Synthesis and in vitro activities of new anticancer duplex drugs linking 2′-deoxy-5-fluorouridine (5-FdU) with 3′-C-ethynylcytidine (ECyd) via a phosphodiester bonding

Article abstract of DOI:10.1016/j.bmc.2009.08.033

Two isomeric cytostatic duplex drugs 2′-deoxy-5-fluorouridylyl-(3′→5′)-3′-C-ethynylcytidine [5-FdU(3′→5′)ECyd] and 2′-deoxy-5-fluorouridylyl-(5′→5′)-3′-C-ethynylcytidine [5-FdU(5′→5′)ECyd] were designed and synthesized at gram scale according to the hydro

Full text of DOI:10.1016/j.bmc.2009.08.033

Bioorganic & Medicinal Chemistry 17 (2009) 6824–6831
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro activities of new anticancer duplex drugs linking
20-deoxy-5-fluorouridine (5-FdU) with 30-C-ethynylcytidine (ECyd)
via a phosphodiester bonding
b
c
Herbert Schott a, , Sarah Schott , Reto A. Schwendener
*
a Institute of Organic Chemistry, University Tuebingen, Auf der Morgenstelle 18, D-72076 Tuebingen, Germany
b Department of Gynecology and Obstetrics, University of Heidelberg Medical School, D-69120 Heidelberg, Germany
c Institute of Molecular Cancer Research, University of Zuerich, CH-8057 Zuerich, Switzerland
a r t i c l e i n f o
a b s t r a c t
Two isomeric cytostatic duplex drugs 20-deoxy-5-fluorouridylyl-(30?50)-30-C-ethynylcytidine [5-
FdU(30?50)ECyd] and 20-deoxy-5-fluorouridylyl-(50?50)-30-C-ethynylcytidine [5-FdU(50?50)ECyd] were
designed and synthesized at gram scale according to the hydrogenphosphonate method in an overall
yield of about 40%. The in vitro evaluation of the anticancer effects indicated highly varying sensibilities
of the panel of 60 tested tumor cell lines against the duplex drugs. 5-FdU(30?50)ECyd had a 50% growth
inhibition (IC50 6 10ꢀ8 M) in 44/58 cell lines. However, only 25/53 of those cell lines showed correspond-
ing IC50 values when the isomeric 5-FdU(50?50)ECyd was tested. Total growth inhibition was achieved
using micromolar concentrations of the duplex drugs. The 5-FdU residue of the duplex drug can cause
very different effects like additive, synergistic, antagonistic as well as sequence-depending activities,
which drastically changed efficiency as well as specificity of the anticancer activities of the duplex drugs,
in comparison to those of the monomeric drugs.
Article history:
Received 29 May 2009
Revised 15 August 2009
Accepted 18 August 2009
Available online 21 August 2009
Keywords:
20-Deoxy-5-fluorouridine (5-FdU)
30-C-Ethynylcytidine (ECyd)
Antimetabolites
Cytostatic duplex drugs
Hydrogenphosphonate method
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
The chemical coupling of two single compounds to a new duplex
drug must go along with a justifiable synthetic effort. Furthermore,
It is known that antitumor chemotherapy is unsuccessful if
resistance against the administered cytostatic drug occurs. Drug
monotherapy, using only one anticancer compound, often leads
to drug resistance. The combination therapy as an alternative,
which is based on a simultaneous or sequential application of var-
ious anticancer drugs, can optimize the therapeutic success and
may prevent resistance. However, combination therapy schedules
are often complex and exhausting for the patients. Another prom-
ising but not yet well-evaluated possibility of anticancer combina-
tion therapy consists in the chemical linkage of two different,
clinically well-characterized cytostatic drugs into one molecule, a
so called duplex drug. After application of a duplex drug as a
monotherapy the molecule should be degraded into a mixture of
several metabolites each possessing different cytostatic profiles
with additive or synergistic properties. This concept could exploit
the advantage of a combination therapy without additional burden
for patients. Cytostatic drugs are suitable for the design of duplex
drugs provided that they vary considerably in their anticancer
mechanisms in order to cause additive or synergistic antitumor
activities and, optimally, a simultaneous reduction of side effects.
the molecular structure of the created duplex drug should allow an
in vivo metabolism resulting in multiple active compounds. Nucle-
oside analogues1–3 have become a major class of successful anti-
metabolites in cancer therapy over many years and fulfill
important preconditions for the preparation of duplex drugs. Two
different nucleoside analogues can be coupled via a natural phos-
phodiester bonding resulting in heterodinucleoside phosphate
analogues that can easily be cleaved in vivo by phosphodiesterases
into the parent nucleosides.
Heterodinucleoside phosphates linking 20-deoxy-5-fluorouri-
dine (5-FdU) with thioinosine were among the first dimers that
inhibited effectively both 6-mercaptopurine sensitive as well as
resistant cancer cells in vitro.4 However, the potential of such di-
mers as possible duplex drugs has not yet been recognized. The di-
rect coupling of 5-FdU with the lipophilic 20-deoxy-5-fluoro-N4-
octadecylcytidine resulted in an amphiphilic heterodinucleoside
phosphate analogue. In in vitro clonogenic growth assays using
the human pancreatic adenocarcinoma cell line MIAPaCa 2, this
duplex drug was significantly more cytotoxic than 5-FdU.5 The
antitumor potential of the duplex drug evaluated in p53-mutated
and androgen-independent DU-145 human prostate tumor cells
showed a 100% eradication of tumor cells whereas 10% of cells
were resistant to 5-FdU.6 In PC-3 cells the duplex drug exerted
* Corresponding author. Fax: +49 7071 65782.
E-mail address: herbert.schott@uni-tuebingen.de (H. Schott).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.033

H. Schott et al. / Bioorg. Med. Chem. 17 (2009) 6824–6831
6825
stronger cytotoxicity and induced more S-phase arrest and apopto-
sis than 5-FdU.7 The direct linkage of arabinofuranosylcytosine
ECyd and 5-FdU, which is described here, will result in new duplex
drugs with a broad spectrum of antitumor activity based on addi-
tive or synergistic effects of 5-FdU and ECyd metabolites arising
from enzymatic degradation of the duplex drug. As a proof of prin-
cipal to this assumption two isomeric duplex drugs were synthe-
sized by combining 5-FdU and ECyd either via a 30?50or a
50?50phosphodiester linkage to evaluate the influence of the direc-
tion of the phosphodiester on the antitumor activity of the dinucle-
oside phosphate.
(araC) with N4-octadecyl-1-b-
D-arabinofuranosylcytosine, a lipo-
philic derivative of the antitumor drug araC resulted in a potent
duplex drug.8 An amphiphilic duplex drug can also be stably incor-
porated in liposomes. This offers a new opportunity for a targeted
anticancer combination therapy that has been proven in mouse
models.9 The indirect linkage of 5-FdU and araC via a phospholipid
backbone resulted in an amphiphilic duplex drug that might be an
additional option for the treatment of 5-FdU sensitive and resistant
colon cancer, human lymphoma and hematological malignan-
cies.10–12
2. Results and discussion
2.1. Chemistry
A new derivative of cytidine, 30-C-ethynylcytidine (ECyd), which
is meanwhile evaluated in clinical studies,13 is highly active and
suitable for the synthesis of cytostatic duplex drugs. Because the
antitumor mechanism of ECyd differs from that of 5-FdU it can
be expected that the synthesis of dinucleoside phosphates linking
The synthesis of the two isomeric duplex drugs summarized in
Figure 1 was performed according to the phosphonate method.
