Anti-cancer ProTides: Tuning the activity of BVDU phosphoramidates related to thymectacin

Article abstract of DOI:10.1016/j.bmc.2005.02.041

Based on our wide ranging knowledge of phosphoramidate ProTides as anti-viral agents we have tuned the lead anti-cancer agent thymectacin in the ester and amino acid regions and revealed a substantial enhancement in in vitro potency versus colon and prostate cancer cell lines. Twelve analogues have been reported, with yields of 29-78%. The compounds are fully characterised and data clearly reveal the presence of two phosphate diastereoisomers, as expected, in roughly equi-molar proportions. The compounds were evaluated in tissue culture versus three different tumour cell lines, using thymectacin as the control. It is notable that minor structural modification of the parent phenyl methoxyalaninyl structure of thymectacin leads to significant enhancements in potency. In particular, replacement of the methyl ester moiety in the lead by a benzyl ester gave a 175-fold boost in potency versus colon cancer HT115. This derivative emerges as a low micromolar inhibitor of HT115 cells and a new lead for further optimisation.

Full text of DOI:10.1016/j.bmc.2005.02.041

Bioorganic & Medicinal Chemistry 13 (2005) 3219–3227
Anti-cancer ProTides: tuning the activity of BVDU
phosphoramidates related to thymectacin
Christopher McGuigan,a,* Jean-Christophe Thiery,a Felice Daverio,a Wen G. Jiang,b
Gaynor Daviesb and Malcolm Masonb,c
aWelsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3XF, UK
bWales College of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK
cVelindre Hospital, Cardiff University, Velindre Road, Cardiff CF14 2TL, UK
Received 10 September 2004; revised 11 February 2005; accepted 18 February 2005
Abstract—Based on our wide ranging knowledge of phosphoramidate ProTides as anti-viral agents we have tuned the lead anti-can-
cer agent thymectacin in the ester and amino acid regions and revealed a substantial enhancement in in vitro potency versus colon
and prostate cancer cell lines. Twelve analogues have been reported, with yields of 29–78%. The compounds are fully characterised
and data clearly reveal the presence of two phosphate diastereoisomers, as expected, in roughly equi-molar proportions. The com-
pounds were evaluated in tissue culture versus three different tumour cell lines, using thymectacin as the control. It is notable that
minor structural modification of the parent phenyl methoxyalaninyl structure of thymectacin leads to significant enhancements in
potency. In particular, replacement of the methyl ester moiety in the lead by a benzyl ester gave a 175-fold boost in potency versus
colon cancer HT115. This derivative emerges as a low micromolar inhibitor of HT115 cells and a new lead for further optimisation.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1.Introduction
phenyl methoxyalanyl phosphate (2) being 10-fold more
potent than the parent agent versus HIV in vitro.3
Moreover (2) retained full activity in thymidine kinase
deficient cells, providing compelling evidence of its
action via intracellular nucleoside monophosphate
release.4
All chemotherapeutic nucleoside analogues, for viral
infections and cancer, exert their effect only after meta-
bolic activation in the target cell to their 50-phosphate
(nucleotide) forms. Often the efficiency of intracellular
phosphorylation of nucleoside analogues can limit their
therapeutic potential, but the pre-formed phosphates are
of limited utility on account of their poor membrane
permeation. This has lead to a wide variety of phos-
phate pro-drug approaches,1 known collectively as ÔPro-
TideÕ methods.
Subsequent applications of this technology by both our
lab and others demonstrated significant potency boosts
on phosphoramidate formation for a wide range of
nucleosides and also phosphonates: >25-fold for 8-aza-
isoddA,5 ca. 400-fold for iso-ddA,5 ca. 2000-fold for
d4A,6 >100-fold for ddA,7 10–20-fold for adenallene8
and 30–100-fold for the phosphonates PMPA and
PMEA.9,10
One such approach which we introduced in 1992 is
based on aryloxy phosphoramidates,2 of general struc-
ture (1) (Fig. 1).
However, the approach did not appear to be successful
in every case. The phosphoramidates derived from
acycloguanosine (3) were found to be less active than
the parent agent versus HSV-2, and only slightly more
active versus HCMV.11
The application of our phosphoramidate approach was
highly successful with the anti-HIV agent d4T, the
Keywords: Nucleosides; Nucleotides; Anti-cancer; ProTides; Thy-
mectacin; Phosphoramidates.
Application of our technology to the anti-VZV agent
BVDU (brivudin) (4) has proved to be more intriguing.
Thus, we reported that phosphoramidates, such as (5a)
*
Corresponding author. Tel./fax: +44 29 2087 4537; e-mail:
mcguigan@cardiff.ac.uk
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.02.041

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C. McGuigan et al. / Bioorg. Med. Chem. 13 (2005) 3219–3227
Figure 1. Some nucleosides and nucleotides.
derived from BVDU were 5–25-fold less potent than the
parent agent versus VZV in tissue culture.12 This was
interpreted as corresponding to poor intracellular deliv-
ery of BVDU monophosphate (BVDUMP), rapid deg-
radation to BVDU or poor onward phosphorylation
of BVDUMP to the bio-active triphosphate.12 However,
at about the same time the NewBiotics group had inde-
pendently prepared (5a) and found it was a potent and
effective anti-cancer agent.13 Further studies on (5a) re-
vealed that it to be selectively toxic to tumour cells
expressing elevated levels of thymidylate synthase (TS),
a key enzyme in DNA synthesis.14 Compound (5a)
entered clinical evaluation against colon cancer, with
only phase I data emerging to date and indicating the
compound to be well tolerated.15
lar protein is the eventual target for (5a) and not DNA
as might have been expected.
In order to further pursue this intriguing situation, and
given our experience of widespread phosphoramidate
modification, we sought to prepare a small family of
analogues of (5a) with modification in the aryl, ester
and amino acid regions, and to evaluate them against
a small panel of tumour cell lines. We herein report
the first data from this project.
2.Chemistry
The target phosphoramidates were all prepared in one
step from BVDU (4) using the phosphorochloridate
chemistry we have extensively described (Scheme 1).17,18
These data are intriguing on account of the detrimental
effect of phosphoramidate formation on anti-VZV po-
tency, implying poor phosphate delivery. Indeed, recent
data from NewBiotics16 indicates that, unusually, cellu-
Thus, according to Scheme 1 the appropriate aryl phos-
phorochloridates was reacted with the requisite amino
Scheme 1. Synthetic route to target phosphoramidates.

