Synthesis and Biological Evaluation of Oral Prodrugs Based on the Structure of Gemcitabine

Article abstract of DOI:10.1111/j.1747-0285.2012.01422.x

A series of oral prodrugs based on the structure of gemcitabine (2′,2′-difluorodeoxycytidine) were synthesised by introducing an amide group at the N4-position of the cytidine ring. A total of 16 compounds were obtained, and their chemical and biological characteristics were evaluated. The half-maximal inhibitory concentrations (IC₅₀s) for most of these compounds were higher than that of gemcitabine in vitro. Compounds 5d and 5m, the representative compounds, were examined in terms of their physiological stabilities and pharmacokinetics. Compound 5d showed good stability in PBS and simulated intestinal fluid, and an analysis of its pharmacokinetics in mice suggested that the introduction of an amide group to gemcitabine could greatly improve its bioavailability. Further evaluation of compound 5din vivo showed that this compound possesses higher activity than gemcitabine against the growth of HepG2 human hepatocellular carcinoma cells and HCT-116 colon adenocarcinoma cells with less toxicity to animals. These results suggest that compound 5d could be further developed as a potential oral anticancer agent for clinical applications in which gemcitabine is currently used. A series of oral prodrugs based on the structure of gemcitabine were synthesized. Physiological and metabolic stabilities, pharmacokinetics and antitumor activities were evaluated for representative compounds.

Full text of DOI:10.1111/j.1747-0285.2012.01422.x

ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01422.x
Chem Biol Drug Des 2012; 80: 479–488
Research Article
Synthesis and Biological Evaluation of Oral
Prodrugs Based on the Structure of Gemcitabine
Cuirong Zhao1, Xiaoxia Xue2, Gang Li2,
deoxycytidine kinase to form mono-, di- and triphosphate nucleo-
tides (4,5). Among these compounds, gemcitabine diphosphate
(dFdCDP) and gemcitabine triphosphate (dFdCTP) are the active
metabolites (6,7). dFdCDP could inhibit ribonucleotide reductase,
Cuicui Sun1, Changjun Sun2, Xianjun Qu1,*
and Wenbao Li2,*
1Department of Pharmacology, School of Pharmaceutical Sciences,
which is essential for DNA repair and synthesis, by decreasing the
production of deoxynucleotides, especially dCTP. dFdCTP is an inhib-
itor of DNA polymerase and may also incorporate into DNA, leading
to chain termination, strand breakage and the trapping of topoisom-
erase I cleavage complexes, which is critical for gemcitabine-
induced apoptosis and cell cycle arrest (8,9). However, in the
plasma and liver, gemcitabine can be rapidly inactivated to form a
uridine metabolite through the deamination by deoxycytidine deami-
nase (10,11). Thus, gemcitabine is not effective against hepatocarci-
noma by injection. Moreover, due to its low bioavailability and
gastrointestinal toxicity, it has to be administered intravenously
instead of orally (2,3,12).
Shandong University, Wen Hua Xi Rd 250012, Jinan, China
2Sanlugen PharmaTech Ltd., 2766 Yingxiu Road, Jinan 250101,
China
*Corresponding authors: Xian Jun Qu, qxj@sdu.edu.cn and Wen
Bao Li, wbli@sanlugen.com
A series of oral prodrugs based on the structure of
gemcitabine (2¢,2¢-difluorodeoxycytidine) were syn-
thesised by introducing an amide group at the
N4-position of the cytidine ring. A total of 16
compounds were obtained, and their chemical and
biological characteristics were evaluated. The half-
maximal inhibitory concentrations (IC50s) for most
of these compounds were higher than that of
gemcitabine in vitro. Compounds 5d and 5m, the
Prodrug approaches have been proven to be effective for the oral
delivery of several nucleoside drugs (13,14). An appropriately
designed gemcitabine prodrug could incorporate strategies that
improve intestinal absorption, decrease gastrointestinal toxicity,
slow metabolic inactivation and avoid chemoresistance (15). In this
study, we introduce a prodrug moiety at the N4-position of the
cytidine ring to form a hydrolysable amide linkage (Figure 1).
Because the amide linkage is postulated to be more stable to both
chemical and enzymatic hydrolysis (16), this series of compounds is
expected to be stable in gastrointestinal fluid, which would improve
its bioavailability, decrease its susceptibility to deamination and
reduce the effects of first-pass metabolism, maintain the active
concentration for a longer time and reduce the intestinal toxicity
(17). In this work, we report the synthesis and biological evaluation
of this series of oral prodrugs that are based on the structure of
gemcitabine.
representative
compounds,
were
examined
in terms of their physiological stabilities and
pharmacokinetics. Compound 5d showed good sta-
bility in PBS and simulated intestinal fluid, and an
analysis of its pharmacokinetics in mice suggested
that the introduction of an amide group to gemcit-
abine could greatly improve its bioavailability. Fur-
ther evaluation of compound 5d in vivo showed
that this compound possesses higher activity than
gemcitabine against the growth of HepG2 human
hepatocellular carcinoma cells and HCT-116 colon
adenocarcinoma cells with less toxicity to animals.
These results suggest that compound 5d could be
further developed as a potential oral anticancer
agent for clinical applications in which gemcita-
bine is currently used.
Key words: anticancer activity, bioavailability, gemcitabine, pharma-
cokinetics, prodrug
Experimental Section
Received 29 January 2012, revised 20 March 2012 and accepted for
publication 15 May 2012
Synthesis
Gemcitabine hydrochloride was obtained from Ningbo Teampharm
Co. Ltd., (Ningbo, Zhejiang, China). PyBop and 4-dimethylaminopyri-
dine (DMAP) were purchased from GL Biochem (Shanghai) Ltd.
(Shanghai, China). All other reagents and solvents were purchased
from commercial sources and were used without further purification
unless otherwise noted. Mass spectra were taken on a Waters
Gemcitabine (Gem) hydrochloride, 2¢,2¢-difluoro-2-deoxycytidine
hydrochloride (b-isomer), is a pyrimidine nucleoside analogue that
has shown anticancer activity against a variety of cancers, such as
non-small cell lung cancer (NSCLC), pancreatic cancer, bladder
cancer and breast cancer. Gemcitabine can be used alone or in
combination with other anticancer agents (1–3). The activation of
gemcitabine requires multiple phosphorylations by intracellular
1
Micromass ZQ instrument (Waters, Milford, Massachusetts, USA). H
NMR and 13C NMR spectra were recorded at 600 MHz (Varian
AS600). Spin multiplets are given as s (singlet), d (doublet), t (triplet)
479

Zhao et al.
