Synthesis of Phosphoramidate Derivatives of 5-flurouracil as a Possible Prodrugs for Targeting Cancer Tissue

5-Fluorouracil (5-FU) is used widely as an anticancer drug to treat solid cancers, such as colon, breast, rectal, and pancreatic cancers; although it s clinical application is limited because 5-FU has gastrointestinal and hematological toxicity. An approach to improve the cancer cell selective properties of 5-fluorouracil is the chemical transformation into reversible derivatives (prodrugs) which are converted to the parent drug by virtue of enzymatic and / or chemical hydrolysis within the cancer tissue. In the present study, three derivatives of 5-fluorouracil has been designed to be synthesized as 5-fluorouracil phosphoramidate prodrugs, compounds (I, II and III) to selectively deliver the 5-fluorouracil into the cancer cells. The generation of the target compounds I, II and III were accomplished following one-pot reaction procedures. The reaction and purity of the products were checked by TLC, the structure of the final compounds was confirmed by their melting points, infra red spectroscopy and elemental microanalysis. The hydrolysis of compounds I, II and III in aqueous buffer solution of pH 6, pH 7.4 and in serum were studied. Compounds I, II and III had acceptable rate of hydrolysis at pH 6 (t = 45.05 min, t = 41.22min and t = 38.80min respectively) and enough stability at pH 7.4 (t = 348.72 min, t = 395.31min and t = 345.38min respectively); and enough stability at serum; therefore these three compounds can selectively deliver 5-fluorouracil into the tumor cells which have pH approximate to (6). According to the results mentioned above, compounds I, II and III can be good candidates as 5-fluorouracil prodrugs that can selectively deliver the parent drug into the cancer cells by the effect of pH and /or enzyme. Keyword: 5-Fluorouracil, Phosphoramidate prodrug, Cancer targeting. Introduction 5-fluorouracil (5-FU) is being widely used in oncology for treatment of various cancers including head and neck, colorectal, breast, and pancreatic tumors [1].The use of 5-fluorouracil accompanied by several disadvantages including sever adverse effects [2,3] drug resistance[4], limitation of uses [5,6], and variable bioavailability [7]. The cytotoxic effect of 5-FU in most systems is attributed primarily to its anabolism to 5-fluoro-2 -deoxyuridine monophosphate (FdUMP), a potent inhibitor of thymidylate synthase [8], a pivotal enzyme in pyrimidine biosynthesis [9]. Prodrugs are defined as per se therapeutically inactive agents but that can be predictably transformed into active metabolites. In other words, prodrugs act as precursors of parent drugs, with no intrinsic activity, and must undergo, by enzymatic and/or chemical, transformation into active agents in vivo. Simple prodrugs contain a covalent link between the drug and the strategically selected chemical/transport moiety or promoiety. [10]. Lowering extracellular pH (pHe) is one of the few well documented physiological differences between solid tumor and normal tissues, with an absolute value as low as 5.8[11]. prodrugs that could be selectively activated at the lower than normal pHe (occurring in tumor tissue) could have some theoretical advantage as drug for cancer therapy[12]. The prodrug strategy for a site-specific or tumor-targeting delivery has been employed, and much effort has been expended in searching for prodrugs that might improve the clinical utility of 5-FU as an important cancer chemotherapeutic agent. Examples include 1-prodrug forms of Mohammad H. Mohammad 2 5-FU such as tegafur (Ftorafur) [1-(2-tetrahydrofuranyl)-5-fluorouracil] derivatives [13], 1-alkylcarbamoyl-5-fluorouracils [14], 5fluoro-2 -deoxyuridine (5-FdUrd) derivatives [15], and polymeric matrix systems for the controlled release of 5-FU [16]; 2the recently advanced tumor-specific targeting of 5-FU prodrugs using tumor specific gene expression such as antibody-directed enzyme prodrug therapy [17] and targeting carcinoembryonic antigen-promoted activity [18]; and 3intratumoral prodrug activation in which a nontoxic drug is converted into 5-FU by intratumorally expressed enzymes [19]. Materials and Methods 5-Fluorouracil was purchased from EBEWE pharma (Austria); Benzyl alcohol and Benzyl amine were purchased from Fluka (Germany); Phosphoroxychloride was purchased from Fluka (Switzerland). All chemicals were reagent grade and obtained from standard commercial sources. Elemental micro analysis were performed using Carlo Euro-vector EA 3000A(Italy); Melting points were measured on Thomas Hoover Electronic melting point apparatus; and are uncorrected; Infra red spectra were recorded as KBr disks on Back IR spectrophotometer (College of Pharmacy, University of Baghdad); and UV spectrophotometer (College of Pharmacy, University of Baghdad). Synthesis of (Compound I): To a stirred solution of Phosphoroxychloride (0.93 ml, 10 mmol) in dry chloroform (50ml) at −20 C, a solution of benzyl alcohol (1.04 ml 10 mmol) and dry triethylamine (1.53 ml, 11 mmol) in dry chloroform (10ml) was added. After 30 min at −20 C a solution of benzyl amine (1.09 ml, 10 mmol) in dry chloroform (10 ml) was added into the reaction mixture. Then, dry triethylamine (1.53 ml, 11 mmol) was added. After 30 min at −20 C a solution of 5-FU(1.31gm,10 mmol) in dry tetrahydrofuran (200ml) was added in three portion into the reaction mixture Then, dry triethyl amine (1.53ml,11mmol) was added again and the mixture was kept at room temperature for 1 hour. The obtained suspension was filtered and the filterate was washed with distilled water (3×20 ml),dried with anhydrous sodium sulphate and the solvent was evaporated. The obtained compound was crystallized from ethyl ether to give White crystal of compound I. Percent yield (38%), melting point (169170 C decomp.).And elemental microanalysis calculated/found: C55.53/54.71, H4.40/4.22, N10.79/10.49. The infrared characteristic bands (cm): 3060: N-H (str.vib.) of pyrimidine and N−H (str.vib.) of 2° amine, 1958, 1892 (str.vib.) of mono substituted benzene, 1724, 1521and1346 NH(C=O)−C= C−amide I, II, and III (str.vib.) of uracil, 1663C=O (str.vib.) of Pyrimidine, 1246 C−F(str.vib.), 1111 P=O (str.vib.), 1600, 1550 and 1467 C=C of benzene ring, 1058 P−O−C (str.vib.), 746 and 694 C−H out-of-plane bending vib. of mono substituted benzene. Synthesis of (Compound II): To a stirred solution of Phosphoroxychloride (0.93 ml, 10 mmol) in dry chloroform (50 ml) at −20 C a solution of benzyl alcohol (2.07 ml, 20 mmol) and dry triethylamine (3.06 ml, 22 mmol) in dry chloroform (20ml) was added. After 30 min at −20 C a solution of 5-FU (1.31 gm, 10 mmol) in dry tetrahydrofuran (200 ml) was added in three portion into the reaction mixture. Then, dry triethylamine (1.53 ml, 11 mmol) was added again and the mixture was kept at room temperature for 1 hour. The obtained suspension was filtered and the filterate washed with distilled water (3×20 ml), dried with anhydrous sodium sulphate and the solvent was evaporated. The obtained compound was crystallized from ethyl ether to give off white powder of compound II. Percent yield (38%), melting point (144-146 C decomp.) and elemental microanalysis calculated/found: C55.39/54.59, H4.13/4.01, N7.18/7.01. The infrared characteristic bands (cm): 3174: N-H (str.vib.)Of pyrimidine, 1892, 1780(str.vib.) of mono substituted benzene, 1722, 1521and1350 