Partially protected ECyd was used as the 50-hydroxyl compound
OBz
O
F
F
N
HN
N
O
N
O
HO
(MeO)TrO
O
O
d,b
a,b,c,b
92%
OBz
d, b
OH
90%
2
1
82 %
OBz
NHBz
O
N
F
F
N
HN
O
N
O
N
N
O
O
O
HO
O
H
P
(MeO)TrO
O
O
TMS
OH
O
P
H
OH
OTBDMS
OBz
HO
O
5
3
4
42 %
39 %
1. condensation: e, b
2. deprotection: f, g, h
O
O
N
NH2
F
HN
O
F
NH
N
O
P
N
O
NH2
N
HO
N
O
O
O
O
O
N
OH
O
O
P
OH
OH
OH
O
HO
O
O
6
O
OH
OH
7
Figure 1. Synthesis of the heterodinucleoside phosphate 20-deoxy-5-fluorouridylyl-(50?50)-30-C-ethynylcytidine (6) and 20-deoxy-5-fluorouridylyl-(30?50)-30-C-ethynylcyti-
dine (7) according to the phosphonate method starting from 50-O-(4-monomethoxytrityl)-20-deoxy-5-fluorouridine (1) and N4-benzoyl-20-O-(tert-butyldimethylsilyl)-30-
(trimethylsilylethynyl)cytidine (5). Reagents and conditions: (a) Benzoylchloride, pyridine, (b) column chromatography on silica gel, (c) p-toluenesulfonic acid, acetone, (d)
salicylchlorophosphite, pyridine/dioxane, (e) (1) pivaloylchloride, pyridine, (2) iodine, THF/pyridine/water, (f) TBAF in THF, (g) satd NH3/MeOH, (h) column chromatography
on RP18
.

6826
H. Schott et al. / Bioorg. Med. Chem. 17 (2009) 6824–6831
5 whereas two derivatives of 5-FdU were used as 50- or 30-phos-
phonate compounds 3, 4. The rationale for this synthesis concept
is that the necessary 50-hydroxyl compound of the partially pro-
tected ECyd derivative N4-benzoyl-20-O-(tert-butyldimethylsilyl)-
30-C-(trimethylsilylethynyl)cytidine 5 occurs as an intermediate
product of the published six step synthesis of ECyd, which used
cytidine as starting material.14 According to the alternative multi-
step synthesis of ECyd13,15,16 which did not use cytidine as starting
compound a fully protected ECyd was obtained as an intermediate
compound. The selective deprotection of the 50-or 30-hydroxyl
function of these fully protected ECyd intermediates cannot easily
be performed in order to obtain the desired partially protected hy-
droxyl compound for the dinucleoside phosphate synthesis. The
synthesis of the sequence isomer duplex drugs with 5-FdU as the
30 and ECyd as the 50-terminal, which cannot be obtained according
to our proposed synthesis route is expected to be more compli-
cated. The postulated selective derivation of one of three hydroxyl
groups of ECyd to obtain a 30-hydroxyl or 30-phosphonate com-
pound of ECyd is more difficult as in the case of 5-FdU, which
has only two hydroxyl groups.
has a sterical advantage for the condensation reaction in respect
to the 30-hydroxyl group of 5.
After chromatographic purification the different protection
groups of the dimers were removed using the following proce-
dures. At first the acid labile 4-monomethoxytrityl group of the di-
mer formed by condensation of 4 + 5 was cleaved with acetic acid.
Trimethylsilyl- and tert-butyldimethylsilyl protecting groups of
both dimers were removed by treatment with tetrabutylammo-
nium fluoride (TBAF) and the deprotected dinucleoside phosphate
analogues were obtained as tetrabutylammonium salts. The alkali
labile benzoyl residue was finally removed with ammonia. After
the chromatographic purification of the deprotected dinucleoside
phosphate analogues using a preparative reversed phase (RP-18)
column and their lyophilization both duplex drugs 20-deoxy-5-flu-
orouridylyl-(50?50)-30-C-ethynylcytidine (6) and the isomeric 20-
deoxy-5-fluorouridylyl-(30?50)-30-C-ethynylcytidine (7) were ob-
tained at about 40% yield. The low yield may be partially explained
by possible side reactions during the condensation as discussed
above. The quantitative exchange of the tetrabutylammonium cat-
ions cannot be achieved using a cation exchanger (H+ form). The
presence of the tetrabutylammonium cation in both dinucleoside
phosphates was detected by elementary analysis and mass spec-
trometry. The course of the synthesis and purification was con-
trolled by thin-layer chromatography (TLC) on silica gel plates.
The chemical structure and the analytical purity of the products
were confirmed by NMR spectra, elementary analysis and high res-
olution mass spectra.
The 5-FdU-30-phosphonate compound 4 can be obtained from 5-
FdU in two steps, whereas the synthesis of the 5-FdU-50-phospho-
nate 3 needs a three step procedure. The additional steps render
the synthesis of 5-FdU-(50?50)ECyd in respect to the preparation
of the isomeric compound 50-FdU(30?50)ECyd more difficult.
The synthesis of both phosphonate compounds 3, 4 started with
50-O-(4-monomethoxytrityl)-20-deoxy-5-fluorouridine (1) which
was obtained from 5-FdU according to published procedures.17
The phosphonylation of the 30-hydroxyl residue of 1 resulted with
90% yield in the desired 30-phosphonate compound 50-O-(4-mono-
methoxytrityl)-20-deoxy-5-fluorouridine-30-hydrogenphosphonate
(4). The 50-phosphonate compound 3 was also obtained from 1 in
two steps. In the first step protection of hydroxyl groups of 1 with
benzoyl residues followed by removing of the 40-monomethoxytri-
tyl protection from the 50hydroxyl group resulted in 92% yield of
30,4-di-O-benzoyl-20-deoxy-5-fluorouridine (2) which was phos-
phonylated at the 50-hydroxyl group without further purification
at 82% yield resulting in the desired 50-phosphonate compound
30-4-di-O-benzoyl-20-deoxy-5-fluorouridine-50-hydrogenphospho-
nate (3). After coupling of the free 50-hydroxyl group of 5 with the
50-or 30-phosphonate compounds 3 or 4 according to the conditions
given in Table 1 the resulted heterodinucleoside phosphonates
were subsequently oxidized with iodine resulting in the protected
heterodinucleoside phosphates. It cannot be excluded that the
unprotected free 30-hydroxyl group of 5 may be involved in the
condensation reaction. In this case undesired side reactions will oc-
cur which reduces the yield of the desired condensation product.
