Synthesis and anticancer activities of amphiphilic 5-fluoro-2′- deoxyuridylic acid prodrugs

Article abstract of DOI:10.1016/j.ejmech.2004.12.006

Amphiphilic anticancer prodrugs of 5′-fluoro-2′-deoxyuridine- 5′-monophosphate (5-FdUMP) were synthesized according to the hydrogen phosphonate method by coupling lipophilic cytosine derivatives or a phospholipid with 5-fluoro-2′-deoxyuridine (5-FdU). Stu

Full text of DOI:10.1016/j.ejmech.2004.12.006

European Journal of Medicinal Chemistry 40 (2005) 494–504
www.elsevier.com/locate/ejmech
Short communication
Synthesis and anticancer activities of amphiphilic
5-fluoro-2′-deoxyuridylic acid prodrugs
Peter S. Ludwig a, Reto A. Schwendener b, Herbert Schott a,*
a Institute of Organic Chemistry, University of Tübingen, Auf der Morgenstelle 18, 72076 Tübingen, Germany
b Paul Scherrer Institut, Molecular Cell Biology, CH-5232 Villigen-PSI, Switzerland
Received 30 April 2004; received in revised form 16 December 2004; accepted 21 December 2004
Available online 09 March 2005
Abstract
Amphiphilic anticancer prodrugs of 5′-fluoro-2′-deoxyuridine-5′-monophosphate (5-FdUMP) were synthesized according to the hydrogen
phosphonate method by coupling lipophilic cytosine derivatives or a phospholipid with 5-fluoro-2′-deoxyuridine (5-FdU). Studies within the
in vitro Anticancer Screen Program of the National Cancer Institute have demonstrated high anticancer activities of the heterodinucleoside
phosphates: N4-palmitoyl-2′-deoxycytidylyl-(3′ → 5′)-3′-O-acetyl-5-fluoro-2′-deoxyuridine (dCpam-5-FdU(Ac), N4-palmitoyl-2′,3′-
dideoxycytidylyl-(5′ → 5′)-3′-O-acetyl-5-fluoro-2′-deoxyuridine (ddCpam-(5′ → 5′)-5-FdU(Ac), 5-fluoro-2′-deoxyuridylyl-(3′ → 5′)-5-fluoro-
N4-hexadecyl-2′-deoxycytidine (5-FdU-5-FdChex), and of the new liponucleotide 1-O-octadecyl-rac-glycerylyl-(3 → 5′)-5-fluoro-2′-
deoxyuridine (Oct1Gro-(3 → 5′)-5-FdU). The anticancer activities of these prodrugs are comparable to those of 5-FdU and the tumor specificities
are modulated by their structures. The highest cytotoxic activity being even superior to 5-FdU was expressed by the dimer 5-FdU-5-FdChex
© 2005 Elsevier SAS. All rights reserved.
.
Keywords: 5-Fluoro-2′-deoxyuridine derivatives; Anticancer prodrugs; Amphiphilic heterodinucleoside phosphates; Liponucleotide; Antimetabolites
1. Introduction
are responsible for the anticancer effect of 5-FU, whereby
inhibition of thymidylate synthase (TS) was found to be the
central mechanism. The nucleobase 5-FU is converted to the
deoxynucleoside 5-FdU by thymidine phosphorylase and sub-
sequent phosphorylation of the nucleoside by thymidine
kinase results in the cytotoxic nucleotide 5-fluoro-2′-
deoxyuridine-5′-monophosphate (5-FdUMP). In the pres-
ence of the reduced folate 5, 10-methylene-tetrahydrofolate,
5-FdUMP forms a complex with TS, inhibiting TS enzyme
activity and leading to depletion of deoxythymidine triphos-
phate, a monomeric unit necessary for DNA synthesis [1].
Alternatively, 5-FU is anabolized to 5-fluorouridine-5′-
triphosphate that can incorporated into RNA or converted to
5-fluoro-2′-deoxyuridine-5′-triphosphate, which, in turn can
be incorporated into DNA [2,3]. The antitumor activity of
5-FU is comparable to that of 5-FdU. Due to extensive hepatic
extraction 5-FdU is a useful drug for hepatic arterial chemo-
therapy of liver metastases [4].
5-Fluorouracil (5-FU) is a widely used antimetabolite for
the treatment of human solid tumors. Moreover, 5-FU and
derivatives thereof have invoked interest because of their syn-
ergistic interaction with other antitumor agents, physiologic
nucleosides and radiotherapy. To exert its cytotoxic activity
5-FU requires intracellular activation. Different mechanisms
Abbreviations: Heterodinucleoside phosphates, dCpam-5-FdU(Ac) = N4-
palmitoyl-2′-deoxycytidylyl-(3′ → 5′)-3′-O-acetyl-5-fluoro-2′-deoxyuridine;
ddCpam-(5′ → 5′)-5-FdU(Ac) = N4-palmitoyl-2′,3′-dideoxycytidylyl-(5′ →
5′)-3′-O-acetyl-5-fluoro-2′-deoxyuridine; 5-FdU-5-FdChex = 5-fluoro-2′-
deoxyuridylyl-(3′ → 5′)-5-fluoro-N4-hexadecyl-2′-deoxycytidine, 5FdU-
5FdCoct = 5-fluoro-2′-deoxyuridylyl-(3′ → 5′)-5-fluoro-N4-octadecyl-2′-
deoxycytidine; 5FdCoct-5FdU = 5-fluoro-N4-octadecyl-2′-deoxycytidylyl-
(3′ → 5′)-5-fluoro-2′-deoxyuridine, dCpam-5-FdU = N4-palmitoyl-2′-
deoxycytidylyl-(3′ → 5′)-5-fluoro-2′-deoxyuridine; of the liponucleotide:
Oct1Gro-(3 → 5′)-5-FdU = 1-O-octadecyl-rac-glycerylyl-(3 → 5′)-5-fluoro-
2′-deoxyuridine and of the monomers; p5-FdCoct = 5′-fluoro-N4-octadecyl-
2′-deoxycytidine-5′-monophosphate; 5-FdChex = 5-fluoro-N4-hexadecyl-2′-
deoxycytidine.
The usefulness of 5-FU and 5-FdU is impaired by the fre-
quent development of resistance in tumor cells. Resistance
can occur through deletion of one of the key enzymes required
for the first phosphorylation step from 5-FdU to 5-FdUMP
* Corresponding author.
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.12.006

P.S. Ludwig et al. / European Journal of Medicinal Chemistry 40 (2005) 494–504
495
catalyzed by thymidine kinase. Depletion of this enzyme is
often the main reason for 5-FU resistance. Consequently, the
direct introduction of 5-FdUMP into cells could circumvent
this resistance. However, because phosphate residues are
strongly acidic and thus negatively charged at physiological
pH, 5-FdUMP is not able to penetrate cell membranes. In
addition, plasma and cell surface phosphohydrolases rapidly
metabolize the nucleotide to the corresponding nucleoside.
In order to overcome the poor cell membrane penetration
of therapeutically useful nucleotides various approaches were
developed with the aim to enhance the intracellular delivery
of anticancer nucleoside monophosphates [5]. Despite suc-
cess and failure of different approaches many questions and
issues remain to be addressed because no single concept has
proven to be generally useful for all nucleotides.
tives. In a previous work [12] we had coupled 5-FdU to the
lipophilic cytidine derivative 5-fluoro-N4-octadecyl-2′-
deoxycytidine yielding the amphiphilic 5-FdUMP prodrug
5-fluoro-2′-deoxyuridylyl-(3′ → 5′)-5-fluoro-N4-octadecyl-
2′deoxycytidine (5-FdU-5-FdCoct, 11a). To investigate struc-
ture–activity relations the two dimers 5-fluoro-2′-
deoxyuridylyl-(3′ → 5′)-5-fluoro-N4-hexadecyl-2′-deoxy-
cytidine (5-FdU-5-FdChex, 10) and 5-fluoro-N4-octadecyl-2′-
deoxycytidylyl-(3′ → 5′)-5-fluoro-2′-deoxyuridine (5-FdCoct
-
5-FdU, 11) the sequence isomer to 11a were synthesized in
analogy to 11 (unpublished data). The structures of these com-
pounds together with the monomeric cytidine derivatives are
shown in Fig. 1. We describe herein two synthetic routes for
the preparation of new amphiphilic prodrugs of 5-FdUMP.
