Synthesis and antitumor activity of a heterodinucleotide of BVDU and gemcitabine

Article abstract of DOI:10.1080/15257770802088787

A heterodinucleotide comprising BVDU and Gemcitabine bound together by a 5′,5′-pyrophospate bridge (BVDUp2dFdC) has been synthesized and evaluated as antitumor agent against AH13 rat sarcoma cells. BVDUp2dFdC showed a cytotoxicity similar to that of Gemcitabine. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770802088787

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Synthesis and Antitumor Activity of
a Heterodinucleotide of BVDU and
Gemcitabine
a
a
a
a
L. Cappellacci , P. Franchetti , P. Vita , R. Petrelli & M.
a
Grifantini
a
Department of Chemical Sciences , University of Camerino ,
Camerino, Italy
Published online: 19 Jun 2008.
To cite this article: L. Cappellacci , P. Franchetti , P. Vita , R. Petrelli & M. Grifantini (2008) Synthesis
and Antitumor Activity of a Heterodinucleotide of BVDU and Gemcitabine, Nucleosides, Nucleotides
and Nucleic Acids, 27:5, 460-468
To link to this article: http://dx.doi.org/10.1080/15257770802088787
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Nucleosides, Nucleotides and Nucleic Acids, 27:460–468, 2008
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770802088787
SYNTHESIS AND ANTITUMOR ACTIVITY OF A
HETERODINUCLEOTIDE OF BVDU AND GEMCITABINE
L. Cappellacci, P. Franchetti, P. Vita, R. Petrelli, and M. Grifantini
Department of Chemical Sciences, University of Camerino, Camerino, Italy
ꢁ
ꢁ
2
A heterodinucleotide comprising BVDU and Gemcitabine bound together by a 5 ,5 -pyrophospate
bridge (BVDUp
sarcoma cells. BVDUp
2
dFdC) has been synthesized and evaluated as antitumor agent against AH13 rat
dFdC showed a cytotoxicity similar to that of Gemcitabine.
2
Keywords BVDU; gemcitabine; dinucleotides; antitumor activity
INTRODUCTION
Nucleoside analogues (NA) constitute an important class of antimetabo-
lites used in the treatment of several types of tumor diseases. These antitu-
mor agents, as mimics of physiological nucleosides, are incorporated after
phosphorylation into newly synthesized DNA resulting in DNA synthesis
inhibition and chain termination. The mechanism of action of some of
these drugs also includes the inhibition of key enzymes involved in the
purine and pyrimidine nucleotide biosynthetic pathways and RNA synthesis,
[
1]
and apoptotic activity through caspase cascade activation. However, the
antitumor efficacy of these nucleosides could be weakened by the devel-
opment of resistant tumor cells. The resistance to NA could arise from an
insufficient intracellular concentration of NA di- or triphosphates, which
[
2]
may result from reduced levels of phosphorylating enzymes. Chemore-
sistance could be overcome by the combination of two or more drugs that
interact with different targets in order to interfere with multiple alterated
[
3]
pathways.
Received 19 December 2007; accepted 27 February 2008.
This research was supported by grant from the Italian Ministry of Research (PRIN 2006). We would
like to acknowledge RESprotect GmbH, Germany for a gift of BVDU and Gemcitabine.
Address correspondance to Mario Grifantini, Department of Chemical Sciences, University of
Camerino 62032, Camerino, Italy. E-mail: mario.grifantini@unicam.it
4
60

A Heterodinucleotide of BVDU and Gemcitabine
461
Various hetero- and homodinucleotides containing nucleosides en-
dowed with antiviral or antitumor activity have been synthesized in the
[
4–15]
last years as an alternative strategy of the standard chemotherapy.
These dinucleotides have some advantages compared to the administration
of nucleoside analogues as single drugs: 1) can act as prodrugs for a
slow delivery of monomers in circulation; 2) can overcome the limiting
phosphorylating activities of target tumor cells; 3) can have the advantage
of a combination therapy with the administration of a single drug.
