Synthesis and cytotoxicity of 5-fluorouracil/diazeniumdiolate conjugates

Article abstract of DOI:10.1016/j.bmc.2003.09.003

5-Fluorouracil/diazeniumdiolate conjugates were first synthesized, and showed greater cytotoxicities than 5-fluorouracil for DU 145 human prostate and HeLa cancer cells.

Full text of DOI:10.1016/j.bmc.2003.09.003

Bioorganic & Medicinal Chemistry 11 (2003) 4971–4975
Synthesis and Cytotoxicity of 5-Fluorouracil/Diazeniumdiolate
Conjugates
Tingwei Bill Cai,a Xiaoping Tang,a Janet Nagorski,b Paul G. Brauschweigerb
and Peng George Wanga,*
aDepartments of Biochemistry and Chemistry, The Ohio State University, OH 43210, USA
bDepartment of Radiation Oncology, University of Miami, Miami, FL 33136, USA
Received 17 June 2003; revised 21 August 2003; accepted 3 September 2003
Abstract—5-Fluorouracil/diazeniumdiolate conjugates were first synthesized, and showed greater cytotoxicities than 5-fluorouracil
for DU 145 human prostate and HeLa cancer cells.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
such as breast, colorrectal, and gastric cancers. To
optimize the efficacy of 5-FU, it is often administrated
by continuous infusion as well as incombination with
other cytotoxic or with biochemical modulators, such as
leucovorin.6 5-FU is poorly tumor selective, so its ther-
apy causes high incidences of toxicity in the bone mar-
row, gastrointestinal tract, central nerve system and
skin, which has prompted the efforts in the development
of derivatives aiming at reducing the adverse effects of
5-FU. Novel prodrugs of 5-FU possessing a broader
spectrum of antitumor activity and fewer toxicity have
been sought diligently by many researchers.7,8 The
common feature of these prodrugs is that they are all
N1-modified derivatives through different biodegradable
linkages.9
Nitric oxide (NO) plays a critical role in a variety of
bioregulatory processes, including normal physiological
control of the blood pressure, neurotransmission, and
microphage-induced cytostatics and cytotoxity.1
Recently, conjugation of NO releasing moieties with
active drugs is of particular interest. Studies showed
that the use of non-steroid anti-inflammatory drug
derivatives (NSAID) with NO releasing properties had
improved gastrointestinal safety.2 There are several NO-
donating NSAIDs, for example, NO-Aspirin and NO-
Flurbiprofen, in clinical trials at present. Moreover, NO
can inhibit metastasis, enhance cancer cell apoptosis
and assist macrophage to kill tumor cells.3
Diazeniumdiolates (NONOates) are compounds con-
taining the [N(O)NO]À structural unit. This class of
compounds is known as an excellent source for a con-
trolled release of NO both in vitro and in vivo.4 Keefer’s
group has recently introduced a new class of diaze-
niumdiolate prodrugs which are acetoxymethylated at the
anionic oxygen of the parent NONOates. These esterase-
sensitive compounds reveal significant antileukemic
activity in vitro.5
Based on the mutual prodrug concept, 5-FU and diaze-
niumdiolate conjugates (Fig. 1) with methylene or acyl-
oxymethylene as the spacers were synthesized with the
aim to improve tumor selectivity, efficiency and safety.
Their NO-releasing ablilties and cytotoxicity were also
evaluated.
Results and Discussion
5-Fluorouracil (5-FU, Fig. 1) is one of the antitumor
agents most frequently used for treating solid tumors,
Synthesis of 5-FU/diazeniumdiolate conjugates
The synthetic scheme toward the conjugate 1 is shown
in Scheme 1. The sodium PYRRO/NO 3, was synthe-
sized according to a modified procedure reported by
Keefer.10 The chloromethyl NONOate 5 was obtained
*Corresponding author. Tel.: +1-614-292-9884; fax: +1-614-688-
3106; e-mail: pgwang@chemistry.ohio-state.edu
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.09.003

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T. B. Cai et al. / Bioorg. Med. Chem. 11 (2003) 4971–4975
from the O-alkylation of sodium PYRRO/NO 3 with
chloromethyl methyl sulfide and subsequent treatment
with sulfuryl chloride.11 Attachment of 5-FU to 5 gave
the desired N1-alkylation product 1 as the major pro-
duct. The N1,N3-bis-alkylation product 1a was also
obtained from the same reaction.