J = 7.8 Hz, 1H), 8.35 (d, J = 7.8 Hz, 1H), 8.76 (d, J = 4.8 Hz, 1H),
11.72 (s, 1H).
Undecyl-3-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-
5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-
1,2-dihydropyrimidin-4-yl)carbamoyl) picolinate
(compound 5c)
Figure 1: Chemical structures of gemcitabine hydrochloride and
The title compound was prepared according to the method
described for compound 5a, except 2,3-pyridinedicarboxylic acid
was used instead of 2-(dodecyloxycarbonyl)benzoic acid and 1-un-
decanol was used instead of 1-dodecanol. A purity of 97% was
determined by UV-VIS spectrophotometry at 254 nm. LC-MS m ⁄ z
567 [M + H]+, calculated MW: 566 for C27H36F2N4O7. 1H NMR
(DMSO, 600 MHz) d: 0.85 (t, J = 7.2 Hz, 3H), 1.18–1.27 (m, 16H),
1.58 (m, 2H), 3.67 (m, 1H), 3.82 (m, 1H), 3.91 (m, 1H), 4.21 (m, 3H),
5.33 (t, J = 5.4 Hz, 1H), 6.19 (t, J = 7.2 Hz, 1H), 6.34 (d, J = 6.6 Hz,
1H), 7.35 (d, J = 7.7 Hz, 1H), 7.70 (m, 1H), 8.13 (d, J = 7.8 Hz, 1H),
8.35 (d, J = 7.8 Hz, 1H), 8.77 (d, J = 4.2 Hz, 1H), 11.72 (s, 1H).
prodrugs of gemcitabine.
and m (multiplet). Coupling constants are reported in Hz. In most
cases, the purities of compounds were >95% as determined by a UV-
VIS spectrophotometer at 254 nm.
Dodecyl-2-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-
5-(hydroxymethyl) -tetrahydrofuran-2-yl)-2-oxo-
1,2-dihydropyrimidin-4-yl)carbamoyl) benzoate
(compound 5a)
A mixture of o-phthalic anhydride (5.0 g, 34 mmol), 1-dodecanol
(7.5 g, 40 mmol) and DMAP (240 mg, 2 mmol) were influxed in THF
for 4 h. The reaction mixture was then cooled down to give 2-
(dodecyloxycarbonyl) benzoic acid, which was used for the next step
without further purification.
Dodecyl-3-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-
5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-1,
2-dihydropyrimidin-4-yl)carbamoyl) pyrazine-2-
carboxylate (compound 5d)
A solution of gemcitabine hydrochloride (0.7 g, 2.34 mmol), 2-(dode-
cyloxycarbonyl) benzoic acid (1.02 g, 3.04 mmol), PyBop (1.34 g,
2.57 mmol) and DMAP (428 mg, 3.51 mmol) in N, N-dimethylforma-
mide (DMF, 10 mL) was stirred overnight at room temperature. The
reaction mixture was poured into water and then extracted three
times with ethyl acetate. The combined organic phases were dried
over anhydrous Na2SO4, filtered and concentrated under reduced
pressure. The residue was purified by column chromatography (silica
gel, DCM:Methanol = 30:1) to give the title compound (362.5 mg).
A purity of 98% was determined by UV-VIS spectrophotometry at
254 nm. LC-MS m ⁄ z 580 [M + H]+, calculated MW: 579 for
C29H39F2N3O7. 1H NMR (DMSO, 600 MHz) d: 0.85 (t, J = 7.2 Hz,
3H), 1.17–1.25 (m, 18H), 1.55 (m, 2H), 3.67 (m, 1H), 3.82 (m, 1H),
3.91 (m, 1H), 4.18 (t, J = 10.8 Hz,3H), 4.23 (m, 1H), 5.33 (m, 1H),
6.18 (t, J = 7.2 Hz, 1H), 6.34 (d, J = 6.0 Hz, 1H), 7.39 (d,
J = 7.8 Hz, 1H), 7.58 (d, J = 7.2 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H),
7.68 (t, J = 7.2 Hz, 1H), 7.88 (d, J = 7.8 Hz, 1H), 8.33 (d,
J = 7.2 Hz, 1H), 11.56 (s, 1H).
N, N-dimethylformamide (2 drops) was added to a solution of 2, 3-
pyrazinedicarboxylic acid (504 mg, 3.0 mmol) in 15 mL of SOCl2.
The reaction mixture was refluxed for 5 h, and then the solvent
was removed under reduced pressure to give pyrazine-2, 3-dicarbon-
yl dichloride, which was used in the next step without further puri-
fication.
A solution of 1-dodecanol (558 mg, 3.0 mmol) and Et3N (2 mL) in
dioxane (5 mL) was added dropwise to a stirred, cooled (ice-water
bath) mixture of pyrazine-2, 3-dicarbonyl dichloride (612 mg,
3.0 mmol) and dioxane (20 mL), and the reaction was stirred at
room temperature for 4 h. The solvent was removed under reduced
pressure, and the residue was then dissolved in water. After adjust-
ing the pH of the solution to 2–3, it was extracted three times with
ethyl acetate. The combined organic phases were dried over anhy-
drous Na2SO4, filtered and concentrated to give 3-(dodecyloxycar-
bonyl) pyrazine-2-carboxylic acid, which was directly used in the
next step without further purification.
A solution of gemcitabine hydrochloride (500 mg, 1.67 mmol), 3-
(dodecyloxycarbonyl) pyrazine-2-carboxylic acid (674 mg, 2.01 mmol),
PyBop (955 mg, 1.84 mmol) and DMAP (244 mg, 2.0 mmol) in DMF
(10 mL) was stirred at room temperature overnight. The reaction
mixture was poured into water and extracted three times with ethyl
acetate. The combined organic phases were dried over anhydrous
Na2SO4, filtered and concentrated under reduced pressure. The resi-
due was purified by column chromatography (silica gel,
DCM:MeOH = 40:1) to give the title compound (225.7 mg). A purity
of 98% was determined by UV-VIS spectrophotometry at 254 nm.
LC-MS m ⁄ z 582 [M + H]+, calculated MW: 581 for C27H37F2N5O7.