On these bases, we designed a new heterodinucleotide comprising the
chemotherapeutic agents BVDU and Gemcitabine as a single drug.
ꢁ
BVDU [(E)-5-(2-bromovinyl)-2 -deoxyuridine, RP101] is a highly potent
antiviral drug endowed with selective activity against herpes simplex virus
type 1 (HSV-1) and varicella-zoster virus (VZV) infections. Moreover, several
other herpes viruses and varicella viruses are sensitive to BVDU. The
high selectivity of BVDU toward HSV-1 and VZV depends primarily on
ꢁ
a specific phosphorylation of BVDU to its 5 -diphosphate by the virus-
encoded thymidine kinase (TK). After further phosphorylation to the
ꢁ
-triphosphate, the drug interferes with the viral DNA polymerase.[16]
5
The specific phosphorylation by the HSV-1 and VZV-induced TK also
explains the marked cytostatic activity of BVDU against tumor cells that
have been transduced by the viral TK gene. It also has been found that
BVDU cotreatment with standard chemotherapy prevents the acquisition
of chemoresistance.[ Moreover, combination therapy of BVDU with the
antitumor agent Gemcitabine improves the efficacy of chemotherapy in
3]
[
17]
pancreas carcinoma cell lines and pancreatic cancer patients.
An im-
provement of chemotherapy in vitro also was showed by the combina-
[
3]
tion of BVDU with Mitomycine C on AH13 sarcoma cells.
So, BVDU
could be used as a co-treatment with cytostatic drugs to give a broader
range of chemotherapy treatment options. This drug has demonstrated
promising results in a clinical Phase I/II pilot study in pancreatic cancer
[
17]
patients.
ꢁ
ꢁ
ꢁ
Gemcitabine (2 ,2 -difluoro-2 -deoxycytidine, dFdC) is a very promising
new anticancer drug that is most commonly used to treat non-small cell lung
[
18]
cancer, pancreatic, bladder and breast cancer.
Gemcitabine is metabo-
ꢁ
lized intracellularly to its active metabolites 5 -diphosphate (dFdCDP) and
triphosphate (dFdCTP).[ dFdCDP is a potent inhibitor of ribonucleotide
reductase (RR), that is increased in several model of acquired resistance to
Gemcitabine.
18]
On the basis of these findings we prepared the heterodinucleotide
BVDUp dFdC (1) comprising BVDU and Gemcitabine bound together by
2
ꢁ
ꢁ
a 5 ,5 -pyrophospate bridge. In this article, we report on the synthesis and
antitumor evaluation of this dinucleotide. The homodinucleotide of BVDU
(
BVDUp BVDU, (2)) also was investigated.
2

4
62
L. Cappellacci et al.
RESULTS AND DISCUSSION
The synthesis of compound 1 (BVDUp dFdC) was performed as re-
2
ꢁ
ported in Scheme 1 by coupling of the 5-bromovinyl-2 -deoxyuridine-
-monophosphate (BVDU-MP) (3) as imidazolide derivative (4) with
ꢁ
5
ꢁ
ꢁ
ꢁ
ꢁ
the mono n-tributylammonium salt of 2 -deoxy-2 ,2 -difluorocytidine-5 -
monophosphate (6). BVDU-MP, was prepared as ammonium salt following
[
19]
the literature procedure of Yoshikawa et al.
This compound was con-
+
verted to free acid by treatment with Dowex H form and then activated
as imidazolide (4) with an excess of 1,1 -carbonyldiimidazole (CDI) in
dimethylformamide in quantitative yield. 2 -Deoxy-2 ,2 -difluorocytidine-5 -
monophosphate (5) was prepared as reported by Bonjouklian et al.