Such syntheses are easier than those previously published.
To overcome these disadvantages we have developed the
strategy of masking mononucleotides by their incorporation
into amphiphilic dimers with the aim to improve cellular
uptake of antiviral and anticancer nucleotide analogues and
to extent the range of their therapeutic applications and cyto-
toxic potency by including them into liposome formulations
[6–15].Amphiphilic dimers can be obtained by condensation
of a lipophilic nucleoside with a hydrophilic nucleotide and
vice versa via phosphodiester linkage. The natural enzymati-
cally easily cleavable phosphodiester bonds warrant that active
mononucleotides are released from the dimers.
2. Chemistry
The synthesis of amphiphilic heterodinucleoside phos-
phates and liponucleotides containing 5-FdUMP started with
3′-O-acetyl-5-fluoro-2′-deoxyuridine (2) which can be
obtained in analogy to a published procedure [16] from
5-fluoro-2′-deoxyuridine in two steps. According to the first
synthesis route (Fig. 2) the free 5′-hydroxyl group of 3′-O-
acetyl-5-fluoro-2′-deoxyuridine (2, hydroxyl compound) was
linked to the phosphonate compounds N4-palmitoyl-2′,3′-
dideoxycytidine-5′-hydrogen phosphonate (3) or to 5′-O-(4-
monomethoxy-trityl)-N4-palmitoyl-2′-deoxycytidine-3′-
The transformation of therapeutically active mononucle-
otides into amphiphilic prodrugs using lipophilic nucleoside
derivatives can be achieved with a large number of deriva-
Fig. 1. Structure formulas of the amphiphilic heterodinucleoside phosphates.
10: 5-fluoro-2′-deoxyurididylyl-(3′ → 5′)-5-fluoro-N4-hexadecyl-2′-deoxycytidine; 11: 5-fluoro-N4-octadecyl-2′-deoxycytidylyl-(3′ → 5′)-5-fluoro-2′-
deoxyuridine; 11a: 5-fluoro-2′-deoxy-uridylyl-(3′ → 5′)-5-fluoro-N4-octadecyl-2′-deoxycytidine and the monomers I: 5-fluoro-N4-octadecyl-2′-deoxycytidine-
5′-monophosphate; II: 5-fluoro-N4-hexadecyl-2′-deoxycytidine.

496
P.S. Ludwig et al. / European Journal of Medicinal Chemistry 40 (2005) 494–504
Fig. 2
.
Synthesis of heterodinucleoside phosphates (5, 6, 7) starting from 3′-O-acetyl-5-fluoro-2′-deoxyuridine (2) and N4-palmitoyl-2′,3′-dideoxycytidine-3′-
hydrogen phosphonate (3) or 5′-O-(4-monomethoxytrityl)-N4-palmitoyl-2′-deoxycytidine-3′-hydrogen phosphonate (4).
The reaction steps are: a = 1. pivaloyl chloride in pyridine, 2. iodine (0.22 M) in THF/pyridine/water (16:1:1). b = p-toluenesulfonic acid (2%) in CHCL3/MeOH.
c = column chromatography on silica gel. d = methanol saturated with ammonia. e = column chromatography on RP18
.
hydrogen phosphonate (4) using the hydrogen phosphonate
method. The internucleoside linkage of the resulting two
dimers was oxidized with iodine.After chromatographic puri-
fication on silica gel the pure amphiphilic heterodinucleoside
phosphate N4-palmitoyl-2′,3′-dideoxycytidylyl-(5′ → 5′)-3′-
O-acetyl-5-fluoro-2′-deoxyuridine (ddCpam-(5′ → 5′)-5-
FdU(Ac), 5) was obtained in 68% yield. The reaction mix-
ture obtained by condensation of 4 with 2 was concentrated
to a syrup and treated with acid to remove the 4-mono-
methoxytrityl protecting group followed by purification on
silica gel. The partially deprotected dimer dCpam-5-FdU(Ac),
6 was obtained in 70% yield. By careful treatment with ammo-
nia the 3′-acetyl group of 6 was completely cleaved whereas
the equally alkali-labile N4-palmitoyl residue was only par-
tially lost. After partial deprotection the reaction mixture was
purified using reversed phase chromatography and the pure
amphiphilic heterodinucleoside phosphate N4-palmitoyl-2′-
deoxycytidylyl-(3′ → 5′)-5-fluoro-2′-deoxyuridine (7) re-
sulted in 48% yield.
As illustrated in Fig. 3, instead of a second nucleoside
derivative the free 5′-hydroxyl group of 2 was coupled to the
lipophilic glyceryl derivative 1-O-octadecyl-2-O-acetyl-rac-
glycerol-3-hydrogen phosphonate (8, phosphonate com-
pound) in analogy to the synthesis described in Fig. 2. The
reaction mixture was chromatographed using silica gel and
the acetyl protecting groups of the fully protected liponucle-
otide were removed by ammonia treatment. The resulting
amphiphilic liponucleotide 1-O-octadecyl-rac-glycerylyl-(3
→ 5′)-5-fluoro-2′-deoxyuridine (Oct1Gro-(3 → 5′)-5-FdU, 9)
was isolated by crystallization in a yield of 82%. The course
of the synthesis and the purification steps were monitored by
thin-layer chromatography (TLC). The chemical structure and
the analytical purity of the products were confirmed by NMR
spectroscopy and high resolution mass spectroscopy
(< 5 ppm).
3. Anticancer activity evaluation
The anticancer activities of the dimers ddCpam-(5′ → 5′)-
5-FdU(Ac) (5), dCpam-5-FdU(Ac) (6), Oct1Gro-(3 → 5′)-5-
FdU (9), 5-FdU-5-FdChex (10), 5-FdCoct-5-FdU (11) and the
monomeric 5-fluoro-2′-deoxycytidine derivatives p5-FdCoct
(I), 5-FdChex (II) were tested in the framework of the in vitro
Anticancer Screen Program of the National Cancer Institute
(USA) on a panel of 60 human cancer cell lines [17]. The
anticancer activities (cf. Tables 1 and 2) were obtained from
the screening data report including the data sheet, dose–
response curves and the mean graphs. Mean graphs facilitate
visual scanning of data for the selection of potential com-
pounds for particular cell lines or for particular tumor sub-
panels with respect to a selected response parameter. The
response parameter GI50 (log10 of molar sample concentra-
tion resulting in 50% growth inhibition) given in the mean

P.S. Ludwig et al. / European Journal of Medicinal Chemistry 40 (2005) 494–504
497
Fig. 3. Synthesis of the amphiphilic liponucleotide (9) containing 5-FdUMP as monomer unit starting from 3′-O-acetyl-5-fluoro-2′-deoxyuridine (2) and
1-O-octadecyl-2-O-acetyl-rac-glycerol-3-hydrogen phosphonate (8).
The reaction steps are: a = 1. pivaloyl chloride in pyridine, 2. iodine (0.2 M) in THF/pyridine/water (16:1:1); b, column chromatography on silica gel. c = methanol
saturated with ammonia.
graphs are listed in Table 1. The average of the GI50-values
for all 60 cell lines is indicated by the mean graphs midpoint.
step syntheses that are very complicated for the preparation
of an enantiomerically pure form. The hydrolytic stability of
the liponucleotides can be varied by different derivatization
of the hydroxyl groups of the glycerol backbone. Alkylation
with long carbon chains produces alkali-stable ether lipids of
high hydrolytic stability, whereas acylation with fatty acid
derivatives yields alkali-labile esters that are easily cleavable
by hydrolysis. The synthesis of ether liponucleotides is of
particular advantage when prodrugs with slow-relase proper-
ties and high stability are desired.