stable in aqueous solution at neutral pH. Both 2 and 11
slowly release NO at pH=8 (Fig. 2), and in the presence
of porcine liver esterase (Fig. 3). The NO release rate for
compound 2 (Scheme 4) is much slower than 11 at the
same condition. Compound 2 should be subjected to
esterase action for longer than 3 min. Scheme 4 shows
the proposed NO releasing pathway. On the contrary,
the produrg 1 was very stable in aqueous solution. No
substantial NO was detected even at either acidic or
basic conditions. There might be other enzymatic path-
ways to release 5-Fu and NO. Further experiments are
necessary to clarify the mechanism.
The stepwise synthesis of conjugate 2 is illustrated in
Scheme 2. The intermediate 10 could be obtained fol-
lowing a similar procedure reported by Menger and
Rourk.12 Alkylation of succinic acid monobenzyl ester
(6) with chloromethyl methyl sulfide and subsequent
treatment with sulfuryl chloride generated the desired
chloromethyl ester 8. Attachment of 5-FU to 8 provided
the compound 9 as the only product, and there was no
sign of the N3-alkylation product. Catalytic transfer
hydrogenation debenzylation of 9 in glacial acetic acid,
using 1,4-cyclohexadiene as the hydrogen source, gave
10 in quantitative yield. Coupling of 10 with chlor-
omethyl PYRRO/NO 5 gave the desired N1-alkylation
product 2, along with the N1,N3-bis-alkylation product
2a.
Cytotoxic effects
The cytotoxicities of 5-FU/diazeniumdiolate conjugates
were assessed by in vitro clonogenic cell survival assays
as described previously (Table 1).13 The median effect
dose (ED50) for compound 1 in DU145 (98 mM) and
HeLa (112 mM) cell lines indicated that this prodrug
was about twice as potent as 5-FU (204 mM in DU145
and 278 mM in HeLa). Compound 2a (257 mM) and 5-
FU (278 mM) exhibited similar cytotoxic effects in HeLa
cells, however compound 2 (50 mM) was twice as potent
as compound 1 and 5-fold more toxic than 5-FU and
compound 2a. In DU145 cells, compound 2a (65 mM)
exhibited the greatest cytotoxicity. It was about twice as
potent as compound 2, and about three fold more
Another previously reported prodrug AcOM-PYRRO
(11) was synthesized from 5 in high yield by a modified
procedure (Scheme 3).5
Hydrolysis of conjugates
The hydrolysis of the prodrugs were studied by mon-
itoring of the NO release with an Electrochemical ISO-
NO marker II isolated Nitric Oxide Meter manu-
factured by World Precision Instruments, Inc. (Sar-
asota, Florida). All of the prodrugs 1, 2, and 11 are
Figure 1.
Scheme 2. (a) ClCH2SCH3, Cs2CO3, DMF, rt (88%); (b) SO2Cl2,
CH2Cl2, rt (70%); (c) 5-FU, NEt3, DMF, rt (54%); (d), 10%Pd/C,
AcOH, 1,4-cyclohexadiene, rt (100%); (e) 5, Cs2CO3, DMF, rt (2,
18%; 2a, 12%).
Scheme 1. (a) ClCH2SCH3, KI, DMF/THF, rt (45%); (b) SO2Cl2,
CH2Cl2, rt ; (c) 5-FU, NEt3, DMF, rt (1, 21%; 1a, 12%).
Scheme 3. (a) NaAc, DMF, rt (51%).

T. B. Cai et al. / Bioorg. Med. Chem. 11 (2003) 4971–4975
4973
Scheme 4. The hydrolysis pathway of conjugate 2.
Figure 2. NO releasing from prodrugs 2 (&) and 11 (^) in a pH=8.0
buffer.