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
[
20]
with some modifications, and treated with n-tributylamine to give the mono
n-tributylammonium salt 6. The coupling of 4 with 6 was performed in dry
dimethylformamide at room temperature affording the heterodinucleotide
SCHEME 1 Synthesis of BVDUp2dFdC

A Heterodinucleotide of BVDU and Gemcitabine
463
O
Br
HN
O
P
O
N
i
+
-
3
nBu NH
O
O
3
O
OH
HO
7
OH
O
Br
HN
N
O
O
O
Br
ii
N
N
O
4
+ 7
P
P
O
O
O
O
O
O
OH
OH
H
HO
2
: BVDUp BVDU
2
Reagents and conditions: i) tributylamine, MeOH a., 30 min; ii) DMF a., 40 °C, 3 h.
SCHEME 2 Synthesis of BVDUp2BVDU.
1
, which was purified on a silica gel column. In the same reaction, homod-
1
2
ꢁ
ꢁ
inucleotide 2 [P ,P -bis(5-bromovinyl-2 -deoxyuridine-5 -yl)pyrophosphate]
also was obtained (5% as determined by HPLC).
Homodinucleotide 2 (BVDUp BVDU) also was synthesized by reaction
2
of BVDU-MP imidazolide (4) with BVDU-MP n-tributylammonium salt (7)
(
Scheme 2).
Antitumor activity of dinucleotides 1 and 2 was evaluated in in vitro
experiments on AH13 rat sarcoma cells. AH13 cells were subjected to
stepwise increasing doses of Gemcitabine and passaged serially. In parallel,
cells were treated with increasing doses of BVDUp dFdC (equimolar).
2
Enhanced dosage of either cytostatic compound resulted in decreasing
cell numbers as expected (Figure 1). An advantage of BVDUp dFdC over
2
Gemcitabine was not observed in this set of experiments.
AH13 cells were subjected to stepwise increasing doses of Mitomycine
C (MMC) (30, 42, 49, 56 ng/ml) and passaged serially. In parallel, cells
were treated with a combination of Mitomycine C + BVDUp BVDU or
2
Mitomycine C + BVDU (30 µM). As BVDUp BVDU was toxic at 30 µM,
2
and still toxic to 15 µM, the dosage was reduced to 7.5 µM. BVDU showed
no toxicity at 30 µM. After 4 passages (17 days of treatment), the following
cell numbers were recorded:
A13H Cells/mL
MMC alone
846667
609333
846333
MMC + BVDU
MMC + BVDUp2BVDU

4
64
L. Cappellacci et al.
FIGURE 1 In vitro experiments. Effect of Gemcitabine (A) and BVDUp2dFdC (B) on cell numbers in
AH13 rat sarcoma cells.
Once the dose of BVDUp BVDU was reduced to non-toxic levels no
2
more enhancement of the MMC effect was observed.
In conclusion, no advantages of heterodinucleotide BVDUp dFdC over
2
Gemcitabine alone and of homodinucleotide BVDUp BVDU over BVDU
2
against AH13 tumor cells were observed.

A Heterodinucleotide of BVDU and Gemcitabine
465
EXPERIMENTAL
Chemistry
Thin layer chromatography (TLC) was run on silica gel 60 F254 plates
Merck); silica gel 60 (70–230 Merck) for column chromatography was used.
(
Ion exchange chromatography was carried out on (diethylamino)ethyl
DEAE)-A25 Sephadex column. Nuclear magnetic resonance spectra were
recorded on a Varian Mercury AS400 spectrometer (Palo Alto, CA, USA)
(
1
31
with TMS as the internal standard for H NMR and external H PO for
P
3
4
NMR. Chemical shift values are expressed in δ values (parts per million)
as s (singlet), d (doublet), t (triplet), dd (double doublet), q (quartet),
m (multiplet), or brs (broad singlet). High-resolution mass spectra were
recorded on an Agilent TOF II TOF/MS instrument equipped with ei-
ther an ESI or APCI interface (Agilent Technologies, Santa Clara, CA,
USA). HPLC analysis was carried out on a HP-1100 (Agilent Technologies
GmbH, Waldbroon, Germany), using a LiChroCART 125–3 (Merck) RP18
endcapped (5 mm) Purospher column with a flow rate of 1mL/min of
buffer A (250 mM CH COONH , 10 mM tetrabutylammonium phosphate
3
4
monobasic and NH OH till to pH 7.2) 60%, and B (MeOH) 40%.
