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

Authors


Corresponding author: Xian Jun Qu, qxj@sdu.edu.cn and Wen Bao Li, wbli@sanlugen.com

Abstract

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.

Gemcitabine (Gem) hydrochloride, 2′,2′-difluoro-2-deoxycytidine hydrochloride (β-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 deoxycytidine kinase to form mono-, di- and triphosphate nucleotides (4,5). Among these compounds, gemcitabine diphosphate (dFdCDP) and gemcitabine triphosphate (dFdCTP) are the active metabolites (6,7). dFdCDP could inhibit ribonucleotide reductase, which is essential for DNA repair and synthesis, by decreasing the production of deoxynucleotides, especially dCTP. dFdCTP is an inhibitor of DNA polymerase and may also incorporate into DNA, leading to chain termination, strand breakage and the trapping of topoisomerase 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 deaminase (10,11). Thus, gemcitabine is not effective against hepatocarcinoma by injection. Moreover, due to its low bioavailability and gastrointestinal toxicity, it has to be administered intravenously instead of orally (2,3,12).

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.

Figure 1.

 Chemical structures of gemcitabine hydrochloride and prodrugs of gemcitabine.

Experimental Section

Synthesis

Gemcitabine hydrochloride was obtained from Ningbo Teampharm Co. Ltd., (Ningbo, Zhejiang, China). PyBop and 4-dimethylaminopyridine (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 Micromass ZQ instrument (Waters, Milford, Massachusetts, USA). 1H NMR and 13C NMR spectra were recorded at 600 MHz (Varian AS600). Spin multiplets are given as s (singlet), d (doublet), t (triplet) 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.

A solution of gemcitabine hydrochloride (0.7 g, 2.34 mmol), 2-(dodecyloxycarbonyl) 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-dimethylformamide (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) δ: 0.85 (t, = 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, = 10.8 Hz,3H), 4.23 (m, 1H), 5.33 (m, 1H), 6.18 (t, = 7.2 Hz, 1H), 6.34 (d, = 6.0 Hz, 1H), 7.39 (d, = 7.8 Hz, 1H), 7.58 (d, = 7.2 Hz, 1H), 7.62 (t, = 7.8 Hz, 1H), 7.68 (t, = 7.2 Hz, 1H), 7.88 (d, = 7.8 Hz, 1H), 8.33 (d, = 7.2 Hz, 1H), 11.56 (s, 1H).

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) δ: 0.85 (t, = 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, = 4.8 Hz, 1H), 6.19 (t, = 6.6 Hz, 1H), 6.34 (d, = 6.6 Hz, 1H), 7.35 (d, = 7.2 Hz, 1H), 7.70 (m, 1H), 8.13 (d, = 7.8 Hz, 1H), 8.35 (d, = 7.8 Hz, 1H), 8.76 (d, = 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)

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-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]+, calculated MW: 566 for C27H36F2N4O7. 1H NMR (DMSO, 600 MHz) δ: 0.85 (t, = 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, = 5.4 Hz, 1H), 6.19 (t, = 7.2 Hz, 1H), 6.34 (d, = 6.6 Hz, 1H), 7.35 (d, = 7.7 Hz, 1H), 7.70 (m, 1H), 8.13 (d, = 7.8 Hz, 1H), 8.35 (d, = 7.8 Hz, 1H), 8.77 (d, = 4.2 Hz, 1H), 11.72 (s, 1H).

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)

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-dicarbonyl dichloride, which was used in the next step without further purification.