1H NMR (DMSO, 600 MHz) d: 0.85 (t, J = 6.6 Hz, 3H), 1.22–1.30
(m, 18H), 1.62 (m, 2H), 3.67 (m, 1H), 3.82 (m, 1H), 3.92 (m, 1H), 4.21
(m, 1H), 4.30 (t, J = 6.0 Hz, 2H), 5.33 (t, J = 5.4 Hz, 1H), 6.18 (m,
Dodecyl-3-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-
5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-
1,2-dihydropyrimidin-4-yl)carbamoyl) picolinate
(compound 5b)
The title compound was prepared according to the method
described for compound 5a, except 2, 3-pyridinedicarboxylic acid
was used instead of 2, 3-pyrazinedicarboxylic acid. A purity of
97% was determined by UV-VIS spectrophotometry at 254 nm. LC-
MS m ⁄ z 581 [M + H]+, calculated MW: 580 for C28H38F2N4O7. 1H
NMR (DMSO, 600 MHz) d: 0.85 (t, J = 6.6 Hz, 3H), 1.18–1.27 (m,
18H), 1.58 (m, 2H), 3.67 (m, 1H), 3.82 (m, 1H), 3.91 (m, 1H), 4.25
(m, 3H), 5.33 (t, J = 4.8 Hz, 1H), 6.19 (t, J = 6.6 Hz, 1H), 6.34 (d,
J = 6.6 Hz, 1H), 7.35 (d, J = 7.2 Hz, 1H), 7.70 (m, 1H), 8.13 (d,
480
Chem Biol Drug Des 2012; 80: 479–488

Synthesis and Evaluation of Gemcitabine Oral Prodrugs
1H), 6.34 (d, J = 6.6 Hz, 1H), 7.28 (s, 1H), 8.38 (d, J = 7.8 Hz, 1H),
8.94 (dd, J1 = 10.8 Hz, J2 = 1.8 Hz, 2H), 11.50 (s, 1H). 13C NMR
(DMSO,600MHz) d: 14.61, 22.76, 26.04, 28.53, 29.28, 29.37, 29.54,
29.60, 29.66, 29.67, 31.96, 59.28, 66.80, 68.92, 81.755, 81.863,
96.58, 143.01, 146.18, 146.60, 146.74, 154.55, 162.98, 164.34,
165.83.
Undecyl-6-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-
5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-
1,2-dihydropyrimidin-4-yl)carbamoyl) nicotinate
(compound 5h)
The title compound was prepared according to the method
described for compound 5d, except 2, 5-pyridinedicarboxylic acid
was used instead of 2, 3-pyrazinedicarboxylic acid, and 1-undecanol
was used instead of 1-dodecanol. A purity of 99% was determined
by UV-VIS spectrophotometry at 254 nm. LC-MS m ⁄ z 567 [M + H]+,
Undecyl-3-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-
5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-
1,2-dihydropyrimidin-4-yl)carbamoyl) pyrazine-
2-carboxylate (compound 5e)
1
calculated MW: 566 for C27H36F2N4O7. H NMR (DMSO, 600 MHz)
d: 0.84 (t, J = 6.6 Hz, 3H), 1.24–1.44 (m, 16H), 1.73 (m, 2H), 3.68
(m, 1H), 3.82 (m, 1H), 3.93 (m, 1H), 4.22 (m, 1H), 4.35 (t, J = 6.6 Hz,
2H), 5.38 (t, J = 4.8 Hz, 1H), 6.21 (t, J = 6.6 Hz, 1H), 6.38 (d,
J = 6.0 Hz, 1H), 7.45 (d, J = 7.2 Hz, 1H), 8.32 (d, J = 8.4 Hz, 1H),
8.44 (d, J = 7.8 Hz, 1H), 8.57 (dd, J1 = 8.4 Hz, J2 = 1.8 Hz, 1H),
9.20 (s, 1H), 10.64 (s, 1H).
The title compound was prepared according to the method
described for compound 5d, except 1-undecanol was used instead
of 1-dodecanol, and tetrahydrofuran was used instead of dioxane. A
purity of 97% was determined by UV-VIS spectrophotometry at
254 nm. LC-MS m ⁄ z 568 [M + H]+, calculated MW: 567 for
C26H35F2N5O7. 1H NMR (DMSO, 600 MHz) d: 0.85 (t, J = 6.6 Hz,
3H), 1.22–1.30 (m, 16H), 1.62 (m, 2H), 3.67 (m, 1H), 3.82 (m, 1H),
3.92 (m, 1H), 4.21 (m, 1H), 4.29 (t, J = 6.6 Hz, 2H), 5.35 (m, 1H),
6.18 (m, 1H), 6.35 (d, J = 6.6 Hz, 1H), 7.28 (d, J = 6.6 Hz, 1H), 8.38
(d, J = 7.2 Hz, 1H), 8.94 (dd, J1 = 10.8 Hz, J2 = 2.4 Hz, 2H), 11.51
(s, 1H).
Undecyl-5-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-
5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-
1,2-dihydropyrimidin-4-yl)carbamoyl) picolinate
(compound 5i)
The title compound was prepared according to the method
described for compound 5a, except 2, 5-pyridinedicarboxylic acid
was used instead of 2-(dodecyloxycarbonyl)benzoic acid, and 1-un-
decanol was used instead of 1-dodecanol. A purity of 97% was
determined by UV-VIS spectrophotometry at 254 nm. LC-MS m ⁄ z
567 [M + H]+, calculated MW: 566 for C27H36F2N4O7. 1H NMR
(DMSO, 600 MHz) d: 0.85 (t, J = 6.0 Hz, 3H), 1.23–1.39 (m, 18H),
1.73 (m, 2H), 3.68 (m, 1H), 3.83 (m, 1H), 3.93 (m, 1H), 4.22 (m, 1H),
4.33 (t, J = 6.6 Hz, 2H), 5.35 (m, 1H), 6.22 (t, J = 6.6 Hz, 1H), 6.35
(d, J = 6.0 Hz, 1H), 7.39 (s, 1H), 8.15 (t, J = 8.4 Hz, 1H), 8.36 (s,
1H), 8.50 (m, 1H), 9.20 (s, 1H), 11.81 (s, 1H).
Dodecyl-6-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-
5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-
1,2-dihydropyrimidin-4-yl)carbamoyl) nicotinate
(compound 5f)
The title compound was prepared according to the method
described for compound 5d, except 2, 5-pyridinedicarboxylic acid
was used instead of 2, 3-pyrazinedicarboxylic acid, and the reaction
time was 3 h. A purity of 97% was determined by UV-VIS spectro-
photometry at 254 nm. LC-MS m ⁄ z 581 [M + H]+, calculated MW:
580 for C28H38F2N4O7. 1H NMR (DMSO, 600 MHz) d: 0.84 (t,
J = 6.6 Hz, 3H), 1.23–1.30 (m, 18H), 1.72 (m, 2H), 3.67 (m, 1H), 3.82
(m, 1H), 3.92 (m, 1H), 4.22 (m, 1H), 4.35 (t, J = 6.6 Hz, 2H), 5.37 (s,
1H), 6.21 (t, J = 7.2 Hz, 1H), 6.37 (d, J = 6.6 Hz, 1H), 7.45 (d,
J = 7.2 Hz, 1H), 8.32 (d, J = 8.4 Hz, 1H), 8.44 (d, J = 7.8 Hz, 1H),
8.57 (dd, J1 = 8.4 Hz, J2 = 1.8 Hz, 1H), 9.21 (s, 1H), 10.65 (s, 1H).