The prevalent condensation reaction, however, should be the cou-
pling to the 50-hydroxyl group of 5 because this alcohol function
2.2. In vitro anticancer activities
The in vitro antitumor activities of the duplex drugs 5-
FdU(30?50)ECyd, 5-FdU(50?50)ECyd and of the parent monomeric
drugs 5-FdU and ECyd were evaluated in the framework of the
anticancer screen program of the National Cancer Institute (NCI,
USA). The NCI anticancer screen consists of 60 human tumor cell
lines. In those 60 cell lines the drugs are tested at a minimum of
five concentrations at 10-fold dilutions. A 48 h drug exposure pro-
tocol and a sulforhodamine B (SRB) protein assay were used to esti-
mate cell viability or growth.18 Additional details can be found at
http://dtp.nci.nih.gov. The anticancer activities were obtained from
the data screening report including the data sheet, dose response
curves and the mean graphs. Mean graphs facilitated visual scan-
ning of data for the selection of potential compounds for particular
cell lines or for particular tumor subpanels with respect to a sus-
pected response parameter. The response parameter GI50 (log10
of molar sample concentration resulting in 50% growth inhibition)
of 5-FdU and ECyd given in the mean graphs are listed in Table 2.
The average of the GI50 values for all 60 cell lines is indicated by the
mean graphs midpoint. The comparison of the in vitro tumor cell
growth inhibition activity which is based on the mean graphs mid-
point of ECyd [log10 GI50 (M) = ꢀ7.6] and of 5-FdU [log10 GI50
(M) = ꢀ6.4] shows that ECyd was active against all 60 tested tumor
cell lines at high nanomolar concentrations and that it was in gen-
eral about 10-times more cytostatic than 5-FdU. In many cases
however, the response of ECyd and 5-FdU to the same cell line sig-
nificantly differs as follows. More than a 100-fold difference of the
GI50-values of ECyd and 5-FdU was observed in 3/9 non small cell
lung; 2/8 melanoma; 2/6 ovarian; 3/8 renal; 5/7 colon and 6/8
breast cancer cell lines. These data demonstrated a pronounced
drug specific antitumor activity of ECyd and 5-FdU against several
cancer cell lines, predominantly in colon and breast tumor cell
lines. Corresponding high differences of GI50 values were not ob-
served in the 6 leukemias, 6 CNS and 2 prostate tumor cell lines in-
cluded in the screen.
Table 1
Experimental data for the synthesis according to the hydrogenphosphonate method
yielding the fully protected isomeric heterodinucleoside phosphates of 20 -deoxy-5-
fluorouridylyl-(50?50-)-30-C-ethynylcytidine (6) and of 20deoxy-5-fluorouridylyl-
(30?50)-30-C-ethynylcytidine (7)
Experimental data for the condensation
Obtained protected crude
heterodinucleoside phosphates
6
7
Hydroxyl compound number, (g/mmol)
Phosphonate compound number, (g/mmol) +3, 11.0/21.2
Pyridine (mL)
5, 9.5/17
5, 18.4/33
+4, 19.2/33
100
80
Pivaloylchloride (mL/mmol)
H2O (mL)
13/106
10
24/195
20
Iodine in THF (mL) + H2O (mL)
CHCl3/MeOH (9:1) (mL)
H2O (mL)
80 + 10
200
150
160 + 20
300
250
The response parameter GI50 was also used to compare the anti-
tumor activities of ECyd and both isomeric duplex drugs. Half of

H. Schott et al. / Bioorg. Med. Chem. 17 (2009) 6824–6831
6827
Table 2
In vitro anticancer activities resulting in 50% growth inhibition (log10 GI50) of the monomeric drugs 5-FdU and ECyd and the 100% growth inhibition (log10 TGI) of the duplex
drugs 5-FdU(30?50)ECyd, 5-FdU(50?50)ECyd and ECyd which were screened twofold on 60 human cancer cell lines (panel/cell line) and expressed by sample concentration (M)
Panel/Cell line
Log10 GI50 (M)
Log10 TGI (M)
5-FdU
ECyd
ECyd
5-FdU(30?50)ECyd
5-FdU(50?50)ECyd
Leukemia
CCRF-CEM
HL-60
ꢀ8.2
ꢀ6.7
ꢀ6.1
ꢀ7.4
ꢀ6.1
ꢀ7.9
<ꢀ8.0
<ꢀ8.0
<ꢀ8.0
<ꢀ8.0
<ꢀ8.0
<ꢀ8.0
ꢀ5.90
ꢀ5.65
ꢀ5.98
>ꢀ4.00
ꢀ5.76
A
>ꢀ4.00
ꢀ7.91
ꢀ7.78
>ꢀ4.00
>ꢀ4.00
>ꢀ4.00
ꢀ4.99
K-562
MOLT-4
RPMI-8226
SR
ꢀ4.82
a
ꢀ6.89
a
ꢀ6.85
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
ꢀ7.9
ꢀ5.0
ꢀ7.6
ꢀ6.1
ꢀ5.0
ꢀ6.3
ꢀ6.3
ꢀ8.7
ꢀ5.6
<ꢀ8.0
ꢀ7.2
ꢀ4.00
>ꢀ4.53
ꢀ5.20
>ꢀ4.00
ꢀ5.66
ꢀ4.89
ꢀ4.84
ꢀ4.94
ꢀ4.99
ꢀ5.94
>ꢀ4.00
ꢀ5.05
ꢀ6.77
ꢀ4.67
>ꢀ4.00
ꢀ6.92
A
ꢀ5.40
a
<ꢀ8.0
ꢀ7.2
ꢀ5.36
a
ꢀ7.8
ꢀ7.5
ꢀ6.59
ꢀ5.94
ꢀ7.00
ꢀ7.45
ꢀ7.7
<ꢀ8.0
<ꢀ8.0
ꢀ5.34
>ꢀ4.00
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
ꢀ5.8
ꢀ9.0
ꢀ6.9
ꢀ5.7
ꢀ5.6
ꢀ5.2
ꢀ5.0
<ꢀ8.0
<ꢀ8.0
<ꢀ8.0
<ꢀ8.0
<ꢀ8.0
<ꢀ8.0
