Iraqi J Pharm Sci, Vol.25(1) 2016 Coumarin- based mutual prodrug of 5-fluorouracil 6 Design, Synthesis and Kinetic Study of Coumarin-Based Mutual Prodrug of 5-Fluorouracil and Dichloroacetic acid Yasser F. Mustafa * and Nohad A. Al-Omari *,1 * Department of Pharmaceutical Chemistry, College of Pharmacy,University of Mosul, Mosul, Iraq. Abstract On the basis of known coumarin-based prodrug system, a novel coumarin-based mutual prodrug of 5-fluorouracil and dichloroacetic acid was designed, synthesized and evaluated as a promising oral chemotherapeutic agent basing on in vitro stability study in HCl buffer (pH 1.2) and in phosphate buffer (pH 7.4), as well as in vitro release study in human serum. The chemical structure of prodrug was confirmed by analyzing its FTIR, 1 H NMR, 13 C NMR and MS-ESI spectra. The results of in vitro kinetic study indicated that the prodrug was significantly stable in HCl and in phosphate buffers, and was hydrolyzed in human serum followed pseudo first order kinetics. Keywords: Coumarin-based prodrug, 5-fluorouracil, Dichloroacetate, kinetics. تصمٍم، تصنٍع ودراسة حركٍة بادئ الذواء التبادلً المرتكز على الكىمارٌن الخلٍك حامض الكلىر وثنائً فلىروٌراسٍل -٥لـ ٌاسر فخري مصطفى * نهاد عبذ الىهاب العمريو ،*1 * ، انؼراق . جايؼة انًىصم،كهٍة انصٍذنةفرع انكًٍٍاء انصٍذالٍَة ، الخالصة -٥ اػحًادا ػهى َظاو بادئ انذواء انًرجكز ػهى انكىيارٌٍ، جى جصًٍى وجصٍُغ بادئ دواء جبادنً غٍر يأنىف يكىٌ يٍ فهىروٌراسٍم وثُائً انكهىر حايض انخهٍك، وجقًٍه كؼالج كًٍٍائً فًىي واػذ اسحُادا انى دراسة االسحقرارٌة خارج جسى انكائٍ انحً (، باالضافة انى ٧,٤( ويحهىل انفىسفٍث انبفري )أس هٍذوجًٍُ ١,٢نبفري )أس هٍذوجًٍُ فً يحهىل حايض انهاٌذروكهىرٌك ا دراسة انححرر فً يصم انذو انبشري خارج جسى انكائٍ انحً. انحأكذ يٍ انشكم انكًٍٍائً نبادئ انذواء يٍ خالل جحهٍم اطٍافه نألشؼة جحث انحًراء، انرٍٍَ انُىوي انًغُاطٍسً نههٍذروجٍٍ جى كربىٌ باالضافة نطٍف انكحهة. نقذ أشارت َحائج انذراسة انحركٍة خارج جسى انكائٍ انحً انى اسحقرارٌة بادئ انذواء انحبادنً بشكم ونه كبٍر فً يحهىل حايض انهاٌذروكهىرٌك انبفري ويحهىل انفىسفٍث انبفري وجحههه فً يصم انذو انبشري يحبؼا حركٍات انرجبة انزائفة األونى. .، ثنائً الكلىر حامض الخلٍك ، الحركٍة فلىروٌراسٍل --٥الذواء المرتكز على الكىمارٌن ، المفتاحٍة : لكلماتا Introduction The classical antimetabolite, 5- fluorouracil (5-FU), exerts its antitumor effect by inhibiting thymidylate synthase (1) and by misincorporating into DNA or RNA to inhibit its normal function (2) . It is widely used since its discovery in 1957 as a single agent in treatment of colorectal cancer and as a part of various regimens in treatment of breast, head, neck and upper gastrointestinal tumors (3) . However, 5-FU suffers from many drawbacks; first, it can cause many toxic effects as gastrointestinal disorders, myelosuppression, oral mucositis and cardiotoxicity (4) .Secondly, its efficiency is limited by the short plasma half-life after intravenous administration due to the activity of dihydropyrimidine dehydrogenase (DPD), and irregular absorption with unpredictable plasma level after oral administration (5) . Thirdly, the resistance of some tumors to chemotherapeutic effect of 5-FU; such resistance is a result of high expression of thymidine phosphorylase or low reserves of reduced folates (6) . To tackle these problems, many approaches have been developed and evaluated; the most important one is the use of prodrug strategy to yield 5-FU prodrugs that can avoid certain routes of degradation [7] or can target the tumor cells (8) . 