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 adjusting the pH of the solution to 2–3, it was extracted three times with ethyl acetate. The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated to give 3-(dodecyloxycarbonyl) 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 residue 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) δ: 0.85 (t, = 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, = 6.0 Hz, 2H), 5.33 (t, = 5.4 Hz, 1H), 6.18 (m, 1H), 6.34 (d, = 6.6 Hz, 1H), 7.28 (s, 1H), 8.38 (d, = 7.8 Hz, 1H), 8.94 (dd, J1 = 10.8 Hz, J2 = 1.8 Hz, 2H), 11.50 (s, 1H). 13C NMR (DMSO,600MHz) δ: 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-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)

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) δ: 0.85 (t, = 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, = 6.6 Hz, 2H), 5.35 (m, 1H), 6.18 (m, 1H), 6.35 (d, = 6.6 Hz, 1H), 7.28 (d, = 6.6 Hz, 1H), 8.38 (d, = 7.2 Hz, 1H), 8.94 (dd, J1 = 10.8 Hz, J2 = 2.4 Hz, 2H), 11.51 (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 spectrophotometry at 254 nm. LC-MS m/z 581 [M + H]+, calculated MW: 580 for C28H38F2N4O7. 1H NMR (DMSO, 600 MHz) δ: 0.84 (t, = 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, = 6.6 Hz, 2H), 5.37 (s, 1H), 6.21 (t, = 7.2 Hz, 1H), 6.37 (d, = 6.6 Hz, 1H), 7.45 (d, = 7.2 Hz, 1H), 8.32 (d, = 8.4 Hz, 1H), 8.44 (d, = 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-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)

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 [M + H]+, calculated MW: 580 for C28H38F2N4O7. 1H NMR (DMSO, 600 MHz) δ: 0.84 (t, = 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, = 6.6 Hz, 2H), 5.35 (s, 1H), 6.22 (t, = 6.6 Hz, 1H), 6.35 (d, = 5.4 Hz, 1H), 7.39 (s, 1H), 8.15 (d, = 7.8 Hz, 1H), 8.36 (d, = 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-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]+, calculated MW: 566 for C27H36F2N4O7. 1H NMR (DMSO, 600 MHz) δ: 0.84 (t, = 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, = 6.6 Hz, 2H), 5.38 (t, = 4.8 Hz, 1H), 6.21 (t, = 6.6 Hz, 1H), 6.38 (d, = 6.0 Hz, 1H), 7.45 (d, = 7.2 Hz, 1H), 8.32 (d, = 8.4 Hz, 1H), 8.44 (d, = 7.8 Hz, 1H), 8.57 (dd, J1 = 8.4 Hz, J2 = 1.8 Hz, 1H), 9.20 (s, 1H), 10.64 (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-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]+, calculated MW: 566 for C27H36F2N4O7. 1H NMR (DMSO, 600 MHz) δ: 0.85 (t, = 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, = 6.6 Hz, 2H), 5.35 (m, 1H), 6.22 (t, = 6.6 Hz, 1H), 6.35 (d, = 6.0 Hz, 1H), 7.39 (s, 1H), 8.15 (t, = 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) 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 m/z 581 [M + H]+, calculated MW: 580 for C28H38F2N4O7. 1H NMR (DMSO, 600 MHz) δ: 0.84 (t, = 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, = 6.6 Hz, 2H), 5.37 (s, 1H), 6.23 (t, = 6.6 Hz, 1H), 6.37 (d, = 6.6 Hz, 1H), 7.48 (d, = 7.8 Hz, 1H), 8.33 (t, = 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, = 7.8 Hz, 1H), 10.47 (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 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]+, calculated MW: 566 for C27H36F2N4O7. 1H NMR (DMSO, 600 MHz) δ: 0.84 (t, = 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, = 6.6 Hz, 2H), 5.37 (s, 1H), 6.21 (t, = 6.6 Hz, 1H), 6.36 (s, 1H), 7.47 (d, = 7.8 Hz, 1H), 8.17 (s, 1H), 8.43 (d, = 7.2 Hz, 1H), 8.49 (s, 1H), 8.98 (s, 1H), 10.62 (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]+, calculated MW: 566 for C27H36F2N4O7. 1H NMR (DMSO, 600 MHz) δ: 0.85 (t, = 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, = 4.8 Hz, 1H), 6.19 (t, = 4.8 Hz, 1H), 6.35 (d, = 6.6 Hz, 1H), 7.37 (d, = 7.8 Hz, 1H), 7.63 (d, = 4.8 Hz, 1H), 8.36 (d, = 7.2 Hz, 1H), 8.87 (d, = 5.4 Hz, 1H), 8.89 (d, = 4.8 Hz, 1H), 11.71 (s, 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) δ: 0.84 (t, = 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, = 6.6 Hz, 2H), 5.36 (s, 1H), 6.21 (t, = 6.6 Hz, 1H), 6.36 (s, 1H), 7.47 (d, = 7.8 Hz, 1H), 8.17 (d, = 7.8 Hz, 1H), 8.43 (d, = 7.2 Hz, 1H), 8.49 (s, 1H), 8.98 (s, 1H), 10.62 (s, 1H).