4
ꢁ
ꢁ
5
-Bromovinyl-2 -deoxyuridine-5 -monophosphate (3). To a cooled mixture
of BVDU (275 mg, 0.82 mmol) and (CH O) PO (4.7 mL), P(O)Cl (750
3
3
3
◦
µL, 8.2 mmol) was added dropwise and the mixture was stirred at -5 C for 20
hours. Ice deionized water (5.0 mL) was added, the solution was neutralized
with concentrated NH OH and then washed with CHCl (3 × 15 mL).
4
3
Concentration of the aqueous layer in vacuo gave the crude compound,
which was purified by DEAE-Sephadex chromatography eluting with a linear
gradient of NH HCO (0–0.5 M). The pure compound (308 mg, 0.69
4
3
+
mmol) was dissolved in H O (11 mL) and treated with Dowex 50W-X8 (H
2
form) ion exchange resin (580 mg) stirring for 30 minutes. The resin was
filtered off, the filtrate was concentrated in vacuo and coevaporated with
1
anhydrous EtOH (3 × 10 mL) to give a solid (270 mg, 79% yield). H NMR
ꢁ
ꢁ
ꢁ
(
4
400 MHz, D O): δ 2.20 (m, 2H, H-2 ); 3.87 (m, 2H, H-5 ); 4.0 (m, 1H, H-4 );
2
ꢁ
ꢁ
.40 (m, 1H, H-3 ); 6.13 (t, J = 7.0 Hz, 1H, H-1 ); 6.77 (d, J = 14.1 Hz, 1H,
31
vinyl); 7.08 (d, J = 13.7 Hz, 1H, vinyl); 7.78 (s, 1H, H-6). P NMR (162
MHz, D O): δ 1.87 (s).
2
ꢁ
ꢁ
ꢁ
ꢁ
2
3
-Deoxy-2 ,2 -difluorocytidine-5 -monophosphate (5). To a solution of
◦
(
2
CH O) PO (3.4 mL), and P(O)Cl (2.3 mL, 25 mmol) at 5 C, was added
3
3
ꢁ
ꢁ
ꢁ
-deoxy-2 ,2 -difluorocytidine (150 mg, 0.57 mmol) in small portions with
rapid stirring (reaction time 2 hours). Ice deionized water (15 mL) was
added, and the reaction mixture was extracted with CHCl (2 × 15 mL).
3
The aqueous layer was treated with concentrated NH OH to pH 6.5, taking
4
◦
care to keep the solution below 30 C and then extracted a third time with
CHCl . The aqueous layer was concentrated under vacuo at 30 C or less.
◦
3
The residue was stirred with 30 mL of MeOH for 1 hour and filtered.

4
66
L. Cappellacci et al.
The filtrate was evaporated under vacuo to a solid which was purified by
chromatography on a silica gel column eluting with iPrOH/NH OH/H O
4
2
(
7:2:1). The obtained solid (153 mg, 0.4 mmol) was dissolved in H O (1.5
2
+
mL) and treated with Dowex 50W-X8 (H form) ion exchange resin (325
mg) stirring for 30 minutes. The resin was filtered off, the filtrate was
concentrated in vacuo and coevaporated with anhydrous EtOH (3 × 10 mL)
1
to a solid (100 mg, 53% yield). H NMR (400 MHz, D O): δ 3.94 (2dd, J =
2
ꢁ
ꢁ
ꢁ
3
2
1
.0, 5.6 Hz, 2H, H-5 ); 4.10 (m, 1H, H-4 ); 4.32 (m, 1H, H-3 ); 6.10 (d and t,
H, H-5, H-1 ); 7.92 (d, J = 8.1 Hz, 1H, H-6). P NMR (162 MHz, D O): δ
ꢁ
31
2
.11 (s).
