells.
DNA polymerase reactions. 5¢-End-labeled primer was an-
nealed to its template by combining primer and template at a molar
ratio of 1 : 2 and heating the mixture to 70 ◦C for 10 min, followed
by slow cooling to room temperature over a period of 2 h. The
complex of P1T1 was used for the single nucleotide incorporation
study of compounds 1–4. A series of 20-mL-batch reactions were
performed for the enzyme HIV-1 RT (Ambion, Inc.; 10 U mL-1
stock solutions). The final mixture with HIV-1 RT contained
125 nM primer-template complex, RT buffer (50 mM Tris-HCl,
50 mM KCl, 10 mM MgCl2, 0.5 mM spermidine, 10 mM DTT;
pH 8.3), 0.025 U/mL HIV-1 RT, and different concentrations of
phosphoramidate substrates. Mixtures were incubated at 37 ◦C,
and aliquots (2.5 mL) were removed and quenched after 10, 20, 30,
60, 120 min. In the control reaction with the natural nucleotides,
10 mM d4TTP was used.
Polyacrylamide electrophoresis. All polymerase reaction (2.5
mL) were quenched by the addition of 10 mL of loading buffer
(90% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol,
and 50 mM ethylenediaminetetraacetic acid (EDTA)). Samples
were heated at 75 ◦C for 5 min prior to analysis by electrophoresis
for 2.5 h at 2000 V on a 0.4-mm 20% denaturing gel in the
presence of a 100 mM Tris-borate, 2.5 mM EDTA buffer, pH
CEM cells were infected as previously described. Briefly, 4 ¥ 105
cells mL-1 were infected with HIV-2 at ª 100 CCID50 (50% cell
culture infective dose) per mL of cell suspension. The thymidine
kinase-deficient CEM (CEM/TK-) cell cultures were also infected
with HIV-2. Then, 100 mL of the infected cell suspensions was
transferred into 96-well microtiter plate wells and mixed with 100
mL of the appropriate dilutions of the test compounds. After 4 days
152 | Org. Biomol. Chem., 2012, 10, 146–153
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of incubation at 37 ◦C, the giant cell formation was recorded
microscopically in the HIV-infected cell cultures.
13 J. Deval, K. Alvarez, B. Selmi, M. Bermond, J. Boretto, C. Guerreiro,
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Acknowledgements
This work was financed by a grant from K. U. Leuven (GOA) and
from ESF. We would like to thank Dr Jef Rozenski for HRMS
and Chantal Biernaux for editorial help. We are indebted to
Kristien Erven, Cindy Heens and Kris Uyttersprot for evaluating
the compounds for their antiviral activity. Luc Baudemprez is
acknowledged for running the NMR spectra. Present address of
Shiqiong Yang is Department of Chemistry, Fudan University,
Shanghai 200433, P.R. China.
20 X. P. Song, C. Bouillon, E. Lescrinier and P. Herdewijn, ChemBioChem,
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