Synthesis and activity evaluation of 3'-floxuridinyl 4-[3-(3, 5-di-t-butyl-4-methoxyphenyl)-3-oxo-propenyl]benzoate: In vitro and in vivo as a potential dual-acting antitumor prodrug

Article abstract of DOI:10.1002/ddr.20463

BOBA (4-[3-(3, 5-di-tert-butyl-4-methoxyphenyl)-3-oxo-propenyl]benzoic acid), a substituted chalcone derivative, exhibits an excellent inducing differentiation on neoplastic cellular differentiation. FUDR (5-fluoro-2'-deoxyuridine, floxuridine) inhibits D

Full text of DOI:10.1002/ddr.20463

DRDURGUDGEDVEELVOELPOMPEMNETNRTESREEASERACRHC7H3:7273: –18–19 ((22001121))
Research Article
Synthesis and Activity Evaluation of 30-Floxuridinyl 4-[3-(3,
5-di-t-butyl-4-methoxyphenyl)-3-oxo-propenyl]benzoate:
In Vitro and In Vivo as a Potential Dual-Acting
Antitumor Prodrug
Ã
Lixue Du,1 Juqing Jin,2 Wei Wu,1 Jiangang Chen,2 Ailin Shan,3 and Sanqi Zhang3
1Department of Hepatobiliary Surgery, Shaanxi Provincial People’s Hospital, Xi’an, 710068
People’s Republic of China
2Department of Chemistry, School of Science, Xi’an Jiaotong University, Xi’an, 710061 People’s
Republic of China
3Department of Pharmacy, School of Medicine, Xi’an Jiaotong University, Xi’an, 710061 People’s
Republic of China
Strategy, Management and Health Policy
Enabling
Preclinical Development Clinical Development
Toxicology, Formulation Phases I-III
Technology,
Genomics,
Proteomics
Preclinical
Research
Postmarketing
Phase IV
Drug Delivery,
Regulatory, Quality,
Manufacturing
Pharmacokinetics
ABSTRACT
BOBA (4-[3-(3, 5-di-tert-butyl-4-methoxyphenyl)-3-oxo-propenyl]benzoic acid), a sub-
stituted chalcone derivative, exhibits an excellent inducing differentiation on neoplastic cellular
differentiation. FUDR (5-fluoro-20-deoxyuridine, floxuridine) inhibits DNA biosynthesis and has been
used extensively to treat various cancers. In our efforts to find a new dual-action antitumor prodrug, 30-
floxuridinyl 4-[3-(3, 5-di-t-butyl-4-methoxyphenyl)-3-oxo-propenyl] benzoate (30-O-BOBA- FUDR) was
synthesized, and its antiproliferative activity in vitro and antitumor efficacy in vivo were evaluated.
Compared with FUDR, the antiproliferative activity of 30-O-BOBA-FUDR was improved by 3–7-fold. In rat
hepatocellular carcinoma xenografts, 30-O-BOBA-FUDR-treated rats had smaller tumors than were found
in controls. In addition, the expression of Bcl-2 protein was significantly downgraded, whereas the
expression of Bax protein was upregulated in neoplastic tissues. The early apoptotic ratio of 30-O-BOBA-
FUDR-treated rat group was increased dose-dependently. These findings strongly support the concept that
330’--OO--BBOOBBAA--FFUUDDRR mmaayy bbeeaannoovveellananddefefeffcetcivtievedudaula-al-catciotinonanatnittuitmumoroprropdrorudgru.gD.rDugruDgeDveRvesR7e3s:7723:1–8–91,,
2011. r 2011 Wiley Periodicals, Inc.
2012.
© 2011 Wiley Periodicals, Inc.
Key words: inducing differentiation; FUDR; antitumor activity; prodrug
INTRODUCTION
Cancer therapy is a great challenge in the fields of
Grant sponsors: Shaanxi Provincial Department of Health;
medicine and immunology. Finding novel and effica-
cious compounds has been a high priority for research
in pharmaceutical science. Apart from surgery, radia-
tion, immunotherapy, and hormonal therapy, therapies
for cancer treatment include cytotoxic drugs as the
main form of chemotherapy for cancer. 20-Deoxy-5-
fluorouridine (FUDR) is cytotoxic in vitro against a
People’s Republic of China; Grant number: 08A01.