ꢁ
ꢁ
ꢁ
ꢁ
2
-Deoxy-2 ,2 -difluorocytidine-5 -monophosphate tri-butylammonium salt (6).
A mixture of 5 (92 mg, 0.27 mmol) and tributylamine (1 eq.) in dry
MeOH (1.9 mL) was stirred for 30 minutes. Evaporation in vacuo gave
1
the title compound as a white amorphous solid (125 mg, 88% yield). H
NMR (400 MHz, D O): δ 0.74 (t, J = 7.5 Hz, 9H, CH CH CH CH ); 1.18
2
3
2
2
2
(
m, 6H, CH CH CH CH ); 1.48 (m, 6H, CH CH CH CH ); 2.95 (m, 6H,
3 2 2 2 3 2 2 2
ꢁ
CH CH CH CH ); 3.94 (2dd, J = 3.0, 5.6 Hz, 2H, H-5 ); 4.10 (m, 1H,
3
ꢁ
2
2
2
ꢁ
H-4 ); 4.28 (m, 1H, H-3 ); 5.96 (d, J = 7.7 Hz, 1H, H-5); 6.06 (t, J = 7.3
ꢁ
31
Hz, 1H, H-1 ); 7.74 (d, J = 7.7 Hz, 1H, H-6). P NMR (162 MHz, D O): δ
2
1
.22 (s).
1
ꢁ
ꢁ
2
ꢁ
ꢁ
ꢁ
ꢁ
P -5 -[5-Bromovinyl-2 -deoxyuridine]-P -5 -[2 -Deoxy-2 ,2 -difluorocytidine]
pyrophosphate (1). To a solution of 3 (94 mg, 0.23 mmol) in anhydrous
DMF (4.6 mL), CDI (184 mg, 1.13 mmol) was added and the mixture was
stirred at room temperature for 4 hours. Dry MeOH (95.2 µL) was added
to hydrolyze excess CDI stirring for 30 minutes. To obtained imidazolide
4, compound 6 (120 mg, 0.23 mmol) was added and the mixture was
stirred at room temperature for 18 hours. The solvent was removed in
vacuo and the residue was dissolved in H O (10 mL) and extracted with
2
CHCl (5 × 5 mL). The aqueous layer was concentrated in vacuo and the
3
residue was purified by chromatography on a silica gel column eluting with
iPrOH/NH OH/H O (8:1:1). Evaporation of the appropriate fractions
4
2
gave the desired compound as a white solid (diammonium salt, 56 mg, 32%
1
ꢁ
yield). H NMR (400 MHz, D O): δ 2.14 (m, 2H, H-2 (BVDU)); 4.0 (m,
2
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
5
(
H, H-4 (Gem), H-5 (Gem), H-5 (BVDU)); 4.28 (m, 2H, H-3 (Gem), H-4
ꢁ
BVDU)); 4.40 (m, 1H, H-3 (BVDU)); 5.90 (d and t, 2H, H-5 (Gem), H-1
ꢁ
(
Gem)); 6.12 (t, J = 6.8 Hz, 1H, H-1 (BVDU)); 6.70 (d, J = 13.7 Hz, 1H,
vinyl); 7.10 (d, J = 13.7 Hz, 1H, vinyl); 7.60 (d, J = 7.7 Hz, 1H, H-6 (Gem));
31
7
.66 (s, 1H, H-6). P NMR (162 MHz, D O): δ -10.35 (dd, J = 20.74, 40.18
2
Hz). HRMS (ESI-): calcd for C H BrF N O P (M-H) 735.9868, found
20
23
2
5
14 2
7
359872.