C. McGuigan et al. / Bioorg. Med. Chem. 13 (2005) 3219–3227
3221
acid ester hydrochloride to give the phosphorochlori-
dates (6) as key synthons. These were used as crude oils
without further purification. The reagents (6) were
allowed to react with (4) in THF in the presence of
N-methylimidazole (NMI) at low temperature, rising
to room temperature over 18 h. Evaporation, extraction
and flash column chromatography on silica yielded the
desired compounds (5a–l) in yields of 29–78%. In every
case the products were isolated as mixtures of diastereo-
isomers corresponding to roughly 50:50 mixed stereo-
chemistry at the phosphate centre. These isomers do
not easily separate by column chromatography, but
are readily distinguished by P-31 NMR(e.g., 5a, dP
4.72, 4.40). The compounds were further characterised
by H-1 and C-13 NMR. The presence of phosphate dia-
stereoisomers was also apparent in the H-1 and, particu-
larly C-13 data where several peaks were ÔsplitÕ in a 1:1
ratio due to the diastereoisomeric mixture. Common
examples include the H-6 signal (dca. 7.6 and the C-20
signal (dca. 42). Phosphorus-31 coupling was also noted
in the C-13 NMRto several of the carbon atoms within
three bonds of the phosphate.
the full biological and therapeutic potential of the deriva-
tives herein described. However, it does appear that the
biological profile of parent phosphoramidates may be
tuned on a nucleoside by nucleoside basis to enhance
potency and perhaps selectivity for particular cellular
and/or tumour targets.
The proposed mechanism of action of the ProTides (5) is
outlined in Scheme 2. Thus, following esterase-mediated
carboxyl ester hydrolysis, a spontaneous cyclisation, loss
of phenol and ring opening would yield the putative
amino acid metabolite (AAM).17,18 Structures of the lat-
ter general type are known to arise from phosphorami-
dates of a range of nucleosides on exposure to
carboxyl esterase18 and in tissue culture. Moreover, the
replacement of a methyl by a benzyl ester leads to an
enhancement in the anti-viral potency of dideoxynucleo-
side phosphoramidates and to more rapid esterase-medi-
ated hydrolysis.19 Whether this is the origin of the
enhanced activity we note for 5c over 5a is currently un-
clear. However, it is also notable that 5c is estimated to
be roughly 50-fold more lipophilic than 5a (ClogP: 5a
0.11, 5c 1.82, Chemdraw 7.0), which may lead to more
rapid membrane permeation for 5c.
3.Results and discussion
The phosphoramidates 5a–l were evaluated for their
anti-cancer effect against a panel of three tumour cell
lines in vitro. These were breast MDA MB231, colon
(HT115) and prostate (PC-3) tumour cells. Data are pre-
sented in Table 1.
4.Conclusions
We have successfully applied the phosphoramidate
based ProTide methodology to the preparation of a ser-
ies of analogues of thymectacin, the BVDU-derived
phosphoramidate of NewBiotics. Twelve analogues
have been reported, with yields of 29–78%. The com-
pounds are fully characterised and data clearly reveal
the presence of two phosphate diastereoisomers, as
expected, in roughly equi-molar proportions. The com-
pounds were evaluated in tissue culture versus three
different tumour cell lines, using thymectacin as the con-
trol. It is notable that minor structural modification of
the parent phenyl methoxyalaninyl structure of thymec-
tacin leads to significant enhancements in potency. In
particular, replacement of the methyl ester moiety in
the lead by a benzyl ester gave a 175-fold boost in po-
tency versus colon cancer HT115. This derivative, 5c,
emerges as a low micromolar inhibitor of HT115 cells
and a new lead for further optimisation.
In our hands the lead clinical candidate thymectacin (5a)
is only moderately active in vitro, with EC50 values of
79–245 lM in our assays. However, relatively modest
tuning of the structure reveals a significant boost in po-
tency. For example, replacement of the methyl ester (5a)
by a benzyl ester (5c) gave a 2-fold boost in potency ver-
sus breast, on 8-fold boost versus prostate and a 175-
fold boost in potency versus the colon cancer cell line.
Use of the alternative, often highly effective, amino acid
moieties a,a-dimethylglycine (5d–f) and cyclopentyl gly-
cine (5g–i) did not yield any further improvement in
potency. However, the para-chloro phenyl analogue of
alanine (5l) did show increased potency versus the breast
cancer cell line. Further assays are underway to probe
Table 1. Anti-cancer effect of test compounds
Compound
Aryl
Ester
Amino
EC50/lM
Breast MDAMB2.31
Colon HT115
Prostate PC-3
5a (Thymectacin)
Ph
Ph
Me
Et
Ala
Ala
79
56
245
52
155
36
5b
5c
5d
5e
5f
5g
5h
5i
Ph
Ph
Bn
Me
Et
Ala
34
1.4
19
Me2Gly
Me2Gly
Me2Gly
cPntGly
cPntGly
cPntGly
Ala
41.1
218
19
77.9
39.7
14.5
77
1.5
76.1
5.1
16
Ph
Ph
Bn
Me
Et
Ph
Ph
79
44
81.3
9.7
41
33
Ph
Bn
Bn
Bn
Bn
78
17
5j
p-FPh
p-NO2Ph
p-ClPh
3.5
ND
3.4
16
5k
5l
Ala
Ala
28
9
2.4
6.2

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C. McGuigan et al. / Bioorg. Med. Chem. 13 (2005) 3219–3227
Scheme 2. Proposed mechanism of action of BVDU ProTides.
5.Experimental
and after 1 h the reaction was left to rise to room tem-
perature. The formation of phosphochloridate was moni-
tored by 31P NMR. After 2–5 h the solvent was
removed under reduced pressure and the solid obtained
washed with anhydrous ether (2 · 20 mL), filtered and
the filtrate reduced to dryness to give the products as
crude oil. These oils were usually used without further
purification.
Note on NMRmultiplicity descriptions: standard
descriptors are used (s, d, t, etc.) with a Ô2Õ prefix if a sig-
nal is split due to the presence of phosphate diastereo-
mers, if this is clear from the signals.
5.1. Standard procedure: synthesis of phosphoro-
dichloridate
5.5. Synthesis of phenyl-(methoxy-L-alaninyl)-phosphoro-
chloridate
Phosphorus oxychloride (1.0 mol equiv) and the appro-
priate substituted phenol (1.0 mol) were stirred with
anhydrous diethylether (31 mol equiv). To this was
added anhydrous triethylamine (1.0 mol equiv) at
ꢀ78 ꢁC and allowed to warm to room temperature over
16 h. The triethylamine hydrochloride salt was filtered
off, and the filtrate reduced to dryness to give the crude
product as a clear liquid.