Dodecyl-6-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-
5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-
1,2-dihydropyrimidin-4-yl)carbamoyl) picolinate
(compound 5j)
The title compound was prepared according to the method
described for compound 5d, except 2, 6-pyridinedicarboxylic acid
was used instead of 2, 3-pyrazinedicarboxylic acid. A purity of 96%
was determined by UV-VIS spectrophotometry at 254 nm. LC-MS
Dodecyl-5-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-
5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-
1,2-dihydropyrimidin-4-yl)carbamoyl) picolinate
(compound 5g)
1
m ⁄ z 581 [M + H]+, calculated MW: 580 for C28H38F2N4O7. H NMR
(DMSO, 600 MHz) d: 0.84 (t, J = 7.2 Hz, 3H), 1.23–1.28 (m, 18H),
1.73 (m, 2H), 3.68 (m, 1H), 3.82 (m, 1H), 3.92 (m, 1H), 4.24 (m, 1H),
4.40 (t, J = 6.6 Hz, 2H), 5.37 (s, 1H), 6.23 (t, J = 6.6 Hz, 1H), 6.37
(d, J = 6.6 Hz, 1H), 7.48 (d, J = 7.8 Hz, 1H), 8.33 (t, J = 7.2 Hz, 1H),
8.36 (dd, J1 = 7.8 Hz, J2 = 1.2 Hz, 1H), 8.41 (dd, J1 = 7.2 Hz,
J2 = 1.2 Hz, 1H), 8.44 (d, J = 7.8 Hz, 1H), 10.47 (s, 1H).
The title compound was prepared according to the method
described for compound 5a, except 2, 5-pyridinedicarboxylic acid
was used instead of 2-(dodecyloxycarbonyl)benzoic acid, and
pyridine was used instead of dioxane. A purity of 95% was
detected by UV-VIS spectrophotometry at 254 nm. LC-MS m ⁄ z 581
1
[M + H]+, calculated MW: 580 for C28H38F2N4O7. H NMR (DMSO,
600 MHz) d: 0.84 (t, J = 6.6 Hz, 3H), 1.23–1.33 (m, 18H), 1.72 (m,
2H), 3.67 (m, 1H), 3.83 (m, 1H), 3.93 (m, 1H), 4.22 (m, 1H), 4.33 (t,
J = 6.6 Hz, 2H), 5.35 (s, 1H), 6.22 (t, J = 6.6 Hz, 1H), 6.35 (d,
J = 5.4 Hz, 1H), 7.39 (s, 1H), 8.15 (d, J = 7.8 Hz, 1H), 8.36 (d,
J = 6.0 Hz, 1H), 8.50 (dd, J1 = 7.8 Hz, J2 = 2.4 Hz, 1H), 9.20 (s,
1H), 11.82 (s, 1H).
Undecyl-2-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-
5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-
1,2-dihydropyrimidin-4-yl)carbamoyl)
isonicotinate (compound 5k)
The title compound was prepared according to the method
described for compound 5d, except 2, 4-pyridinedicarboxylic acid
Chem Biol Drug Des 2012; 80: 479–488
481

Zhao et al.
was used instead of 2, 3-pyrazinedicarboxylic acid, and 1-undecanol
was used instead of 1-dodecanol. A purity of 95% was determined
by UV-VIS spectrophotometry at 254 nm. LC-MS m ⁄ z 567 [M + H]+,
Dodecyl-5-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-
5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-
1,2-dihydropyrimidin-4-yl)carbamoyl) thiophene-
2-carboxylate (compound 5o)
1
calculated MW: 566 for C27H36F2N4O7. H NMR (DMSO, 600 MHz)
d: 0.84 (t, J = 6.6 Hz, 3H), 1.22–1.43 (m, 16H), 1.73 (m, 2H), 3.67
(m, 1H), 3.83 (m, 1H), 3.93 (m, 1H), 4.22 (m, 1H), 4.36 (t, J = 6.6 Hz,
2H), 5.37 (s, 1H), 6.21 (t, J = 6.6 Hz, 1H), 6.36 (s, 1H), 7.47 (d,
J = 7.8 Hz, 1H), 8.17 (s, 1H), 8.43 (d, J = 7.2 Hz, 1H), 8.49 (s, 1H),
8.98 (s, 1H), 10.62 (s, 1H).
The title compound was prepared according to the method
described for compound 5d, except thiophene-2, 5-dicarboxylic acid
was used instead of 2, 3-pyrazinedicarboxylic acid. A purity of 95%
was determined by UV-VIS spectrophotometry at 254 nm. LC-MS
1
m ⁄ z 586 [M + H]+, calculated MW: 585 for C27H37F2N3O7S. H NMR
(DMSO, 600 MHz) d: 0.84 (t, J = 6.6 Hz, 3H), 1.18–1.29 (m, 18H),
1.68 (m, 2H), 3.67 (m, 1H), 3.81 (m, 1H), 3.92 (m, 1H), 4.20 (m, 1H),
4.28 (t, J = 6.6 Hz, 2H), 5.35 (t, J = 4.2 Hz, 1H), 6.20 (s, 1H), 6.35
(d, J = 6.6 Hz, 1H), 7.36 (brs, 1H), 7.83 (d, J = 4.2 Hz, 1H), 8.32
(brd, 2H), 11.75 (s, 1H).