4. Results and discussion
Amphiphilic drugs are soluble in aqueous systems as well
as in organic solvents and should be able to penetrate cell
membranes by passive transport mechanisms. The am-
phiphilic nature and the corresponding intrinsic membrane
permeability of a drug can be estimated with the octanol/water
partition coefficient (PC). Based on previous studies with the
amphiphilic [3H]-labeled heterodinucleoside phosphate
N4-palmitoyl-2′-deoxycytidylyl-(3′ → 5′)-3′-azido-2′,3′-
dideoxythymidine that has PC value of 0.62 [13] and pen-
etrates through the membranes of H9 cells [11] we estimate
that passive membrane diffusion of the new 5-FdUMP pro-
drugs should also occur because they have higher PC-values
ranging from 1.38 to 8.15.
Through coupling of 5-FdUMP to a lipid the liponucle-
otide Oct1Gro-(3 → 5′)-5-FdU (9) is obtained which has the
highest PC value of 8.15 of all 5-FdUMP prodrugs. The
dimers ddCpam-(5′ → 5′)-5-FdU(Ac) (5) and dCpam-5-
FdU(Ac) (6) have similar PC values of 3.20 and 3.78 but they
are significantly less lipophilic than liponucleotide 9. The
additional polar 5′-hydroxyl group of 6 which is absent in 5
does not seem to influence the partition properties of 6. The
dimer N4-palmitoyl-2′-deoxycytidylyl-(3′ → 5′)-5-fluoro-2′-
deoxyuridine (7) is obtained through cleavage of the 3′-acetyl
group of 6. Compared to 6 the deprotected 7 is significantly
more hydrophilic which is reflected in the low PC value of
1.38.
Cytosine nucleosides were chosen as phosphonate com-
ponents for the synthesis of the N4-palmitoylated heterodi-
nucleoside phosphates. The syntheses of these nucleosides
are simple and the products have long storage stabilities.
Although the use of the expensive 2′,3′-dideoxycytidine as
alternative to the inexpensive 2′-deoxycytidine renders the
synthesis easier requiring less protective groups, however it
allows only 5′-coupling of the 2′,3′-dideoxycytidine deriva-
tives. Introduction of the natural palmitoyl residue at the
N4-position of cytosine contributes not only to the amphiphilic
property of the dimers but it also slows down enzymatic
hydrolysis.A reduced hydrolysis rate may cause a depot effect
in the cell membrane that is considerably less pronounced in
analogous unprotected dimers. Upon enzymatic degradation
in human serum of N4-palmitoylated dinucleoside phos-
phates the palmitoyl residue is preferentially removed before
the hydrolytic cleavage of the phosphodiester bond [13]. This
property might increase the ratio of in vivo cell uptake of
unmetabolized prodrugs.
The dimers ddCpam-(5′ → 5′)-5-FdU(Ac) (5) and dCpam
-
5-FdU(Ac) (6), where N4-palmitoyl-2′,3′-dideoxycytidine or
N4-palmitoyl-2′-deoxycytidine are linked to 5-FdUMP are
new alternative compounds to 5-FdU-5-FdChex (10) and
5-FdCoct-5-FdU (11) which were synthesized before (unpub-
lished data). Instead of the non-toxic N4-palmitoyl cytidine
moieties compounds 10 and 11 contain the cytotoxic N4-
The liponucleotide is significantly more lipophilic than the
heterodinucleoside phosphates. However, this advantage is
counteracted by a relatively complicated synthesis. The lipid
derivatives necessary for the coupling reaction require multi-

498
P.S. Ludwig et al. / European Journal of Medicinal Chemistry 40 (2005) 494–504
Table 1
In vitro anticancer activities of 5FdU, 5-fluoro-2-deoxycytidine derivatives (I, II) and 5-FdUMP-prodrugs (amphiphilic heterodinucleoside phosphates 5, 6, 10,
11; liponucleotide 9) which were screened on a panel of 60 human cancer cell lines (panel/cell line) and expressed by the GI50 (log10 of sample concentration
(molar) resulting in 50% growth inhibition; concentration below 10–7 M are shown in bold face)
Growth inhibition GI50 (M)a
5-FdU
I
II
5c
6c
10
11
9c
Panel/cell line
Leukemias
CCRF-CEM
HL-60
b
–8.2
–6.7
–6.1
–7.4
–6.1
–7.9
–4.42
–
–6.03
–4.95
–5.71
–5.07
–6.23
–6.37
–5.45
–6.20
–5.08
–5.00
–5.42
<–8.00
–6.69
–5.97
–5.72
–7.09
<–8.00
–4.89
–5.29
–4.57
–4.83
–4.95
–5.31
–6.52
–5.13
–5.00
–5.27
–5.66
–6.32
b
–
–5.26
–5.33
K-562
>–4.00
–4.38
b
MOLT-4
–
RPMI–8226
SR
>–4.00
–4.80
–5.40
b
b
–
–
Non-small cell lung cancers
A549/ATCC
EKVX
–7.9
–5.0
–7.6
–6.1
–5.0
–6.3
–6.3
–8.7
–5.6
–4.95
>–4.00
–4.09
–5.59
–4.49
–4.31
>–4.00
–6.03
–4.84
–5.12
–4.99
–5.89
–5.31
–4.89
–4.97
–5.28
–6.01
–4.51
–5.49
–5.69
–4.49
–5.11
–4.52
–6.41
–6.60
–4.43
–6.45
–6.31
–4.66
–5.36
–4.76
–7.18
–5.04
–7.64
–4.65
<–8.00
–5.37
–5.52
–6.33
–7.94
<–8.00
–5.16
–5.14
>–4.00
–4.82
–5.27
–4.01
–4.41
–4.27
–6.36
–4.18
–7.34
–5.95
>–4.00
–5.50
–6.76
–7.38
–4.90
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322 M
NCI-H460
NCI-H522
Colon cancers
COLO205
HCC-2998
HCT-116
HCT-15
b
b
–
–
b
>–4.00
–
–4.52
–5.8
–9.0
–6.9
–5.7
–5.6
–5.2
–5.0
>–4.00
–4.82
–4.90
–7.15
–5.08
–6.06
–5.01
–4.86
–4.89
–4.95
–5.13
–5.21
–4.66
–4.88
<–8.00
–5.41
–4.80
–5.34
–4.90
–4.82
–4.97
–8.00
–6.41
–4.87
–6.52
–4.56
–4.74
4.68
–5.14
–7.33
–5.55
–4.94
–4.97
–4.86
–4.92
–5.22
–4.33
–4.43
–4.78
–4.95
–4.73
–4.03
>–4.00
>–4.00
>–4.00
>–4.00
b
HT29
?