This was synthesised according to Standard procedure
5.4, using L-alanine methyl ester hydrochloride (2.00 g,
14.3 mmol), phenyldichlorophosphate (3.02 g, 2.14 mL,
14.3 mmol) and TEA (2.9 g, 4.0 mL, 28.7 mmol)
in DCM (60 mL), to yield 3.91 g (98%) of crude
product used without further purification. 31P NMR
(CDCl3, 121 MHz): d 9.28, 8.97.
5.2. Synthesis of p-chlorophenyl-phosphodichloridate
5.6. Synthesis of phenyl-(ethoxy-L-alaninyl)-phosphoro-
chloridate
This was synthesised according to Standard procedure
5.1 using phosphorus oxychloride (1533 mg, 10.00
mmol, 932 lL), 4-chlorophenol (1.29 g, 10.03 mmol)
and TEA (1.01 g, 9.98 mmol, 1394 lL) in ethylether
(40 mL) to give an oil (1.90 g, 78%).
This was synthesised according to Standard procedure
5.4, using L-alanine ethyl ester hydrochloride (770 mg,
5.01 mmol), phenyldichlorophosphate (1.12 g, 5.01
mmol, 749 lL) and TEA (1.4 mL, 10.02 mmol) in DCM
(40 mL). The crude was purified by flash chromatogra-
phy (ethyl acetate/petroleum ether 7:3) affording 1.02
(69%) of oil. 31P NMR(CDCl 3, 121 MHz): d 9.49, 9.07.
5.3. Synthesis of p-fluorophenyl-phosphodichloridate
This was synthesised according to Standard procedure
5.1, using phosphorus oxychloride (2.07 g, 13.50 mmol,
1259 lL), 4-fluorophenol (1.52 g, 13.56 mmol, 1884 lL)
and TEA (1.37 g, 13.54 mmol, 1692 lL) in ethylether
(40 mL) to give an oil (2.59 g, 84% yield). 31P NMR
(CDCl3, 121 MHz): d 5.49.
5.7. Synthesis of phenyl-(benzoxy-L-alaninyl)-
phosphorochloridate
This was synthesised according to Standard procedure
5.4, using L-alanine benzyl ester hydrochloride (1.00 g,
4.64 mmol), phenyldichlorophosphate (980 mg, 0.69
mL, 4.64 mmol) and TEA (0.94 g, 1290 lL, 9.27 mmol)
in DCM (40 mL). The crude was purified by flash chro-
matography (ethyl acetate/petroleum ether 6:4) afford-
5.4. Standard procedure: synthesis of phosphochloridate
Phosphodichloridate (1.0 mol equiv) and the appropri-
ate amino ester hydrochloric salt (1.0 mol equiv) were
suspended in anhydrous DCM (123 mol equiv). Anhy-
drous triethylamine was added dropwise at ꢀ80 ꢁC
ing 1.61 (98%) of oil. 31P NMR(CDCl , 121 MHz): d
3
9.41, 9.23.

C. McGuigan et al. / Bioorg. Med. Chem. 13 (2005) 3219–3227
3223
5.8. Synthesis of phenyl-(methyl-2-amino-2-methylpro-
panoate)-phosphorochloridate
(0.577 mL, 3.84 mmol) and TEA (1.08 mL, 7.69 mmol)
in DCM (30 mL), to yield 1.485 g (98%) of crude prod-
uct used without further purification. 31P NMR(CDCl 3,
121 MHz): d 7.85.
This was synthesised according to Standard procedure
5.4, using 2-aminoisobutyrate methyl ester hydrochlo-
ride (583.5 mg, 3.75 mmol), phenyl dichlorophosphate
(791.1 mg, 3.75, 560 lL) and TEA (759 mg, 7.5 mmol,
1045 lL) in DCM (20 mL), to yield 1.04 g (95%) of
crude product used without further purification. 31P
5.14. Synthesis of p-fluorophenyl-(benzoxy-L-alaninyl)-
phosphorochloridate
This was synthesised according to Standard procedure
5.4, using L-alanine benzyl ester hydrochloride (1.08 g,
5.01 mmol), para-fluorophenyl-dichlorophosphate (1.210
mg, 5.01 mmol) and TEA (1.4 mL, 1.4 mmol) in DCM
(40 mL). The crude was purified by flash chromato-
graphy (ethyl acetate/petroleum ether 7:3) affording
1.599 (86%) of oil. 31P NMR(CDCl 3, 121 MHz): d
9.15, 9.06.
NMR(CDCl , 121 MHz): d 6.99.
3
5.9. Synthesis of phenyl-(ethyl-2-amino-2-methylpropano-
ate)-phosphorochloridate
This was synthesised according to Standard procedure
5.4, using 2-aminoisobutyrate ethyl ester hydrochloride
(629 mg, 3.75 mmol), phenyl dichlorophosphate
(791 mg, 3.75, 560 lL) and TEA (759 mg, 7.50 mmol,
1045 lL) in DCM (20 mL), to yield 1.018 g (89%) of
crude product used without further purification. 31P
5.15. Synthesis of p-nitrophenyl-(benzoxy-L-alaninyl)-
phosphorochloridate
NMR(CDCl , 121 MHz): d 7.02.
