Synthesis, anti-HIV and cytostatic evaluation of 3′-deoxy-3′- fluorothymidine (FLT) pro-nucleotides

Article abstract of DOI:10.1016/j.bmcl.2014.03.092

A series of pro-nucleotide phosphoramidates and phosphorodiamidates of the antiviral lead compound 3′-deoxy-3′-fluorothymidine (FLT) have been designed and synthesized. In vitro antiretroviral and cytostatic studies revealed potent (sub-micromolar) inhibition of HIV-1 and HIV-2 replication, with retention of activity in thymidine kinase-negative cell models, as predicted by the ProTide concept.

Full text of DOI:10.1016/j.bmcl.2014.03.092

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2240–2243
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, anti-HIV and cytostatic evaluation
of 30-deoxy-30-fluorothymidine (FLT) pro-nucleotides
Winnie Velanguparackel a, , Nadège Hamon a, , Jan Balzarini b, Christopher McGuigan a,
Andrew D. Westwell a,
⇑
a School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff, CF10 3NB Wales, UK
b Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pro-nucleotide phosphoramidates and phosphorodiamidates of the antiviral lead compound
30-deoxy-30-fluorothymidine (FLT) have been designed and synthesized. In vitro antiretroviral and cyto-
static studies revealed potent (sub-micromolar) inhibition of HIV-1 and HIV-2 replication, with retention
of activity in thymidine kinase-negative cell models, as predicted by the ProTide concept.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 9 March 2014
Revised 26 March 2014
Accepted 27 March 2014
Available online 5 April 2014
Keywords:
Nucleosides
Antiviral
Anticancer
Phosphoramidates
Pro-nucleotides
Pro-drugs
Despite their crucial and widespread role as approved thera-
peutics for antiviral1 and anticancer2 therapy, the activity of nucle-
oside analogues is compromised by their requirement for active
transporter-mediated cell uptake and bioactivation, usually via
three successive phosphorylation steps. Since initial nucleoside
kinase-catalysed phosphorylation is frequently the rate-limiting
step in nucleoside drug activation, a number of pro-drug (pro-
nucleotide) monophosphate strategies have been developed to cir-
cumvent delivery, uptake and metabolic activation issues.
One of the most widely used pro-nucleotide strategies is the
approach pioneered by McGuigan,3,4 in which the monophosphate
is masked as a phosphoramidate,5 or in more recent modalities as a
phosphorodiamidate.6 Following passive diffusion through the cell
membrane, the pro-nucleotide (ProTide) breaks down within the
cell via a well-studied mechanism to release the nucleoside mono-
phosphate, trapped intracellularly and primed for further phos-
phorylation. The ProTide approach has been applied to improve
efficacy for a wide range of antiviral and anticancer nucleosides.
Recent highlights include the clinical trial candidates INX-189
(hepatitis C virus, to Phase 2)7 and a pro-nucleotide of the cancer
chemotherapeutic gemcitabine (NUC-1031, Phase 1, ongoing),8
shown in Figure 1.
Fluorine is well known to impart rather special physicochemical
and pharmacological properties on drug candidates, and is present
in 15–20% of currently approved drugs overall.9 Several fluorinated
nucleosides have been applied as both therapeutic agents and in
diagnostic imaging. Examples of fluorinated nucleoside antivirals
include emtricitabine (HIV),10 clevudine (HBV)11 and trifluridine
(HSV; cancer).12,13 Besides trifluridine, fluorinated nucleoside-
based anticancer drugs include also the 5-fluorouracil pro-drug
such as capecitabine14 and gemcitabine;15 and the related purine
nucleosides fludarabine and clofarabine.16
30-Deoxy-30-fluorothymidine (FLT) is a particularly interesting
fluorinated nucleoside derivative possessing a variety of biological
properties. The major clinical application of FLT is in the non-inva-
sive diagnostic imaging of tumour proliferation using Positron
Emission Tomography (PET), where the 18F radiolabelled analogue
(
18FLT, t1/2 = 110 min.) is routinely employed as a highly sensitive
imaging probe.17 In terms of therapeutic application, FLT (also
known as alovudine) has been shown to be a more potent inhibitor
of HIV replication than the well-known anti-retroviral agent AZT.