1 Corresponding author E-mail: nohad.alomari@gmail.com Received: 3/11/2015 Accepted: 2/1/2016 Iraqi J Pharm Sci, Vol.25(1) 2016 Coumarin- based mutual prodrug of 5-fluorouracil 7 There is no doubt that prodrug strategies play a critical role in the development of drug delivery and drug discovery; one of these strategies is a coumarin-based prodrug system that is important for preparing esterase- sensitive prodrugs of alcohols, amines and peptides (9) . This system (Scheme 1) has several advantages such as the facile lactonization when an acyl group (R) is hydrolyzed by esterase and the safety profile of the final product, coumarin (10) . To date, this system is successfully used to prepare several prodrugs of opioid peptide (11) , non-peptide analgesic (12) and peptidomimetic (13) . O N R''R' O R O Esterase RCOOH O N R''R' OH Spontaneous HNR'R'' O O Scheme( 1): The illustration of a coumarin-based prodrug system for amine. Metabolic abnormity is a phenotypic trait of cancer cells. It is observed that cancer cells generally utilize glycolysis for energy production rather than oxidative phosphorylation. Thus, pyruvate is converted to lactate through anaerobic metabolism rather than its conversion to acetyl-CoA by action of pyruvate dehydrogenase (PDH) in aerobic glucose metabolism, this is called Warburg effect. Pyruvate dehydrogenase kinase (PDK) can inactivate PDH in many glycolytic phenotypes including cancer and switch the metabolism from anaerobic glycolysis to aerobic oxidation which is proved to be detrimental to tumor growth (14-16) . Dichloroacetate (DCA) has been known for many years as an experimental drug for treating certain metabolic disorders (e.g. inborn errors of metabolism) and as a promising agent in cancer therapy (17) . DCA is a well-known inhibitor of mitochondrial PDK; thus it can reverse Warburg effect, restore mitochondrial function, induce apoptosis and diminish the growth advantage of highly glycolytic tumors (18) . It is approved that the co-administration of DCA with 5-FU potentiates the antitumor activity of 5-FU in treatment of different types of cancer (19) . In addition, there are many reports indicated the beneficial effect of DCA in the treatment of glioblastoma, metastatic carcinomas, ovarian cancer, colorectal cancer, endometrial cancer, lung cancer and breast cancer (20- 22) . The aim of this work was to synthesize a novel coumarin - based mutual prodrug of 5- FU and DCA starting from coumarin and to evaluate it as a promising oral chemotherapeutic agent by monitoring its in vitro stability in HCl buffer (pH 1.2) and in phosphate buffer (pH 7.4), as well as its in vitro release in human serum. Experimental Materials All chemicals and solvents used in this work were purchased from commercial sources and used without further purification. Coumarin was purchased from Sigma-Aldrich, DCA and LiAlH4 from Tokyo Chemical Industry (TCI) and the others from Fluka. Instruments The instruments used for structure elucidation of the prodrug were: Bruker Avance DRX-400 MHz (Germany) to scan NMR spectra that were expressed in part per million upfield to TMS as an internal standard, Shimadzu LCMS-2020 Single Quadrupole Liquid Chromatograph Mass Spectrometer (Japan) with electrospray ionization source to measure the mass spectrum. The LCMS and NMR spectra were performed in Japan via Japan Food Research laboratories (JFRL). Bruker-Alpha ATR-FTIR spectrophotometer (Germany) to record the IR spectrum that performed in College of Pharmacy/University of Mosul. Melting points were determined, using open capillary method, on an electrochemical CIA 9300 melting point apparatus (UK) and were uncorrected. The instrument used to identify UV spectra and to follow kinetic study was Carrywinn UV Varian UV/Visible spectrophotometer. The purity of compounds and the completion of reactions were checked by thin layer http://www.google.iq/url?url=http://www.tcichemicals.com/en/gb/&rct=j&frm=1&q=&esrc=s&sa=U&ved=0CCUQjBAwBmoVChMIzYOAgLS8xwIVzD4UCh26iweU&usg=AFQjCNF-KV_P74Jd43OTzNJ1q-nRF4cvqA