This was synthesised according to Standard procedure
5.4, using L-alanine benzyl ester hydrochloride (1.08 g,
5.01 mmol), para-nitrophenyl-dichlorophosphate (1.362 g,
5.01 mmol) and TEA (1.4 mL, 10.02 mmol) in DCM
(40 mL), to yield 1.85 g (93%) of crude product used
3
5.10. Synthesis of phenyl-(benzyl-2-amino-2-methylpro-
panoate)-phosphorochloridate
This was synthesised according to Standard procedure
5.4, using 2-aminoisobutyrate benzyl ester hydrochlo-
ride (861 mg, 3.75 mmol), phenyl dichlorophosphate
(791 mg, 3.75, 560 lL) and TEA (759 mg, 7.50 mmol,
1045 lL) in DCM (30 mL). The crude was purified by
flash chromatography (ethyl acetate/petroleum ether
6:4) affording 580 mg (42%) of colourless oil. 31P
without further purification. 31P NMR(CDCl
,
3
121 MHz): d 9.15, 9.06.
5.16. Synthesis of p-chlorophenyl-(benzoxy-L-alaninyl)-
phosphorochloridate
This was synthesised according to Standard procedure
5.4, using L-alanine benzyl ester hydrochloride (1.00 g,
4.63 mmol), 4-chlorophenylphosphodichloridate (1.14 g,
4.63 mmol) and TEA (937 mg, 9.26 mmol, 1290 lL) in
DCM (40 mL), to yield 1534 mg (87%) of crude product
NMR(CDCl , 121 MHz): d 6.79
3
5.11. Synthesis of phenyl-(methoxy-a,a-cycloleucinyl)-
phosphorochloridate
used without further purification. 31P NMR(CDCl
,
3
This was synthesised according to Standard procedure
5.4, using methyl-1-amino-1-cyclopentanoate hydro-
chloride salt (0.885 g, 5.01 mmol), phenyldichlorophos-
phate (1.12 g, 0.749 mL, 5.01 mmol) and TEA (1.4 mL,
10 mmol) in DCM (40 mL), to yield 1.266 g (81%) of
crude product used without further purification. 31P
121 MHz): d 9.43, 9.16.
5.17. Standard procedure: synthesis of phosphoramidate
derivatives
To a stirring solution of (E)-5-(2-bromovinyl)-20-deoxy-
uridine (1.0 mol equiv) and the appropriate phosphoro-
chloridate (2.0–3.0 mol equiv) in anhydrous THF at
ꢀ80 ꢁC was added dropwise over 1 min NMI (5.0 mol
equiv). After 15 min the reaction was left to rise to room
temperature and stirred at room temperature for 2–19 h.
The solvent was removed under reduced pressure and
the yellow oil obtained was dissolved in DCM, washed
with 0.5 M HCl, and water. The organic layer was dried
over MgSO4, filtered, reduced to dryness and purified by
flash chromatography (chloroform/methanol 97/3,
dichloromethane/methanol 97/3).
NMR(CDCl , 121 MHz): d 7.90.
3
5.12. Synthesis of phenyl-(ethoxy-a,a-cycloleucinyl)-
phosphorochloridate
This was synthesised according to Standard procedure
5.4, using ethyl-1-amino-1-cyclopentanoate hydrochlo-
ride salt (955 mg, 5.01 mmol), phenyldichlorophosphate
(1.12 g, 5.01 mmol, 749 lL) and TEA (1.4 mL, 10.02
mmol) in DCM (40 mL). The crude was purified by flash
chromatography (ethyl acetate/petroleum ether 7:3)
affording 1.457 g (89%) of oil. 31P NMR(CDCl
,
3
121 MHz): d 8.04, 7.97.
5.18. Synthesis of (E)-5-(2-bromovinyl)-20-deoxyuridine-
50-[phenyl methoxy-L-alaninyl]-phosphate, 5a
5.13. Synthesis of phenyl-(benzoxy-a,a-cycloleucinyl)-
phosphorochloridate
This was synthesised according to Standard procedure
5.17 using BVdU (300 mg, 0.90 mmol), phenyl methoxy-
L-alaninyl phosphorochloridate (472 mg, 1.7 mmol),
NMI (4.73 mmol, 378 lL) in THF (9 mL) for 2 h. The
crude product was purified by column chromatography,
This was synthesised according to Standard procedure
5.4, using benzyl-1-amino-1-cyclopentanoate hydrochlo-
ride salt (0.984 g, 3.84 mmol), phenyldichlorophosphate

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C. McGuigan et al. / Bioorg. Med. Chem. 13 (2005) 3219–3227
eluting with dichloromethane/methanol 97/3 to give the
pure product as a white foamy solid (356 mg, yield
69%).
2s, H-6), 7.45–7.14 (11H, m, OPh, CH2Ph, H-5b),
6.75, 6.66 (1H, 2d, J = 14 Hz, H-5a), 6.30–6.25 (1H,
3
m, H-10), 5.18–5.09 (1H, m, CH2Ph), 4.70–4.04 (6H,
m, H-30, H-50, H-40, NH, CHCH3), 2.42 (1H, m, one
of H-20), 2.02 (1H, m, one of H-20), 1.40 (3H, d,
3J = 7 Hz, CH3CH). 13C NMR(CDCl 3, 75 MHz): d
20.7, 20.8 (CH3CH), 40.4 (C-20), 50.4 (CHCH3), 66.0
(C-50), 67.4 (CH2Ph), 70.6 (C-30), 85.4, 85.5, 85.6, 85.8
(C-10, C-40), 109.9 (C-5b), 111.5 (C-5b), 120.2 (ÔoÕ,
OPh), 125.4 (ÔpÕ, OPh), 128.5, 128.6, 129.9 (ÔmÕ OPh,
CH2Ph, C-5a), 135.1 (ÔipsoÕ, CH2Ph) 137.8 (C-6), 149.8
(C-4) 150.2 (ÔipsoÕ, OPh), 161.8 (C-2), 173.6
(COOCH2Ph).
1
31P NMR(CDCl , 121 MHz): d 4.72, 4.40. H NMR
3
(CDCl3; 300 MHz): d 9.90 (1H, br s, H-3), 7.64 (1H,
2s, H-6), 7.44, 7.39 (1H, 2d, J = 14 Hz, H-5b), 7.37–
3
3
7.15 (5H, m, OPh), 6.75, 6.67 (1H, 2d, J = 14 Hz, H-
3
5a), 6.30, 6.21 (1H, 2t, J = 6 Hz, H10), 4.57–4.29 (3H,
m, H-50 + H-30), 4.20–3.96 (3H, H-40, NH, CHCH3),
3.72 (3H, s, CH3O), 2.49–2.40 (1H, m, one of H-20),
3
2.12–2.01 (1H, m, one of H-20), 1.38 (3H, d, J = 7 Hz,
CH3CH). 13C NMR(DMSO; 75 MHz):
d 22.4
(CH3CH), 41.9, 41.8 (C-20), 51.9 (CHCH3), 54.3
(CH3O), 67.5 (C-50), 72.3, 71.9 (C-30), 87.3, 87.2, 86.9,
86.8 (C-10, C-40), 110.6 (C-5b), 113.1 (C-5), 121.7 (ÔoÕ,
OPh), 127.0 (ÔpÕ, OPh), 130.1 (C-5a), 131.5 (ÔmÕ, OPh),
139.2 (C-6), 150.9 (ÔipsoÕ, OPh) 151.9 (C-4), 163.2 (C-
2), 175.7 (COOCH3).