Ã
Correspondence to: San-Qi Zhang, Department of Pharmacy,
School of Medicine, Xi’an Jiaotong University, Xi’an, 710061
People’s Republic of China. E-mail: sqzhang@xjtu.edu.cn
Received 19 August 2011; Accepted 15 September 2011
Published online in Wiley Online Library (wileyonlinelibrary.com).
wide range of cell lines derived from solid tumors and DOI: 10.1002/ddr.20463
ꢀc
© 22001111 WWiilleeyy PPeerriiooddiiccaallss,, IInnc.

74
DU ET AL.
has been used in combination with folinic acid as a
standard treatment in the adjuvant setting for a variety
of carcinomas, including those of the stomach, colon,
liver, and breast [Meyerhardt and Mayer, 2005].
However, FUDR exhibits adverse reactions and is
rapidly metabolized after administration, particularly in
liver [van Laar et al., 1998]. Resistance, rapid elimina-
tion (plasma t1/2 5 7.7 min), and low lipid solubility all
contribute to the low efficacy of FUDR [LaCreta,
1987].
Fig. 1. Structures of BOBA and 30-O-BOBA-FUDR.
To overcome these shortcomings, prodrugs and RA with FUDR may exhibit a dual-action antitumor
colloidal carriers for the delivery and targeting effect. From a series of O-retinoyl esters of FUDR, the
of FUDR have been designed. We prepared solid 30-O-retinoyl FUDR prodrug was the most
lipid nanoparticles (SLN) of FUDR derivatives and potent anticancer agent as assessed by in vitro cell
found that the SLN had a desirable hepatocyte- differentiation and tumor growth delay [Xia et al.,
selective targeting with the concentration of 1999]. From a series of substituted chalcones, BOBA
FUDR in liver enhanced [Li et al., 2008; Lian (4-[3-(3, 5-di-t-butyl-4-methoxyphenyl)-3-oxo-prope-
et al., 2008]. Urokinase-targeting FUDR conjugates nyl] benzoic acid; Fig. 1) displayed differentiation
[Vine et al., 2010] and FUDR duplex prodrugs showed activity comparable to that of RA [Guo et al., 1997].
that anticancer activity was sequence-dependent The EC50 values of BOBA and RA for HL-60 cell line
[Schott et al., 2009]. The lactosaminated human were 86 nM and 100 nM, repectively. Compared with
albumin–FUDR conjugate produces a high concentra- RA, BOBA is easy to synthesize and exhibits more
tion of FUDR in hepatic sinusoids and improves chemical stability. The combination of FUDR and
FUDR efficacy [Fiume and Stefano, 2010], whereas BOBA may represent a novel dual-action prodrug,
the Amidon group has studied amino acid ester functioning as an antimetabolite and differentiation
prodrugs of FUDR for several years [Vig et al., 2003; inducer. The aim of the present research was to
Landowski et al., 2005; Shin et al., 2006; Tsume et al., synthesize 30-O-BOBA-FUDR and evaluate its activity
2008]. These investigators found that these prodrugs in vitro and in vivo as a potential antitumor dual action
could enhance intestinal absorption; were resistant to prodrug.
glycosidic bond metabolism; had good solubility,
stability, and relatively fast enzymic conversion rates;
and improved absorption, tumor selectivity, and
MATERIALS AND METHODS
efficacy.
FUDR was kindly supplied by the Zhejiang
It is well known that lipophilic neutral prodrugs of Haizheng Pharmaceutical Company (Taizhou, China).