KM12
–4.80
–4.61
SW-620
SW-6 CNS CNS cancer
SF-268
–7.9
–7.4
–8.4
–5.7
–6.7
–6.9
–4.26
–5.53
–4.78
–6.55
–4.86
–5.30
–4.93
–6.26
–5.26
–6.43
–5.48
–4.92
–4.96
–6.51
–6.02
–6.86
–4.77
–4.96
–4.84
–8.00
<–8.00
<–8.00
–6.57
–5.80
–5.84
–4.90
–4.87
–5.18
–4.92
–4.43
–4.76
–6.86
–6.37
–7.14
–4.98
–6.07
–5.86
SF-295
>–4.00
–4.96
SF-539
SNB-19
>–4.00
>–4.00
>–4.00
SNB-75
U251
Melanomas
LOXI MVI
MALME-3 M
M14
–7.6
–5.1
–6.8
–5.0
–5.7
–6.7
–5.5
–7.4
–4.18
–4.98
–4.97
–5.07
–4.71
–4.90
–4.94
–4.78
–4.97
–5.18
–4.72
–4.84
–4.64
–4.69
–4.87
–4.80
–4.94
–5.89
–7.37
–5.18
–6.32
–4.37
–4.76
–5.88
–4.88
–6.53
–4.58
–6.13
–4.86
–5.60
–4.60
–4.79
–5.45
–4.95
–6.27
b
b
b
–
–
–
>–4.00
>–4.00
>–4.00
>–4.00
>–4.00
–4.21
–5.60
–4.69
–4.52
–5.15
–4.49
–4.56
–4.71
–4.56
–4.57
–4.46
–4.22
–4.69
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancers
IGROV1
–5.6
–5.6
–5.0
–5.2
–6.9
–5.7
>–4.00
>–4.00
>–4.00
>–4.00
–4.54
–4.94
–4.86
–4.80
–4.74
–5.74
–4.87
–4.69
–4.53
–4.76
–4.31
–5.06
–4.55
–4.71
–4.55
–4.50
–4.76
–5.41
–5.35
–5.52
–5.08
–4.03
–4.35
–6.82
–4.94
–4.33
–4.54
>–4.00
–4.27
–4.76
–4.50
–4.96
–4.82
–4.85
–4.79
–5.53
–4.89
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Renal cancers
786-0
>–4.00
–7.0
–5.9
–7.5
–7.5
–4.15
–5.54
–5.45
–4.31
–5.74
–5.24
–5.51
–4.91
–6.89
–6.19
–6.85
–7.90
–7.84
–6.59
–4.31
–4.89
–4.95
–4.58
–6.09
–6.16
–6.40
–5.67
b
b
A498
–
–
ACHN
–5.20
–5.07
–6.59
–5.68
CAKL-1
(continued on next page)

P.S. Ludwig et al. / European Journal of Medicinal Chemistry 40 (2005) 494–504
499
Table 1
(continued)
Growth inhibition GI50 (M)a
5-FdU
–5.4
–6.7
–5.3
–6.9
I
II
5c
6c
10
11
9c
RXF393
>–4.00
>–4.00
>–4.00
–4.54
–4.87
–4.90
–4.92
–5.85
–4.69
–5.03
–4.45
–5.21
–4.77
–5.38
–4.39
–6.10
–4.88
–6.58
–4.76
–6.54
–4.94
>–4.00
–4.15
–4.87
–4.97
–5.24
–4.99
–6.10
SN12C
TK-10
UO-31
Prostate cancers
PC-3
–6.5
–6.6
–4.67
–5.41
–5.00
–4.74
–5.09
–5.10
–5.71
–5.12
–6.97
–4.65
–4.21
–5.46
–5.88
DU-145
>–4.00
Breast cancers
MCF7
–8.2
–5.9
–5.4
–5.4
–5.5
–5.9
–6.0
–5.9
–6.4
–5.05
–5.88
–5.54
–4.66
–4.76
–4.91
–4.80
–4.79
–4.93
–5.20
–6.30
–5.09
–4.27
–4.46
–4.80
–4.73
–4.87
–4.90
–5.09
>–4.00
–4.94
–4.73
–4.92
–4.76
–4.37
–5.50
–5.06
–5.27
<–8.00
–6.33
–4.55
–4.82
–4.84
–4.86
–4.88
–5.85
–6.05
–5.01
–4.52
>–4.00
–4.45
–4.12
–6.90
–5.44
–4.69
–4.69
–4.62
–4.74
–4.89
–5.11
–5.51
b
NCI /ADR-RES
MDA-MB-231
HS 578T
–
>–4.00
>–4.00
>–4.00
>–4.00
>–4.00
–4.37
MDA-MB-435
MDA-N
b
–
b
BT-549
–
T-47D
–4.51
–4.62
Mean graphs midpoint
–4.23
a Screening data (mean graphs) obtained from the National Cancer Institute Developmental Therapeutics Program, USA.
b not screened.
c octanol/water PC of 5 = 3.20; 6 = 3.78 and 9 = 8.15.
octadecyl(hexadecyl)-5-fluoro-2′-deoxycytidine monomer
units. The synthesis of the required N4-alkylated 5-fluoro-
cytidine nucleosides entails more steps than the acylated com-
pounds rendering their preparation very cumbersome [18].
The major difference between dimers 5 and 6 where
5-FdUMP is linked to an inactive nucleoside and compounds
10 and 11 is that they contain a second cytotoxic nucleoside
moiety. The cytotoxic activity of the N4-alkylated
5-fluorocytidine derivatives which was observed, e.g. with
p5-FdCoct (I) and 5-FdChex (II) (cf. Table 1) is probably
caused by their intrinsic cytotoxic activity or by dealkylation
followed by enzymatic deamination to 5-FdU.
The amphiphilic properties of the 5-FdUMP prodrugs are
not only based on the concomitant presence of octadecyl- or
palmitoyl residues with hydrophilic hydroxyl groups and a
polar phosphodiester linkage. Rather, the structural align-
ment of these functional groups seems to be responsible for
the amphiphilic nature of the molecules. This is supported by
the different PC-values found for dimer dCpam-5-FdU (7)
(PC = 1.38) and the liponucleotide Oct1Gro-(3 → 5′)-5-FdU
(9) (PC = 8.15). Despite the fact that both prodrugs contain
the same number of functional groups (2 OH groups, a phos-
phodiester link, a long chain hydrocarbon residue and a
5′-linked 5-FdUMP moiety) structural differences must be
responsible for their different amphiphilic properties as
reflected in the significantly different PC-values.
Table 2
Cell lines showing specific sensitivities to 5-FdU, 5-FdUMP prodrugs (5, 6,
9, 10) and 5-fluoro-2′-desoxycytidine derivatives (I, II). Compounds with
GI50 values 10- to 100-fold below average (mean graphs midpoint) of all the
tested 60 cell lines are listed under sensitivity I, GI50 values > 100-fold under
sensitivity II
Panel/cell line
Sensitivity I
compound
Sensitivity II
compound
Leukemias
CCRF-CEM
MOLT-4
RPMI-8226
SR
5-FdU, 9
5-FdU
10
5, 10
5-FdU, 5
10
10
Non-small cell lung cancers
A549/ATCC
HOP-62
5-FdU, 6, 10
5-FdU, 6, 9
6, I
9, 10
5-FdU, 5, 9
HOP-92
NCI-H322M
NCI-H460
Colon cancers
HCC-2998
CNS cancers
SF-268
6, 10
9, II
5-FdU, 6, 10
5-FdU, 5, 6, 9, 10
5-FdU
SF-295
10
SF-539
5, 6, 9, II
5-FdU, 10
Melanoma
LOXI MVI
UACC-62
Renal cancers
A498
After successful synthesis of the 5-FdUMP prodrugs their
antitumor activities were tested in vitro on a panel of 60 human
tumor cell lines. Thereby it was analyzed to what extent the
chemical structure would influence the antitumor activity. As
a measure for the anticancer activity the GI50 values were
used (Table 1). The values show that all tested compounds
exert cytotoxic activities, however with large differences in
activity and tumor type specificity. The mean overall GI50
5-FdU, 10
5-FdU
10
ACHN
5-FdU, 6, II, 10
5-FdU
CAKL-1
Breast cancers
MCF7
5-FdU, 5, 9
10

500
P.S. Ludwig et al. / European Journal of Medicinal Chemistry 40 (2005) 494–504
value of 5-FdU was the lowest one with –6.4, followed by
dimer 5-FdU-5-FdChex (10) with a value of –6.05. The GI50
values of the other prodrugs were –5.51 (Oct1Gro-(3 → 5′)-
5-FdU (9), –5.27 (dCpam-5-FdU(Ac) (6), –5.20 (5-FdChex (II),
–5.09 (ddCpam-(5′ → 5′)-5-FdU(Ac) (5), –4.62 (5-FdCoct-5-
FdU (11), –4.23 (p5-FdCoct (I). Compounds I and 11 are not
suited as antitumor drugs because their activity is approxi-
mately 100-fold lower than that of 5-FdU. The marked dif-
ference of the GI50-values of 10 (–6.05) and 11 (–4.62) is
surprising because of their high structural similarity (cf.