5.21. Synthesis of (E)-5-(2-bromovinyl)-20-deoxyuridine-
50-[phenyl methoxy-a,a-dimethylglycinyl]-phosphate, 5d
This was synthesised according to Standard procedure
5.17, using BVdU (200 mg, 0.60 mmol), phenyl
(methyl-2-amino-2-methylpropanoate) phosphorochlo-
ridate (437.5 mg, 1.5 mmol), NMI (3.0 mmol, 239 lL)
in THF (5 mL) for 4 h. The crude product was purified
by column chromatography, eluting with chloroform/
methanol 97/3 to give the pure product as a white foamy
solid (117 mg, yield 33%).
5.19. Synthesis of (E)-5-(2-bromovinyl)-20-deoxyuridine-
50-[phenyl ethoxy-L-alaninyl]-phosphate, 5b
This was synthesised according to Standard procedure
5.17, using BVdU (150 mg, 0.45 mmol), phenyl ethoxy
L-alaninyl phosphorochloridate (249 mg, 0.9 mmol),
NMI (2.38 mmol, 190 lL) in THF (4 mL) for 2 h. The
crude product was purified by column chromatography,
eluting with dichloromethane/methanol 97/3 to give the
pure product as a white foamy solid (145 mg, yield
55%).
1
31P NMR(CDCl , 121 MHz): d 3.36, 3.14. H NMR
3
(CDCl3; 300 MHz): d 9.91 (1H, br s, H-3), 7.73, 7.65
(1H, 2s, H-6), 7.48–7.45 (1H, 2d, 3J = 14 Hz, H-5b),
7.41–7.02 (5H, m, OPh), 6.81, 6.71 (1H, 2d,
3J = 14 Hz, H-5a), 6.34–6.28 (1H, m, H10), 4.55–4.17
(6H, m, H-50, H-40, H-30, NH, OH-30), 3.78 (3H, s,
CH3O), 2.53–2.39 (1H, m, one of H-20), 2.25–1.99
(1H, m, one of H-20), 1.60 (6H, s, [CH3]2C). 13C
1
31P NMR(CDCl , 121 MHz): d 4.48, 4.86. H NMR
3
(CDCl3, 300 MHz): d 7.68, 7.64 (1H, 2s, H-6), 7.47,
7.41 (1H, 2d, 3J = 13 Hz, H-5b), 7.35–7.10 (5H, m,
OPh), 6.73, 6.69 (1H, 2d, J = 13 Hz, H-5a), 6.31, 6.27
NMR(CDCl ; 75 MHz): d 27.5, 27.4, 27.2 ([CH3]2C),
3
3
40.7, 40.6 (C-20), 53.5 (CH3O), 57.6 (C[CH3]2), 66.5,
66.2 (C-50), 70.7, 71.1 (C-30), 85.4, 85.6, 85.5, 85.9
(C-10, C-40), 110.4 (C-5b), 111.9 (C-5), 120.5, 120.6
(ÔoÕ, OPh), 125.7 (ÔpÕ, OPh), 128.9 (C-5a), 130.3 (ÔmÕ,
OPh), 138.0, 138.3 (C-6), 149.8 (ÔipsoÕ, OPh) 150.9,
150.8 (C-4), 162.0, 162.1 (C-2), 176.4, 176.2
(COOCH3).
3
(1H, 2t, J = 6 Hz, H10), 4.62–3.95 (8H, m, H-50, H-30,
H-40, CHCH3, NH, CH3CH2O), 2.49–2.40 (1H, m,
one of H-20), 2.10–2.00 (1H, m, one of H-20), 1.40
(3H, d, 3J = 7 Hz, CH3CH), 1.25 (3H, 2t, 3J = 7 Hz,
CH3CH2O). 13C NMR(CDCl 3, 75 MHz): d 14.5
(CH3CH2O), 21.2, 21.1 (CH3CH), 40.9, 40.7 (C-20),
50.8, 50.7 (CHCH3), 62.2, 62.1 (CH3CH2O), 66.5, 66.3
(C-50), 70.9, 70.6 (C-30), 86.0, 85.6 (C-10, C-40), 110.1
(C-5b), 111.8 (C-5), 120.6 (ÔoÕ, OPh), 125.0 (ÔpÕ, OPh),
129.0 (C-5a), 130.2 (ÔmÕ, OPh), 138.2 (C-6), 149.9 (C-
4), 150.7 (ÔipsoÕ, OPh), 162.3 (C-2), 174.2, 174.1
(COOCH2CH3).
5.22. Synthesis of (E)-5-(2-bromovinyl)-20-deoxyuridine-
50-[phenyl ethoxy-a,a-dimethylglycinyl]-phosphate, 5e
This was synthesised according to Standard procedure
5.17, using BVdU (200 mg, 0.60 mmol), phenyl (ethyl-
2-amino-2-methylpropanoate) phosphorochloridate (458.0
mg, 1.5 mmol), NMI (3.0 mmol, 239 lL) in THF (5 mL)
for 5 h. The crude product was purified by column chro-
matography, eluting with chloroform/methanol 97/3 to
give the pure product as a white foamy solid (106 mg,
yield 29%).
5.20. Synthesis of (E)-5-(2-bromovinyl)-20-deoxyuridine-
50-[phenyl benzoxy-L-alaninyl]-phosphate, 5c
This was synthesised according to Standard procedure
5.17, using BVdU (150 mg, 0.45 mmol), phenyl benzyl-
oxy-L-alaninyl phosphorochloridate (249 mg, 0.9 mmol),
NMI (2.38 mmol, 190 lL) in THF (5 mL) for 2 h. The
crude product was purified by column chromatography,
eluting with dichloromethane/methanol 97/3 to give the
pure product as a white foamy solid (228 mg, yield
78%).