FUDR can cross the cell membrane and potentially BOBA was prepared in our laboratory. Trityl chloride
liberate the nucleotide intracellularly. Therefore, a was purchased from Alfa Aesar. 3-[4, 5-dimethylthiazol-
number of carboxylic acid ester of FUDR were 2-yl]-2, 5-diphenyltetrazolium bromide (MTT) was
synthesized and their effects evaluated [Nishizawa purchased from Sigma (St. Louis, MO). Solvents were
et al., 1965; McGuigan et al., 1993; Wang et al., 2002]. purchased from SCRC (Xi’an, China). Flash column
Among these, O-butanoyl esters were more effective chromatography was performed on silica gel.
anticancer agents than FUDR based on oral administra-
All melting points were determined on a Beijing
tion experiments that indicated a relative potency order: micromelting-point apparatus, and the thermometer
1
30,50-di-O-butanoyl 430-O-butanoyl 450-O-butanoyl. was uncorrected. H-NMR spectra were recorded in
The greater antitumor efficiency of these esters was CDCl3 at 300 MHz on a Bruker NMR spectrometer
due in part to their slower rate of hydrolysis by with tetramethylsilane (TMS) as an internal reference.
nonspecific esterases [Yamashita et al., 1988].
All chemical shifts are reported in parts per million
Agents that induce cell differentiation or inhibit (ppm). Mass spectra were performed on a Shimadzu
cell proliferation represent another approach for GC-MS-QP2010 instrument.
cancer treatment [Sachs, 1987]. All-trans-retinoic acid
The SD male rats weighing 220725 g were
(RA) can induce cell differentiation or suppress cell purchased from Experiment Animal Center of Xi’an
proliferation in tumor cell lines, including melanoma, Jiaotong University College of Medicine and were
neuroblastoma, leukemia, breast tumor, epithelial raised in the same place. The experimental protocol
cancer, and papillomavirus-induced cancer [Smith was approved by the Ethics Committee of Xi’an
et al., 1992; Khan et al., 1993]. A prodrug combining Jiaotong University.
Drug Dev. Res.

FLOXURIDINYL AS A POTENTIAL DUAL-ACTING ANTITUMOR PRODRUG
75
Synthesis of 30-O-BOBA-FUDR
The synthetic route for 30-O-BOBA-FUDR is
concentrated in vacuo to give a residue, which was
purified by silica gel column chromatography (chloro-
form/methanol 5 100:1) to produce 0.44 g of the title
outlined in Figure 2.
1
compound as an oil. Yield 5 51.0%. H NMR d 9.55
(d, 1H, -NH), 8.10 (d, 2H, Ar-H), 7.95 (s, 1H, Ar-H),
7.90 (d, 1H, 5 CH), 7.80 (d, 1H, 5 CH, J 5 15.6 Hz),
7.44 (d, 2H, Ar-H), 7.34 (d, 1H, 5 CH, J 5 15.0 Hz),
7.29–7.36 (m, 15H, Ar-H), 6.44 (t, 1H, H-10), 5.74
(m, 1H, H-30), 3.74 (s, 3H, -OCH3), 3.69 (m, 1H, H-40),
3.45 (d, 2H, H-50), 2.53–2.78 (2m, 2H, H-20), 1.48
(s, 18H, t-Bu). MS: m/z 5 865 [M11]1, 377, 243.
50-O-tritylfloxuridine (1)
To a three-necked flask equipped with a thermo-
meter were added floxuridine (0.98 g, 3.99 mmol) and
pyridine (20 ml), trityl chloride (1.56 g, 5.60 mmol), and
4-dimethylaminopyridine (0.05 g). The mixture was
stirred under N2 at room temperature for 12 h, then
heated to 1001C and stirred for another 5 h, cooled to
room temperature, and poured into ice water (100 ml).
The resulting mixture was extracted with chloroform
(40 ml ꢁ 4). The combined organic layers were washed
sequentially with hydrochloric acid (2 M, 40 ml ꢁ 2),
saturated sodium bicarbonate solution (40 ml ꢁ 2), and
brine (40 ml ꢁ 2), dried over sodium sulfate and were
concentrated in vacuo to give a residue that was
purified by silica gel column chromography (chloro-
form/methanol 5 20:1) to give 1.65 g of the title com-
pound as a white solid. Mp 5 115–1161C ([Xia
et al., 1999], Mp 5 149–1511C); Yield 5 84.4%. 1H
NMR d: 8.32 (s, 1H, -NH), 7.81 (d, 1H, 5 CH),
7.30–7.43 (m, 15H, Ph-H), 6.27 (t, 1H, H-10), 4.55 (m,
1H, H-30), 4.15 (m, 1H, H-40), 4.06 (d, 2H, H-50),
2.24–2.49 (m, 2H, H-20). MS: m/z 5 489 [M11]1, 243.