Fig. 1). It is conceivable that the nucleoside sequence of the
dimers is responsible for the low activity detected for 11 caus-
ing a very slow release of the active compounds 5-FdU and
5-FdUMP. This depot effect might have prevented the forma-
tion of the active metabolites in the time course of the cyto-
toxicity test, avoiding the release of enough free 5-FdU to
exert antitumor effects. This example shows that by variation
of the nucleoside sequence of the heterodinucleotide phos-
phates the rate of enzymatic degradation and thus the depot
effect can be influenced. This assumption finds support in an
earlier finding that prodrug 5-FdU-5-FdCoct (11a), the
sequence isomer to 5-FdCoct-5-FdU (11), was significantly
more effective than 5-FdU in an in vitro clonogenic growth
assay using the human pancreatic adenocarcinoma cell line
MIA/PaCa 2 [12]. Furthermore, the investigation of the anti-
tumor activity of 11a and dCpam-5-FdU (7) (Fig. 2) demon-
strated that both compounds have comparably high antican-
cer activities against DU-145 human prostate cancer cells and
are effective prodrugs of 5-FdU. Dimer 11a was capable of
eradicating 100% of cancer cells, whereas 10% of the cells
remained resistant to 5-FdU [14,19].
the prodrugs does not only affect cytotoxic activity but also
tumor cell specificity. Monomer 5-FdChex (GI50 –5.20) had a
comparable mean activity as dimer dCpam-5-FdU(Ac) (GI50
–5.27) but only three cell lines were highly sensitive against
5-FdChex as compared to eight highly sensitive cell lines with
dimer dCpam-5-FdU(Ac). Of all tested prodrugs the com-
pound 5-FdU-5-FdChex (A) has the highest similarity to the
cytotoxic activity of 5-FdU (B) which is reflected in the ratio
of the highly sensitive cell lines to the total number of the cell
lines in the panel: leukemias A: 3/6, B: 3/6; non-small cell
lung cancer A: 4/9; B: 3/9; CNS cancer A: 3/6; B 3/6; renal
cancer A: 2/8, B: 3/8; melanoma A: 1/8, B: 2/8; colon cancer
A: 1/8, B 1/8 and breast cancer A: 1/8, B: 1/8.
The amphiphilic character of the prodrugs facilitates their
cell uptake. Due to the natural phosphodiester bond that func-
tions as potential cleavage site intracellular enzymatic degra-
dation leads to 5-FdUMP. The frequently occurring develop-
ment of drug resistance that is caused by reduced or absent
5′-phosphorylation of 5-FdU can be circumvented by the
described prodrugs. However, since the panel of tumor cells
in which the prodrugs were tested did not include 5-FU or
5-FdU resistant cells it will be important to analyze their
activities on such cells. Moreover, as further advantage the
prodrugs can be formulated in small antibody-tagged lipo-
somes for specific delivery to corresponding targets [20].
Thus, the advantages of the 5-FdUMP prodrugs over 5-FdU
warrant their further development as potential new antican-
cer drugs.
5. Conclusion
For compounds 5, 6, 9 the mean GI50 values (–5.09 to
–5.51) are higher than the value of 5-FdU (–6.4). The reduced
activity can also probably be explained by depot effects caused
by structural differences. On the other hand, the high cyto-
toxic activity of 5-FdU-5-FdChex (GI50 –6.05) that is compa-
rable to that of 5-FdU (GI50 –6.4) can be explained by the
coupling of two cytotoxic nucleosides, where upon enzy-
matic cleavage the two active compounds 5-FdU and 5-FdChex
which have high GI50 values (–6.4 for 5-FdU and –5.2 for
(5-FdChex), are released as single compounds. The depot effect
of 5-FdU-5-FdChex is probably concealed by the fast release
of 5-FdChex. Conversely, it is also possible that the slow
hydrolysis of compound 5-FdU-5-FdChex is prevents that a
higher in vitro activity than that of 5-FdU is obtained.
The therapeutic application of the prodrugs requires not
only high cytotoxic activity but also high specificity which is
reflected in the differing sensitivities against the tumor cell
lines investigated. In Table 2 the cell lines are listed which
had sensitivities more than 10-fold below the mean GI50-
value of all 60 cell lines tested. Thereby, we found that
16/60 cell lines were more sensible towards prodrugs 5, 6, 9
and 10 as the mean of all cells. Of these 16 cell lines 12 were
also highly sensible towards 5-FdU, allowing the conclusion
that the 5-FdUMP-derivatives are prodrugs of 5-FdU. As
shown in the following example, the chemical structure of
The coupling of lipophilic cytosine or phospholipid deriva-
tives with 5-FdU affords amphiphilic antitumor prodrugs that
contain a caged 5-FdUMP moiety. The concept presented here
is not restricted to 5-FdU, it can be adapted in a general man-
ner for the transformation of antimetabolites into amphiphilic
prodrugs. The linkage of 5-FdU with N4-palmitoylated cyti-
dine derivatives instead of the N4-hexadecyl- (or octadecyl-)
5-fluoro-2′-deoxycytidine derivatives simplifies the synthe-
sis but also reduces the activity of the resulting prodrugs.
Therefore, it is important to carefully evaluate properties and
nucleoside sequences of the individual compounds to obtain
prodrugs of high activity. The structure–activity parameters
analyzed in in vivo experiments will finally determine which
prodrug will have the most favorable properties in regard of
low unwanted toxicity, bioavailability and metabolism. Such
tests will finally determine the suitability of the prodrugs for
further development.
6. Experimental protocols
6.1. Chemistry
Commercially available were: 5-fluoro-2′-deoxyuridine
(Pharma-Waldhof, Düsseldorf, Germany), pivaloyl chloride,

P.S. Ludwig et al. / European Journal of Medicinal Chemistry 40 (2005) 494–504
501
pyridine and silica gel 60 (Merck, Darmstadt, Germany). Pre-
pared as described were: salicylchlorophosphite [21],
4-monomethoxytrityl chloride [22], 1-O-octadecyl-2-O-
acetyl-rac-glycerol [23], N4-palmitoyl-2′,3′-dideoxycytidine-
5′-hydrogen phosphonate [13], 5′-O-(4-monomethoxytrityl)-
N4-palmitoyl-2′-deoxycytidine [7]. Prepared after a published
method [16] with major modifications were: 5′-O-(4-
monomethoxytrityl)-5-fluoro-2′-deoxyuridine and 3′-O-
acetyl-5-fluoro-2′-deoxyuridine. Pyridine was refluxed over
potassium hydroxide, distilled and stored over molecular
sieves (4 Å). Diethylether was dried over potassium hydrox-
ide, distilled and stored over molecular sieves (4 Å).
mined and the PC-value was calculated as the quotient:
[A260(I)–A260(II)]/A260(II).
6.1.1. General condensation procedure using the hydrogen
phosphonate method
All reactions were performed at room temperature if not
stated differently. The concentration of the reaction mix-
tures, solutions, organic layers and eluted fractions was done
in vacuum at a bath temperature of 45 °C. Equimolar amounts
of a hydroxyl- and a hydrogen phosphonate compound (cf.
Table 3) were dissolved in dry pyridine. This solution was
cooled to 0 °C before the fivefold amount of pivaloylchloride
was added under the exclusion of moisture. After stirring for
3 min (7 min in case of glyceryl phosphonates) the reaction
mixture was cooled to 0 °C before the reaction was stopped
by the addition of water. The obtained phosphonic diester was
oxidized immediately by addition of iodine in THF (0.2 M).
After 1 h stirring at room temperature excess iodine was
removed by addition of solid sodium hydrogen sulfite before
the reaction mixture was concentrated to a syrup that was
dissolved in chloroform and extracted twice with water. The
organic layer was concentrated to a syrup which was co-
evaporated three times with some toluene yielding the crude
condensation product which was purified and isolated as
described below.