In vitro studies have shown that FLT inhibits replication of nucleo-
side analogue reverse transcriptase inhibitor (NRTI)-resistant HIV
strains.18 Further clinical development of FLT was halted, however,
due to toxicological safety concerns.19 Previous preliminary work
⇑
Corresponding author. Tel.: +44 (0)2920 875800; fax: +44 (0)2920 874149.
E-mail address: WestwellA@cf.ac.uk (A.D. Westwell).
Denotes equal contributions to the experimental work.
http://dx.doi.org/10.1016/j.bmcl.2014.03.092
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

W. Velanguparackel et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2240–2243
2241
NH2
under basic conditions to generate an intermediate dichloridate
(4) which was not isolated. Reaction of FLT dichloridate with fur-
O
N
N
N
N
ther triethylamine, followed by excess
a-amino acid ester, gave
O
N
N
NH2
the required FLT phosphorodiamidates after purification by column
Me
F
F
O
O
O
O
O
O
O
O
chromatography and preparative TLC (Scheme 1).25,26
P
P
The resulting FLT Pro-Tides were evaluated for their in vitro
inhibitory effect on the replication of HIV-1 and HIV-2 in human
T-lymphocyte (CEM) cell cultures, according to previously
described methods.24,27 Importantly, TKꢀ mutant CEM cells were
included to examine the hypothesis that the ProTide derivatives,
unlike the parent compound FLT, would be able to by-pass the
requirement for TK-directed phosphorylation and would be active
in this test. The results of the HIV assays are shown in Table 1. FLT
(1) was used as a positive control, confirming potent anti-HIV-1
and -HIV-2 activity in the TK-expressing CEM cells (EC50 = 6.2
and 18 nM, respectively), but not in the TK knockout CEM mutant
NH
NH
OH
OH OH
O
O
O
O
INX-189
NUC-1031
Figure 1. Chemical structures of ProTides INX-189 and NUC-1031.
on FLT pro-nucleotides as HIV inhibitors suggested the possibility
to retain the potent activity of the parent compound using a Pro-
Tide-based strategy.20 In this Letter, we describe the synthesis
and in vitro evaluation of both phosphoramidate (3a–m) and
phosphorodiamidate (5a–b) pro-nucleotide derivatives of FLT as
antiretroviral and anticancer agents. These studies test the
hypothesis that application of the ProTide delivery concept might
generate highly potent new nucleotide analogues that are able to
by-pass the requirement for a functional thymidine kinase (TK).
Synthesis of the FLT phosphoramidates was accomplished
through application of known pro-nucleotide chemistry.5 Reaction
of the commercially available FLT (1, Carbosynth U.K.) with a range
of previously reported aryloxyphosphorochloridates (2a–m), pro-
moted by tert-butylmagnesium chloride as a hindered base in
THF, provided the target phosphoramidate products in low to mod-
erate isolated yield (high yield based on recovered starting mate-
rial, BORSM) following column chromatography (Scheme 1).21,22
We then tried to synthesize diamidate prodrugs of FLT according
to the trimethyl phosphate method used previously in our labora-
tory.23 Unfortunately, after 5 h of reaction at ꢀ78 °C in the presence
of TMP and POCl3, the signal of the desired intermediate 4 on 31P
NMR was barely visible. After a further 15 h in the presence of L-
Ala neopentyl ester and DIPEA, no signal of the desired diamidate
prodrug was observed on 31P NMR. We can explain this by a lack
of solubility of FLT in the organic solvents used. Eventually symmet-
rical FLT phosphorodiamidates (5a–b) were synthesized according
to literature methods6,24 by treating FLT with phosphoryl chloride
cells (EC50 >50
assay is notable.
l
M). The >1000 fold loss of activity of (1) in the TKꢀ
Examination of the in vitro antiretroviral activity indicates
some interesting and novel findings. Although the new pro-
nucleotides were not generally found to be as active as FLT in
TK-positive cells (CEM/0), some phosphoramidates (e.g., 3j–k)
and phosphorodiamidates (e.g., 5a–b) gave EC50 values in the mid-
dle (6100 nM) nanomolar concentration range for HIV-1 and/or
HIV-2. Phosphorodiamidate (5b) (R = neopentyl) was found to be
the most potent compound in the assays. Importantly, the test
compounds were able to retain moderate activity in the CEM/TKꢀ
(TK mutant) cell cultures in the lower micromolar range, unlike
FLT (with the exception of ProTides 3d, 3h–i, 3m and 5b). Based
on the overall profile across these three test assays, FLT
phosphoramidate (3k, R = naphthyl; R0 = neopentyl) and FLT
phosphorodiamidate (5a, R = Bn) are the most potent and promis-
ing compounds for further study. Importantly, compound (3k)
shows only a 10-fold loss of activity in the TK knockout CEM
mutant cell assay, versus >1000-fold for compound (1).