1
31P NMR(MeOD, 121 MHz): d 3.91, 3.85. H NMR
(MeOD, 300 MHz): d 7.84, 7.81 (1H, 2s, H-6), 7.44–
7.20 (6H, m, OPh, H-5b), 6.86, 6.84 (1H, 2d,
3J = 14 Hz, H-5a), 6.34–6.28 (1H, m, H-10), 4.50–4.34
(3H, m, H-50, H-30), 4.23–4.15 (3H, m, H-40,
CH3CH2O), 2.38–2.28 (1H, m, one of H-20),
2.22–2.09 (1H, m, one of H-20), 1.51 (6H, s,
[CH3]2C), 1.29 (3H, t, 3J = 7 Hz, CH3CH2O). 13C
1
31P NMR(CDCl , 121 MHz): d 4.74, 4.44. H NMR
3
(CDCl3, 300 MHz): d 10.31 (1H, br s, H-3), 7.63 (1H,

C. McGuigan et al. / Bioorg. Med. Chem. 13 (2005) 3219–3227
3225
NMR(MeOD, 75 MHz): d 14.9 (CH3CH2O) 27.9, 28.3
([CH3]2C), 41.5 (C-20), 58.51 (C[CH3]2), 63.1
(CH3CH2O), 68.2 (C-50), 72.6 (C-30), 87.1, 87.4 (C-10,
C-40), 109.6 (C-5b), 112.7 (C-5b), 122.0, 122.1, 122.2,
(ÔoÕ, OPh), 126.7 (ÔpÕ, OPh), 131.0, 131.2 (C-5a, ÔmÕ
OPh), 140.4 (C-6), 151.4 (ÔipsoÕ, OPh), 152.5 (C-4),
164.0 (C-2), 177.2 (COOCH2CH3).
138.5 (C-6), 149.9 (C-4), 150.9, 150.8 (ÔipsoÕ, OPh),
162.3 (C-2), 176.3, 176.2 (COOCH3).
5.25. Synthesis of (E)-5-(2-bromovinyl)-20-deoxyuridine-
50-[phenyl ethoxy-a,a-cyclopentylglycinyl]-phosphate, 5h
This was synthesised according to Standard procedure
5.17, using BVdU (250 mg, 0.75 mmol), Phenyl ethoxy-
a,a-cyclopentylglycinyl phosphorochloridate (642 mg,
1.87 mmol), NMI (5.2 mmol, 415 lL) in THF (7 mL)
for 2 h. The crude product was purified by column chro-
matography, eluting with dichloromethane/methanol
97/3 to give the pure product as a white foamy solid
(258 mg, yield 55%).
5.23. Synthesis of (E)-5-(2-bromovinyl)-20-deoxyuridine-
50-[phenyl benzoxy-a,a-dimethylglycinyl]-phosphate, 5f
This was synthesised according to Standard procedure
5.17, using BVdU (242 mg, 0.73 mmol), phenyl (benzyl-
2-amino-2-methylpropanoate)
phosphorochloridate
(533.0 mg, 2.0 mmol), NMI (3.63 mmol, 289 lL) in
THF (5 mL) for 4 h. The crude product was purified
by column chromatography, eluting with chloroform/
methanol 97/3 to give the pure product as a white foamy
solid (129 mg, yield 27%).
1
31P NMR(CDCl , 121 MHz): d 4.23, 4.10. H NMR
3
(CDCl3, 300 MHz): d 10.22 (1H, br s, H-3), 7.73, 7.63
(1H, 2s, H-6), 7.51 (1H, d, 3J = 14 Hz, H-5b), 7.45–
7.10 (5H, m, OPh), 6.74, 6.71 (1H, 2d, 3J = 14 Hz,
H-5a), 6.22 (1H, t, J = 4 Hz, H10), 4.55–4.05 (7H,
3
1
31P NMR(CDCl , 121 MHz): d 3.39, 3.12. H NMR
m, H-50, H-30, H-40, NH, CH3CH2O), 2.50–2.40 (1H,
m, one of H-20), 2.35–1.95 (5H, m, one of H-20, 4H
cyclopentane), 1.95–1.75 (4H, m, 4H cyclopentane),
1.27, 1.26 (3H, 2t, 3J = 7 Hz, CH3CH2O). 13C NMR
(CDCl3, 75 MHz): d 14.5 (CH3CH2O), 24.5, 24,4
(2CH2 cyclopentane), 39.2, 38.9 38.8, 38.4 (2CH2 cyclo-
pentane), 40.6 (C-20), 62.2, 62.1 (CH3CH2O), 66.2 (Cq
cyclopentane), 66.6 (C-50), 70.8 (C-30), 85.7, 85.5 (C-10,
C-40), 110.2 (C-5b), 111.5 (C-5), 120.7, 120.6 (ÔoÕ,
OPh), 125.6 (ÔpÕ, OPh), 129.7 (C-5a), 130.2 (ÔmÕ, OPh),
138.5, 138.3 (C-6), 149.7 (C-4), 150.9, 150.8 (ÔipsoÕ,
OPh), 162.3 (C-2), 176.3 (COOCH2CH3).
3
(CDCl3, 300 MHz): d 9.92 (1H, br s, H-3), 7.67, 7.60
(1H, 2s, H-6), 7.46, 7.44 (1H, 2d, J = 14 Hz, H-5b),
3
7.40–7.16 (10H, m, OPh, CH2Ph), 6.76, 6.70 (1H, 2d,
3J = 14 Hz, H-5a), 6.31–6.25 (1H, m, H-10), 5.18 (1H,
s, CH2Ph), 4.50–4.09 (6H, m, H-30, H-50, H-40, NH,
OH-30), 2.48–2.25 (1H, m, one of H-20), 2.16–1.82
(1H, m, one of H-20), 1.60 (6H, s, [CH3]2C). 13C NMR
(CDCl3, 75 MHz): d 27.3, 27.4, 28.5 ([CH3]2C), 40.6,
40.7 (C-20), 57.6, 57.6 (C[CH3]2), 66.2, 66.5 (C-50), 68.1
(CH2Ph), 70.6, 71.1 (C-30), 85.4, 85.5, 85.6, 85.8 (C-10,
C-40), 110.4 (C-5b), 112.0 (C-5), 120.4, 120.5, 120.6,
125.7, 128.4, 128.5, 128.8, 128.9, 130.3 (OPh, C-5a),
135.7 (ÔipsoÕ, CH2Ph) 138.1, 138.3 (C-6), 149.8, 150.8,
150.9 (ÔipsoÕ OPh, C-4), 162.1 (C-2), 177.5, 175.7
(COOCH2Ph).
5.26. Synthesis of (E)-5-(2-bromovinyl)-20-deoxyuridine-
50-[phenyl benzoxy-a,a-cyclopentylglycinyl] phosphate, 5i
This was synthesised according to Standard procedure
5.17, using BVdU (200 mg, 0.6 mmol), phenyl benzyl-
oxy-a,a-cyclopentylglycinyl phosphorochloridate (589
mg, 1.5 mmol), NMI (4.16 mmol, 332 lL) in THF
(5 mL) for 10 h. The crude product was purified by col-
umn chromatography, eluting with dichloromethane/
methanol 97/3 to give the pure product as a white foamy
solid (127 mg, yield 31%).