30-floxuridinyl 4-[3-(3,5-di-t-butyl-4-
methoxyphenyl)-3-oxo-propenyl] benzoate
(30-O-BOBA-FUDR)
Compound 2 (0.44 g) was added to a solution of
80% aqueous solution of acetic acid (10 ml). The mixture
was heated at reflux for 30 min, cooled to room
temperature. Ethyl acetate (100 ml) was added. The
organic layer was washed with water, saturated sodium
bicarbonate solution (30 ml ꢁ 3) and brine (30 ml ꢁ 3),
dried over sodium sulfate, and concentrated in vacuo to
give a residue, which was purified by silica gel column
chromatography (chloroform/methanol 5 30:1) to give
the title compound as an oil, which was recrystallized
from ethanol to produce a light yellow solid (0.25 g).
Mp5128–1301C; yield578.8%; purity 597.8% (HPLC).
IR (KBr): 3465, 2960, 2358, 1716, 1704, 1652, 1606,
50-O-trityl-30-floxuridinyl 4-[3-(3,5-di-t-butyl-4-
methoxyphenyl)-3-oxo-propenyl] benzoate (2).
1
1558 cmꢂ1. H NMR d 8.58 (s, 1H, NH), 8.10 (d, 2H,
To a solution of compound 1 (0.49 g, 1 mmol) in Ar-H), 7.94 (s, 2H, Ar-H), 7.87 (d, 1H, 5 CH), 7.79
anhydrous THF (10 ml) was added BOBA (0.44 g, (d, 1H, 5 CH, J 515.9 Hz), 7.73 (d, 2H, Ar-H), 7.57
1.12 mmol), N, N0-dicyclohexylcarbodimide (0.41 g, (d, 1H, 5CH, J 5 15.6 Hz), 6.45 (t, 1H, H-10), 5.62
1.99 mmol) and 4-dimethylaminopyridine (0.05 g). (m, 1H, H-30), 4.32 (m, 1H, H-40), 4.06 (m, 2H, H-50),
The mixture was stirred under N2 at room temperature 3.74 (s, 3H, -CH3), 2.26 (m, 2H, H-20), 1.48 (s, 18H,
for 11 h. The solid was filtered off and the filtrate t-Bu). MS: m/z 5623 [M11]1, 377.
Fig. 2. Synthesis of 30-O-BOBA-FUDR. Conditions: (a) rt, 3 h; 1001C, 5 h; 84.4% (b) rt, 11 h; 51.0%; (c) reflux, 30 min, 78.8%.
Drug Dev. Res.

76
DU ET AL.
Antiproliferative Activity In Vitro
Inhibitory rates for tumor volume and tumor weight
Vernier calipers were used to determine the
longest diameter (a) and shortest diameter (b) of the
tumor. The formula
Two different human cancer cell lines, SMMC-
7721 (human hepatoma cell line) and MDA-MB-231
(human breast cancer cell line), were used in the in
vitro assay. Approximately 5 ꢁ 104 cells, containing
RPMI 1640 medium supplemented with 10% fetal
bovine serum (FBS), were added into each well of a
96-well plate and incubated in 5% CO2 at 371C for
24 h. Test compounds were added to each well at the
indicated final concentrations. The concentrations
ranged from 10 nM to 100 mM. The plate was
incubated at 371C for 48 h. Fresh MTT was added to
each well at a final concentration of 5 mg/ml and
incubated with cells at 371C for 4 h. The medium was
removed and 150 ml of DMSO was added. Optical
densities (OD570) were recorded in a microplate
reader. Results were expressed as IC50 values (n 5 5)
and were calculated by using the Bacus Laboratories
Incorporated Slide Scanner (Bliss) software.
v ¼ ab2=2
ð1Þ
was used to calculate the tumor volume.