In vitro examination of the cytostatic potential of the FLT
ProTides was studied in the human T-lymphocyte CEM cells (both
wild-type TK and TK-deficient (TKꢀ)), and the murine leukaemia
cell line L1210, according to previously reported protocols.24,28
The results of these studies are shown in Table 2.
R
R'
Me
Et
a
b
c
d
e
Ph
Ph
O
N
O
Ph
Ph
i-Pr
t-Bu
Bn
Me
Me
HN
O
O
HN
Ph
RO
P
Cl
O
O
N
(i)
1-Naph
1-Naph
1-Naph
Me
Et
f
g
NH
+
O
O
O
HO
R'O
P
NH
h
i
i-Pr
RO
O
1-Naph t-Bu
O
F
F
c-Hex
t-BuCH2
Bn
j
1-Naph
1-Naph
2a-m
1 (FLT)
OR'
3a-m
k
l
1-Naph
Me
aReagents and conditions: (i) t-BuMgCl, anhydrous THF, 16h
m
Bn
O
O
N
O
N
Me
HN
Me
(i)
Me
HN
O
HN
O
O
N
O
O
O
O
(ii)
O
O
O
P
N
RO
HO
O
H
P
NH
Cl
Cl
F
F
F
OR
5a (R=Bn)
5b (R=tBuCH2)
O
1 (FLT)
4
bReagents and conditions: (i) POCl3, Et3N, anhydrous THF; (ii) amino ester (5 eq), Et3N (10 eq), anhydrous CH2Cl2
Scheme 1. Synthesis of FLT pro-nucleotides..

2242
W. Velanguparackel et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2240–2243
Table 1
support (to N.H.). We thank Ms. Helen Murphy for excellent secre-
Anti-HIV-1 and -HIV-2 activity (EC50, lM) values in human T-lymphocyte (CEM) cell
cultures. Results are expressed as mean values of at least 2–3 independent
experiments
tarial assistance, and Mrs. Leen Ingels and Mrs. Lizette van Berckel-
aer for excellent technical assistance with the biological assay (KU
Leuven grant GOA 10/014). We thank the EPSRC National Mass
Spectrometry Centre (Swansea, U.K.) for provision of accurate mass
spectrometric analysis.
Compd
EC50
/
l
M (CEM/0)
EC50
/
l
M (CEM/TKꢀ)
HIV-1
0.55 0.21
0.10 0.068
0.75 0.0
0.29 0.16
0.13 0.11
0.43 0.35
0.16 0.028
0.56 0.23
2.3 1.4
HIV-2
HIV-2
3a
3b
3c
3d
3e
3f
3g
3h
3i
1.3 0.14
0.24 0.23
1.1 0.23
0.90 0.71
0.18 0.049
0.65 0.35
0.18 0.071
1.0 0.27
4.7 4.8
1.3 0.71
28 2.1
>50
2.0 0.0
3.0 1.3
5.2 0.92
>50
Supplementary data
Supplementary data (1H, 13C, 19F and 31P NMR, mass spectrom-
etry and HPLC data for FLT pro-nucleotide analogues) associated
with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmcl.2014.03.092.
4.7 2.4
>50
3j
3k
3l
3m
5a
5b
1
0.048 0.078
0.032 0.0057
0.12 0.014
0.14 0.0071
0.080 0.0
0.18 0.078
1.6 0.38
0.13 0.0071
0.045 0.012
0.018 0.014
2.0 1.1
0.87 0.33
2.1 1.2
89 8.5
1.0 0.87
20 0.71
>50
References and notes
1. De Clercq, E. J. Clin. Virol. 2004, 30, 115.
2. Damaraju, V. L.; Damaraju, S.; Young, J. D.; Baldwin, S. A.; Mackey, J.; Sawyer,
M. B.; Cass, C. E. Oncogene 2003, 22, 7524.
0.65 0.21
0.036 0.0042
0.017 0.0092
0.0062 0.0021
3. Cahard, D.; McGuigan, C.; Balzarini, J. Mini. Rev. Med. Chem. 2004, 4, 371.
4. Mehellou, Y.; Balzarini, J.; McGuigan, C. ChemMedChem 2009, 11, 1779.
5. McGuigan, C.; Murziani, P.; Slusarczyk, M.; Gonczy, B.; Vande Voorde, J.;
Liekens, S.; Balzarini, J. J. Med. Chem. 2011, 54, 7247.