Figure 3. NO releasing from prodrugs 2 (&) and 11 (^) in the pre-
sence of esterase.
Table 1. Median effect dose (mM) for HeLa and DU145 cell lines
obtained from a Kratos MS 80 spectrometer using elec-
tronspray ionization mode (ESI). UV–vis spectroscopy
measurements were carried out on a HP 8453 spectro-
meter. Silica gel F254 plates (Merck) and Silica Gel 60
(70–230 mesh) Merck) were used in analytical thin-layer
chromatography (TLC) and silica gel column chroma-
tography, respectively.
Compd
HeLa
DU145
5-FU
1
2
2a
278
112
50
204
98
129
65
257
potent than 5-FU. Compound 11 had little or no effect
in the tested cell lines.
Sodium 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (3). A
modified procedure was adopted from ref 5. A solution
of 16.5 mL (0.20 mol) of pyrrolidine in a mixture of
diethyl ether (40 mL) and acetonitrile (12 mL) was
placed in a reaction flask, then bubbled with nitrogen
for 20 min, and cooled to À78 ꢀC using a dry ice/ace-
tone bath. Then NO gas was slowly bubbled into the
flask for 1 h. The bath was removed, and the reaction
vessel was flushed with nitrogen to remove the NO gas.
After 10 min, 100 mL of dry diethyl ether was added
into the mixture, and a white precipitate was obtained
after the filtration under nitrogen. To the white solid
was added 10 mL of aqueous NaOH solution (10 M).
The mixture was stirred and 100 mL of diethyl ether was
added. The white solid was collected by filtration and
washed with ether. The product was dried under
vacuum to give 5.6 g (37%) yield of 3. 1H NMR was the
same as the literature.5
Since both of the conjugate 1 and 2 have greater activity
than 5-FU, the anti-tumor nucleoside and NO could
have synergetic effects in biological systems. The
apparent difference between the cytotoxic activities of
the two conjugates, 1 and 2, could be explained by the
appreciation of the individual design. The N–O ketal
linker in compound 1 could not be hydrolyzed as easy
as the spacer in compound 2.
Experimental
Chemistry
All reagents were purchased from commercial suppliers
and were used without further purification. Melting
points were measured on an Electrothermal IA 9100
1
digital apparatus without correction. H and 13C NMR
O2-Methylthiomethyl
1,2-diolate (4). A slurry of compound 3 (3.20 g, 20.9
mmol) and anhydrous sodium carbonate (2.22 g, 20.9
1-(pyrrolidin-1-yl)diazen-1-ium-
were recorded on a Varian Unity-300, a Mercury-400,
or a Varian unity-500 spectrometer. MS spectra were

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T. B. Cai et al. / Bioorg. Med. Chem. 11 (2003) 4971–4975
mmol) in 40 mL of dry THF was cooled with an ice-
water bath, to which 2.28 mL (27.2 mmol) chloromethyl
methylsulfide was slowly added via a syringe, followed
by 15 mL of DMF. After 5 min, the bath was removed,
and catalytic amount of KI (200 mg) was added. The
reaction mixture was stirred under nitrogen overnight.
Then it was diluted with 200 mL of ether, washed with
cold water. The separated aqueous layer was extracted
with ether again. The combined ether solution was
washed with brine, dried over anhydrous sodium sul-
fate, and concentrated under vacuum. The residue was
purified by silica gel chromatograph with 1:4 ethyl ace-
tate/hexane, resulting in 1.80 g (45%) of 4 as colorless
oil, which solidified when kept inside refrigerator. Rf
0.38 (EtOAc/hexane: 1/3). 1H NMR (CDCl3, 300 MHz):
d 5.09 (s, 2H), 3.44 (t, 4H), 2.15 (s, 3H), 1.83 (m, 4H);
13C NMR (CDCl3, 75 MHz): d 78.06, 50.94, 23.02,
15.61. MS 214 (M+Na), 405 (2M+Na). HRMS calcd
for C6H13N3O2S 191.0728, found 191.0727.
ester (4.0 g, 19.0 mmol) in 25 mL of DMF was added
cesium carbonate (6.2 g, 19.0 mmol), the resulting sus-
pension was stirred for 30 min at room temperature.