Volume inhibition rate
¼ ð1 ꢂ mean tumor volume of test group
=saline group mean tumor volumeÞ ꢁ 100%:
ð2Þ
Tumor weight inhibition rate
¼ ð1 ꢂ mean tumor weight of experimental
group=average tumor weight of saline groupÞꢁ100%:
ð3Þ
Immunohistochemistry
Tumor tissues were removed from each group
and fixed overnight in 10% neutral formalin, followed
by gradient alcohol dehydration, and paraffin-embed-
ding, and were cut into slices. All slices were blinded.
Bcl-2 and Bax protein immunohistochemical staining
was performed according to the manufacturer’s kit
instructions. A medical pathology image analysis system
was used for data collection and analysis.
Antitumor Activity In Vivo
Establishment of experimental animal models
SD rat were inoculated sc in the bilateral armpit
and groin with hepatocellular carcinoma cells (Walker-
256) (1.5 ꢁ 107 cells per ml, 0.5 ml for each rat). Three
weeks after inoculation, solid tumors had grown to
ꢃ2 cm in diameter. The sc tumor tissue was removed,
cut into small pieces of 1-mm size, and kept in saline
for future use. Male rats were anesthetized, and an
abdominal incision was made exposing the left lateral
lobe of liver. On the liver surface, a 1-cm-long tunnel
was cut under the liver capsule, and tumor pieces
inserted. A wet cotton swab was pressed on the cut for
3 min to prevent the tumor pieces from overflowing.
The lobe of the liver was placed in the abdominal
cavity, which was sutured. Ten days after inoculation,
solid tumors grew in the liver to ꢃ1-cm diameter. The
rats were used in the experiment.
Identification of Apoptosis
Approximately 1 g of fresh tumor tissue was added
to a Petri dish containing phosphate buffered saline (PBS)
solution, digested for 30 min (0.25% trypsin), and the
process terminated by FBS. Treated tissues were filtered
through 200-mesh nylon, centrifuged at 1,000 rpm for
5 min, washed twice with PBS, and filtered again through
with 300-mesh nylon mesh and centrifuged at 1,000 rpm
for 5 min. The supernatant was discarded. RPMI 1640
culture medium was added and single-cell suspensions
made. These were centrifugated at 41C at 1,000 rpm for
5 min and washed twice with 250 ml ice-cold PBS. The
number of the cells was adjusted to 1 ꢁ 106/ml. DNA
Grouping and administration
All tumor-bearing rats were randomly divided staining of cells was performed with Annexin-V/PI double
into five groups, 10 rats per group. In the positive staining according to the manufacturer’s kit instructions.
group, FUDR was administered i.p. at 30 mg/kg once a
Statistical Analysis
day for 5 days. In the negative control group, the same
volume of saline was administered i.p. 30-O-BOBA-
All data were statistically analyzed by the SPSS10.0
software; monofactor analysis of variance and the q-test
FUDR was dissolved in DMSO/alcohol/water
(30:30:40) and administered i.p. at 3.75, 7.5, 15 mg/kg
were carried out.
for the low-, middle-, and high-dosage groups once a
day for 5 days, respectively. Rats were sacrificed on day
12. Body weight, neoplasm, and volume of neoplasm
were measured and inhibitory rates for tumor volume
and weight calculated.
RESULTS AND DISCUSSION
Synthesis of 30-O-BOBA-FUDR
To synthesize 30-BOBA ester of FUDR, the
50-hydroxy of FUDR requires selective protection.
Drug Dev. Res.

FLOXURIDINYL AS A POTENTIAL DUAL-ACTING ANTITUMOR PRODRUG
77
Reaction of trityl chloride and FUDR produced measured on day 12 (Table 2). 30-O-BOBA-FUDR
50-O-trityl FUDR (1) in the presence of pyridine and reduced the average volume and weight of transplanted
DMAP. Esterification of BOBA and 1 was carried out tumors with a consistent inhibition ratio for effects on
in the presence of dicyclohexylcarbodiimide (DCC) tumor weight and volume within groups. The anti-
and DMAP. In this process, DCC was converted to tumor efficacy of 30-O-BOBA-FUDR at 7.5 mg/kg was
dicyclohexylurea, which proved difficult to remove greater than that of FUDR at 30 mg/kg, and the
from the reaction mixture. As a result, compound 2 was efficacy was dose-dependent.