6. McGuigan, C.; Madela, K.; Aljarah, M.; Bourdin, C.; Arrica, M.; Barrett, E.; Jones,
S.; Kolykhalov, A.; Bleiman, B.; Bryant, K. D.; Ganguly, B.; Gorovits, E.; Henson,
G.; Hunley, D.; Hutchins, J.; Muhammad, J.; Obikhod, A.; Patti, J.; Walters, C. R.;
Wang, J.; Vernachio, J.; Ramamurty, C. V. S.; Battina, S. K.; Chamberlain, S. J.
Med. Chem. 2011, 54, 8632.
7. Vernachio, J. H.; Bleiman, B.; Bryant, K. D.; Chamberlain, S.; Hunley, D.;
Hutchins, J.; Ames, B.; Gorovits, E.; Ganguly, B.; Hall, A.; Kolykhalov, A.; Liu, Y.;
Muhammad, J.; Raja, N.; Walters, C. R.; Wang, J.; Williams, K.; Patti, J. M.;
Henson, G.; Madela, K.; Aljarah, M.; Gillies, A.; McGuigan, C. Antimicrob. Agents
Chemother. 2011, 55, 1843.
8. Slusarczyk, M.; Huerta Lopez, M.; Balzarini, J.; Mason, M.; Jiang, W. G.; Blagden,
S.; Thompson, E.; Ghazaly, E.; McGuigan, C. J. Med. Chem. 2014, 57, 1531.
9. Shah, P.; Westwell, A. D. J. Enz. Inhib. Med. Chem. 2007, 22, 527.
10. Frampton, J. E.; Perry, C. M. Drugs 2005, 65, 1427.
11. Asselah, T.; Lada, O.; Moucari, R.; Marcellin, P. Exp. Opin. Invest. Drugs 2008, 17,
1963.
12. Skevaki, C. L.; Galani, I. E.; Pararas, M. V.; Giannopoulou, K. P.; Tsakris, A. Drugs
2011, 71, 331.
13. Peters, G. J.; Bijnsdorp, I. V. Lancet Oncol. 2012, 13, e518.
14. Li, Q. Y.; Jiang, Y.; Wei, W.; Yang, H. W.; Liu, J. L. PLoS One 2013, 8, e53403.
15. Moysan, E.; Bastiat, G.; Benoit, J. P. Mol. Pharm. 2013, 10, 430.
16. Robak, P.; Robak, T. Cancer Treat. Rev. 2013, 39, 851.
17. Daniels, S.; Tohid, S. F. M.; Velanguparackel, W.; Westwell, A. D. Exp. Opin. Drug
Disc. 2010, 5, 291.
Table 2
Cytostatic activity (IC50
leukaemia (L1210) cells. Results are expressed as mean values of at least 2–3
,
lM) values in human T-lymphocyte (CEM) and murine
independent experiments
Compd
IC50/lM
CEM/TK-
CEM/0
L1210
8.2 3.7
4.4 1.4
23
6.3 0.6
5.8 0.6
3.0 1.2
6.0 0.9
24
36
2.9 1.2
1.9 1.1
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
5a
5b
1
>250
>250
145 38
124 58
117 88
66 10
>250
>250
>250
>250
172 59
112 52
1
81
1
138
5
206 62
148 17
>250
>250
2
3
13
14
54
2
1
5
20
20
57
3
3
8
5.2 3.3
43 13
1.2 1.1
3.8 2.7
100 12
17
>250
22
6
5
66 11
39 26
43 34
>250
0.081 0.037
18. Herdewijn, P.; Balzarini, J.; De Clercq, E.; Pauwels, R.; Baba, M.; Broder, S.;
Vanderhaeghe, H. J. Med. Chem. 1987, 30, 1270.
