Design And Synthesis Of 5-fluorouracil-Galactoside Derivatives And The Study Of Their Antitumor Activities

Background:Malignant tumors, as common diseases and frequently encountered diseases, still seriously threaten the health and lives of human beings and affect people’s quality of life greatly. Malignant tumors are still public health problems globally. The global number of new cancer cases and deaths in 2008 has been increasing primarily.Based on the GLOBOCAN 2008 estimates, approximately 12.7 million cancer cases were diagnosed and 7.6 million people died of cancer in 2008 all over the world. About 1.5 million cancers were diagnosed and 0.8 million people died of it each year in China.Although there are various kinds of treatments for malignant tumors, the cure rates of the great majority of malignant tumors are very low and the recurrence rates of malignant tumors are still high. Accordingly, the treatments for malignant tumor are attracting more and more serious public concern.5-fluorouracil (5-FU), as a thymidylate synthase (TS) inhibitor, has been used against cancer for about 50 years. 5-FU has a good antitumor activity, which is a first-line treatment for solid tumors. Although 5-FU is one of the most widely-used antitumor agents worldwide, it has some therapeutic drawbacks and limitations. Because the bioavailability of 5-FU is low orally, it has to be given by vein. Wide individual differences in its metabolism, low selectivity for tissues and serious side effects are produced by 5-FU in patients. 5-FU has influences on general systems seriously. The cells of rapid differentiation were inhibited by fluorouracil, such as gastrointestinal mucosa and bone marrow. 5-fluorouracil could lead to myelosuppression, mucositis, diarrhea, alopecia and damages of liver and kidney. The search for find 5-fluorouracil derivatives with favorable tumor targeting, good antitumor activities and low toxicities, therefore, is the focus of research recently.Galactose, as a monosaccharide, exists inside the body abundantly. Galactose can be recognized and bound by galectins and asialoglycoprotein receptor (ASGP-R) by means of carbohydrate recognition domain (CRD) specifically. Recent studies have demonstrated the relationships between the galectin species and tumors. Among the family, galectin-3 is one of the most extensively studied members, and is usually considered as a potential tumor marker because its expression correlates with tumor progression and metastasis. ASGP-R, as a transmembrane protein, can lead to receptor-mediated endocytosis. It is expressed exclusively on the surface of cell membrane of hepatocytes. It could bind to galactose-terminal molecules and transports them to lysosomes inside the cell, and then is recycled. ASGP-R has been researched extensively since it can be considered as a target site. The enhancing effects of tumor targeting by means of modifying antitumor agents with galactose are paid attention by people more and more.AimTo design and synthesize a series of 5-fluorouracil-galactoside derivatives, targeting by galectins and ASGP-R, by means of binding galactose to 5-fluorouracil. These 5-fluorouracil-galactoside derivatives are presumed to reach a high local concentration with reduced systematic toxicity by releasing 5-fluorouracil in tumor tissues.Methods1. Based on the molecular structure of 5-fluorouracil, both of carbonyl groups of 5-fluorouracil were silanizated using hexamethyldisilazane (HMDS) and trimethyl chlorosilane (TMSCl). Peracetylated galactose was bound to N1 of modified 5-fluorouracil under the effect of tin (IV) chloride. Acetyl groups were removed using the ammonia (2M in methanol). 1-galactopyranosyl- 5-fluorouracil and 1-peracetylated galactopyranosyl-5-fluorouracil were synthesized and identified using MS, 1H NMR and 13C NMR.2. 2-benzyloxy-5-fluoro-4(3H)-pyrimidone, as an intermediate product was synthesized by means of binding of a benzyl group to C2 of 5-fluorouracil. Because of steric hindrance effect, N1 was inactivated due to the binding between the big size groups and oxygen atoms, while N3 was activated. Under the condition of tetrabutylammonium bromide as a phase-transfer catalyst, peracetylated galactose with the substitution of bromide on C1 and 2-benzyloxy-5-fluoro-4(3H)-pyrimidone were reacted for the purpose of the binding of modified galactose and modified fluorouracil on N3. 3-galactopyranosyl-5-fluorouracil was synthesized by two ways: one way is deacetylation using the ammonia (2M in methanol) following by debenzylation using Pd-C, and the other way is debenzylation following by deacetylation. 3-galactopyranosyl-5-fluorouracil and 3-peracetylated galactopyranosyl-5- fluorouracil were synthesized and identified using MS, 1H NMR and 13C NMR. 3. The growth inhibiting rates of 5-fluorouracil and these 5-fluorouracil -galactoside derivatives on human hepatocarcinoma cells (Hep G2), human colon cancer cells (SW-620、HT-29、LoVo、SW-1116) and human intestinal epithelial cells (HIEC) were evaluated using MTT assay. The antitumor activities and effects of 5-fluorouracil-galactoside derivatives on the growth of human intestinal epithelial cells (HIEC) were also assessed. The tumor targeting of 5-fluorouracil-galactoside derivatives were investigated.ResultsThese 5-fluorouracil-galactoside derivatives were synthesized successfully and the structures of which were determined by MS, 1H NMR and 13C NMR as 1-galactopyranosyl-5-fluorouracil (1C), 1-peracetylated galactopyranosyl-5- fluorouracil (1B) and 3-peracetylated galactopyranosyl-5-fluorouracil (2E). And in vitro the growth inhibiting rates of 2E (0.01~100μmol?L-1) on Hep G2, SW-620, HT-29, LoVo, SW-1116 and HIEC cells for 48 h were 0.88%~44.3%, 0.55%~45.82%, 0.87%~81.16%, 0.82%~48.64%, 8.45%~64.04% and 6.59%~54.13%, respectively. 2E had a good antitumor activity in vitro. There was no statistical significance between 5-FU and 2E. Besides, 2E had a lower inhibiting effect on HIEC than that of 5-FU. 1-peracetylated galactopyranosyl- 5-fluorouracil (1B) and 1-galactopyranosyl-5-fluorouracil (1C) did not have good antitumor activities on several tumor cells in vitro.ConclusionsThese 5-fluorouracil-galactoside derivatives, 1-galactopyranosyl-5- fluorouracil (1C), 1-peracetylated galactopyranosyl-5-fluorouracil (1B) and 3-peracetylated galactopyranosyl-5-fluorouracil (2E), were synthesized successfully in our lab. 2E has a good antitumor activity in vitro. Furthermore, it is less toxic than 5-fluorouracil on human intestinal epithelial cells (HIEC). The results reported herein demonstrated that 3-peracetylated galactopyranosyl -5-fluorouracil (2E) has a potential tumor-targeting effect. Nevertheless, further studies on the targeting characterisitcs are needed.