<ꢀ8.0
ꢀ6.71
ꢀ7.40
ꢀ4.94
ꢀ6.06
ꢀ6.96
>ꢀ4.00
ꢀ4.82
>ꢀ4.00
ꢀ5.08
>ꢀ4.00
ꢀ6.02
A
>ꢀ4.00
>ꢀ4.00
>ꢀ4.00
ꢀ5.25
>ꢀ4.00
>ꢀ4.00
a
ꢀ6.06
ꢀ5.89
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
ꢀ7.9
ꢀ7.4
ꢀ8.4
ꢀ5.7
ꢀ6.7
ꢀ6.9
ꢀ7.8
<ꢀ8.0
ꢀ7.4
ꢀ7.2
ꢀ7.8
-7.7
ꢀ5.89
ꢀ6.24
ꢀ5.17
ꢀ5.11
ꢀ5.97
ꢀ6.78
>ꢀ4.00
ꢀ6.05
>ꢀ 4.00
ꢀ5.54
ꢀ6.06
ꢀ6.86
>ꢀ4.00
A
ꢀ5.24
a
ꢀ5.78
>ꢀ4.00
Melanomas
LOXI MVI
MALME-3 M
M14
SK-MEL-2
SK-MEL-28
SK-MRL-5
UACC-257
UACC-62
ꢀ7.6
ꢀ5.1
ꢀ6.8
ꢀ5.0
ꢀ5.7
ꢀ6.7
ꢀ5.5
ꢀ7.4
<ꢀ8.0
ꢀ7.8
ꢀ7.4
ꢀ7.9
ꢀ7.3
ꢀ8.0
ꢀ7.1
ꢀ7.5
ꢀ7.42
ꢀ6.28
ꢀ5.57
ꢀ6.60
ꢀ5.76
ꢀ6.46
ꢀ5.27
ꢀ6.51
ꢀ6.46
ꢀ5.89
ꢀ6.54
ꢀ6.37
ꢀ5.85
ꢀ6.80
ꢀ5.66
ꢀ5.89
A
ꢀ6.49
ꢀ6.14
ꢀ5.24
ꢀ6.42
ꢀ7.39
ꢀ5.27
ꢀ6.53
Ovarian cancers
IGROV1
ꢀ5.6
ꢀ5.6
ꢀ5.0
ꢀ5.2
ꢀ6.9
ꢀ5.7
ꢀ7.9
ꢀ7.1
ꢀ6.8
ꢀ7.3
<ꢀ8.0
ꢀ7.1
ꢀ4.84
ꢀ5.37
ꢀ4.89
ꢀ5.54
ꢀ5.70
ꢀ4.95
ꢀ5.54
>ꢀ4.00
>ꢀ4.00
ꢀ6.13
>ꢀ4.00
ꢀ4.46
ꢀ5.68
>ꢀ4.00
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
ꢀ5.81
a
>ꢀ4.00
>ꢀ4.00
Renal cancers
786-0
A489
a
ꢀ7.0
ꢀ5.9
ꢀ7.5
ꢀ7.5
ꢀ5.4
ꢀ6.7
ꢀ5.3
ꢀ6.9
<ꢀ8.0
<ꢀ8.0
<ꢀ8.0
<ꢀ8.0
ꢀ7.6
>ꢀ4.00
ꢀ6.12
ꢀ5.82
ꢀ7.10
ꢀ5.83
>ꢀ4.00
>ꢀ4.00
ꢀ7.08
>ꢀ4.00
A
ꢀ7.03
ꢀ6.58
ꢀ7.53
ACHN
CAKI-1
RXF 393
SN12C
TK-10
ꢀ5.68
ꢀ6.36
a
<ꢀ8.0
ꢀ7.8
A
ꢀ6.12
ꢀ6.40
ꢀ4.74
A
a
UO-31
<ꢀ8.0
>ꢀ4.00
Prostate cancers
PC-3
DU-145
ꢀ6.5
ꢀ6.6
ꢀ7.5
ꢀ6.51
ꢀ6.02
ꢀ4.62
ꢀ6.36
ꢀ4.32
<ꢀ8.0
>ꢀ4.00
(continued on next page)

6828
H. Schott et al. / Bioorg. Med. Chem. 17 (2009) 6824–6831
Table 2 (continued)
Panel/Cell line
Log10 GI50 (M)
Log10 TGI (M)
5-FdU
ECyd
ECyd
5-FdU(30?50)ECyd
5-FdU(50?50)ECyd
Breast cancers
MCF7
NCI/ADR-RES
MDA-MB-231
HS 578T
MDA-MB-435
MDA-N
BT-549
ꢀ8.2
ꢀ5.9
ꢀ5.4
ꢀ5.4
ꢀ5.5
ꢀ5.9
ꢀ6.0
ꢀ5.9
ꢀ6.4
<ꢀ8.0
<ꢀ8.0
ꢀ7.4
>ꢀ4.00
>ꢀ4.00
>ꢀ4.00
ꢀ4.80
ꢀ6.86
ꢀ5.53
ꢀ4.42
ꢀ4.90
ꢀ6.60
—
ꢀ6.64
ꢀ4.75
ꢀ5.28
>ꢀ4.00
ꢀ5.01
ꢀ6.56
ꢀ5.82
ꢀ6.74
—
ꢀ7.9
<ꢀ8.0
ꢀ7.9
ꢀ6.77
ꢀ6.76
ꢀ7.4
ꢀ5.23
ꢀ6.26
a
T-47D
Mean graphs midpoint
<ꢀ8.0
ꢀ7.6
A
ꢀ5.56
ꢀ5.39
Results of one representative screen are shown. The lowest GI50 value of ECyd and 5-FdU being more than 100-fold different in the same cell lines and the lowest TGI values of
ECyd and of the two duplex drugs being more than 10-fold different in the same cell lines are shown by bold face.
a
TGI values are not listed when the screening resulted in more than 100-fold different values.
the tested tumor cell lines had GI50 values of 610ꢀ8 M after ECyd
or 5-FdU(50?50)ECyd. The double screened TGI values of 18 tests of
the 60 tested cell lines could not be evaluated because the values
obtained differed more than 100-fold.
treatment. Correspondingly, 44/58 cell lines produced low GI50 val-
ues with 5-FdU(30?50)ECyd. However, GI50 values of 610ꢀ8
M
were observed only in 25/53 cell lines when the same cell lines
were treated with isomeric 5-FdU(50?50)ECyd. On the basis of
these results it could be speculated that the antitumor activities
of 5-FdU(30?50)ECyd linking 5-FdU with ECyd via the natural
30?5 phosphodiester bonding is generally higher in respect to
those of the 50?50 isomer. This assumption is in agreement with
the published results that 30?50linked arabinofuranosylcytidine
dimers were found to be more active than 50?50 linked dimers.19
For the evaluation of the antitumor activities of the duplex drugs
the cell growth inhibition was compared only with that of ECyd be-
cause ECyd was 10-fold more active than 5-FdU. For this compar-
ison the too low and similar GI50 values of ECyd and the duplex
drugs were less suitable. Therefore, instead of the GI50 the response
parameter TGI (log10 of molar sample concentration resulting in
100% growth inhibition) listed in Table 2 was used to demonstrate
the different sensibility of the same tumor cell lines against ECyd
and the duplex drugs.
The mean graphs midpoint of ECyd [log10 TGI (M) = ꢀ5.56
corresponds to the values of 5-FdU-(30?50)ECyd [log10 TGI (M) =
ꢀ5.28 and of 5-FdU(50?50)ECyd [log10 TGI (M) = ꢀ5.39. In respect
to the similar TGI mean graphs midpoint of the three compounds
it could be assumed that the cytostatic potential of ECyd and both
duplex drugs would be similar. However, the TGI values of the
drugs obtained after the parallel treatment of the same tumor cell
line clearly demonstrated that the main part of the 60 tested cell
lines showed significantly different sensibilities against ECyd, 5-
FdU(30?50)ECyd and 5-FdU(50?50)ECyd because their responses
against single cell lines varied in a broad range.