purified by using silica gel column chromatography
twice. The O-Trityl group can be removed in acidic
Tumor Bcl-2, Bax protein immunohistochemical
staining
Bcl-2 and Bax proteins in the cell cytoplasm
were yellow or brown in color. Bcl-2 protein was
condition. Refluxing of 2 in 80% acetic acid gave
30-O-BOBA-FUDR as a white solid. The structure of
30-O-BOBA-FUDR was characterized by 1H NMR,
MS, and IR.
expressed in a high percentage in the saline group.
In their studies, Xia et al. [1999] indicated that
With increasing doses of 30-O-BOBA-FUDR, Bcl-2
30-O-retinoyl-FUDR was the most promising antic-
expression was significantly diminished. Expression
ancer agent with a broad spectrum of anticancer
of Bax protein was the lowest in the saline group.
activities relative to 30, 50-di-O-retinoyl-FUDR and
With the increasing doses of 30-O-BOBA-FUDR,
50-O-retinoyl-FUDR, as well as to FUDR, RA alone, or
Bax expression gradually increased. The integral
optical density value of Bcl/Bax expression in the
in physical combination. Thus, 30-O-BOBA-FUDR
was the only compound synthesized in this work.
tumor tissue is summarized in Table 3. The results
30-O-BOBA-FUDR and FUDR were compared under
demonstrate that the differentiation activity of 30-O-
similar conditions.
BOBA-FUDR is significantly higher than that of
FUDR. This is likely because 30-O-BOBA-FUDR is
Antiproliferative Activity In Vitro
hydrolyzed to BOBA and FUDR in rats and BOBA has
FUDR was used as positive control. The results
excellent differentiation-inducing properties. Immuno-
expressed as IC50 values are summarized in Table 1. As
histochemistry results (Figs. 3 and 4) showed that 30-O-
listed in Table 1, the antiproliferative activity of 30-O-BOBA-
BOBA-FUDR decreased the expression of Bcl-2
protein and increased the expression of Bax protein
FUDR was improved over that of FUDR by 3–7-fold.
in tumor cells.
Antitumor Activity In Vivo
Inhibitory rate of tumor volume and weight
No significant weight loss was observed in all the
dosage groups. Tumor volume and weight were
TABLE 3. Integral Optical Density of Bcl/Bax Expression in Tumor
Tissue (xꢀ ꢄ s, n 5 10)
TABLE 1. Antiproliferative Activity of 30-O-BOBA-FUDR (xꢀ ꢄ s,
Group
Dosage (mg/kg)
Bcl (OD)
Bax (OD)
n 5 5)
Saline
FUDR
30-O-BOBA-
FUDR
5 ml/kg
30.0
3.75
7.50
15.0
37.5877.17
23.4674.91
13.8473.76
29.3676.64
Drug
Cell
IC50 (mM)
Ã
Ã
Ã
Ã
30-O-BOBA-FUDR
SMMC-7721
MDA-MB-231
SMMC-7721
MDA-MB-231
12.473.2
7.172.1
86.078.5
23.274.5
28.3476.39 ,z
22.8675.39 ,z
Ã
Ã
12.0474.34 ,y
37.8678.65 ,z
Ã
Ã
6.2072.24 ,y
48.0879.71 ,y
FUDR
Ã
Po0.01 vs saline group; yPo 0.01; zPo 0.05 vs FUDR group.
TABLE 2. Inhibitory Effect on Tumor Volume and Weight (xꢀ ꢄ s, n 5 10)
Average
vol (cm3)
Inhibition in
vol (%)
Inhibition
in wt (%)
Groups
Dosage (mg/kg)
Average wt (mg)
Saline
FUDR
30-O-BOBA-FUDR
5 ml/kg
30.0
3.75
7.50
15.0
2.9370.45
–
2515.57452.5
1747.67405.3
–
Ã
Ã
2.0070.38
2.5270.45
31.7
13.9
46.3
67.6
30.5
13.9
46.0
66.5
2164.67368.3
Ã
Ã
1.5770.42 ,z
1358.27343.8 ,z
Ã
Ã
0.9570.23 ,y
842.47237.8 ,y
Ã
Po 0. 01 vs saline group; yPo 0.01; zPo 0.05 vs FURR group.