All reactions were monitored by TLC on pre-coated silica
gel 60 F254 plates (0.25 mm, Merck) using UV light for visu-
alization and spray reagents as developing agents. Sugar moi-
eties were developed by spraying the plates with perchloric
acid (60%) followed by heating. Compounds protected by
the 4-monomethoxytrityl group were detected as yellow spots
when acid was used as spraying reagent. Compounds con-
taining hydrogen phosphonate or phosphodiester groups were
detected as blue spots on the TLC-plates when a molybdic
acid spraying reagent was used for the detection. Long alkyl
chains were detected as orange fluorescent spots under UV
(366 nm) when ethanolic 2,7-dichlorofluorescein was used
as spray reagent. Multi-step flash chromatography was car-
ried out on dry packed silica gel 60 (0.040–0.063 mm) col-
umns using binary solvent mixtures prepared by volume ratios
(v/v) as eluent. NMR spectra were recorded with BrukerAMX
250 (250 MHz) and AMX400 (400 MHz) instruments and
calibrated using DMSO-d6 or CDCl3 as solvent and internal
standards. 2D-Cosy and Dept135-spectra were recorded on a
Bruker AMX 400 instrument and used when necessary to
determine the shifts of protons and carbons. FD mass spectra
of intermediate products were recorded on a Finnigan MAT
711A HRMS (ESI, negative mode) mass spectra of the deriva-
tives were recorded on a BrukerAPEX II. Melting points (not
corrected) were determined in a Stuart Scientific SMP3 cap-
illary melting point apparatus. PC in 1-octanol/water were
determined using UV-absorbance. The UV-absorbance
(A260(I)) of an aqueous solution of the test compound was
determined. Equal volumes of this solution and 1-octanol were
mixed and vigorously shaken at room temperature for 15 min,
followed by centrifugation (10,000 rpm, 20 min). The
UV-absorbance (A260(II)) of the aqueous layer was deter-
6.1.2. 5′-O-(4-Monomethoxytrityl)-5-fluoro-2′-deoxyuridine
(1)
To a solution of 5-fluoro-2′-deoxyuridine (10 g, 41 mmol)
in dry pyridine (50 ml) 4-monomethoxytrityl chloride (14 g,
46 mmol) was added under the exclusion of moisture. The
reaction vessel was sealed air tight and shaken for 5 h The
reaction mixture was cooled to 0 °C and the obtained precipi-
tate collected by filtration, washed with dry ether (20 ml) and
discarded. To the filtrates methanol (20 ml) was added, fol-
lowed by 10 min shaking before concentration to a syrup that
was co-evaporated with toluene (50 ml). This syrup was dis-
solved in chloroform (150 ml) and extracted three times with
a saturated aqueous solution of sodium hydrogen carbonate
(50 ml). The organic layer was concentrated to a syrup that
was co-evaporated twice with toluene (50 ml) before being
dissolved in ether (200 ml) and chromatographed on a silica
gel column (16 × 9 cm) using ether (6 l) as eluent. The desired
fractions were pooled, concentrated and dried, affording 5′-(4-
Table 3
Experimental data for the synthesis of the liponucleotide (9) and the heterodinucleoside phosphates (5, 6) using the hydrogen phosphonate method
Condensation reaction
Obtained crude condensation products
9
5
6
Hydroxyl compound number (g/mmol)
+ Phosphonate compound number (g/mmol)
Pyridine (ml)
2, 3.5/12.2
+ 8, 5.5/12.2
90
2, 1.5/5.2
+ 3, 2.7/5.2
40
2, 1.5/5.2
+ 4, 4.0/5.2
40
Pivaloyl chloride (ml/mmol)
Water (ml)
7.5/61
4
3.5/28.4
2
3.5/28.4
2
Iodine in THF (ml)
55
24
24
Chloroform (ml)
200
50
100
25
100
25
Water (ml)

502
P.S. Ludwig et al. / European Journal of Medicinal Chemistry 40 (2005) 494–504
monomethoxytrityl)-5-fluoro-2′-deoxyuridine (1) as a color-
less foam (19.8 g, 94%) that could be precipitated from
n-pentane under sonification yielding a white powder. M.p.
90–93 °C (foam), 95–97 °C (solid). TLC (ether) Rf = 0.15.
MS (FD) m/z: 541.0 (M + Na+), 518.1 (M + H+).
1H-NMR (DMSO-d6): d 7.90 (d, J = 6.82, 1H, H6), 6.88–
7.44 (m, 14H, aromatic H of trityl), 6.16 (t, J = 5.79, 1H, H1′),
5.37 (bs, 1H, H3′OH), 4.30 (m, 1H, H3′), 3.90 (m, 1H, H4′),
3.74 (s, 3H, OCH3), 3.13–3.32 (m, 2H, H5′, H5′′), 2.08–2.32
(m, 2H, H2′, H2′′).
on a silica gel column (16 × 9 cm) using a three step
chloroform/methanol gradient: Step 1: 95/5 (4 l); step 2: 9/1
(4 l) and step 3: 8/2 (8 l). The fractions containing the desired
product were pooled, concentrated and dried, affording 5′-O-
(4-monomethoxytrityl)-N4-palmitoyl-2′-deoxycytidine-3′-
hydrogen phosphonate (4) as a colorless foam (16.3 g, 75%).
M.p. 159–164 °C. TLC (CHCl3/MeOH 8:2) Rf = 0.34.
1H-NMR (DMSO-d6, 250.134 MHz): d 8.09 (d,
J = 7.46 Hz, 1H, H5), 7.22–7.39 (m, 12H, aromatic-H), 7.09
(d, J = 7.42 Hz, 1H, H6), 6.89 (d, J = 8.89 Hz, 2H, aromatic-
H), 6.59 (d, J = 604.18 Hz, 1H, P-H), 6.11 (t, J = 5.79 Hz,
1H, H1′), 4.73 (m, 1H, H3′), 4.15 (m, 1H, H4′), 3.73 (s, 3H,
OCH3), 3.18–3.37 (m, 2H, H5′ + H5′′), 2.35–2.41 (m, 2H, H2′
+ H2′′), 1.52 (m, 2H, –CH2–), 1.22 (s, 26H, –(CH2)n–), 0.83
(t, J = 6.56 Hz, 3H, –CH3).
13C-NMR (DMSO-d6): d 158.28, 144.21, 144.06, 134.80,
130.06, 127.95, 126.94, 123.88, 113.28, 86.15 (trityl-group),
157.02 (d, J = 25.91 Hz, C4), 148.94 (C2), 140.00 (d,
J = 231.52 Hz, C5), 124.49 (d, J = 33.90 Hz, C6), 85.58 (C4′),
84.57 (C1′), 70.06 (C3′), 64.91, 63.75 (C5′), 55.04 (OCH3),
39.41 (C2′).
13C-NMR (DMSO-d6, 100.624 MHz): d 173.84 (NHCO),
162.33 (C4), 154. 28 (C2), 144.24 (C6), 158.20, 143.87,
143.73, 134.63, 129.94, 127.90, 126.92, 113.23 (aromatic-
C), 86.24 (C(C6H5)3), 86.24 (C1′), 86.06 (C4′), 71.66 (C3′),
62.77 (C5′), 54.94 (OCH3), 36.32 (C2′), 31.27, 29.02, 28.99,
28.86, 28.72, 28.69, 28.43, 24.41, 22.06 ((CH2)n), 13.87
((CH2)nCH3). HRMS calculated for C45H59N3O8P [M – H+]:
800.40453, found: 800.40471.
6.1.3. 3′-O-Acetyl-5-fluoro-2′-deoxyuridine (2)
To a solution of 1 (9.1 g, 17.5 mmol) in dry pyridine (25 ml)
acetic anhydride (5 ml, 53 mmol) was added under the exclu-
sion of moisture. The reaction vessel was sealed and shaken
for 5 h before methanol (6 ml) was added under cooling (0 °C).
After 10 min of shaking the reaction mixture was concen-
trated to a syrup which was co-evaporated tree times with
toluene (20 ml). This syrup was dissolved in acetic acid (80%,
50 ml) and refluxed for 20 min followed by another concen-
tration step and co-evaporation with toluene (3 × 50 ml). The
crude product was dissolved in hot ethyl acetate (80 ml) and
crystallized at –25 °C. The obtained precipitate was collected
by filtration and recrystallized form ethyl acetate (80 ml) at
0 °C yielding 3′-O-acetyl-5-fluoro-2′-deoxyuridine (2) as col-
orless crystals (4.2 g, 83%). M.p. 201 °C. TLC (CHCl3/MeOH
9:1) Rf = 0.44. MS (FD) m/z: 289.1 (M + H+).
6.1.5. N4-Palmitoyl-2′,3′-dideoxycytidylyl-(5′ → 5′)-3′-O-
acetyl-5-fluoro-2′-deoxyuridine (5)
The syrup, obtained by condensation of compound 3 with
compound 2 (Table 3), was crystallized form methanol (50 ml)
at –25 °C. The crude product was dissolved in CHCl3/MeOH
(9:1, 50 ml) and purified by flash chromatography on a silica
gel column (20 × 5 cm) using a three step CHCl3/MeOH gra-
dient: step 1: 9/1 (3 l); step 2: 8/2 (3 l); and step 3: 7/3 (3 l).