The following 14 cell lines showed TGI values of ECyd (see Table
2), which are more than 10-fold lower than those of one or both
duplex drugs: Melanomas (SK-Mel-2); ovarian (OVAR4); colon
(SW-620); CNS (SNB-19, U251); renal (ACHN, TK-10, UO-31); non
small lung (NCI-H23, NCI-H322M, NCI-H460, NCI-H522) and the
prostate cancer cell lines (PC-3, DU-145). On the basis of these
TGI values it can be concluded that the equimolar coupling of 50-
FdU with ECyd results in an antagonistic effect, thus reducing the
cytostatic activity of the duplex drug, in respect to that of ECyd.
In contrast to these results the following 9 cell lines were more
than 10-times more sensitive to one or both duplex drugs in com-
parison to ECyd: HL-60, Molt-4 (leukemia); SF-539 (CNS); OVCAR-
8 (ovarian); MCF7, NCI/ADR-RES, MDA-MB-231, HS 578T; BT-549
(breast). In these examples the linked 5-FdU acts additively or syn-
ergistically when coupled with ECyd. Of the tested cell lines 27
showed only little to unchanged growth inhibition in comparison
to that of ECyd independently of treatment with 5-FdU(30?50)ECyd
Besides the change of the cytostatic activities of ECyd, which
was observed after coupling with 5-FdU it was surprising that
the direction of the phosphodiester linkage sequence in which 5-
FdU and ECyd were coupled has a significant influence on the anti-
tumor activity and specificity of the synthesized duplex drugs.
However, 25 of the tested 60 cell lines demonstrated a marked
structure–activity relationship against the duplex drugs. The se-
quence dependent antitumor activity can be observed from the dif-
ference of the TGI values resulting after parallel treatment of the
same cell line with 5-FdU(30?50)ECyd and 5-FdU(50?50)ECyd.
One half of these cell lines were more sensitive against 5-
FdU(30?50)ECyd whereas the other half showed higher growth
inhibition with the isomeric 5-FdU(50?50)ECyd duplex. The follow-
ing 7 cell lines showed a significant high sequence activity rela-
tionship expressed by an about 100-fold difference of the TGI
values obtained with the same cell line after parallel treatment
with both duplex drugs: Molt-4, SR (leukemia); HOP-62, NCI-H23
(non small cell lung cancer), DU-145 (prostate); MCF7; MDA-MB-
231(breast).
The results demonstrate that the chemical coupling of 5-FdU
and ECyd caused, depending on the cell lines resulted either in in-
creased, decreased or unchanged antitumor activities of the corre-
sponding duplex drug compared to monomeric ECyd. Activity and
specificity of the antitumor effects of the duplex drugs depend on
the cytostatic potential of the coupled monomeric drugs as well as
on the structure of the obtained dimer, for example the direction of
the phosphodiester bonding in a dinucleoside phosphate. Thus, the
described transformation of cytostatic antimetabolites to dinucle-
oside phosphate analogues represents a valuable alternative to
the intensive search for new anticancer drugs.
2.2.1. Possible reasons of the antitumor activities of the duplex
drugs
The principal mechanism for the cytostatic effect of 5-FdU is the
inhibition of thymidylate synthase (TS).20 The inhibition of TS en-
zyme activity leads to depletion of deoxythymidine triphosphate
which is necessary for DNA synthesis. ECyd can contribute to the
antitumor activity through its action mechanisms which are differ-
ent from those of 5-FdU.21–25
By coupling ECyd and 5-FdU the resulting duplex drug should
be a potent inhibitor of tumor cell growth by simultaneous inhibi-
tion of both DNA and RNA synthesis. It can be supposed that the
intact duplex drugs do not act as active compounds. However,
the several cytostatic metabolites, which can be formed by
enzymatic degradation of the duplex drugs should initiate the

H. Schott et al. / Bioorg. Med. Chem. 17 (2009) 6824–6831
6829
antitumor effects. Because the phosphodiester cleavage can either
be initiated at the 30- as well as the 50-terminal, a mixture of differ-
ent active metabolites can be expected. The cleavage of 5-FdU
(30?50)ECyd produces an equimolar mixture of ECyd-50-mono-
phosphate and 5-FdU if the phosphodiesterase cleaves at the
30-terminal. Cleavage at the 50-terminal results in a mixture of 5-
FdU-30-monophosphate and ECyd. The degradation of the isomeric
5-FdU(50?50)ECyd results in a 1:1 molar mixture of 5-FdU-50-
monophosphate and ECyd if cleavage occurs at the 5-FdU residue.
An equimolar mixture of ECyd-50-monophosphate and 5-FdU will
be formed if cleavage occurs at the ECyd residue of the duplex
drugs. However, certain steps of the metabolic pathway can be fa-
vored, if for example the recognition and cleavage of the ECyd will
be hindered by the 30-C-ethynyl group of the carbohydrate residue,
whereas the unmodified carbohydrate residue of 5-FdU is more
accessible to the enzyme.
The antitumor activity of ECyd depends essentially on its phos-
phorylation. The first phosphorylation step from ECyd to ECyd-50-
monophosphate is unnecessary if ECyd-50-monophosphate is pro-
duced by metabolic action. In this example the nucleotide sequence
of the duplex drug which favors the metabolic pathway resulting in
ECyd-50-monophosphate instead of ECyd can result in increased
antitumor activity of the duplex. Under this aspect the other se-
quence isomeric dinucleoside phosphate ECyd(30?50)5-FdU with
ECyd as the 50- and 5-FdU as the 30-terminal coupling partner is less
favored to be metabolized by phosphodiesterase to ECyd-50-mono-
phosphate in comparison to 5-FdU(30?50)ECyd. This duplex drug
was therefore not synthesized.
pared as described. All solvents were of technical grade and used
without further purification unless stated otherwise. Dioxane
was dried with sodium, distilled and stored over 5 Å molecular
sieve and pyridine was refluxed over KOH, distilled and stored over
4 Å molecular sieve. The TBAF cleaving solution was obtained by
dissolving tetrabutylammonium fluoride trihydrate (157.8 g) in
THF (500 mL). For the oxidation reaction a solution of iodine
(25 g) in THF (500 mL) was used. All reactions were monitored
by TLC on precoated Silica Gel 60 F254 plates (0.25 mm, Merck)
using UV light for visualization and spray reagents as developing
agents.17 Multi step flash chromatography was carried out on self
packed Silica Gel 60 (0.040–0.0063 mm, Merck) columns using elu-
ent mixtures prepared by volume ratios. All reactions were per-
formed at room temperature, if not stated differently. The
concentration of the reaction mixtures, solutions, organic layers
and eluted fractions was done in vacuum at a bath temperature
of 40 °C. 1H- and 13C NMR spectra were obtained on a Bruker AC
250 spectrometer at 250 MHz and 62.9 MHz, respectively or on a
Bruker Avance 400 spectrometer at 400 MHz and 100 MHz, respec-
tively. DMSO-d6 was used as solvents. Me4Si was used as an inter-
nal standard. 31P NMR spectra were obtained on a Bruker Avance
400 spectrometer at 161 MHz, using H3PO4 as an external stan-
dard. Mass spectra were measured on a Finnigan TSQ 70 or a
MAT 95 instrument. For FAB mass-spectra, all compounds were
measured in a NBA-or glycerine-matrix. HRMS were measured on
a Bruker Apex II FT-ICR instrument.