19. Ghosn, J.; Quinson, A. M.; Sabo, N. D.; Cotte, L.; Piketty, C.; Dorleacq, N.; Bravo,
M. L.; Mayers, D.; Harmenberg, J.; Mardh, G.; Valdez, H.; Katlama, C. HIV Med.
2007, 8, 142.
20. McGuigan, C.; Jones, B. C. N. M.; Devine, K. G.; Nicholls, S. R.; O’Connor, T. J.;
Kinchington, D. Bioorg. Med. Chem. Lett. 1991, 1, 729.
21. General method for synthesis of FLT phosphoramidates (3a–m). A solution of tert-
butylmagnesium chloride (1 M in THF, 1.3 equiv) was added dropwise to a
stirring solution of 30-deoxy-30-fluorothymidine (FLT, 1, 0.1 M) in anhydrous
Examination of Table 2 reveals few compounds with signifi-
cantly potent cytostatic activity within these cell line models.
The most potent activity was observed in the L1210/0 leukaemia
cell line, where low micromolar IC50 values was found for a variety
of compounds, but where cytostatic activity was lower than for the
parental FLT (1). Gratifyingly, compounds with moderate activity
in the CEM/0 cell line (such as 3j, 3k and 5a) were largely able to
retain activity within the CEM/TKꢀ tumour cell model (unlike the
parent FLT) and were non-toxic at antiviral concentrations.
In conclusion, the preparation of a series of phosphoramidate
and phosphorodiamidate ProTides of the antiviral lead compound
FLT have been prepared. In vitro screening with anti-HIV and anti-
cancer model systems revealed compounds with generally lower
activity than the parent lead, but with the ability to retain activity
within TKꢀ models. This retention of activity across the test panels
provides further evidence for the potential of ProTides to, at least
in part, by-pass the need of thymidine kinase-catalysed phosphor-
ylation and deliver the intact nucleoside monophosphate ready for
further metabolic conversion to the biologically active form.
THF at room temperature, followed by stirring for 30 min.
A solution of
phosphochloridate (2a–m, 2.05 equiv) in anhydrous THF (1 mL) was then
added dropwise to the reaction mixture followed by stirring for 14–22 h. THF
was then removed in vacuo and the residue purified by column
chromatography (CHCl3/MeOH 100/0 to 95/5) followed by preparative TLC
(CHCl3/MeOH 95/5) to give the required FLT phosphoramidates (3a–m) as
colorless oils.
22. Representative characterisation data for FLT phosphoramidates: 30-Deoxy-30-
fluorothymidine 50-O-1-naphthyl-(neopentoxy-alaninyl)-phosphate (3k). This
compound was synthesized according to the procedure above in 57% yield. The
ratio of the diastereoisomers (d/s) at the phosphorous atom was 1/0.7 (1H NMR
analysis). 1H NMR (500 MHz, CDCl3): d 8.62 (br s, 1H, NH of one d/s), 8.58 (br s,
1H NH of one d/s), 8.05 (m, 1.7H, ArH), 7.86 (m, 1.7H, ArH), 7.68 (m, 1.7H, ArH),
7.56–7.49 (m, 5.1H, ArH), 7.42–7.35 (m, 2.7H, ArH+H5 of both d/s), 7.29 (dd,