Then chloromethyl sulfide (1.59 mL, 19.0 mmol) was
added, and the mixture was stirred at room temperature
overnight and at 70 ꢀC for 10 min. The mixture was
cooled to room temperature, diluted with water, and
extracted with ethyl acetate. The separated organic layer
was washed with brine, dried over sodium sulfate, and
concentrated to give 4.5 g (88%) yellow oil. Rf 0.35
1
(EtOAc/hexane: 1/3); H NMR (CDCl3, 400 MHz): d
7.34 (m, 5H), 5.12 (4H), 2.69 (4H), 2.21 (s, 3H); 13C
NMR (CDCl3, 100 MHz): d 171.88, 135.62, 128.50,
128.24, 128.18, 68.43, 66.54, 29.12, 28.96, 15.31.
Succinic acid benzyl ester chloromethyl ester (8). Fol-
lowing similar procedure for the synthesis of 5. 70%
yield after flash column chromatography. Rf 0.38
1
(EtOAc/hexane: 1/3); H NMR (CDCl3, 500 MHz): d
7.35 (m, 5H), 5.68 (s, 2H), 5.14 (s, 2H), 2.72 (4H); 13C
NMR (CDCl3, 125 MHz): d 171.88, 170.70, 135.83,
128.83, 128.59, 128.52, 69.00, 66.96, 66.91, 64.22, 29.19,
28.98. MS (ESI) 279 (M+Na+).
O2-Chloromethyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-dio-
late (5). A solution of compound 4 (91 mg, 0.48 mmol)
in 10 mL of dry CH2Cl2 was cooled with an ice-water
bath, to which 0.48 mL of sulfuric chloride (1.0 M in
CH2Cl2) was added dropwise. After 10 min, the ice-
water bath was removed, and the mixture was stirred
for another 30 min. Evaporation under vacuum gave 5
as yellow oil, which was immediately used in the fol-
lowing reactions without further purification. Rf 0.43
Succinic acid benzyl ester (5-fluro-2,4-dioxo-di-hydro-
2H-pyrimidin-1-yl)methyl ester (9). Following similar
procedure for the synthesis of 1. 54% yield after flash
column chromatography; white solid, mp 125–127 ꢀC;
Rf: 0.72 (EtOAc/CH2Cl2: 1/1); 1H NMR (CDCl3,
500 MHz): d 9.04 (br, 1H), 7.56 (d, J=4.5 Hz, 1H), 7.35
(m, 5H), 5.62 (s, 2H0, 5.12 (s, 2H), 2.70 (s, 4H); 13C
NMR (CDCl3, 125 MHz): d 172.54, 171.73, 156.83,
149.09, 141.09, 139.18, 135.50, 128.59, 128.39, 128.29,
128.23, 69.75, 66.78, 28.80. MS (ESI) 373 (M+Na+),
723 (2M+Na+).
1
(EtOAc/hexane: 1/3). H NMR (CDCl3, 400 MHz): d
5.80 (s, 2H), 3.60 (t, 4H), 1.95 (m, 4H); 13C NMR
(CDCl3, 100 MHz): d 79.86, 50.86, 23.32.