3.2. General chemistry methods
In addition to phosphodiesterase cleavage phosphohydrolases
can convert the phosphorylated metabolites to the corresponding
nucleosides. The main part of the duplex drug will be metabolized
before reaching the cytoplasm because the negatively charged
phosphodiester renders the dinucleoside phosphate too hydro-
philic to penetrate the lipid rich cell membrane easily.26 The extra-
cellular release of active metabolites may cause a depot effect
which does not occur by application of a mixture of 5-FdU and
ECyd. The suspected extracellular metabolism could be a disadvan-
tage if duplex drugs are synthesized as hydrophilic heterodinucle-
oside phosphate analogues.
In case that the metabolism of both isomeric duplex drugs
would produce an identical mixture of active metabolites the ob-
served structure–activity relationship would be difficult to explain.
Thus, it can be speculated that the sequence dependent metabolic
pathways which produce in a time dependent manner different
amounts and species of cytotoxic metabolites can modulate their
antitumor activity and tumor cell specificity.
The results of the in vitro cytotoxicity test screens provide a pre-
liminary orientation of expected in vivo antitumor activities. How-
ever, a wide variety of biochemical and physiological processes
can drastically influence thepatterns of the antitumoreffects in vivo.
For example, it has been reported that a duplex drug tested on mur-
ine leukemia cells with the highest cytotoxicity in vitro exerted the
lowest therapeutic effect in vivo.12 Nevertheless, the excellent
in vitro antitumor activities of the described new duplex drugs jus-
tify further evaluation in in vivo tumor models.
3.2.1. Condensation procedure using the
hydrogenphosphonate method
The corresponding hydroxyl- and hydrogenphosphonate com-
pounds were dissolved together in dry pyridine and rigorously
dried by repetitive evaporation and addition of pyridine before
the condensation reaction was started by addition of pivaloyl chlo-
ride. After drying and addition of the required amount of pyridine
(see Table. 1) the solution was cooled to 0 °C before pivaloyl chlo-
ride was added under exclusion of moisture. After stirring for
5 min the reaction mixture was cooled again to 0 °C before the
reaction was stopped by addition of water. The obtained phos-
phonic diester was immediately oxidized by addition of iodine in
THF and H2O. After 1 h stirring excess iodine was reduced by addi-
tion of solid sodium hydrogensulfite before the reaction mixture
was concentrated to a syrup that was dissolved in CHCl3/MeOH
and extracted with water. The organic layer was concentrated to
a syrup, which was co-evaporated three times with toluene yield-
ing the crude protected heterodinucleoside phosphates, which
were purified and de-protected as described below.
3.3. Synthesis of 30, 4-Di-O-benzoyl-20-deoxy-5-fluorouridine-
50-hydrogenphosphonate (3)
To a solution of 50-O-(4-monomethoxytrityl)-20-deoxy-5-fluoro-
uridine (1) (26 g, 50 mmol) in dry pyridine (150 ml) benzoylchlo-
ride (56 g, 400 mmol) was added under cooling. The reaction
vessel was sealed airtight and shaken for 8 h at room temperature
before saturated (satd) aq Na2CO3 (130 mL) was added under cool-
ing. The resulting reaction mixture was concentrated to a syrup
which was diluted with CHCl3 (300 mL) and then extracted with
satd aq Na2CO3 (130 mL). The CHCl3 layer was concentrated to a
syrup which was dissolved in a mixture of CHCl3/petroleum ether
(1:1, 200 mL) followed by chromatography on a silica gel column
using a CHCl3/petroleum ether gradient with increasing percentage
of CHCl3. The fractions containing the desired product were con-
centrated to a foam (46 g) which was dissolved in acetone
(100 mL) containing p-toluenesulfonic acid monohydrate (16 g)
3. Experimental
3.1. General chemistry
3.1.1. Reagents
Pivaloyl chloride, benzoyl chloride, tetrabutylammonium fluo-
ride trihydrate, p-toluenesulfonic acid monohydrate were obtained
commercially. Salicylchlorophosphite27 50-O-(4-monomethoxytri-
tyl)-20-deoxy-5-fluorouridine (1),17 N4-benzoyl-20-O-(tert-butyldi-
methylsilyl)-30-(trimethylsilylethynyl) cytidine (5)14 were pre-

6830
H. Schott et al. / Bioorg. Med. Chem. 17 (2009) 6824–6831
and stirred for 20 min at room temperature, before satd aq Na2CO3
(50 mL) was added. The resulting solution was concentrated to a
syrup which was dissolved in CHCl3 (500 mL) and extracted with
H2O (100 mL). The organic layer was separated and chromato-
graphed on a silica gel column using a two step gradient with step
1; CHCl3/petroleum ether with increasing percentage of CHCl3 and
step 2; ether. The fractions containing the desired product were
concentrated, affording crude 30,4-di-O-benzoyl-20-deoxy-5-flu-
orouridine (2) as a colorless foam (21 g, 46 mmol) at 92% yield.
In the following step 2 was dissolved without further purification
in dry pyridine (90 mL) and diluted with dry dioxane (180 mL).
To this solution dioxane (75 mL) was added in which salicyl-
chlorophosphite (13 g, 64 mmol) was dissolved. After stirring of
the reaction mixture at room temperature for 2 h satd aq NaHCO3
(12 mL) was added and the solution was evaporated to a syrup
which was dissolved in CHCl3 (500 mL) and extracted with a mix-
ture of H2O/satd aq NaCl/MeOH (1:1:2) (3 ꢁ 100 mL). The sepa-
rated CHCl3-phase was concentrated and the resulting syrup
dissolved in CHCl3 (150 mL). By slow addition of the obtained solu-
tion to vigorously stirred ether (1.5 L) the desired 3 precipitated.
The isolated and dried precipitate was then extracted during 70 h
with ether. After drying of the remaining precipitate, 3 (20 g,
39 mmol) was obtained at 82% yield as a white powder. TLC
(CHCl3/MeOH 70:30) Rf = 0.46; MS (FABꢀ) m/z: 517.1 [MꢀH]ꢀ; 1H
chromatographed on a silica gel column using a CHCl3/MeOH gra-
dient with increasing percentages of MeOH. The fractions contain-
ing the desired protected heterodinucleoside phosphate were
concentrated to a syrup which changed to a fine solid by vigorously
shaking after addition of ether. To the isolated solid, dissolved in THF
(45 mL) a solution (22 mL) of TBAF in THF was added. The sealed
reaction mixture was stirred for three days whereby the trimethyl-
silyl- and tert-butyldimethylsilyl protecting groups were cleaved.