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) δ: 0.85 (t, = 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, = 6.6 Hz, 2H), 5.37 (s, 1H), 6.22 (t, = 6.6 Hz, 1H), 6.36 (s, 1H), 7.38 (d, = 7.8 Hz, 1H), 8.05 (d, = 7.8 Hz, 1H), 8.37 (d, = 7.2 Hz, 1H), 8.49 (s, 1H), 8.95 (s, 1H), 11.88 (s, 1H).

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)

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 m/z 586 [M + H]+, calculated MW: 585 for C27H37F2N3O7S. 1H NMR (DMSO, 600 MHz) δ: 0.84 (t, = 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, = 6.6 Hz, 2H), 5.35 (t, = 4.2 Hz, 1H), 6.20 (s, 1H), 6.35 (d, = 6.6 Hz, 1H), 7.36 (brs, 1H), 7.83 (d, = 4.2 Hz, 1H), 8.32 (brd, 2H), 11.75 (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) 1H-pyrazole-3-carboxylate (compound 5p)

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]+, calculated MW: 555 for C25H35F2N5O7. 1H NMR (DMSO, 600 MHz) δ: 0.84 (t, = 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, = 6.6 Hz, 2H), 5.36 (t, = 6.0 Hz, 1H), 6.20 (t, = 7.2 Hz, 1H), 6.35 (d, = 5.4 Hz, 1H), 7.41 (d, = 7.2 Hz, 1H), 7.50–7.91 (brd, 1H), 8.35 (d, = 7.8 Hz, 1H), 10.56–11.61 (brd, 1H), 14.60–14.80 (brd, 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 stabilities were determined by measuring the concentrations of gemcitabine 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 solution 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.

Pharmacokinetics of compound 5d and compound 5m

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 compound 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 hepatoma 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 μg/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).

Inhibition of cell proliferation in vitro by the MTT assay

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 μL 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 (Beijing, China). Animals were housed under pathogen-free conditions according to the institutional guidelines. Cancer cells (1 × 107) suspended in 100 μL of Matrigel (Collaborative Biomedical, Springfield, MA, USA) were injected subcutaneously into the right anterior flank of each mouse (21). After 2 weeks, when tumours were established 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 μmol/kg (namely 0, 7.5, 10, 12.5 mg/kg) daily and HCT-116 xenografts: 0, 17.2, 21.5, 25.8 μmol/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 μmol/kg (namely 10 mg/kg) for HepG2 xenografts and 41.8 μmol/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.

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 considered statistically significant.

Results and Discussion

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 compounds 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-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), according to the previously published papers (26,27).

Figure Scheme 1:.

 Synthesis of gemcitabine derivatives 5a–5p.

Table 1.   Structures of desired prodrugs and IC50m) for five human cancer cell lines (mean ± SD) Thumbnail image of

The desired compounds were synthesised by reacting the substituted acids R1COOH obtained above with gemcitabine hydrochloride 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 characteristics are summarized in Table 1.

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 displayed 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 summarized in Table 1. The IC50s of most compounds were higher than that of gemcitabine in these cell lines, implying that these derivatives exhibit weaker inhibitory activities on cancer cell growth in vitro. 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 incorporates prodrug strategies for gemcitabine, and we selected representative compounds for further study.