5-FU/diazeniumdiolate conjugate (1). To a solution of 5-
fluorouracil (133 mg, 1.02 mmol) in 5 mL of DMF was
added triethyl amine (328 mL, 2.36 mmol), the mixture
was stirred at room temperature for 30 min. Then a
solution of compound 5 in 5 mL DMF, which was
freshly prepared from 4 (150 mg, 0.78 mmol) and sul-
furic chloride (0.78 mL, 1.0 M in CH2Cl2), was added
dropwise, and the mixture was stirred overnight. The
mixture was diluted with 100 mL of water, extracted
with CH2Cl2 twice (50 mLÂ2). The separated organic
phase was combined, washed with brine, dried over
anhydrous sodium sulfate, and concentrated under
vacuum. The residue was loaded on the silica gel chro-
matograph column. Elution with 1:1 ethyl acetate/
methylene chloride provided 45 mg (21%) 1 and 50 mg
(12%) 1a. 1: Colorless solid, mp 172–178 ꢀC; Rf 0.26
(EtOAc/CH2Cl2: 1/1); 1H NMR (acetone-d6, 400 MHz):
d 7.98 (d, 1H, J=6.4 Hz), 5.67 (s, 2H), 3.53 (m, 4H),
1.95 (m, 4H); 13C NMR (acetone-d6, 100 MHz): d
129.53, 129.20, 78.13, 51.26, 23.39. MS (ESI) 296
(M+Na+), 569 (2M+Na+); HRMS calcd for
C9H12FN5O4 274.0952, found 274.0962 (M+H). 1a: Rf
0.45 (EtOAc/CH2Cl2: 1/1); 1H NMR (CDCl3, 500 MHz):
d 7.43 (d, 1H, J=5.0 Hz), 5.99 (s, 2H), 5.67 (s, 2H), 3.55
(m, 4H), 1.95 (m, 4H); 13C NMR (CDCl3, 125 MHz): d
126.36, 126.09, 77.78, 71.17, 71.17, 50.65, 50.56, 23.04,
22.89. MS (ESI) 439 (M+Na+), 855 (2M+Na+).
Succinic acid mono[(5-fluro-2,4-dioxo-di-hydro-2H-pyri-
midin-1-yl)methyl] ester (10). To the stirred solution of
compound 9 (470 mg, 1.34 mmol) in 10 mL of acetic
acid was added 10% Pd/C (0.5 g) and 1,4-cyclohex-
adiene (0.76 mL, 8.0 mmol). The mixture was stirred for
3 h at room temperature, then it was filtered and evap-
1
orated to give 350 mg product 10 (100%). H NMR
(DMSO-d6, 400 MHz): d 8.08 (d, J=6.4 Hz, 1H), 5.55
(s, 2H), 2.48 (m, 4H). MS (ESI) 283 (M+Na+), 543
(2M+Na+).
5-FU/diazeniumdiolate conjugate (2) and (2a). To a
solution of 5-fluorouracil (145 mg, 0.56 mmol) in 5 mL
of DMF was added cesium carbonate (182 mg, 0.56
mmol). The mixture was stirred at room temperature
for 30 min. Then a solution of compound 5 in 5 mL
DMF, which was freshly prepared from 4 (128 mg, 0.67
mmol) and sulfuric chloride (0.67 mL, 1.0 M in
CH2Cl2), was added dropwise, and the mixture was
stirred overnight. After the evaporation of solvent
under vacuum, the residue was loaded onto a silica gel
chromatograph column. Elution with 1:1 ethyl acetate/
methylene chloride provided 40 mg (18%) 2 and 36 mg
1
(12%) 2a. 2: Rf 0.45 (EtOAc/CH2Cl2: 1/1); H NMR
(CDCl3, 500 MHz): d 9.38 (br, 1H), 7.60 (d, J=6.4 Hz,
1H), 5.72 (s, 2H), 5.62 (2H), 3.59 (m, 4H), 2.66–2.73 (m,
4H), 1.96 (m, 4H); 13C NMR (CDCl3, 125 MHz):
Succinic acid benzyl ester (methylsulfanyl)methyl ester
(7). To the stirred solution of succinic acid monobenzyl

T. B. Cai et al. / Bioorg. Med. Chem. 11 (2003) 4971–4975
4975
d 171.2, 170.93, 149.17, 141.03, 139.14, 128.69, 128.42,
87.44, 87.30, 69.93, 50.69, 50.56, 28.78, 28.55, 23.02,
22.94. MS (ESI) 426 (M+Na+), 829 (2M+Na+);
HRMS (FAB) calcd for C14H19FN5O8 (M+H)
404.1218, found 404.1225 (M+H). 2a: Rf: 0.58 (EtOAc/
litres of HBSS were placed on cells for treatment period.