Undecyl-3-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-
5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-
1,2-dihydropyrimidin-4-yl)carbamoyl)
isonicotinate (compound 5l)
The title compound was prepared according to the method
described for compound 5d, except 3, 4-pyridinedicarboxylic acid
was used instead of 2, 3-pyrazinedicarboxylic acid, and 1-undecanol
was used instead of 1-dodecanol. A purity of 97% was determined
by UV-VIS spectrophotometry at 254 nm. LC-MS m ⁄ z 567 [M + H]+,
Undecyl-5-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-
5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-1,
2-dihydropyrimidin-4-yl)carbamoyl) 1H-pyrazole-
3-carboxylate (compound 5p)
1
calculated MW: 566 for C27H36F2N4O7. H NMR (DMSO, 600 MHz)
The title compound was prepared according to the method
described for compound 5d, except 3, 5-pyrazoledicarboxylic acid
was used instead of 2, 3-pyrazinedicarboxylic acid, and 1-undecanol
was used instead of 1-dodecanol. A purity of 99% was determined
by UV-VIS spectrophotometry at 254 nm. LC-MS m ⁄ z 556 [M + H]+,
d: 0.85 (t, J = 6.6 Hz, 3H), 1.17–1.26 (m, 16H), 1.56 (m, 2H), 3.67
(m, 1H), 3.82 (m, 1H), 3.92 (m, 1H), 4.24 (m, 3H), 5.33 (t, J = 4.8 Hz,
1H), 6.19 (t, J = 4.8 Hz, 1H), 6.35 (d, J = 6.6 Hz, 1H), 7.37 (d,
J = 7.8 Hz, 1H), 7.63 (d, J = 4.8 Hz, 1H), 8.36 (d, J = 7.2 Hz, 1H),
8.87 (d, J = 5.4 Hz, 1H), 8.89 (d, J = 4.8 Hz, 1H), 11.71 (s, 1H).
1
calculated MW: 555 for C25H35F2N5O7. H NMR (DMSO, 600 MHz)
d: 0.84 (t, J = 6.6 Hz, 3H), 1.16–1.40 (m, 16H), 1.68 (m, 2H), 3.67
(m, 1H), 3.82 (m, 1H), 3.91 (m, 1H), 4.22 (m, 1H), 4.26 (t, J = 6.6 Hz,
2H), 5.36 (t, J = 6.0 Hz, 1H), 6.20 (t, J = 7.2 Hz, 1H), 6.35 (d,
J = 5.4 Hz, 1H), 7.41 (d, J = 7.2 Hz, 1H), 7.50–7.91 (brd, 1H), 8.35
(d, J = 7.8 Hz, 1H), 10.56–11.61 (brd, 1H), 14.60–14.80 (brd, 1H).
Dodecyl-2-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-
5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-
1,2-dihydropyrimidin-4-yl)carbamoyl)
isonicotinate (compound 5m)
The title compound was prepared according to the method
described for compound 5d, except 2, 4-pyridinedicarboxylic acid
was used instead of 2, 3-pyrazinedicarboxylic acid. A purity of 96%
was detected by UV-VIS spectrophotometry at 254 nm. LC-MS m ⁄ z
581 [M + H]+, calculated MW: 580 for C28H38F2N4O7. 1H NMR
(DMSO, 600 MHz) d: 0.84 (t, J = 6.6 Hz, 3H), 1.22–1.43 (m, 18H),
1.73 (m, 2H), 3.67 (m, 1H), 3.83 (m, 1H), 3.93 (m, 1H), 4.22 (m, 1H),
4.36 (t, J = 6.6 Hz, 2H), 5.36 (s, 1H), 6.21 (t, J = 6.6 Hz, 1H), 6.36
(s, 1H), 7.47 (d, J = 7.8 Hz, 1H), 8.17 (d, J = 7.8 Hz, 1H), 8.43 (d,
J = 7.2 Hz, 1H), 8.49 (s, 1H), 8.98 (s, 1H), 10.62 (s, 1H).
Physiological stability in vitro
Gemcitabine and its prodrug derivatives were dissolved in PBS
(pH = 7.4) and simulated intestinal fluid (pH = 6.8) (18). Their stabil-
ities were determined by measuring the concentrations of gemcita-
bine and its prodrugs after 0, 1, 2, 4, 6, 8, 12 and 24 h using an
LC-10ATVP-ODS HPLC with an SPD-10A VP UV detector (Shimadzu).
Experiments were performed in triplicate.
Pharmacokinetics of gemcitabine
Gemcitabine was dissolved in physiological saline to prepare a
standard solution at a concentration of 6.25 mg ⁄ mL. A total of 120
healthy Kunming mice were randomly divided into two groups; one
group received the drug orally, and the other group was injected
intravenously through the tail vein. Before administration, the solu-
tion was diluted with a physiological buffer to a dosage of
50 mg ⁄ kg (equivalent to 0.2 mL per mouse). The mice were food-
restricted for 12 h before administration but allowed to drink water.
Five mice were tested in parallel at each time point. Blood samples
were drawn from the eyebase veniplex at 5, 15, 30, 45 min, 1, 2,
4, 6, 8, 12, 24 and 48 h after administration. The plasma samples
were treated for HPLC analysis according to the method described
previously (19). The chromatographic peak areas were used to
determine the plasma concentrations of gemcitabine and its
pharmacokinetic parameters.
Dodecyl-4-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-
5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-
1,2-dihydropyrimidin-4-yl)carbamoyl) picolinate
(compound 5n)
The title compound was prepared according to the method
described for compound 5a, except 2, 4-pyridinedicarboxylic acid
was used instead of 2-(dodecyloxycarbonyl)benzoic acid. A purity of
96% was determined by UV-VIS spectrophotometry at 254 nm. LC-
MS m ⁄ z 581 [M + H]+, calculated MW: 580 for C28H38F2N4O7. 1H
NMR (DMSO, 600 MHz) d: 0.85 (t, J = 6.6 Hz, 3H), 1.23–1.39 (m,
18H), 1.73 (m, 2H), 3.67 (m, 1H), 3.83 (m, 1H), 3.93 (m, 1H), 4.22 (m,
1H), 4.35 (t, J = 6.6 Hz, 2H), 5.37 (s, 1H), 6.22 (t, J = 6.6 Hz, 1H),
6.36 (s, 1H), 7.38 (d, J = 7.8 Hz, 1H), 8.05 (d, J = 7.8 Hz, 1H), 8.37
(d, J = 7.2 Hz, 1H), 8.49 (s, 1H), 8.95 (s, 1H), 11.88 (s, 1H).
482
Chem Biol Drug Des 2012; 80: 479–488

Synthesis and Evaluation of Gemcitabine Oral Prodrugs
Pharmacokinetics of compound 5d and
compound 5m
(Beijing, China). Animals were housed under pathogen-free conditions
according to the institutional guidelines. Cancer cells (1 · 107)
suspended in 100 lL of Matrigel (Collaborative Biomedical, Spring-
field, MA, USA) were injected subcutaneously into the right anterior
flank of each mouse (21). After 2 weeks, when tumours were estab-
lished with a mean volume of 200–300 mm3, mice were given an oral
dose of compound 5d suspended in 0.5 mL of 5% amylum (HepG2
xenografts: 0, 12.9, 17.2, 21.5 lmol⁄ kg (namely 0, 7.5, 10, 12.5 mg ⁄kg)
daily and HCT-116 xenografts: 0, 17.2, 21.5, 25.8 lmol ⁄ kg (namely 0,
10, 12.5, 15 mg ⁄ kg) daily). Other mice were injected with gemcitabine
via the tail vein once every 3 days (33.4 lmol ⁄ kg (namely 10 mg ⁄ kg)
for HepG2 xenografts and 41.8 lmol ⁄ kg (namely 12.5 mg ⁄ kg) for
HCT-116 xenografts) to serve as a positive control. Administration
was performed for two or three consecutive weeks. Tumour volumes
were determined by measurement of the two perpendicular diameters
with vernier callipers and calculated with the formula: 1 ⁄ 2 (large
diameter) · (small diameter) (2,22). Inhibition of tumour growth was
defined as a percentage of the control tumour weight.