Physiological stability in vitro

Compounds 5d and 5m were selected as representative gemcitabine 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%, 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 bioavailability in the intestine than the parent drug. Additionally, it is logical to suggest that these prodrugs might also exhibit less intestinal toxicity than gemcitabine.

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)
HoursGemcitabineCompound 5dCompound 5m
0100.0 ± 0.0100.0 ± 0.0100.0 ± 0.0
198.4 ± 9.695.0 ± 9.194.7 ± 9.3
299.1 ± 8.999.5 ± 8.598.1 ± 8.8
498.5 ± 9.093.5 ± 9.298.2 ± 8.9
694.1 ± 8.490.1 ± 8.1103.4 ± 10.6
896.6 ± 9.790.6 ± 9.597.5 ± 10.5
1291.3 ± 8.896.6 ± 9.5102.6 ± 11.0
2491.8 ± 7.996.9 ± 8.9100.2 ± 9.3
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)
HoursGemcitabineCompound 5dCompound 5m
0100.0 ± 0.0100.0 ± 0.0100.0 ± 0.0
172.3 ± 6.580.0 ± 7.998.2 ± 10.4
263.8 ± 7.867.9 ± 7.5104.0 ± 9.9
440.0 ± 5.685.0 ± 9.7104.0 ± 9.5
630.6 ± 3.085.8 ± 8.9106.0 ± 10.6
823.3 ± 3.683.0 ± 8.8109.0 ± 11.0
121.5 ± 0.485.7 ± 7.799.8 ± 9.4
240.9 ± 0.188.9 ± 7.995.7 ± 9.6

Pharmacokinetics of gemcitabine, compound 5d and compound 5m in mice

The pharmacokinetic parameters of gemcitabine (Table 4), compound 5d and compound 5m (Table 5) were analysed in Kunming strain mice. Table 5 shows a longer mean retention time for compounds 5d and 5m than for gemcitabine. The maximum plasma concentration (Cmax) of gemcitabine and the area under the concentration curve (AUC0–t) produced by compound 5d after oral administration 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 compound 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 compared 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 compound 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 maintain an active concentration of the drug for a longer time. We hypothesised 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 candidate for further evaluation of its pharmacological activity in vivo.

Table 4.   Pharmacokinetic parameters of gemcitabine in mice (n = 5; mean ± SD)
ParametersIntravenous groupOral group
  1. MRT, mean retention time.

  2. Absolute bioavailability = AUCoral/AUCiv.

Cmaxm)43.9 ± 1.79.9 ± 0.6
MRT0–t (h)1.1 ± 0.11.3 ± 0.2
t1/2 (h)1.1 ± 0.21.5 ± 0.3
Tmax (h)0.08 ± 0.00.5 ± 0.1
AUC0–tm*h)39.2 ± 1.119.3 ± 0.8
Absolute bioavailability49.2%
Table 5.   Pharmacokinetic parameters of compound 5d and 5m in mice (n = 5; mean ± SD)
ParametersProduced gemcitabine
Compound 5dCompound 5m
  1. MRT, mean retention time.

  2. Absolute bioavailability of compound 5d or compound 5m versus gemcitabine = AUCproduced gemcitabine by compound 5d (compound 5m) × Div gemcitabine/(AUCgemcitabine, iv × Dcompound 5d (compound 5m)).

  3. Relative bioavailability of compound 5d or compound 5m versus gemcitabine = AUCproduced gemcitabine by compound 5d (compound 5m) × Doral gemcitabine/(AUCgemcitabine,oral × Dcompound 5d (compound 5m)).