5.24. Synthesis of (E)-5-(2-bromovinyl)-20-deoxyuridine-
50-[phenyl methoxy-a,a-cyclopentylglycinyl] phosphate, 5g
This was synthesised according to Standard procedure
5.17, using BVdU (250 mg, 0.75 mmol), phenyl methoxy-
a,a-cyclopentylglycinyl phosphorochloridate (589 mg,
1.87 mmol), NMI (5.2 mmol, 415 lL) in THF (7 mL)
for 3 h. The crude product was purified by column chro-
matography, eluting with dichloromethane/methanol
97/3 to give the pure product as a white foamy solid
(234 mg, yield 51%).
1
31P NMR(CDCl , 121 MHz): d 4.11, 4.01. H NMR
3
(CDCl3, 300 MHz): d 10.20 (1H, br s, H-3), 7.69, 7.60
(1H, 2s, H-6), 7.45, 7.44 (1H, 2d, 3J = 14 Hz, H-5b),
7.40–7.10 (10H. m, OPh+CH2Ph), 6.74, 6.71 (1H, 2d,
3J = 14 Hz, H-5a), 6.20 (1H, m, H-10), 5.15 (1H, s,
CH2Ph), 4.4–4.2 (3H, m, H-30, H-40, NH), 4.1 (2H, m,
H-50), 2.45–2.35 (1H, m, one of H-20), 2.35–1.95 (5H,
m, one of H-20, 4H cyclopentane), 1.95–1.75 (4H, m,
4H cyclopentane). 13C NMR(CDCl 3, 75 MHz): d
24.4, 24,3, 24.2 (2CH2 cyclopentane), 39.9, 39.7 38.6,
38.5 (2CH2 cyclopentane), 40.5 (C-20), 66.2 (Cq cyclo-
pentane), 66.5 (C-50), 67.8 (CH2Ph), 70.8, 70.7 (C-30),
85.7, 85.6, 85.5, 85.4 (C-10, C-40), 110.2 (C-5b), 111.8,
118.7 (C-5b), 120.7, 120.5 (ÔoÕ, OPh), 125.7 (ÔpÕ, OPh),
130.2, 129.0, 128.8, 128.7, 128.5 (ÔmÕ OPh, CH2Ph, C-
5a), 135.8 (ÔipsoÕ, CH2Ph) 138.4, 138.2 (C-6), 149.8 (C-
4), 150.9, 150.8 (ÔipsoÕ, OPh), 162.2 (C-2), 175.7, 175.5
(COOCH2Ph).
1
31P NMR(CDCl , 121 MHz): d 3.87, 3.82. H NMR
3
(CDCl3; 300 MHz): d 10.28 (1H, br s, H-3), 7.75, 7.72
(1H, 2s, H-6), 7.42 (1H, d, 3J = 13 Hz, H-5b), 7.37–
7.15 (5H, m, OPh), 6.73, 6.71 (1H, 2d, J = 13 Hz, H-
3
3
5a), 6.30 (1H, t, J = 6 Hz, H10), 4.4–4.2 (4H, m, H-50,
H-30, NH), 4.1 (1H, m, H-40), 3.72 (3H, s, CH3O),
2.49–2.40 (1H, m, one of H-20), 2.35–2.01 (5H, m,
one of H-20 + 4H cyclopentane), 1.76–1.70 (4H, m, 4H
cyclopentane). 13C NMR(CDCl 3; 75 MHz): d 24.4,
24,3, 24.2 (2CH2 cyclopentane), 39.2, 38.6, 38.5 (2CH2
cyclopentane), 40.0 (C-20), 53.2 (CH3O), 66.4 (Cq cyclo-
pentane), 66.6 (C-50), 70.9 (C-30), 85.8, 85.6, 85.4, 85.3
(C-10, C-40), 110.2 (C-5b), 111.9 (C-5), 120.7–120.6 (ÔoÕ,
OPh), 125.7 (ÔpÕ, OPh), 129.0 (C-5a), 130.2 (ÔmÕ, OPh),

3226
C. McGuigan et al. / Bioorg. Med. Chem. 13 (2005) 3219–3227
5.27. Synthesis of (E)-5-(2-bromovinyl)-20-deoxyuridine-
50-[para-fluorophenyl benzoxy-L-alaninyl]-phosphate, 5j
1.80 mmol), NMI (369.5 mg, 4.5 mmol, 358.7 lL) in
THF (10 mL) for 2 h. The crude product was purified
by column chromatography, eluting with dichloro-
methane/methanol 95:5 to give the pure product as a
white foamy solid (310 mg, yield 50%). 31P NMR
This was synthesised according to Standard procedure
5.17, using BVdU (200 mg, 0.60 mmol), para-fluorophen-
yl benzyloxy-L-alaninyl phosphorochloridate (556 mg,
1.5 mmol), NMI (4.16 mmol, 332 lL) in THF (5 mL)
for 2 h. The crude product was purified by column chro-
matography, eluting with dichloromethane/methanol
97/3 to give the pure product as a white foamy solid
(256 mg, yield 64%).
(CDCl3, 121 MHz): d 4.81, 4.53. 1H NMR(CDCl
,
3
300 MHz): d 10.10 (1H, br s, H-3), 7.65, 7.63 (1H, 2s,
H-6), 7.44, 7.43 (1H, 2d, J = 14 Hz, H-5b), 7.40–7.17
3
3
(9H, m, OPh), 6.73, 6.68 (1H, 2d, J = 14 Hz, H-5a),
6.31, 6.25 (1H, 2t, 3J = 6 Hz, H-10), 5.17 (2H, s, CH2Ph),
4.60–4.23 (4H, m, H-30, H-50, NH), 4.20–3.97 (2H, m, H-
40, CHCH3), 2.48–2.44 (1H, m, one of H-20), 2.15–2.05
(1H, m, one of H-20), 1.43, 1.40 (3H, d, 3J = 7.0
Hz, CHCH3). 13C NMR(CDCl 3, 75 MHz): d 21.2 (CH-
CH3), 40.7 (C-20), 50.8, 50.9 (CHCH3), 66.6 (C-50), 67.9
(CH2Ph), 70.7, 71.1 (C-30), 85.4, 85.5, 85.8, 86.1 (C-10, C-
40), 110.5 (C-5b), 111.9, 112.0 (C-5), 122.0 (ÔoÕ, OPh),
128.7, 129.0, 129.1, 130.3 (ÔmÕ, OPh + C-5a), 131.1 (ÔipsoÕ,
CH2Ph), 135.4 (ÔpÕ, OPh), 138.2 (C-6), 149.1 (ÔipsoÕ, OPh),
150.0 (C-4), 162.1 (C-2), 173.9, 174.0 (COOCH2Ph).
