Synthesis and Preliminary Kinetic Study of 5-fluorouracil Derivatives for Targeting Colon Tumor

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 greatly limited by its short plasma half-life, poor tumor affinity, myelosuppression, and gastrointestinal toxicity. To tackle these problems, numerous modifications of the 5Fu structure have been performed.Thus, 5-Fu as possible colon specific prodrugs were synthesized in which 5-Fu is attached to amino acids (alanine & phenylalanine) using succinate group as a spacer via amide or ester bond. An approach to improve the properties of 5-fluorouracil is the chemical transformation into bioreversible derivatives (prodrugs) which are converted to the parent drug by enzymatic and / or chemical hydrolysis. The synthesis of the target compounds were accomplished following multistep reaction procedures, the chemical reaction followed up and the purity of the products were checked by TLC.


Introduction
Colon cancer is the second cause of cancer related deaths in the world. Although improvements have been made in surgical techniques and in chemotherapies, the survival rate is still low [1]. 5-fluorouracil is an antimetabolite of the pyrimidine analogue type, which is frequently used for treating solid tumors, such as colorectal, gastric tract, and liver carcinomas [2,3].
However, the clinical applications of 5-FU are greatly limited by its short plasma half-life, poor tumor affinity, myelosuppression, and strong intestinal toxicity. To tackle these problems, numerous modifications of the 5-Fu structure have been performed, thus, a series of 5-Fu prodrugs in which 5-Fu is attached to amino acids, peptides, phospholipids, and polymers have been reported [4][5][6][7].
The term "prodrug", first introduced by Albert, refers to a chemically modified form of a drug [8] that is devoid of pharmacological activity, but that can be converted to the active form of the drug in a biological system, where it exerts the desired action. This strategy can improve the limitations associated with the effective transport into tumor cells, catabolic inactivation before the cytotoxic entity can reach the tumor, and short plasma half-1ife [9,10].
As the site of drug absorption, the colon holds several drawbacks, such as small surface area, low fluidity of the lipid membrane, existence of tighter junction compared to the small intestine, low motility, and high viscosity of intestinal contents. On the other hand, long transit time, low proteolytic activity, and responsiveness to the action of absorption enhancers are the positive aspects of the large intestine as the site of drug absorption compared to the small intestine [11].
The objective of the colon-targeted drug delivery is to avoid absorption and degradation of drugs in the upper GI tract allowing them act site-specifically in the colon and this achieved by various approaches such as pH dependent utilizing the changes in pH along the GI tract [12], coated dosage forms [13], time-controlled release systems [14], pressure-controlled colon delivery systems [15], coating drugs with bacterially degradable polymer [16] and prodrug based drug delivery system [17]. The colonic drug delivery systems are mainly based on degradation of a prodrug by microorganisms of the colon flora [18] and colonic enzymes [19].
To design a colon-specific prodrug, a parent drug is usually linked to a nonabsorbable carrier such as a polymeric matrix or a hydrophilic small molecule to prevent the absorption of the colon-specific prodrug in the upper intestine [20].
Ho et al. [21] reported that the liver has the highest dihydrouracil dehydrogenase activity with minimal activity in the colon and colon tumor. These studies suggest that bioactivation of 5-FU to 5-fluoro-2 ' -deoxyuridine is most active and bioinactivation of 5-FU to dihydro-5-FU is the least active in colon tumor. If 5-FU is delivered specifically to colon, the active metabolite of 5-FU should be most available in colon tumor, and the systemic side effects of 5-FU will be minimized compared with oral administration.
Spacer or linkers can be used if the desired prodrug promoiety cannot be attached directly to the parent molecule due to a sterical hindrance or functional properties of the parent drug. The spacers increase the distance between the parent molecule and the promoiety and are usually cleaved spontaneously after the enzymatic or chemical decomposition of the prodrug bond between the promoiety and the spacer [22].

Synthesis of methyl ester hydrochlorides of L-phenylalanine and L-Alanine:
A suspension of L-Phenylalanine (9 mmole) dissolved in (15 mL) of absolute methanol, was cooled down to -15 0 C then thionyl chloride was added drop wise (9 mmole), (the temperature should be keep below -10 0 C), the reaction mixture was left at 40 0 C for 1hr, then refluxed for 4hr and left at room temperature overnight, , It was titrated with 20 ml portion of cold ether at 0 0 C until the excess of dimethyl sulphate was removed, the excess solvent was evaporated to dryness under vacuum, re-crystallize the product from methanol-petroleum ether to get pure L-Phenylalanine methyl ester HCl. The same procedure was followed to synthesize L-Alanine methyl ester HCl [23].