J = 6.0, 0.9 Hz, 0.7H, ArH), 6.29 (dd, J = 9.5, 5.2 Hz, 0.7H, H10 of one d/s), 6.23
(dd, J = 9.4, 5.2 Hz, 1H, H10 of one d/s), 5.17 (m, dd, J = 5.0, 3.5 Hz, 0.7H, H30 of
one d/s), 5.11 (m, dd, J = 5.0, 3.5 Hz, 1H, H30 of one d/s), 4.50–4.45 (m, 0.7H, H40
of one d/s), 4.43–4.30 (m, 4.4H, H40 of one d/s, H50 of both d/s), 4.19–4.10 (m,
1.7H, CH Ala of both d/s), 4.00–3.96 (dd, J = 9.0, 11.2 Hz, 1H, NH of one d/s),
3.88, 3.76 (AB system, J = 10.5 Hz, 2H, CH2 neopentyl of one d/s), 3.84–3.80 (m,
0,7H, NH of one d/s), 3.86, 3.69 (AB system, J = 10.5 Hz, 1.4H, CH2 neopentyl of
one d/s), 2.46–2.35 (m, 2H, H20a of both d/s), 1.82 (d, J = 0.9 Hz, 2.1H, CH3 base
of one d/s), 1.80 (d, J = 1.0 Hz, 3H, CH3 base of one d/s), 1.70–1.62 (m, 1.7H,
H20b of both d/s), 1.42 (d, J = 7.0 Hz, 3H, CH3 Ala of one d/s), 1.37 (d, J = 7.1 Hz,
Acknowledgments
The authors are grateful to Cancer Research Wales for the award
of a Ph.D. Scholarship (to W.V.) and to Cardiff University for

W. Velanguparackel et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2240–2243
2243
2.1H, CH3 Ala of one d/s), 0.93 (s, 9H, CH3 neopentyl of one d/s), 0.90 (s, 6.3H,
CH3 neopentyl of one d/s). 13C NMR (126 MHz, CDCl3): d 173.5 (d, J = 7.3 Hz,
C@O of one d/s), 173.3 (d, J = 7.3 Hz, C@O of one d/s), 163.39 (Cq), 163.32 (Cq),
150.07 (Cq), 150.05 (Cq), 146.3 (d, J = 6.9 Hz, Cq), 146.1 (d, J = 6.8 Hz, Cq), 135.0
(C6 of one d/s), 134.8 (C6 of one d/s), 134.7 (Cq), 128.06, 128.04, 126.8, 126.56,
126.55 (CAr), 126.28, 126.23, 126.21, 126.16 (Cq), 125.46, 125.42, 125.40,
125.3, 125.2, 121.0, 120.9, 115.40, 115.3, 115.1, 115.08 (CAr), 111.44 (C5 of one
d/s), 111.39 (C5 of one d/s), 93.4 (d, J = 179.3 Hz, C30 of one d/s), 93.2 (d,
J = 179.7 Hz, C30 of one d/s), 85.0 (C10 of one d/s), 84.8 (C10 of one d/s), 82.9 (dd,
J = 7.7, 27.0 Hz, C40 of one d/s), 82.8 (dd, J = 7.7, 26.8 Hz, C40 of one d/s), 74.98
(CH2 neopentyl), 74.95 (CH2 neopentyl), 66.1 (dd, J = 5.1, 11.3 Hz, C50 of one d/
s), 65.9 (dd, J = 5.2, 10.9 Hz, C50 of one d/s), 50.58 (d, J = 1.5 Hz, CH Ala of one d/
s), 50.50 (CH Ala of one d/s), 37.9 (d, J = 20.9 Hz, C20 of one d/s), 37.8 (d,
J = 20.9 Hz, C20 of one d/s), 31.3 (Cq of one d/s), 31.4 (Cq of one d/s), 26.27 (CH3
neopentyl of one d/s), 26.26 (CH3 neopentyl of one d/s), 21.2 (d, J = 4.6 Hz, CH3
Ala of one d/s), 21.1 (d, J = 5.2 Hz, CH3 Ala of one d/s), 12.42 (CH3 base of one d/
s), 12.24 (CH3 base of one d/s). 19F NMR (471 MHz, CDCl3): d ꢀ174.92, ꢀ175.21.
31P NMR (202 MHz, CDCl3): d 3.18, 3.03. MS (ES+) m/z: 614.20 (M+Na+).
Reverse HPLC eluting with H2O/MeOH from 90/10 to 0/100 over 25 min:
tR = 39.91 and 58.53 min (98%).
by a preparative TLC (CHCl3/MeOH 95/5) gave the desired diamidate prodrug
as a colorless oil.