A total of 125 mg of compound 5d or compound 5m were weighed
into a 10 mL volumetric flask. The flask was then filled with water
(containing 0.1% Tween 80) and mixed to obtain uniform suspensions
of compound 5d or compound 5m. A total of 120 healthy Kunming
mice were randomly divided into two groups. Compound 5d and com-
pound 5m were administered by gavages at a dosage of 100 mg ⁄ kg
(equivalent to 0.2 mL per mouse). Five mice were tested in parallel at
each time point. Blood samples were taken from the eyebase
veniplex at 5, 15, 30, 45 min, 1, 2, 4, 6, 8, 12, 24 and 48 h after
administration. The blood samples were processed for HPLC analysis
according to the method described in (19). The peak areas were used
to calculate the plasma concentrations of compound 5d or compound
5m, and mean drug-time curves were drawn based on these values.
Cell lines and cell culture
The human NSCLC cell lines NCI-H460 and A549, the human hepa-
toma cell lines Bel7402 and HepG2 and the human colon cancer
cell lines HCT-116 were purchased from Shanghai Cell Bank, China
Academy of Sciences (Shanghai, China). Cells were maintained in
RPMI-1640 medium supplemented with 10% (v ⁄ v) heat-inactivated
fetal bovine serum, penicillin-streptomycin (100 IU ⁄ mL–100 lg ⁄ mL),
2 mM glutamine and 10 mM HEPES buffer at 37 ꢀC in a humidified
atmosphere of 95% air ⁄ 5% carbon dioxide. Culture medium was
changed every 2 days. Cells were harvested by brief incubation in
0.02% (w ⁄ v) trypsin in PBS (ICN, Aurora, OH, USA).
Statistical analysis
Data were expressed as the mean € SD and analysed by Student's
two-tailed t-test. AUC was calculated by the trapezoidal rule using
NCSS software. Statistical analysis was performed with SPSS ⁄ WIN13.0
software (SPSS Inc., Chicago, IL, USA). A p value < 0.05 was consid-
ered statistically significant.
Results and Discussion
Inhibition of cell proliferation in vitro by the
MTT assay
Chemistry
The synthetic route to gemcitabine derivatives is shown in
Scheme 1. The substituted acids R1COOH were obtained either by
reactions between acid anhydrides (R2CO)2O and various alcohols
R3OH in a 1:1.2 molar ratio or by reactions of acyl chlorides R2COCl
with alcohols R3OH in a 1:1 molar ratio. To make isomeric pairs,
such as compounds 5f and 5g, compounds 5h and 5i, or com-
pounds 5m and 5n (Table 1), two different methods were used.
The substituted acids in which the N atom of the pyridine ring is
closer to the R3 group were synthesised by the reactions of acyl
chlorides R2COCl with R3OH (23–25), whereas the substituted acids
in which the N atom of the pyridine ring is closer to the amide
bond were synthesised by the reactions of R2COOH with coupling
reagents, such as dicyclohexylcarbodiimide or benzotriazole-1-yl-oxy-
tripyrrolidinophosphonium hexafluorophosphate (PyBop), according to
the previously published papers (26,27).
Cancer cells seeded in 96-well plates (3 · 103 per well) were
exposed to various concentrations of prodrugs or gemcitabine for
72 h. All wells were treated with an equal volume of vehicle DMSO.
Cell viability was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay by adding 20 lL of MTT
(5 mg ⁄ mL; Sigma, St. Louis, Missouri, USA) and incubating for 4 h
(20). The absorbance of the solution was measured at 570 nm on a
microplate reader (Perkin-Elmer, Waltham, Massachusetts, USA), and
the IC50s were calculated. Experiments were performed in triplicate.
Inhibition of tumour growth in vivo
Compound 5d was further evaluated in terms of its inhibitory effects
on the growth of human hepatocellular carcinoma HepG2 xenografts
and human colon adenocarcinoma HCT-116 xenografts in mice. Balb ⁄ c
athymic (nu+ ⁄ nu+) female mice at 5–6 weeks of age were purchased
from the Animal Centre of China Academy of Medical Sciences
The desired compounds were synthesised by reacting the substi-
tuted acids R1COOH obtained above with gemcitabine hydrochloride
Scheme 1: Synthesis of gemcitabine derivatives 5a–5p.
Chem Biol Drug Des 2012; 80: 479–488
483

Zhao et al.
Table 1: Structures of desired prodrugs and IC50 (lM) for five human cancer cell lines (mean € SD)
Compounds
R2
R3
A549
NCI-H460
ND
Bel7402
20 € 1.7
HepG2
ND
HCT-116
13 € 1.0
O
5a
C12H25
23 € 1.9
N
H
O
O
O
O
5b
5c
5d
5e
C12H25
C11H23
C12H25
C11H23
ND
ND
60 € 5.9
4 € 3.3
8 € 0.6
16 € 1.4
ND
20 € 1.9
20 € 1.8
20 € 1.4
30 € 2.2
N
H
N
N
N
N
3 € 2.1
20 € 1.8
ND
1 € 0.1
20 € 1.7
2 € 0.3
4 € 2.9
6 € 0.7
4 € 0.6
N
H
N
H
N
N
N
H
O
5f
C12H25
4 € 0.3
4 € 0.2
20 € 2.5
2 € 0.3
23 € 2.8
13 € 1.5
7 € 0.9
6 € 0.7
6 € 0.5
3 € 0.1
N
H
N
O
5g
C12H25
N
H
N
O
O
5h
5i
C11H23
13 € 1.6
3 € 0.5
ND
ND
30 € 2.6
4 € 0.3
ND
ND
4 € 0.3
2 € 0.2
N
H
N
C11H23
N
H
N
N
O
O
5j
C12H25
C11H23
C11H23
15 € 1.3
8 € 1.1
8 € 1.2
ND
ND
ND
16 € 1.9
8 € 1.6
ND
ND
ND
7 € 0.9
2 € 0.4
N
H
5k
5l
N
H
N
O
16 € 2.1
30 € 3.4
N
H
N
O
5m
C12H25
3 € 0.5
10 € 1.2
8 € 1.1
2 € 0.3
20 € 2.4
N
H
N
484
Chem Biol Drug Des 2012; 80: 479–488

Synthesis and Evaluation of Gemcitabine Oral Prodrugs
Table 1: Continued
Compounds
R2
R3
A549
NCI-H460
1 € 0.6
Bel7402
4 € 0.6
HepG2
1 € 0.2
HCT-116
2 € 0.3
O
O
5n
C12H25
4 € 3.9
N
H
N
5o
5p
C12H25
13 € 1.6
14 € 1.8
ND
ND
14 € 1.3
16 € 1.9
ND
ND
6 € 0.8
4 € 0.7
S
N
H
O
C11H23
H
N
N
N
H
Gemcitabine
–
–
<2
<1
4 € 0.5
<1
7 € 0.5
ND, not detected.
