Design and Synthesis of azo Derivatives of 5-Fluorouacil for Targeting Colon Cancer

1257 | P a g e J a n u a r y 2 7 , 2 0 1 4 Design and Synthesis of azo Derivatives of 5-Fluorouacil for Targeting Colon Cancer Mohammad Abdul Amir Ulaiwy , Mohammed Hassan Mohammed 1 Department of Pharmaceutical Chemistry, College of Pharmacy, University of Baghdad, Baghdad, Iraq Corresponding Author; College of Pharmacy, Baghdad University, Baghdad, Iraq. E-mail: dr.mhassanm666666@yahoo.com Abstract


Introduction
The aim of most cancer chemotherapeutic drugs currently in clinical use is to kill malignant tumor cells by inhibiting some of the mechanisms implied in cellular division. However, the knowledge of tumor biology has exploded during the past decades and this may pave the way for more active, targeted anticancer drugs (1) . It is obvious that cancer chemotherapy is a very difficult task (2) . One of its main associated problems is the nonspecific toxicity of most anticancer drugs due to their biodistribution throughout the body, which requires the administration of a large total dose to achieve high local concentrations in a tumor. Drug targeting was aimed at preferred drug accumulation in the target cells independently on the method and route of drug administration (3) . One approach that allows improving the selectivity of cytotoxic compounds is the use of prodrugs that are selectively activated in tumor tissues, taking advantage of some unique aspects of tumor physiology, such as selective enzyme expression, hypoxia, and low extracellular pH (4) . Colorectal cancer (CRC) is a type of cancer that starts in the colon or rectum portion of large intestine in the gastrointestinal (GI) tract (5) .Occurrence of CRC may be dependent on various factors, namely diet, life style and other environmental parameters, including environmental and food-borne mutagens (6) . It is estimated that chronic inflammation, likely to be caused by a dysregulated intestinal microbiota, contributes to approximately 20% of all CRC cases (7,8) . 5-Fluorouracil (5-FU) is still a widely used anticancer drug. Since 1957, it has played an important role in the treatment of colon cancer and is used for patients with breast and other cancers, like those of the head and neck (9) . Due to its structure, 5-FU interferes with nucleoside metabolism and can be incorporated into RNA and DNA, leading to cytotoxicity and cell death (10,11) . .It is administered parenterally because it shows highly variable oral bioavailability due to the unpredictable and incomplete absorption after ingestion of the drug and a marked bioinactivation by dihydropyrimidine dehydrogenase inthe liver and the mucosal membraneof thegastrointestinal tract (12,13) . Once administrated to a patient, 5-FU tends to distribute randomly through the body with low selectively and concentration in the Lumina of the colorectal tissue, resulted in significant toxicities. If an appropriate large molecular carrier is chemically linked to the 5-FU [1] with specificity for colorectal cancer cells, this will enhance the therapeutic index as well as allow the dosage of the drug to be reduced (if so desired) to minimize toxicities. In addition, with the higher specificity and lower toxicity of such prodrug, the oncologist could also titrate the dosage to the level that a patient could maximally tolerate, thus maximizing the efficacy and the likelihood appositive outcome for the patient. Large molecule carriers include ethylene or acrylic acid polymers, polysaccharides, hydroxyl acid polymers, and amino acid polymers, etc . (14) . [1] Because of this specific linkage, the 5-FU prodrug cannot be digested or hydrolyzed in the upper gastrointestinal tract and therefore is delivered specifically to the colorectal area. Once arriving at the colorectal area, the 5-FU will be released by hydrolysis of the prodrug by bacterial enzymes in the lower intestinal tract (15) . An association between inflammation and cancer has been suggested for a long time and it is now well-recognized that inflammation is involved in carcinogenesis in several tissues (16) . The development of hepatocellular carcinoma (HCC) caused by hepatitis B virus or hepatitis C virus (HCV) infection , gastric cancer caused by H. pylori infection , and colitis associated cancer caused by ulcerative colitis are representative examples of inflammation-associated carcinogenesis. Various inflammatory mediators are highly expressed in inflammatory tissues, and inflammation-triggered anti-apoptosis activity and cell growth cause cancer development (17) . Inflammatory bowel disease, such as ulcerative colitis and Crohn"s disease is a longstanding inflammatory disease of intestine with increased risk for colorectal cancer (CRC). Several molecular events involved in chronic inflammatory process are reported to contribute to multi-step carcinogenesis of CRC in the inflamed colon. They include over-production of free radicals, reactive oxygen and nitrogen species, up-regulation of inflammatory enzymes in arachidonic acid biosynthesis pathway, up-regulation of certain cytokines, and intestinal immune system dysfunction (18) .Many compounds belonging to diverse chemical classes have been identified as potential chemopreventive agents, including vitamins and minerals, naturally occurring phytochemicals, and synthetic compounds. Understanding the molecular mechanisms of cancer chemoprevention is not only important for the safe application of these compounds in populations of patients at high risk for cancer, but also allows for further development of novel treatment regimens for cancer patients (19) . The most frequently used chemopreventive agents in UC patients are 5-aminosalicylic acid (5-ASA) compounds (mesalazine and sulfasalazine) as well as ursodeoxycholic acid (UDCA) (20) .Mesalazine or 5-aminosalicylic acid (5-ASA) is a drug widely used in IBDs, mainly UC, for the treatment of mild relapses and maintenance of remission. Mesalazine is structurally related to NSAIDs, but unlike these compounds, it is safe and free of serious adverse effects. Epidemiological observations indicate that mesalazine can be also chemopreventive for IBD-associated CRC (21) . Moreover studies conducted in experimental models of carcinogenesis have shown that the drug has many targets in J a n u a r y 2 7 , 2 0 1 4 cancer cells and modulates multiple biological pathways that sustain CRC (22) . The colon has recently received great attention as a potential site for the delivery of pharmaceutical moieties. Delivery of drugs via the colon offers numerous therapeutic advantages. The successful delivery of drugs to the colon via the gastrointestinal (GI) tract requires the protection of a drug from being released in the stomach and small intestine. This can be achieved by the use of a special drug delivery system that can protect the drug during its transfer to the colon (23) . Prodrug approach is one of the important approaches for targeting drugs to colon. Colon-specific drug delivery through colon-specific prodrug, activation may be accomplished by the utilization of high activity of certain enzymes at the target site, relative to non-target tissues (24) . In view of these observations, three new derivatives of 5-fluorouracil have been designed, synthesized and characterized.