26. Representative characterisation data for FLT phosphorodiamidates: FLT-50-O-
bis[(benzoxy-L-alaninyl)] phosphate (5a). This compound was synthesized
according to the procedure above in 10% yield. 1H NMR (500 MHz, MeOD): d
7.54 (d, J = 1.2 Hz, 1H, H6), 7.37–7.30 (m, 10H, ArH), 6.26 (dd, J = 9.3, 5.5 Hz, 1H,
H10), 5.24 (dd, J = 5.0, 3.5 Hz, 1H, H30), 5.14–5.11 (m, 4H, 2 ꢁ CH2Ph), 4.33–4.26
(m, 1H, H40), 4.14–4.06 (m, 2H, H50), 3.98–3.92 (m, 2H, 2 ꢁ CH Ala), 2.50–2.41
(m, 1H, H20-a), 2.30–2.17 (m, 1H, H20-b), 1.87 (d, J = 1.2 Hz, 3H, CH3 base), 1.38
(dd, J = 7.2, 0.6 Hz, 3H, CH3 Ala), 1.34 (dd, J = 7.2, 0.8 Hz, 3H, CH3 Ala). 13C NMR
(126 MHz, MeOD): d 175.3 (d, J = 4.7 Hz, C@O), 175.2 (d, J = 6.0 Hz, C@O), 166.2
(C@O), 152.2 (C@O), 137.3 (C6), 137.2 (Cq), 129.61, 129.59, 129.38, 129.34
(CAr), 112.15 (C5), 95.1 (d, J = 176.6 Hz, C30), 86.3 (C10), 84.6 (dd, J = 8.3,
26.3 Hz, C40), 67.98 (CH2Ph), 67.96 (CH2Ph), 66.0 (dd, J = 11.0, 5.2 Hz, C50), 51.1
(CH Ala), 38.5 (d, J = 20.9 Hz, C20), 20.7 (d, J = 6.2 Hz, CH3 Ala), 20.6 (d, J = 6.5 Hz,
CH3 Ala), 12.6 (CH3 base). 19F NMR (471 MHz, CDCl3): d ꢀ176.58. 31P NMR
(202 MHz, CDCl3): d 13.69. MS (ES+) m/z: 669.23 (M+Na+). Reverse HPLC
eluting with H2O/MeOH from 90/10 to 0/100 in 25 min: tR 20.97 min (95%).
27. Anti-HIV activity assays: Inhibition of HIV-1(IIIB)- and HIV-2(ROD)-induced
cytopathicity in CEM cell cultures was measured in microtiter 96-well plates
containing ꢂ3 ꢁ 105 CEM cells/mL infected with 100 CCID50 of HIV per
milliliter and containing appropriate dilutions of the test compounds. After
4–5 days of incubation at 37 °C in a CO2-controlled humidified atmosphere,
CEM giant (syncytium) cell formation was examined microscopically. The EC50
(50% effective concentration) was defined as the compound concentration
required to inhibit HIV-induced giant cell formation by 50%.
23. Jones, B. C. N. M.; McGuigan, C.; O’Connor, T. J.; Jeffries, D. J.; Kinchington, D.
Antivir. Chem. Chemother. 1991, 2, 35.
24. McGuigan, C.; Bourdin, C.; Derudas, M.; Hamon, N.; Hinsinger, K.; Kandil, S.;
Madela, K.; Meneghesso, S.; Pertusati, F.; Serpi, M.; Slusarczyk, M.;
Chamberlain, S.; Kolykhalov, A.; Vernachio, J.; Vanpouille, C.; Introini, A.;
Margolis, L.; Balzarini, J. Eur. J. Med. Chem. 2013, 70, 326.
25. General method for synthesis of FLT phosphorodiamidates (5a–b). Et3N
(1.05 equiv) was added dropwise to a solution of FLT (1) in anhydrous THF
(0.2 M). The reaction mixture was stirred at rt for 30 min and was then cooled
down to ꢀ78 °C before addition of POCl3 (1.05 equiv) dropwise. The reaction
mixture was stirred at ꢀ78 °C for 1 h before addition of the appropriate amino
acid (5 equiv), DCM (4–5 mL) and Et3N (10 equiv). The reaction mixture was
stirred at rt for 16–27 h before evaporation of solvents to dryness. Purification
of the crude by column chromatography (CHCl3/MeOH 100/0 to 95/5) followed
28. Cytostatic activity assays: All assays were performed in 96-well microtiter
plates. To each well were added (5–7.5) ꢁ 104 tumour cells and a given amount
of the test compound. The cells were allowed to proliferate for 48 h (murine
lymphocytic CEM and human leukaemia L1210 cells) at 37 °C in a humidified
CO2-controlled atmosphere. At the end of the incubation period, the cells were
counted in a Coulter counter. The IC50 (50% inhibitory concentration) was
defined as the concentration of the compound that inhibited cell proliferation
by 50%.