Treatment time was 2 h at 37 ꢀC and 5% CO2. Stock
solutions were prepared at a 0.01 M concentration and
sterilized by passing through a 0.2 micron filter. Sol-
vents used to prepare the solutions are as follows: 5-FU
in distilled sterile water; TXP30 in DMSO; other com-
pounds in ethanol.
1
CH2Cl2: 1/1); H NMR (CDCl3, 500 MHz): d 7.63 (d,
1H, J=5.0 Hz), 5.96 (s, 2H), 5.74 (s, 2H), 5.66 (s, 2H),
3.56 (m, 4H), 3.52 (m, 4H), 1.95 (m, 4H), 1.91 (m, 4H);
13C NMR (CDCl3, 125 MHz): d 172.19, 170.55, 128.17,
127.77, 127.51, 87.40, 71.17, 70.78, 50.63, 28.74, 28.65,
28.52, 23.00, 22.92, 22.85. MS (ESI) 569 (M+Na+),
1115 (2M+Na+). HRMS (FAB) calcd for
C19H27FN8O10Na (M+Na) 569.1732, found 569.1752.
Cells were washed one time with HBSS, removed from
flasks with trypsin, counted and plated at 50, 100, 250
cells into six-well tissue culture plates, containing cell
culture media. Cells were incubated for seven to ten
days at 37 ꢀC and 5% CO2, fixed with methanol, stained
with Giemsa, and colonies greater than 50 cells counted.
Several concentrations of the compounds were used to
assess the dose dependent cytoxicity by clonogenic cell
survival, calculated as described previously.13 Experi-
ments were run two times and averages calculated.
Graphs and statistics were calculated using Sigma Plot
software.
O2-(Acetoxymethyl) 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-
diolate (11). To a DMF (5 mL) solution of freshly pre-
pared compound 5, which was obtained from 4 (110 mg,
0.58 mmol) and sulfuric chloride (0.58 mL, 1.0 M in
CH2Cl2), was added sodium acetate (158 mg, 1.16
mmol) in one portion. The mixture was stirred at room
temperature for 7 h, then it was diluted with 100 mL
of water, extracted with CH2Cl2 twice (50 mLÂ2).
The separated organic phase was combined, washed
with brine, dried over anhydrous sodium sulfate, and
concentrated under vacuum to give 60 mg (51%)
solid. 1H NMR was the same as the literature.5 13C
NMR (CDCl3, 100 MHz): d 129.99, 87.36, 50.71,
22.98, 20.91.
Acknowledgements
This work is generously supported by a grant from NIH
(GM 54074).
References and Notes
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Booth, M. N.; Citro, M. L.; Keefer, L. K. J. Med. Chem. 2000,
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NO measurement was carried out with an Electro-
chemical ISO-NO Mark II Isolated Nitric Oxide Meter,
a product of World Precision Instruments, Inc. (Sar-
asota, FL, USA). For the test in pH 8.0 buffer, the
prodrug was dissolved in methanol and added to 10 mL
of 1.0 M Tris–Cl buffer (pH=8.0) to a final concen-
tration of 0.10 mM. For the enzymatic test, the prodrug
was dissolved in methanol and added to 10 mL of 0.1 M
Tris–Cl buffer (pH=7.0) to a final concentration of 0.10
mM. Then, esterase (EC.31.1.1, procine liver, crude,
Sigma) was added to a final concentration of 150 mg/mL.
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Clonogenic cell survival assay
Cell lines. DU-145, human prostate adenocarcinoma
and HeLa, human cervical adenocarcinoma were main-
tained in RPMI 1640, supplemented with 15% heat
inactivated fetal bovine serum and 1% penicillin-strep-
tomycin liquid (Life Technologies). 2Â105 cells were
seeded into T-25 tissue culture flasks 24 h before treat-
ment with the compounds.
13. Braunschweiger, P. G.; Basrur, V. S.; Cameron, D.;
Sharpe, L.; Santos, O.; Perras, J. P.; Sevin, B. U.; Markoe,
A. M. Biotherapy 1997, 10, 129.
Media was removed from the cells then washed one time
with Hanks balanced salt solution (HBSS). Three milli-