The chemistry
The designated compounds were synthesized according to schemes (1 to 3). A series of intermediates were synthesized as precursors of the target compounds by several experimental steps. The physical properties of the synthesized compound are showed in table 1. The structure of the final compounds and their intermediates were characterized by elemental microanalysis (table2) and infrared spectroscopy (table J a n u a r y 2 7 , 2 0 1 4 2: The synthesis of compound 2. J a n u a r y 2 7 , 2 0 1 4  The stability of compounds 2&3 was studied in phosphate buffer solution (pH 1.5) and (pH 7.8) incubated at 37C. The rate of hydrolysis was followed spectrophotometrically by recording the decreases in the absorbance of compounds 2&3 accompanying the hydrolysis at their max (305nm). The solutions were kept in a water bath at 37C and samples (3ml) were withdrawn at appropriate time interval (15,30,60,120,240 min.) and the absorbances were recorded at the max of each compound. The observed pseudo first rate constants were determined from the slopes of the linear plots of log. Concentration versus time. The result are showed in tables (4,5,6 and7) and figures (1, 2,3 and4).  The half-life was calculated using equation (2), which is derived from the pseudo first order kinetic law.
Therefore; t 1/2=7.86 hrs. J a n u a r y 2 7 , 2 0 1 4  The half-life was calculated using equation (2), which is derived from the pseudo first order kinetic law.
Therefore; t 1/2=5.35 hrs.  The half-life was calculated using equation (2), which is derived from the pseudo first order kinetic law.

Figure (4): Log concentration of compound 3 incubated in phosphate buffer (pH 7.8) vs. time.
The half-life was calculated using equation (2), which is derived from the pseudo first order kinetic law.