After the concentration of the reaction mixture 33% aq ammonia
(80 mL) was added to the resulting syrup and the sealed solution
was stirred for five days to cleave the benzoyl protecting groups.
When the reaction mixture was concentrated to about 250 mL, a fine
solid precipitate was removed by centrifugation and the superna-
tant liquid concentrated and lyophilized. The resulting lyophilisate
of crude 6 was dissolved in H2O (60 mL) and chromatographed on
a preparative RP-18 column using a H2O/MeOH gradient with
increasing percentage of methanol. The desired 6 eluted at 15–40%
CH3OH. The product containing fractions were pooled, the pH value
adjusted to 5.8 by addition of a cation exchanger resin (H+), that was
removed before the solution was concentrated and lyophilized
yielding 6 as a white powder (5.8 g, 41.8%).
HRMS calcd for C20H22FN5O12P [MꢀH]ꢀ: 574.09921, found:
574.09907; 1H NMR (DMSO-d6, 250 MHz): d = 11.,8 (br, s, 1H,
NH–5FdU), 8.10 (d, J = 6.64 Hz, 1H, H6–5FdU), 7.85 (d, J = 7.25 Hz,
1H, H6–ECyd), 6.14 (t, J = 6.42 Hz, 1H, H1–5FdU), 5.85 (d, J =
6.19 Hz), 1H, H1–ECyd) 5.81 (d, J = 7.07 Hz, 1H, H5–ECyd), 5.64–
0
NMR, 400 MHz, DMSO-d6: d = 2.44–2.75 (m, 2H, H2 ), 3.48–3.89
0
(m, 5H, PH, H2O (DMSO-d6)), 4.0–4.425 (m, 2H, H5 ), 4.34–4.47
0
0
0
0
0
0
(m, 1H, H4 ), 5.47–5.64 (m, 1H, H3 ), 6.22–6.4 (m, 1H, H1 ), 7.35–
5.75 (m, 2H, H2 –OH, H3 –OH–ECyd), 4.29 (m, 1H, H3 –5FdU), 4.08
8.27 (m, 10H, Haromat.), 8.51 (s, 1H, H6), 12.0 (s, br, 1H, P-OH); 13C
NMR, 100 MHz, DMSO-d6: d = 36.7 (C2 ), 62.8 (C5 ), 75.5 (C3 ), 83.6
(C1 ), 85.4 (C4 ), 128.6–147.9 (Caromat.), 155.7 (C6), 156.0 (C5),
165.1 (C@O), 168.0 (C@O); 31P NMR, 161 MHz, DMSO-d6:
d = 1.23 ppm(–O–PH(O)–OH). Anal. Calcd for C23H20FN2O9PꢂNaꢂ1/
2 H2O: C, 50.28; H, 3.67; N, 5.10. Found: C, 50.30; H, 4.21; N, 5.35.
(m, 1H, H2 –ECyd), 3.83–3.97 (m, 6H, H4 –,H5 –,H5 –ECyd/5FdU),
3.46 (s, 1H, „CH–ECyd) 3.16 (m, 8H, NCH2–TBA), 2.10 (m, 2H,
H20, H200-5FdU) 1.56 (m, 8H, CH2–C2H5–TBA), 1.30 (m, 8H, CH2–
CH2–TBA), 0.92 (m, 12H, CH3–TBA); 13C NMR (DMSO-d6,
62 MHz): d = 165.3 (C4–ECyd), 158.0 (C4–5FdU), 156.9 (C2–ECyd),
149.1 (C2–5FdU), 142.2 (C6–ECyd), 137.8 (C5–5FdU), 124.6 (C6–
0
0
0
00
0
0
0
0
0
0
0
0
5FdU), 94.4 (C5–ECyd), 87.3 (C1 –ECyd), 86.3 (C4 –ECyd), 84.6 (C4 -
3.4. Synthesis of 50-O-(4-monomethoxytrityl)-20-deoxy-5-
5FdU), 82.9 („CH–ECyd), 78.7 (C2 –ECyd), 77.3 (C„–ECyd), 72.7
0
fluorouridine-30-hydrogenphosphonate (4)
(C3 –ECyd), 71.1 (C3 –5FdU), 64.6 (C5 –ECyd, C5 –5FdU), 57.6 (C1–
TBA), 23.1 (C2–TBA), 19.2 (C3–TBA), 13.5 (C4–TBA). Anal. Calcd for
C20H22FN5O12PꢂC16H36N: C, 52.93; H, 7.16; N, 10.29. Found: C,
52.58; H, 6.86; N, 9.56; 31P NMR, 161 MHz DMSO-d6: d = ꢀ1.5 ppm.
0
0
0
0
50-O-(4-Monomethoxytrityl)-20-deoxy-5-fluorouridine (1) (20 g,
39 mmol) was dissolved in dry pyridine (50 mL) and the resulting
solution diluted with dry dioxane (90 mL) followed by addition of
salicylchlorophosphite (11 g, 54 mmol). After stirring the reaction
mixture at room temperature for 1.5 h the formed precipitate
was removed by filtration and washed with cold ether. To the com-
bined filtrate and wash liquid satd aq Na2CO3 (50 mL) was added.
The obtained mixture was concentrated to a foam which was then
dissolved in a mixture of CHCl3/MeOH (95:5) and chromato-
graphed on a silica gel column using a CHCl3/MeOH elution gradi-
ent with increasing percentage of MeOH. After the evaporation of
the product containing fractions compound 4 was obtained as a
foam (20 g, 35 mmol) at 90% yield. TLC (CHCl3/MeOH 80:20)
Rf = 0.17; MS (FABꢀ) m/z: 581.2 [MꢀH]ꢀ; 603.2 [M+Na]; 1H NMR,
3.6. Synthesis of 20-deoxy-5-fluorouridylyl-(30?50)-30–C-
ethynylcytidine (7)
The syrup, obtained after condensation of 5 with 4 according to
the experimental data of Table 1 was dissolved in CHCl3 (300 mL)
and chromatographed on a silica gel column using mixtures of
CHCl3/MeOH with increasing percentages of MeOH as the eluent.
Fully protected 7 was eluted first, followed by fractions containing
7 without the monomethoxytrityl protection group. Fractions con-
taining fully protected as well as partially deprotected 7 were pooled
and concentrated to a foam which was dissolved in MeOH (60 mL).
Then 80% aq acetic acid (60 mL) was added, the reaction mixture
stirred for 24 h and then concentrated to a syrup. The syrup changed
to a fine solid after adding ether and vigorous shaking. The solid was
isolated by centrifugation, dissolved in CHCl3 (250 mL) and re-chro-
matographed on a silica gel column as described above. After the
second chromatographic purification the resulting solid of the par-
tially protected 7 was dissolved in THF (170 mL), followed by addi-
tion of TBAF (8 mL). The sealed reaction mixture was stirred for
three days and concentratedto a syrup. The obtained syrup was trea-
ted another five days with 33% aq ammonia (300 mL) and chromato-
graphed on a RP-18 column as described above for the purification of
6 affording pure 7 (10.6 g, 39.3%).