IC50 > 200 lM.
in the presence of PyBop and DMAP. The reactants were dissolved
in an organic solvent and stirred at room temperature for 12–24 h.
The reaction solutions were then poured into water and extracted
with an organic solvent. The isolated organic phase was dried and
purified to give the desired products. Ultimately, 16 gemcitabine
derivatives were obtained, and their chemical and biological charac-
teristics are summarized in Table 1.
Table 2: Physiological stability of gemcitabine, compound 5d
and 5m in PBS (pH 7.4) (expressed as percent remaining drugs at
indicated time, mean € SD)
Hours
Gemcitabine
Compound 5d
Compound 5m
0
1
2
4
6
8
12
24
100.0 € 0.0
98.4 € 9.6
99.1 € 8.9
98.5 € 9.0
94.1 € 8.4
96.6 € 9.7
91.3 € 8.8
91.8 € 7.9
100.0 € 0.0
95.0 € 9.1
99.5 € 8.5
93.5 € 9.2
90.1 € 8.1
90.6 € 9.5
96.6 € 9.5
96.9 € 8.9
100.0 € 0.0
94.7 € 9.3
98.1 € 8.8
98.2 € 8.9
103.4 € 10.6
97.5 € 10.5
102.6 € 11.0
100.2 € 9.3
Biological activity in vitro
Five human cancer cell lines, including two lung carcinoma cell
lines (NCI-H460 and A549), two hepatocellular carcinoma cell lines
(Bel7402 and HepG2) and one colon adenocarcinoma cell line (HCT-
116), were employed to evaluate the inhibitory activity of these
gemcitabine derivatives using the MTT assay. The compounds dis-
played various inhibitory activities on the proliferation of cancer
cells. The half-maximal inhibitory concentrations (IC50s) of these
compounds after 72 h incubation with the cancer cells are summa-
rized in Table 1. The IC50s of most compounds were higher than
that of gemcitabine in these cell lines, implying that these deriva-
tives exhibit weaker inhibitory activities on cancer cell growth in vi-
tro. However, a strong inhibitory activity was observed in the
presence of liver microsomal enzymes (data not shown), suggesting
that these prodrugs might be metabolised to gemcitabine by the
enzymes. The design and synthesis of these compounds incorpo-
rates prodrug strategies for gemcitabine, and we selected represen-
tative compounds for further study.
Table 3: Physiological stability of gemcitabine, compound 5d
and 5m in the simulated intestinal fluid (expressed as percent
remaining drugs at indicated time, mean € SD)
Hours
Gemcitabine
Compound 5d
Compound 5m
0
1
2
4
6
8
12
24
100.0 € 0.0
72.3 € 6.5
63.8 € 7.8
40.0 € 5.6
30.6 € 3.0
23.3 € 3.6
1.5 € 0.4
100.0 € 0.0
80.0 € 7.9
67.9 € 7.5
85.0 € 9.7
85.8 € 8.9
83.0 € 8.8
85.7 € 7.7
88.9 € 7.9
100.0 € 0.0
98.2 € 10.4
104.0 € 9.9
104.0 € 9.5
106.0 € 10.6
109.0 € 11.0
99.8 € 9.4
0.9 € 0.1
95.7 € 9.6
Physiological stability in vitro
respectively, after 4 or 12 h of incubation in the simulated intestinal
fluid. These results confirmed the idea that introducing an amide
linkage into the structure of gemcitabine might render it stable to
both chemical and enzymatic hydrolysis (16). Thus, we hypothesised
that compounds 5d and 5m would retain higher levels of bioavail-
ability in the intestine than the parent drug. Additionally, it is logi-
cal to suggest that these prodrugs might also exhibit less intestinal
toxicity than gemcitabine.
Compounds 5d and 5m were selected as representative gemcita-
bine prodrug derivatives, and their stabilities in phosphate buffered
solution (PBS, pH = 7.4) and simulated intestinal fluid (pH = 6.8)
were examined using gemcitabine as a control. The results showed
that all three compounds were stable in PBS (Table 2). Compounds
5d and 5m were also stable in the simulated intestinal fluid
(Table 3). However, gemcitabine was degraded by 60% or 98.5%,
Chem Biol Drug Des 2012; 80: 479–488
485

Zhao et al.
Table 4: Pharmacokinetic parameters of gemcitabine in mice
(n = 5; mean € SD)
Pharmacokinetics of gemcitabine, compound
5d and compound 5m in mice
The pharmacokinetic parameters of gemcitabine (Table 4), com-
pound 5d and compound 5m (Table 5) were analysed in Kunming
strain mice. Table 5 shows a longer mean retention time for com-
pounds 5d and 5m than for gemcitabine. The maximum plasma
concentration (Cmax) of gemcitabine and the area under the concen-
tration curve (AUC0–t) produced by compound 5d after oral adminis-
tration were much higher than those produced by gemcitabine
when administered orally. The Cmax values of both 5d and 5m
were lower than that of gemcitabine administered by intravenous
injection. Importantly, the t1 ⁄ 2 of gemcitabine released from com-
pound 5d was found to be much longer than that of the parent
drug administered either intravenously or orally. Further analysis
showed that the absolute bioavailability and relative bioavailability
of compound 5d were 59.2% and 120.1%, respectively, when com-
pared to gemcitabine. These results imply that compound 5d might
retain a high level of bioavailability in the body. The Cmax and
AUC0–t values were both lower for compound 5m than for com-
pound 5d (Table 5). These results suggest that compound 5d could
prove to be a useful oral prodrug of gemcitabine that exhibits
improved intestinal absorption, decreased first-pass metabolism in
the liver, decreased gastrointestinal toxicity and the ability to main-
tain an active concentration of the drug for a longer time. We hy-
pothesised that compound 5d might gradually release gemcitabine
in the liver or plasma and maintain a steady-state concentration in
the body. Thus, compound 5d was considered as a potential candi-
date for further evaluation of its pharmacological activity in vivo.