NH(C=O)−C= C−amide I, II, and III (str.vib.) of uracil and C=C of benzene ring, 1654 C=O (str.vib.), 1275 P=O (str.vib.), 1248 C−F (str.vib.), 995 P−O−C (str.vib.). Journal of Al-Nahrain University Vol.13 (1), March, 2010, pp.1-10 Science 3 Synthesis of (Compound III): To a stirred solution of Phosphoroxychloride (0.93ml, 10mmol) in dry chloroform (50ml) at −20 C a solution of benzyl alcohol (1.04 ml, 10 mmol) and dry triethylamine (1.53 ml, 11 mmol) in dry chloroform (10 ml) was added. After 30 min at −20 C a solution of benzyl amine (1.09 ml, 10mmol) in dry chloroform (10ml) was added into the reaction mixture. Then, dry triethylamine (1.53 ml, 11 mmol) was added.After 30 min at room temperature a suspension of 5-fluorouracil sodium salt (1.53 g, 10 mmol) in freshly distilled acetonitrile (10ml) was added and stirred mixture for 10 hr at room temperature. The obtained suspension was filtered and the filterate washed with distilled water (3×20 ml), dried with anhydrous sodium sulphate and the solvent was evaporated. The obtained compound was crystallized from ethyl etherpetroleum ether to give Pale yellow powder of compound III. Percent yield (60.5%), melting point (90-91 C) and elemental microanalysis: C55.53/54.62, H4.40/4.23,N10.79/10.47. The infrared characteristic bands (cm): 3316: N−H (str.vib.) of 2° amine, 3160: N-H (str.vib.)of pyrimidine, 1953,1894 (str.vib.) of mono substituted benzene, 1723, 1500 and 1344 NH(C=O)−C=C− amide I, II, and III (str.vib.) of uracil and C=C of benzene ring, 1670C=O (str.vib.), 1250 C−F (str.vib.), 1201 P=O (str.vib.), 1028 P−O−C (str.vib.), 744 and 696 C−H out-of-plane bending vib. of mono substituted benzene. Hydrolysis of compounds I, II and III at pH 6, pH 7.4 and serum: The hydrolysis of compounds I, II and III were carried out for the equivalent of (0.01mg/ml) in aqueous phosphate buffer solution of pH 6, pH 7.4 and serum at 37 C. The total buffer concentration was 0.1M and the ionic strength ( ) of 1 was maintained by adding calculated amount of NaCl. Different sample were taken for analysis at specific time interval (15, 30, 60, 120, 240 min) and the rate of hydrolysis was followed spectrophotometrically by recording 5-fluorouracil absorbance increase accompanying the hydrolysis at 266nm pH6, pH7.4 and serum. The observed pseudo-first order rate constant was determined from the slope of the linear plot of log concentration of compound vs. time. Results and Discussion The synthetic procedures for the designed target compounds I, II and III are illustrated in [schemes 1, 2 and 3]. Benzyl Compounds I was obtained in one pot-reaction of benzyl alcohol with phosphorusoxychloride in the presence of triethylamine. The obtained dichlorophosphate intermediate was reacted, without isolation, with benzyl amine in the presence of triethylamine, then the obtained monochlorophosphate intermediate was reacted, without isolation, with 5-flourouracil. The Benzyl Compound II was synthesized from reaction in one pot-reaction of benzyl alcohol with phosphorus oxychloride in the presence of triethylamine. The obtained monochlorophosphate intermediate was reacted, without isolation, with 5-flourouracil. This method was previously established by us for the synthesis of isotopically labeled IPAM [20]. Compound III was obtained in one potreaction of benzyl alcohol with phosphorus oxychloride in the presence of triethylamine. The obtained dichlorophosphate intermediate was reacted, without isolation, with benzyl amine in the presence of triethylamine [21], then the obtained monochlorophosphate intermediate was reacted, without isolation, with an acetonitrile solution of 5-flourouracil sodium salt [22]. Mohammad H. Mohammad 4 OH + POCl3 TEA O P O