0
400 MHz, DMSO-d6: d = 2.20–2.65 (m, 2H, H2 ), 3.18–3.43 (m, 2H,
0
H5 ), 3.45–3.68 (m, 3H, NH, PH, POH), 3.73 (s, 4H, OCH3, H2O
0
0
0
(DMSO-d6)), 4.21 (m, 1H, H4 ), 4.91 (m, 1H, H3 ), 6.23 (m, 1H, H1 ),
6.79–7.48 (m, 14H, Haromat.), 7.79 (d, 1H, J = 6.2 Hz, H6); 13C NMR,
0
0
100 MHz, DMSO-d6: d = 23.4 (C2 ), 60.0 (OCH3), 61.6, (C5 ), 68.4
0
0
0
(C3 ), 89.9 (C4 ), 91.8 (C1 ), 128.9 (C6), 132.0.148.9 (Caromat. + C5),
154.2 (C@O), 163.5 (C@O); 31P NMR, 161 MHz, DMSO-d6:
d = 7.3 ppm, (–O–PH(O)–OH). Anal. Calcd for C29H28FN2O8Pꢂ2Naꢂ
H2O: C, 53.96; H, 4.53; N, 4.34. Found: C, 53.55; H, 4.44; N, 4.17.
3.5. Synthesis of 20-deoxy-5-fluorouridylyl-(50?50)-30-C-
ethynylcytidine (6)
MS (FABꢀ) m/z: 574.0 [MꢀH]ꢀ; 815.3 [M+C16H36N]ꢀ; 1H NMR
(DMSO-d6, 250 MHz):d = 11.82 (br, s, 1H, NH–5FdU), 8.20 (d,
J = 7.1 Hz, 1H, H6–5FdU), 7.92 (d, J = 7.5 Hz, 1H, H6–ECyd), 7.83
The syrup obtained after the condensation of 5 with 3 according
to the experimental data of Table 1 was dissolved in CHCl3 and

H. Schott et al. / Bioorg. Med. Chem. 17 (2009) 6824–6831
6831
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Oncol. 2000, 126, 311.
9. Marty, C.; Odermatt, B.; Schott, H.; Neri, D.; Ballmer-Hofer, K.; Klemenz, R.;
Schwendener, R. A. Br. J. Cancer 2002, 87, 106.
10. Saiko, P.; Horvath, Z.; Bauer, W.; Hoechtl, T.; Grusch, M.; Krupitza, G.; Rauko, P.;
Mader, R. M.; Jaeger, W.; Schott, H.; Novotny, L.; Fritzer-Szekeres, M.; Szekeres,
T. Int. J. Oncol. 2004, 25, 357.
11. Saiko, P.; Horvath, Z.; Illmer, C.; Madlener, S.; Bauer, W.; Hoechtl, T.; Erlach, N.;
Grusch, M.; Krupitza, G.; Mader, R. M.; Jaeger, W.; Schott, H.; Agarwal, R. P.;
Fritzer-Szekeres, M.; Szekeres, T. Leukocyte Res. 2005, 29, 785.
12. Rauko, P.; Novotny, L.; Mego, M.; Saiko, P.; Schott, H.; Szekeres, T. Neoplasma
2007, 54, 68.
0
(d, J = 7.5 Hz, 1H, H5–ECyd), 6.09–6.14 (m, 2H, 2ꢁH1 –ECyd/5FdU)
0
5.69–5.85 (m, 6H, OH/NH–ECyd/5FdU), 4.65 (m, 1H, H3 –5FdU),
3.87–4.01 (m, 4H, H5 –ECyd/H3 –5FdU), 3.59 (m, 2H, H5 –5FdU),
3.48 (s, 1H, C„C–H), 3.16 (m, 8H, N–CH2–TBA), 2.50 (m, 1H, H2 –
0
0
0
0
0
ECyd), 2.28–2.1 (m, 2H, H2 –5FdU), 1.53 (m, 8H, CH2–TBA), 1.30
(m, 8H, CH2–TBA), 0.92 (t, J = 7.4, 12H, CH3–TBA); 13C NMR
(DMSO-d6, 62 MHz): d = 165.0 (C4–ECyd), 158.0 (C4-5FdU), 157.29
(C2–ECyd), 149.0 (C2–5FdU), 142.2 (C6–ECyd), 137.8 (C5–5FdU),
0
0
124.6 (C6-5FdU), 94.4 (C5–ECyd), 87.5 (C1 –ECyd), 86.5 (C4 –ECyd),
84.5 (C4 -5FdU), 82.9 („CH–ECyd), 78.7 (C2 –ECyd), 77.4 (C„–
0
0
13. Hattori, H.; Tanaka, M.; Fukushima, M.; Sasaki, T.; Matsuda, A. J. Med. Chem.
1996, 39, 5005.
14. Ludwig, P. S.; Schwendener, R. A.; Schott, H. Synthesis 2002, 16, 2387.
15. Nomura, M.; Sato, T.; Washinosu, M.; Tanaka, M.; Asao, T.; Shuto, S.; Matsuda,
A. Tetrahedron 2002, 58, 1279.
0
0
0
ECyd), 74.5 (C3 –5FdU), 72.6 (C3 –ECyd), 65.2 (C5 –ECyd), 61.5
0
(C5 –5FdU) 57.6 (C1–TBA), 23.1 (C2–TBA), 19.2 (C3–TBA), 13.5 (C4–
TBA); 31P NMR, 161 MHz, DMSO-d6: d = ꢀ1.85 ppm. Anal. Calcd
for C20H22FN5O12PꢂC16H36N: C, 52.93; H, 7.16; N, 10.29. Found: C,
52.62; H, 7.00; N, 9.60.
16. Hrdlicka, P. J.; Jepsen, J. S.; Nielsen, C.; Wengel, J. Bioorg. Med. Chem. 2005, 13,
1249.
17. Ludwig, P. S.; Schwendener, R. A.; Schott, H. Eur. J. Med. Chem. 2005, 40, 494.
18. Grever, R. M.; Schepartz, S. A.; Chabner, B. A. Sem. Oncol. 1992, 19, 622.
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20. Sommer, H.; Santi, D. V. Biochem. Biophys. Res. Commun. 1974, 57, 689.
21. Takatori, S.; Kanda, H.; Takenaka, K.; Wataya, Y.; Matsuda, A.; Fukushima, M.;
Shimamoto, Y.; Tanaka, M.; Sasaki, T. Cancer Chemother. Pharmacol. 1999, 44,
97.
Acknowledgments
For the performed NMR analysis we thank Dr. Ludwig. The
authors thank the National Cancer Institute (Bethesda, USA) for
the in vitro anticancer testing of ECyd and both duplex drugs.
22. Azuma, A.; Matsuda, A.; Sasaki, T.; Fukushima, M. Nucleosides Nucleotides
Nucleic Acids 2001, 20, 609.
23. Murata, D.; Endo, Y.; Obata, T.; Sakamoto, K.; Syouji, Y.; Kadohira, M.; Matsuda,
A.; Sasaki, T. Drug Metab. Dispos. 2004, 32, 1178.
References and notes
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