Drug Dev. Res.

78
DU ET AL.
Fig. 3. Expression of Bcl-2 in rat transplanted tumor tissues. a: saline control (SP 100); b: saline control (SP 400); c: FUDR (SP 100); d: FUDR
(SP 400); e: 30-O-BOBA-FUDR (low-dosage) (SP 100); f: 30-O-BOBA-FUDR (low-dosage) (SP 400); g: 30-O-BOBA-FUDR (middle-dosage) (SP 100);
h: 30-O-BOBA-FUDR (middle-dosage) (SP 400); i: 30-O-BOBA-FUDR (high-dosage) (SP 100); j: 30-O-BOBA-FUDR (high-dosage) (SP 400) [Color
figure can be viewed in the online issue which is available at wileyonlinelibrary.com].
Drug Dev. Res.

FLOXURIDINYL AS A POTENTIAL DUAL-ACTING ANTITUMOR PRODRUG
79
Fig. 4. Expression of Bax in rat transplanted tumor tissues. a: saline control (SP 100); b: saline control (SP 400); c: FUDR (SP 100); d: FUDR
(SP 400); e: 30-O-BOBA-FUDR (low-dosage) (SP 100); f: 30-O-BOBA-FUDR (low-dosage) (SP 400); g: 30-O-BOBA-FUDR (middle-dosage) (SP 100);
h: 30-O-BOBA-FUDR (middle-dosage) (SP 400); i: 30-O-BOBA-FUDR (high-dosage) (SP 100); j: 30-O-BOBA-FUDR (high-dosage) (SP 400) [Color
figure can be viewed in the online issue which is available at wileyonlinelibrary.com].
Drug Dev. Res.

80
DU ET AL.
Apoptosis Detected by Flow Cytometry
(Po0.01) with the apoptotic ratio in saline controls
being lower than the FUDR group (Po0.01). In the
three different 30-O-BOBA-FUDR dose groups, the
ratio was improved with increasing dose. In middle-
and high-dose groups, the ratio was higher than the
FUDR group. In addition, the ratio appeared to be
dose-dependent. Figure 5 shows the apoptotic ratio of
transplanted tumor tissues in each group.
The early apoptotic ratios of transplanted tumor
cells in each group were detected by flow cytometry
(Table 4). Early apoptotic ratios are not fully identical
TABLE 4. Apoptotic Ratio of Transplanted Tumor Cells (xꢀ ꢄ s,
n 5 10)
Group
Dosage (mg/kg)
Apoptosis (%)
CONCLUSIONS
Saline
FUDR
30-O-BOBA-FUDR
5 ml/kg
30.0
3.75
7.50
15.0
2.2771.13
Compared with FUDR, 30-O-BOBA-FUDR
exhibits a higher inducing differentiation action and
antitumor efficacy in vivo and antiproliferative activity
in vitro. These findings strongly support the assump-
tion that 30-O-BOBA-FUDR is an effective dual-action
antitumor prodrug.
Ã
29.3476.24
20.0375.31z
34.46710.48y
56.67715.14y,y
Ã
Po 0.01 vs saline group; yPo 0.01 vs FUDR group; zPo 0.05 vs
y
FUDR group; Po 0.01 vs 30-O-BOBA-FUDR (middle-dosage) group.
Fig. 5. Apoptotic ratios of rat transplanted tumor tissues. a: saline control; b: FUDR (SP 100); c: 30-O-BOBA-FUDR (low-dosage); d: 30-O-
BOBA-FUDR (middle-dosage); e: 30-O-BOBA-FUDR (high-dosage) [Color figure can be viewed in the online issue which is available at
wileyonlinelibrary.com].
Drug Dev. Res.

FLOXURIDINYL AS A POTENTIAL DUAL-ACTING ANTITUMOR PRODRUG
81
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