The fractions containing the desired product were pooled, con-
centrated and crystallized from methanol yielding N4-
palmitoyl-2′,3′-dideoxycytidylyl-(5′ → 5′)-3′-O-acetyl-5-
fluoro-2′-deoxyuridine (5) as colorless crystals (2.9 g, 68%).
M.p. 195–205 °C. TLC (CHCl3/MeOH, 6:4) Rf = 0.71.
1H-NMR (DMSO-d6, 250.134 MHz): d 11.78 (bs, 1H,
NH-5FdU), 10.80 (bs, 1H, NHCO), 8.34 (d, J = 7.50 Hz, 1H,
H5-ddC), 8.18 (m, 1H, H6-5FdU), 7.20 (d, J = 7.48, H6-ddC),
6.16 (m, 1H, H1′-5FdU), 5.95 (m, 1H, H1′-ddC), 5.18–5.25
(m, 1H, H3′-5FdU), 4.13–4.25 (m, 1H, H4′), 3.85–4.10 (m,
5H, H5′ + H5′′ + H4′), 2.04 (s, 3H, COCH3), 1.80–2.40 (m,
6H, H2′; H2′′ (5FdU; ddC) + H3′; H3′′(ddC)), 1.51 (m, 2H,
CH2–(CH2)13–CH3), 1.23 (s, 26H (CH2)13–CH3) 0.85 (t,
J = 6.81, 3H (CH2)13–CH3).
1H-NMR (DMSO-d6): d 8.20 (d, J = 7.18, 1H, H6), 6.15
(t, J = 7.12, 1H, H1’), 5.30 (bs, 1H, H5′OH), 5.21 (m, 1H, H3′),
4.01 (m, 1H, H4′), 3.64 (m, 2H, H5′,H5′′), 2.25–2.30 (m, 2H,
H2′,H2′′), 2.06 (s, 3H, CH3).
13C-NMR (DMSO-d6): d 170.00 (CO-acetyl), 156.97 (d,
J = 26.35 Hz, C4), 149.03 (C2), 140.10 (d, J = 230.64 Hz,
C5), 124.42 (d, J = 34.59 Hz, C6), 84.94 (C4′), 84.46 (C1′),
74.68 (C3′), 36.89 (C2′), 20.78 (CH3-acetyl).
6.1.4. 5′-O-(4-Monomethoxytrityl)-N4-palmitoyl-2′-deoxy-
cytidine-3′-hydrogen phosphonate (4)
To a solution of 5′-O-(4-monomethoxytrityl)-N4-palmi-
toyl-2′-deoxycytidine (20 g, 27 mmol) in dry pyridine (40 ml)
and dry dioxane (160 ml) salicylchlorophosphite (6.6 g,
32.5 mmol) was added under the exclusion of moisture. The
reaction mixture was shaken for 1 h before a saturated solu-
tion of sodium hydrogen carbonate (50 ml) was added at 0 °C,
followed by additional shaking for 15 min. The reaction mix-
ture was concentrated to a syrup which was dissolved in chlo-
roform (200 ml) and extracted with a saturated aqueous solu-
tion of sodium hydrogen carbonate (3 × 100 ml). The organic
layer was concentrated to a syrup which was dissolved in
chloroform/methanol (95:5, 150 ml) and chromatographed
13C-NMR (DMSO-d6, 250.134 MHz): d 173.70 (NHCO),
169.81 (COCH3), 162.17 (C4-ddC), 156.90 (d, J = 26.71 Hz,
C4-5FdU), 154.43 (C2-ddC), 148.98 (C2-5FdU), 144.72 (C6-
ddC), 140.07 (d, J = 232.70 Hz, C5-5FdU), 124.46 (d,
J = 36.24 Hz, C6-5FdU), 94.95 (C5-ddC), 86.93, 84.44 (C1′),
83.12, 80.64 (C4′), 74.76 (C3′-5FdU), 65.24, 64.65 (C5′),
36.27, 32.47 (C2′), 24.43 (C3′-ddC), 31.21, 28.95, 28.62,
28.40, 27.66, 26.94, 26.02, 25.84, 22.00 (–(CH2)–), 20.68
(COCH3), 13.84 ((CH2)n–CH3).
HRMS calculated for C36H54FN2O12P [M – H+]:
798.34961, found: 798.34987.

P.S. Ludwig et al. / European Journal of Medicinal Chemistry 40 (2005) 494–504
503
6.1.6. N4-Palmitoyl-2′-deoxycytidylyl-(3′ → 5′)-3′-O-
acetyl-5-fluoro-2′-deoxyuridine (6)
1H, H3′), 4.08 (m, 1H, H4′), 3.88 (m, 3H, H4′ + H5′ + H5′′),
3.59 (m, 2H, H5′ + H5′′), 2.36 (t, J = 7.20 Hz, COCH2–), 2.06–
2.16 (m, 4H, H2′ + H2′′), 1.52 (bs, 2H, CH2(CH2)12CH3), 1.23
(s, 24H, CH2(CH2)12CH3), 0.84 (t, J = 6.65 Hz, 3H,
CH2(CH2)12CH3).
The syrup, obtained by condensation of compound 4 with
compound 2 (Table 3), was dissolved in chloroform (35 ml)
and after the addition of a methanolic p-toluene-sulfonic acid
solution (4.0%, 35 ml) the resulting mixture was stirred for
5 min. The reaction mixture was then poured into a half satu-
rated aqueous solution (100 ml) of sodium hydrogen carbon-
ate. The organic layer was separated, concentrated to a syrup
which was dissolved in CHCl3/MeOH (9:1, 100 ml) and puri-
fied on a silica gel column (15 × 5 cm) using a three step
CHCl3/MeOH gradient: Step 1: 9/1 (2 l); step 2: 8/2 (2 l) and
step 3: 7/3 (3 l). The fractions containing the desired product
were pooled, concentrated and dried yielding N4-palmitoyl-
2′-deoxycytidylyl-(3′ → 5′)-3′-O-acetyl-5-fluoro-2′-deoxy-
uridine (6) as a colorless foam (2.9 g, 70%). M.p. 185–
190 °C. TLC (CHCl3/MeOH, 7:3) Rf = 0.33.
13C-NMR (DMSO-d6, 62.896 MHz): d 173.87 (NHCO),
162.35 (C4-dC), 156.94 (d, J = 26.05 Hz, C4-5FdU), 154.49
(C2-dC), 149.05 (C2-5FdU), 144.78 (C6-dC), 140.11 (d,
J = 232.12 Hz, C5-5FdU), 124.66 (d, J = 34.28 Hz, C6-5FdU),
95.46 (C5-dC), 86.81 (C4′), 86.14 (C4′), 84.48 (C1′ + C1′),
74.58 (C3′), 70.67 (C3′), 65.02 (C5′), 61.22 (C5′), 39.08 (C2′),
36.38 (C2′), 33.71, 31.29, 29.02, 28.88, 28.70, 28.49, 24.44,
22.08 (–(CH2)14–), 13.92 ((CH2)14CH3).
HRMS calculated for C34H52FN5O12P [M – H+]:
772.33373, found: 772.33360.
6.1.8. 1-O-Octadecyl-2-O-acetyl-rac-glycerol-3-hydrogen
phosphonate (8)
1H-NMR (DMSO-d6, 400.136 MHz): d 11.89 (bs, 1H,
NH-5FdU), 10.83 (bs, 1H, NHCO), 8.29 (d, J = 7.40 Hz, 1H,
H5-dC), 8.14 (m, 1H, H6-5FdU), 7.21 (d, J = 7.40 Hz, 1H,
H6-dC), 6.07–6.16 (m, 2H, H1’), 5.37 (bs, 1H, 5′OH), 5.21
(m, 1H, H3′), 4.71 (m, 1H, H3′), 4.10 (m, 2H, H4′), 3.93–3.98
(m, 2H, H5′ + H5′′), 3.59 (m, 2H, H5′ + H5′′), 2.33–2.37 (m,
2H, COCH2), 2.05–2.30 (m, 4H, H2′ + H2′′), 2.03 (s, 3H,
COCH3), 1.50 (m, 2H, COCH2CH2), 1.21 (s, 26H (CH2)12),
0.82 (t, J = 6.64 Hz (CH2)n–CH3).