General
Chemicals used in the synthesis were of analytical grade. The melting points of the compounds and their intermediates (uncorrected) were determined by capillary tube method on Barnstead Electrothermal (USA) and FT-IR spectra were recorded on FT-IR spectrophotometer Shimadzu (Japan) at the college of pharmacy /University of Baghdad. The CHNO analysis was carried out using Euro-vector EA3000A(Italy).Ascending thin layer chromatography(TLC)was run on silica gel 60 F254 pre-coated aluminum sheets, Merck (Germany) to check the purity and the reaction , sprogress.The detection of derivatives was done using UV254nm lamp and the chromatograms were eluted by three solvent systems: A) Chloroform: methanol (9:1) (25) . B) Chloroform: ethyl acetate: methanol (2:2:1). C) Toluene: ethyl acetate: formic acid (5:4:1).

Synthesis of 3-carboxy-4-hydroxybenzenediazonium chloride (compound A1) (23) .
In 500ml conical flask, 5-aminosalicylic acid (3.06g, 20mmol) was dissolved in a mixture of concentrated HCl (20ml), water (50ml) the resulting solution was stirred and cooled in an ice bath to 0С°. Then a cold solution of sodium nitrite (6.9g, 100mmol) in water (15ml) was placed in a dropping funnel and added drop wise into the first solution during 15min.the temperature should not be allowed to rise above 10С°.The last quantity of the sodium nitrite solution was added more slowly and after stirring for 3-4 minutes, a drop of the solution diluted with 4 drops of water was tested with potassium iodide-starch; if no immediate blue color was obtained at point of contact with the paper, a further amount of sodium nitrite solution was added. The testing was continued every 5 minutes until an immediate blue color was obtained. The reaction mixture was further stirred for 20min. in an ice bath at 0-5С°. The diazonium salt formed was used immediately in the next step.

Synthesis of 5, 5'-(diazene-1, 2-diyl)bis(2-hydroxybenzoic acid) (compound A) (Azo bond formation) ( 23) .
Salicylic acid (2.76g, 20mmol) was dissolved in (50ml) of (10%) NaOH solution in a suitable beaker immersed in an ice bath. The solution was stirred vigorously and the temperature was kept below 5С°. The cold diazonium salt solution from the previous step (compound A1) was placed in a dropping funnel, then it was added drop by drop to the cooled, stirred salicylic acid solution, a brown color was developed and a brown crystals soon separated in a good yield, at the end of the addition the mixture was stirred in an ice bath for further 1hr. The product was collected by filtration and washed with water (3x50ml) and dried under vaccum overnight at room temperature.

Synthesis of4-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-oxobutanoylchloride. (Compound1b).
Compound 1a (1.15g, 5mmol) was suspended in dry chloroform (20ml) in around bottom flask, the mixture was stirred in an ice bath where the temperature should be below 0С°, then thionyl chloride (0.7ml, 10mmol) was added drop wise over a period of 30minutes with continuous stirring. After completing the addition the temperature was allowed to reach room temperature and reflux was then started at about 75-80С° for 3hours. The solvent and the excess thionyl chloride were evaporated by rotary evaporator followed by re-dissolving in chloroform and re-evaporation several times to ensure getting rid of the side products (HCl and SO2 gases), leaving a white oily residue which was used immediately in the next step without further purification. (27) .

Synthesis of 1-(2-chloroacetyl)-5-fluoropyrimidine-2, 4(1H, 3H)-dione. (Compound 2a)
A mixture of 5-FU (1.3g, 10mmol) and dry TEA (1.01g, 10mmol) in dry dichloromethane (25ml) was cooled in an ice salt mixture to -10С°. To this reaction mixture, chloroacetyl chloride (0.8ml, 10mmol) in dry chloroform (25ml) was added drop wise through a dropping funnel with constant stirring over a period of 1hour maintaining the temperature constant. The reaction mixture was stirred further for 5hrs. The temperature then was allowed to reach room temperature and reflux was J a n u a r y 2 7 , 2 0 1 4 started at 80С° for 3hours, Solvents were removed and the residue was washed with sodium bicarbonate solution (5% w/v, 30ml) and subsequently with cold water (50ml). The crude product was dried and on recrystallization from ethanol afforded white fluffy powder (compound 2a).