Parameters
Intravenous group
Oral group
Cmax (lM)
MRT0–t (h)
t1 ⁄ 2 (h)
Tmax (h)
AUC0–t (lM*h)
Absolute bioavailability
43.9 € 1.7
1.1 € 0.1
1.1 € 0.2
0.08 € 0.0
39.2 € 1.1
49.2%
9.9 € 0.6
1.3 € 0.2
1.5 € 0.3
0.5 € 0.1
19.3 € 0.8
MRT, mean retention time.
Absolute bioavailability = AUCoral ⁄ AUCiv.
Table 5: Pharmacokinetic parameters of compound 5d and 5m
in mice (n = 5; mean € SD)
Produced gemcitabine
Parameters
Compound 5d
Compound 5m
Cmax (lM)
MRT0–t (h)
t1 ⁄ 2 (h)
Tmax (h)
AUC0–t (lM*h)
Absolute bioavailability
versus gemcitabine
Relative bioavailability
versus gemcitabine
17.0 € 1.3
3.3 € 0.8
4.6 € 0.8
0.1 € 0.0
21.0 € 2.1
59.2%
4.9 € 1.0
2.1 € 0.5
2.7 € 0.4
0.1 € 0.0
10.4 € 1.1
29.2%
120.1%
59.4%
MRT, mean retention time.
Absolute bioavailability of compound 5d or compound 5m versus gemcita-
bine = AUCproduced · Div
⁄
gemcitabine
gemcitabine by compound 5d (compound 5m)
Anticancer activity of compound 4 in mice
The inhibitory effects of compound 5d and the parent drug gemcit-
abine on growth of human carcinoma xenografts in mice are sum-
marized in Table 6 and Figure 2. Compound 5d effectively inhibited
human hepatocellular carcinoma HepG2 xenograft growth after
(AUCgemcitabine, iv · Dcompound 5d (compound 5m)).
Relative bioavailability of compound 5d or compound 5m versus gemcita-
bine = AUCproduced · Doral
⁄
gemcitabine
gemcitabine by compound 5d (compound 5m)
(AUCgemcitabine,oral · Dcompound 5d (compound 5m)).
D, dosage.
Table 6: Inhibitory effect of compound 5d on the growth of human HepG2 and HCT-116 xenografts in Balb ⁄ c athymic (nu+ ⁄ nu+) (n = 7)
Cell lines
HepG2
Dosage (lmol ⁄ kg)
Body weight (g)a (initial ⁄ ultimate day)
Tumour weightb (mean € SD, g)
Tumour growth inhibition (%)
–
Vehicle
Gemcitabine
33.4
18.17 € 0.97 ⁄ 19.61 € 1.49
0.88 € 0.44
18.00 € 0.98 ⁄ 17.80 € 1.25*
0.56 € 0.34*
36.3
Compound 5d
21.5
17.2
18.64 € 0.79 ⁄ 19.42 € 1.35
18.55 € 1.21 ⁄ 19.14 € 2.17
18.61 € 0.66 ⁄ 19.72 € 0.38
19.15 € 1.75 ⁄ 20.30 € 2.87
0.37 € 0.07**
0.51 € 0.09**
0.60 € 0.10*
1.67 € 0.40
58.0
42.1
31.8
12.9
HCT-116
Vehicle
Gemcitabine
41.8
18.31 € 1.33 ⁄ 18.20 € 1.41*
0.42 € 0.12**
74.7
Compound 5d
25.8
21.5
18.32 € 0.92 ⁄ 19.31 € 2.38
19.19 € 1.63 ⁄ 19.18 € 2.48
17.84 € 0.70 ⁄ 19.25 € 1.70
0.51 € 0.19**
0.87 € 0.32**
1.02 € 0.61*
69.3
47.8
39.1
17.2
Established tumours were treated by tail vein injection with gemcitabine once every 3 days or oral administration with compound 5d once a day for 2 or 3
consecutive weeks.
aBody weight was measured before and after drug administration.
bTumours were measured after mice were sacrificed.
*p < 0.05, **p < 0.01 versus vehicle control.
486
Chem Biol Drug Des 2012; 80: 479–488

Synthesis and Evaluation of Gemcitabine Oral Prodrugs
A
a
b
Figure 2: Inhibitory effects of
compound 5d or gemcitabine on
human hepatocellular carcinoma
HepG2 xenografts (A) and human
colon cancer HCT-116 xenografts
(B) in nude mice. (a) Tumour cells
were injected subcutaneously into
the right anterior flank of the
mice. Gemcitabine was injected
into the tail vein once every
B
a
b
3 days. Compound 5d was admin-
istered orally once a day. (b)
Tumour volumes were measured
on the indicated days.
20 days of oral administration (Table 6 and Figure 2A). The rates of
inhibition by 21.5, 17.2 and 12.9 lmol ⁄ kg of compound 5d once
per day were 58.0%, 42.1% and 31.8%, respectively. Injections of
gemcitabine at 33.4 lmol ⁄ kg inhibited HepG2 xenograft growth by
36.3%. A significant difference existed between 21.5 lmol ⁄ kg com-
pound 5d and 33.4 lmol ⁄ kg gemcitabine (p < 0.05), suggesting
that compound 5d might exhibit a higher activity against human
hepatocellular carcinoma cells.
and in vivo results, compound 5d is proposed as a potent oral anti-
cancer agent that may supplant the use of gemcitabine in clinic.
Acknowledgment
This project was supported by the Ministry of Science and Technol-
ogy of China (No. 2010ZX09401-302-2-07 and 2011ZX09102-001-03).
In human colon adenocarcinoma HCT-116 xenografts (Table 6 and
Figure 2B), the rates of inhibition by compound 5d were 69.3%,
47.8% and 39.1% after 16 days of oral administration at 25.8, 21.5
and 17.2 lmol ⁄ kg, respectively (Table 6). Injections of gemcitabine
at 41.8 lmol ⁄ kg once every 3 days inhibited the growth of HCT-116
xenografts by 74.7%.
Conflicts of interest
We declare that there is no conflict of interest.
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of administration (Table 6). Compound 5d was well tolerated by
mice with no significant loss in body mass (p > 0.05 versus control
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oral anticancer agent, potentially replacing gemcitabine for clinical
use. Future toxicological studies of compound 5d in animals need
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