  4. D, dosage.

Cmaxm)17.0 ± 1.34.9 ± 1.0
MRT0–t (h)3.3 ± 0.82.1 ± 0.5
t1/2 (h)4.6 ± 0.82.7 ± 0.4
Tmax (h)0.1 ± 0.00.1 ± 0.0
AUC0–tm*h)21.0 ± 2.110.4 ± 1.1
Absolute bioavailability versus gemcitabine59.2%29.2%
Relative bioavailability versus gemcitabine120.1%59.4%

Anticancer activity of compound 4 in mice

The inhibitory effects of compound 5d and the parent drug gemcitabine on growth of human carcinoma xenografts in mice are summarized in Table 6 and Figure 2. Compound 5d effectively inhibited human hepatocellular carcinoma HepG2 xenograft growth after 20 days of oral administration (Table 6 and Figure 2A). The rates of inhibition by 21.5, 17.2 and 12.9 μmol/kg of compound 5d once per day were 58.0%, 42.1% and 31.8%, respectively. Injections of gemcitabine at 33.4 μmol/kg inhibited HepG2 xenograft growth by 36.3%. A significant difference existed between 21.5 μmol/kg compound 5d and 33.4 μmol/kg gemcitabine (p < 0.05), suggesting that compound 5d might exhibit a higher activity against human hepatocellular carcinoma cells.

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 linesDosage (μmol/kg)Body weight (g)a (initial/ultimate day)Tumour weightb (mean ± SD, g)Tumour growth inhibition (%)
  1. 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.

  2. aBody weight was measured before and after drug administration.

  3. bTumours were measured after mice were sacrificed.

  4. *p < 0.05, **p < 0.01 versus vehicle control.

HepG2Vehicle18.17 ± 0.97/19.61 ± 1.490.88 ± 0.44
Gemcitabine
33.418.00 ± 0.98/17.80 ± 1.25*0.56 ± 0.34*36.3
Compound 5d
21.518.64 ± 0.79/19.42 ± 1.350.37 ± 0.07**58.0
17.218.55 ± 1.21/19.14 ± 2.170.51 ± 0.09**42.1
12.918.61 ± 0.66/19.72 ± 0.380.60 ± 0.10*31.8
HCT-116Vehicle19.15 ± 1.75/20.30 ± 2.871.67 ± 0.40 
Gemcitabine
41.818.31 ± 1.33/18.20 ± 1.41*0.42 ± 0.12**74.7
Compound 5d
25.818.32 ± 0.92/19.31 ± 2.380.51 ± 0.19**69.3
21.519.19 ± 1.63/19.18 ± 2.480.87 ± 0.32**47.8
17.217.84 ± 0.70/19.25 ± 1.701.02 ± 0.61*39.1
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 3 days. Compound 5d was administered orally once a day. (b) Tumour volumes were measured on the indicated days.

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 μmol/kg, respectively (Table 6). Injections of gemcitabine at 41.8 μmol/kg once every 3 days inhibited the growth of HCT-116 xenografts by 74.7%.

The body weights of the mice were measured throughout the term of administration (Table 6). Compound 5d was well tolerated by mice with no significant loss in body mass (p > 0.05 versus control animals) or other apparent signs of toxicity. In contrast, a notable decrease in body weight was observed in gemcitabine-treated animals (p < 0.05 versus control animals). These results indicated that compound 5d might be less toxic to animals than gemcitabine, suggesting that this compound could be further developed as an oral anticancer agent, potentially replacing gemcitabine for clinical use. Future toxicological studies of compound 5d in animals need to be conducted.

Conclusion

This work presented the development of a series of orally active compounds based on the structure of gemcitabine. Compound 5d, the representative prodrug, displayed stability to both chemical and enzymatic hydrolysis, showed improved bioavailability, maintained an active concentration for a longer period of time and exhibited higher efficacy against the growth of human carcinoma xenografts in mice when compared with gemcitabine. Based on these in vitro and in vivo results, compound 5d is proposed as a potent oral anticancer agent that may supplant the use of gemcitabine in clinic.

Acknowledgment

This project was supported by the Ministry of Science and Technology of China (No. 2010ZX09401-302-2-07 and 2011ZX09102-001-03).

Conflicts of interest

We declare that there is no conflict of interest.

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