1
31P NMR(CDCl , 121 MHz): d 4.74, 4.44. H NMR
3
(CDCl3, 300 MHz): d 7.66, 7.64 (1H, 2s, H-6), 7.45–
7.00 (9H, m, OPh, CH2Ph, H-5b), 6.75 (1H, 2d,
3
3J = 14 Hz, H-5a), 6.31, 6.26 (1H, 2t, J = 6 Hz, H-10),
5.19–5.16 (1H, 2s, CH2Ph), 4.85–4.00 (6H, m, H-30, H-
50, H-40, NH, CHCH3), 2.47 (1H, m, one of H-20), 2.0–
2.15 (1H, m, one of H-20), 1.38 (3H, d, 3J = 7 Hz,
CH3CH). 13C NMR(CDCl 3, 75 MHz): d 21.2, 21.1
(CH3CH), 40.7 (C-20), 50.4 (CHCH3), 66.7, 66.4 (C-50),
67.8 (CH2Ph), 71.1, 70.7 (C-30), 86.0, 85.7, 85.4, 85.3
(C-10, C-40), 110.4 (C-5b), 111.9 (C-5), 117.0 (ÔoÕ, OPh),
122.0 (ÔmÕ, OPh), 128.7, 128.6 (CH2Ph, C-5a), 135.4
(ÔipsoÕ, CH2Ph) 138.2 (C-6), 146.5 (ÔipsoÕ, OPh), 149.9
(C-4), 158.5 (ÔpÕ OPh), 162.2 (C-2), 173.9 (COOCH2Ph).
5.30. Biological evaluation
Human breast cancer cell lines MDA MB 231, human
colon cancer cell line HT115 and prostate cancer cell line
PC-3, were purchased from the European Collection of
Animal Cell Cultures (ECACC, Salisbury, England).
Cytoxicity assay was based on MTT assay as we previ-
ously reported.20,21 The method is based on the ability
of viable mitochondria to convert MTT, a soluble tetra-
zolium salt (3-[4,5-dimethylthiazd-2-yl]-2,5-diphenyl-
tetrazolium bromide) into an insoluble formazan
precipitate that is dissolved and quantified by spectro-
photometry.22 A 96-well culture cell culture plate was
used. Cells were counted with a haemocytomete counting
chamber and a specific number (4000 per well) of cells
were seeded to each well with culture medium (DMEM).
Compounds, dissolved in DMSO, were series diluted
(1:5) in culture medium, to cover a final concentration
range between 0.128 and 2000 lM. The culture plate
was incubated for 72 h at 37 ꢁC. The cells were washed
twice with BSS. A solution of MTT in 0.5 mg/mL in cul-
ture medium was added into each well. The culture plate
was then incubated at 37 ꢁC for 4 h. MTT was then
removed by aspiration. The crystals produced by MTT
reagent within the cells were then extracted by the addi-
tion of 100 lL of Triton X100 (10% in water). The cells
were incubated at 4 ꢁC for 24 h. The absorbance of the
colorimetric products was then measured at a wave-
length of 540 nm using a spectrophotometer (Titertecek).
5.28. Synthesis of (E)-5-(2-bromovinyl)-20-deoxyuridine-
50-[para-nitrophenyl benzoxy-L-alaninyl]-phosphate, 5k
This was synthesised according to Standard procedure
5.17, using BVdU (200 mg, 0.60 mmol), para-nitro-
phenyl benzyloxy-L-alaninyl phosphorochloridate (597
mg, 1.5 mmol), NMI (4.16 mmol, 332 lL) in THF
(5 mL) for 2 h. The crude product was purified by col-
umn chromatography, eluting with dichloromethane/
methanol 97/3 to give the pure product as a white foamy
solid (228 mg, yield 55%).
1
31P NMR(CDCl , 121 MHz): d 4.74, 4.44. H NMR
3
(CDCl3, 300 MHz): d 10.29 (1H, br s, H-3), 8.20–8.14
(2H, m, OPh), 7.64, 7.59 (1H, 2s, H-6), 7.38, 7.22 (1H,
2d, 3J = 14 Hz, H-5b), 7.37–7.00 (7H, m, OPh, CH2Ph),
6.71, 6.62 (1H, 2d, 3J = 14 Hz, H-5a), 6.25–6.15 (1H, 2t,
3J = 6Hz, H-10), 5.17 (2H, d, CH2Ph), 4.87 (1H, m,
H-30), 4.6–4.2 (3H, m, H-50, CHCH3) 4.20–4.00 (2H,
m, H-40, NH), 2.55–2.45 (1H, m, one of H-20), 2.2–
2.05 (1H, m, one of H-20), 1.38 (3H, d, 3J = 7 Hz,
CH3CH). 13C NMR(CDCl 3, 75 MHz): d 21.2, 21.1
(CH3CH), 40.6 (C-20), 50.9 (CHCH3), 67.1, 67.0 (C-
50), 68.0 (CH2Ph), 71.3, 70.9 (C-30), 86.3, 86.0, 85.3,
85.2 (C-10, C-40), 110.4 (C-5b), 111.9, 111.8 (C-5),
121.3 (ÔoÕ, OPh), 126.2–126.1 (ÔmÕ, OPh), 129.1, 128.7,
128.6 (CH2Ph, C-5a), 135.4 (ÔipsoÕ, CH2Ph), 138.3 (C-
6), 145.1 (ÔipsoÕ, OPh), 149.9 (C-4), 155.6 (ÔpÕ OPh),
162.2 (C-2), 173.8, 173.7 (COOCH2Ph).
Acknowledgements
The authors thank Helen Murphy for excellent secretar-
ial assistance.
5.29. Synthesis of (E)-5-(2-bromovinyl)-20-deoxyuridine-
50-[para-chlorophenyl benzoxy-L-alaninyl]phosphate, 5l
References and notes
This was synthesised according to Standard procedure
5.17, using BVdU (300 mg, 0.90 mmol), para-chlorophen-
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