Synthesis of 2-(chlorocabonyl) phenyl acetate. (Compound 3a).
Acetylsalicylic acid (4.5g, 25mmol) was dissolved in dry chloroform (30ml) in around bottom flask; the mixture was stirred in an ice bath where the temperature should be below 0С°. Then thionyl chloride (3.5ml, 50mmol) was added drop wise over a period of 30min. with continuous stirring. After completing the addition the temperature was allowed to reach room temperature and reflux was then started at about 75-80С° for 4hrs. the solvent and the excess thionyl chloride were evaporated with rotary evaporator followed by re-dissolving in chloroform and re-evaporation several times to ensure getting rid of the side products (HCl and SO2 gases) leaving a colorless oily residue (3.48g) which was used immediately in the next step without further purification. (30) .

Synthesis of 2-(2-acetoxybenzamido) acetic acid. (Compound 3b)
The whole quantity of compound 3a obtained (3.48g, 17.5mmol) was dissolved in dry chloroform (50ml) and added drop wise to a stirred solution of glycine (1.3g, 17.5mmol) in dry pyridine (50ml) with continuous stirring at ice bath. After the addition was complete, stirring was continued at room temperature for further 3hours. The reaction mixture was then poured into cold water (200ml) followed by addition of few drops of concentrated HCl. The precipitate was collected by filtration, washed with D.W (50ml x2) and recrystallized from ethanol.

Synthesis of 2-((2-chloro-2-oxoethyl) carbamoyl) phenyl acetate.(Compound 3c).
Compound (3b) (2.37g, 10mmol) was dissolved in dry chloroform (30ml) in around bottom flask; the mixture was stirred in an ice bath where the temperature should be below 0С°. Then thionyl chloride (1.4ml, 20mmol) was added drop wise over a period of 30min. with continuous stirring. After completing the addition the temperature was allowed to reach room temperature and then reflux was started at about 75-80С° for 4hrs. the solvent and the excess thionyl chloride were evaporated with rotary evaporator followed by re-dissolving in chloroform and re-evaporation several times to ensure getting rid of the side products (HCl and SO2 gases) leaving a yellowish oily residue (1.73g) which was used immediately in the next step without further purification.
To a stirring solution of compound (3d) (1.75g, 5mmol) in a mixture of absolute ethanol : DMF (4:1), kept on 18-22С°, an aqueous solution of 1N NaOH (12.5ml) was added drop wise over a period of 30min. where the stirred reaction mixture was continued for additional 5hrs. then the solvents evaporated, and the residue was dissolved in water and acidified with 1N HCl to get PH 1. After cooling a white precipitate was appeared which was separated by filtration, washed on the filter paper with water and recrystallized from ethanol.

Synthesis of compound 3.
This compound was synthesized by following procedures similar to the procedures followed in the synthesis of compound A.

Synthesis of Compound A1
Compound A1 has been synthesized for preparing compound A and it was prepared again since it was needed in in the synthesis of compound 3.
In 500ml conical flask, 5-ASA (0.77g, 5mmol) was dissolved in a mixture of conc. HCl (5ml) and water (12.5ml). The resulting solution was stirred and cooled in an ice bath to 0С°. Then a cold solution of sodium nitrite (1.73g, 25mmol) in water (10ml) was placed in a dropping funnel and added drop wise into the first solution during 15min. The last quantity of the sodium nitrite solution was added more slowly and after stirring for 3-4 minutes, a drop of the solution diluted with 4 drops of water was tested with potassium iodide-starch; if no immediate blue color was obtained at point of contact with J a n u a r y 2 7 , 2 0 1 4 the paper, a further amount of sodium nitrite solution was added. The testing was continued every 5 minutes until an immediate blue color was obtained. After completing the addition of sodium nitrite solution, the reaction mixture was further stirred for 20min. in an ice bath at 0-5С°. The diazonium salt formed was used immediately in the next step.

Synthesis of 5-((3-((2-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-oxoethyl)carbamoyl)-4hydroxyphenyl)diazenyl)-2-hydroxybenzoic acid. (Compound 3) (Azo bond formation).
Compound (3e) (1.53g, 5mmol) was dissolved in (12.5ml) of 10% NaOH solution in a suitable beaker immersed in an ice bath. The solution was stirred vigorously and the temperature was kept below 5С°. The cold diazonium salt solution from the previous step (compound A1) was placed in a dropping funnel, then it was added drop by drop to the cooled, stirred solution of compound (3e), an orange color was developed and orange crystals soon separated. At the end of the addition the mixture was stirred in an ice bath for further 1 hour. The product was collected by filtration and washed with water (3x50ml) and dried at room temperature overnight.