Synthesis of Intermediate (I):
L-Phenylalanine methyl ester HCl (2mmole) was treated with (2mmole) of succinic anhydride in tetrahydrofuran and (2mmole) of triethylamine at room temperature for 10 min. The precipitate was filtered and dried without further purification to get Intermediate (IA). The same procedure was followed with L-Alanine methyl ester HCl to synthesize Intermediate (IB) [24]. J a n u a r y 2 3 , 2 0 1 4

Synthesis of Intermediate (II):
A suspension of intermediate (IA) (2mmole, 0.56g) dissolved in (10ml) of dry chloroform, was cooled down to -15 0 C then thionyl chloride (2mmole, 0.15mL) was added slowly to it. The mixture was refluxed for 4hr at 60-70 0 C with continuous stirring. Then evaporate the excess solvent by using rotary evaporator. The viscous liquid was slowly added drop wise into a beaker contain of 2,4-dinitrophenol(2mmole, 0.368g) dissolved in (5ml) of dry chloroform and triethylamine (2mmole, 0.28ml) for 1hr on an ice bath, then continuous stirring overnight at room temperature. The solid that separated out was filtered off and washed with dry chloroform to give Intermediate (IIC). The same procedure was followed with Intermediate (IB) to give Intermediate (IID) [25].

Synthesis of compound (III) & (IV):
A suspension of intermediate (IA) (2mmole, 0.56g) dissolved in (10ml) of dry chloroform, and was cooled down to -15 0 C then thionyl chloride (2mmole, 0.15mL) was added slowly to it. The mixture was refluxed for 4hr at 60-70 0 C with continuous stirring. Then evaporate the excess solvent by using rotary evaporator. The viscous liquid was slowly added drop wise into a beaker contain 5-fluorouracil (2mmole, 0.26g) dissolved in a dry 1,4 dioxane (5mL) and triethylamine (2mmole, 0.28ml) for 1hr on an ice bath, then stirring overnight at room temperature. The obtained suspension was filtered and the filtrate was washed with distilled water (20ml), dried with anhydrous magnesium sulfate and the solvent was evaporated by vacuum. The obtained compound was crystallized from diethyl ether-petroleum ether to give compound (III). The same procedure was followed with Intermediate (IB) to give compound (IV) [26].

Synthesis of Compound (V) & (VI):
The reaction of Intermediate (IIC) (1 mmole, 0.446g) and 5-fluorouracil as sodium salt (2mmole, 0.306g) in absolute ethanol (10ml) was refluxed for three hours at 65 0 C. The formed precipitate was filtered, washed with cold ethanol and recrystallized from ethanol as a pale yellow crystal of compound (V). The same procedure was followed with Intermediate (IID) to give pale brown crystals of compound (VI) [27]. J a n u a r y 2 3 , 2 0 1 4

5-fluoro-6-oxo-1,6-dihydropyrimidine-2-yl4(1-methoxy-1-oxo-3-phenylpropan-2-ylamino)-4-oxobutanoate. Compound (V)
As The rate of hydrolysis was followed spectrophotometrically (UV method) by recording the decreases in the absorbance of 5-fluorouracil derivatives accompanying the hydrolysis. The reactions were initiated by adding 1 ml of stock solutions (1mg ml) of the derivatives in methanol to preheated buffer solution at 37C to give final concentration of derivatives (0.02mg ml). The solutions were kept in a water bath at 37C and samples (3ml) were withdrawn at appropriate time interval (15,30,60,120 and 240 min.) and the absorbances were recorded. The observed first rate constants were determined from the slopes of the linear plots of log. concentration remaining versus time [28].

Results and Discussion
The synthetic pathways for the designed target compounds (III-VI) are illustrated in (scheme 1)

Scheme1. Synthesis of target compounds (III-VI) & their intermediates.
Intermediate (IA, IIC) (If R= Phenylalanine) Compound (III, V) J a n u a r y 2 3 , 2 0 1 4 Compound (IV, VI) Kinetic study: The max of fluorouracil 265 nm was the same max of compounds III, IV, V and VI, but they differ in molar absorpitivity (E) according to the Beer-Lambert equation [29]: (1) Thus making UV method relevant for studying the hydrolysis of these analogues, so the amount of fluorouracil alone was calculated from the following equation [29]:  One of the crucial requirements for a prodrug is that used, it should show a good stability in aqueous solutions and in gastrointestinal fluid, and it should be readily hydrolyzed following gastrointestinal absorption to release the parent drug [30].

Conclusion:
The synthetic procedure for the designed target compound was successfully achieved and the structural formula for the synthetic compound was characterized using IR spectroscopy, 1 H-NMR, melting points and Rf values. Preliminary kinetic study for compounds III, IV, V and VI revealed that these compounds were stable at pH 1.2 and pH 7.4.