To a solution of 1-O-octadecyl-2-O-acetyl-rac-glycerol
(10 g, 26 mmol) dissolved in dry pyridine (30 ml) and dry
dioxane (120 ml) salicylchlorophosphite (7.9 g, 39 mmol)
was added and the reaction mixture was shaken for 1 h under
the exclusion of moisture. After hydrolysis with water (9 ml)
the reaction mixture was concentrated to a syrup which was
dissolved in chloroform (150 ml) and extracted with a satu-
rated aqueous solution of sodium hydrogen carbonate (2 ×
100 ml). The organic layer was concentrated to a syrup that
was co-evaporated with toluene (3 × 50 ml) before being dis-
solved in hot ethyl acetate (80 ml) and crystallized at –25 °C.
The obtained precipitate was collected by filtration and
re-crystallized from ethyl acetate (80 ml) at 0 °C, yielding
1-O-octadecyl-2-O-acetyl-rac-glycerol-3-hydrogen phospho-
nate (8) as colorless crystals (8.9 g, 76%). M.p. 63 °C. TLC
(CHCl3/MeOH, 8:2) Rf = 0.22.
13C-NMR (DMSO-d6, 100.624 MHz): d 173.87 (NHCO),
169.96 (COCH3), 162.35 (C4-dC), 157.01 (d, J = 26.16 Hz,
C4-5FdU), 154.51 (C2-dC), 149.12 (C2-5FdU), 144.82 (C6-
dC) 140.19 (d, J = 232.24 Hz, C5-5FdU), 124.72 (d,
J = 34.31 Hz, C6-5FdU), 95.47 (C5-dC), 86.89 (C4′), 86.20
(C1′), 84.43 (C1′), 83.18 (C4′), 74.90 (C3′, C3’), 64.66 (C5′),
61.28 (C5′), 36.37 (C2′), 36.20 (C2′), 31.33, 29.09, 29.05,
28.93, 28.80, 28.75, 28.53, 24.46, 23.09, 22.12 ((CH2)n),
20.79 (COCH3), 13.94 ((CH2)nCH3).
1H-NMR (CDCl3, 400.136 MHz): d 6.74 (d, J = 629.41 Hz,
1H, PH), 4.13–4.48 (m, 3H, CH–,CH2-glycerol), 3.41–3.56
(m, 4H, O–CH2– +CH2-glycerol), 2.03 (s, 3H, COCH3), 1.50
(bs, 2H, O–CH2–CH2–), 1.22 (bs, 30H, –(CH2)15–), 0.84 (t,
J = 6.80 Hz, 3H, –(CH2)15–CH3).
HRMS calculated for C36H54FN5O13P [M – H+]:
814.34453, found: 814.34350.
6.1.7. N4-Palmitoyl-2′-deoxycytidylyl-(3′ → 5′)-5-fluoro-
2′-deoxyuridine (7)
13C-NMR (CDCl3, 100.624 MHz): d 171.40 (COCH3),
71.26 (CH2-glycerol), 70.24 (O–CH2–(CH2)16), 68.63 (CH-
glycerol), 64.21 (CH2-glycerol), 31.85, 29.89, 29.71, 29.66,
29.61, 29.48, 29.38, 29.30, 25.96, 22.61, 21.13, 20.83
(–(CH2)16–), 20.77 (–COCH3), 14.02 (–(CH2)16–CH3).
HRMS calculated for C23H46O6P [M – H+]: 449.30375,
found: 449.30567.
To a solution of 6 (1.5 g, 1.8 mmol) in CHCl3 (20 ml)
methanolic ammonia (50 ml) was added and the reaction mix-
ture was kept for 1 h After concentration the crude product
was dissolved in H2O/MeOH (3:7, v/v, 5 ml) and purified by
reversed phase chromatography on a LiChroprep RP-18 (40–
63 nm) column (Merck) (310 × 25 mm) using a binary
NH4OAc (aq., 0.05 N)/MeOH-gradient (2 l) from 70% MeOH
to 100% MeOH. The product containing fractions were
pooled, concentrated and twice lyophilized, yielding N4-
palmitoyl-2′-deoxycytidylyl-(3′ → 5′)-5-fluoro-2′-deoxy-
uridine (7) as colorless powder (0.68 g, 48%). M.p. 180–
185 °C. TLC (CHCl3/MeOH, 7:3) Rf = 0.11.
6.1.9. 1-O-Octadecyl-rac-glycerylyl-(3 → 5′)-5-fluoro-2′-
deoxyuridine (9)
The syrup, obtained by condensation of compound 8 with
compound 2 (Table 3), was dissolved in CHCl3/MeOH (95:5,
50 ml) and purified on a silica gel column (15 × 5 cm) using
a three step chloroform/methanol gradient: Step 1: 9/1 (2 l);
step 2: 8/2 (2 l); step 3: 7/3 (2 l). The fractions containing the
desired product were pooled and concentrated, affording the
fully protected product which was dissolved in chloroform
1H-NMR (DMSO-d6, 250.134 MHz): d 11.82 (bs, 1H,
NH-5FdU), 10.81 (bs, 1H, NHCO), 8.30 (d, J = 7.69 Hz, 1H,
H5-dC), 8.05 (m, 1H, H6-5FdU), 7.23 (d, J = 7.37 Hz, 1H,
H6-dC), 6.09–6.15 (m, 2H, H1′), 4.66 (m, 1H, H3′), 4.27 (m,

504
P.S. Ludwig et al. / European Journal of Medicinal Chemistry 40 (2005) 494–504
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(50 ml). To that solution methanolic ammonia (200 ml) was
added and the resulting solution was kept over night, before
being concentrated to a syrup which was crystallized from
ethyl acetate/ethanol (8:2) at 0 °C yielding 1-O-octadecyl-
rac-glycerylyl-(3 → 5′)-5-fluoro-2′-deoxyuridine (9) as col-
orless crystals (6.5 g, 82%). M.p. 188–193 °C. TLC
(CHCl3/MeOH, 6:4) Rf = 0.28.
1H-NMR (DMSO-d6, 400.136 MHz): d 7.97 (d, J = 6.46,
1H, H6), 6.15 (t, J = 6.34 Hz, 1H, H1′), 4.30 (m, 1H, H3′),
3.78–3.90 (m, 3H, H4′ + CH2-glycerol), 3.61–3.72 (m, 2H,
H5′ + H5′′), 3.25–3.45 (m, 5H, CH-glycerol + CH2-glycerol +
OCH2CH2–), 2.06–2.12 (m, 2H, H2′ + H2′′), 1.41 (m,
2H,OCH2CH2(CH2)15CH3), 1.21 (s, 30H, –(CH2)15), 0.83 (t,
J = 6.46 Hz,3H (CH2)15CH3).
13C-NMR (DMSO-d6, 100.624 MHz): d 157.29 (d;
J=25.79 Hz; C4), 149.33 (C2), 140.04 (d; J = 232.14 Hz; C5),
124.39 (d; J = 34.72 Hz; C6), 86.15 (C4′), 84.53 (C1′), 71.92
(CH2-glycerol), 70.80 (C3′), 70.59 (OCH2CH2(CH2)15CH3),
69.23 (CH-glycerol), 66.48 (C5′), 64.71 (CH2-glycerol), 39.25
(C2′), 31.28, 29.68, 29.22, 29.04, 28.70, 25.63, 22.08
(–(CH2)15–), 13.90 ((CH2)15CH3).
[14] R.M.C. Cattaneo-Pangrazzi, H. Schott, H. Wunderli-Allenspach,
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311–319.
HRMS calculated for C30H53FN2O10P [M – H+]:
651.34273, found: 651.34358.
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622–638.
Acknowledgements
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The authors thank the National Cancer Institute (Bethesda,
MD, USA) for the in vitro testing of the prodrugs.
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