ꢁ
ꢁ
5-Bromovinyl-2 -deoxyuridine-5 -monophosphate tri-butylammonium salt (7).
The title compound was obtained from 3 (110 mg, 0.27 mmol) as de-
1
scribed for 6 (white amorphous solid, 134 mg, 83% yield). H NMR (400
MHz, D O): δ 0.74 (t, J = 7.5 Hz, 9H, CH CH CH CH ); 1.18 (m, 6H,
2
3
2
2
2

A Heterodinucleotide of BVDU and Gemcitabine
467
ꢁ
ꢁ
CH CH CH CH ); 1.48 (m, 6H, CH CH CH CH ); 2.20 (m, 2H, H-2 );
3
2
2
2
3
2
2
2
ꢁ
2
4
.94 (m, 6H, CH CH CH CH ); 3.90 (m, 2H, H-5 ); 4.03 (m, 1H, H-4 );
3 2 2 2
ꢁ
ꢁ
.40 (m, 1H, H-3 ); 6.15 (t, J = 6.8 Hz, 1H, H-1 ); 6.78 (d, J = 13.7 Hz, 1H,
31
vinyl); 7.10 (d, J = 13.7 Hz, 1H, vinyl); 7.78 (s, 1H, H-6). P NMR (162
MHz, D O): δ 1.60 (s).
2
2
1
ꢁ
ꢁ
P ,P -Bis[5-bromovinyl-2 -deoxyuridine]-5 -pyrophosphate (2). To a solu-
tion of 3 (60 mg, 0.14 mmol) in anhydrous DMF (3.0 mL), CDI (117 mg,
0
.72 mmol) was added and the mixture was stirred at room temperature
for 3 hours. Dry MeOH (61 µL) was added stirring for 30 minutes. To the
◦
mixture, compound 7 (130 mg, 0.217 mmol) was added stirring at 40 C for
3
hours. The solvent was removed in vacuo and the residue was dissolved in
H O (10 mL) and extracted with CHCl (5 × 5 mL). The aqueous layer was
2
3
concentrated in vacuo and the residue was purified by chromatography on a
silica gel column eluting with iPrOH/NH OH/H O (8:1:1). Evaporation of
4
2
the appropriate fractions gave compound 2 as a white solid (diammonium
1
ꢁ
ꢁ
salt, 56 mg, 46% yield). H NMR (400 MHz, D O): δ 2.14 (m, 4H, H-2 );
2
ꢁ
ꢁ
ꢁ
4
6
2
.0 (m, 6H, H-4 , H-5 ); 4.37 (m, 2H, H-3 ); 6.0 (t, J = 6.8 Hz, 2H, H-1 );
.72 (d, J = 13.7 Hz, 2H, vinyl); 7.05 (d, J = 13.7 Hz, 2H, vinyl); 7.62 (s,
H, H-6). 3 P NMR (162 MHz, D O): δ -10.39 (s). HRMS (ESI-): calcd for
1
2
C H Br N O P (M-H) 804.9159, found 804.9163.
22
25
2
4
15 2
Biological Evaluation
Treatment of AH13R Sarcoma Cells in Culture. AH13r cells, a subline of
the rat Yoshida sarcoma, were obtained from the Cell and Tumor Bank of
the West German Cancer Center, University Essen, Medical School, Essen,
Germany. Cells were grown in DMEM (FG 0415, Biochrom AG, Berlin,
Germany) supplemented with 10% (v/v) heat-inactivated fetal bovine
serum, 100 U/ml penicillin and 100 µg/ml streptomycin in a humidified
◦
atmosphere containing 5% CO at 37 C. Logarithmically growing cells
2
were seeded at a density of 100,000 cells/ml and incubated with different
cytostatic drugs in combination with or without BVDU. After 2–4 days
(
unless otherwise indicated), cells were counted using the Cell Counter and
Analyser System CASY TT (Sch a¨ rfe System GmbH, Reutlingen, Germany)
and serially passaged.
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