http://www.google.iq/url?url=http://www.tcichemicals.com/en/gb/&rct=j&frm=1&q=&esrc=s&sa=U&ved=0CCUQjBAwBmoVChMIzYOAgLS8xwIVzD4UCh26iweU&usg=AFQjCNF-KV_P74Jd43OTzNJ1q-nRF4cvqA Iraqi J Pharm Sci, Vol.25(1) 2016 Coumarin- based mutual prodrug of 5-fluorouracil 8 chromatography (TLC) using precoated silica gel plates (60G F254, Merck) and the spots on chromatograms were localized via UV light (at 366 nm). Structures, the calculated molecular formula and molecular weights were carried out by using Chemdraw Ultra 2010. Synthesis Dichloroacetyl chloride (23). Freshly distilled thionyl chloride (10 ml) was added dropwise to a cold solution of dichloroacetic acid (2.1 ml, 25 mmol) in 10 ml dry ether with continuous stirring under anhydrous condition. The mixture was heated at 40°C for 30 minutes and then refluxed for two hours. The solvent and the excess of thionyl chloride were removed under reduced pressure. The product was obtained by distillation at 108°C as slightly yellowish liquid with 82% of yield and λmax (ethanol) = 244 nm. O O (1) OH OH (2) LiAlH4, 0°C 15 min OH O Si (3) TBDM S-Cl, DM AP 0°C, 14 h O O Si O (4) ClCl Dichloroacetyl chloride K2CO3, CH2Cl2, 4 h THF: H2O: HOAc (1:1:3) 50 o C, 1 h O Cl Cl O (5) OH O Cl Cl O O (6) M nO2, CHCl3 Reflux, 20 h O Cl Cl O (7) 10 o C, 155 min O OH NaClO2, H2O2 5-FU, DCC, DMAP (Prodrug) TEA, RT, overnight O O N NH O O F Cl Cl O Scheme (2): Synthetic pathway of coumarin-based mutual prodrug. (Z)-2-(3-hydroxypropenyl)phenol (2). A solution of 1 (3.65 g, 25 mmol) in 50 ml dry ether was placed in an ice bath and treated with a solution of 1.0 M of pure LiAlH4 in dry ether (1.9 g of LiAlH4 dissolved in 50 ml, 50 mmol). After stirring for 15 min, 5 % HCl (25 ml) was added to the reaction at 0°C. Then the solution was adjusted to pH 5 with 1M HCl and extracted with ether (3×50 ml). The ether layer was dried over Na2SO4, filtered and evaporated. The residue was dissolved in Iraqi J Pharm Sci, Vol.25(1) 2016 Coumarin- based mutual prodrug of 5-fluorouracil 9 ethanol, filtrated and evaporated to afford the desired product. (Z)-2-(3-(tert-butyldimethylsilyloxy)propenyl) phenol (3). A solution of compound 2 (3.43 g, 22.8 mmol) in 40 ml dry THF was placed in an ice bath. To this solution, tert-butyldimethylsilyl (TBDMS) chloride (3.79 g, 25 mmol) dissolved in 35 ml dry THF was added at 0°C. Then N,N-dimethylaminopyridin (DMAP) (4.18 g, 34 mmol) in 40 ml dry THF was added in a dropwise manner. After stirring for 14 hours at 0°C, the solution was filtered and evaporated to remove the THF. The residue was re-dissolved in ethyl acetate (50 ml) and washed with 1 M HCl (2 × 27 ml), 5 % NaHCO3 (22 ml) and H2O (22 ml). The ethyl acetate layer was dried over Na2SO4, filtered and evaporated. The residue was then crystallized from CHCl3. (Z)-2-(3-(tert-butyldimethylsilyloxy)propenyl) phenyl dichloroacetate (4) To a solution of compound 3 (2.65 g, 10 mmol) in 25 ml dry CH2Cl2 treated with K2CO3 (600mg), solution of dichloroacetyl chloride (1 ml, 10 mmol) in 10 ml dry CH2Cl2 was added dropwise for 30 minutes. The reaction mixture was refluxed for 4 hours. The progress of reaction was monitored by TLC using ether: ethyl acetate (1:1) mixture as a mobile phase. The reaction mixture was washed with water (2×25 ml); the organic layer was dried over anhydrous Na2SO4 and concentrated in rotary evaporator to dryness. The residue was re-dissolved in ethyl acetate, filtrated and evaporated to afford the desired product. ( Z ) -2 - ( 3- hydroxypropenyl )phenyl dichloroacetate (5) To a solution of compound 4 (3 g, 8 mmol) in THF (20 ml), water (20 ml) was added. This was followed by dropwise addition of acetic acid (60 ml). The mixture was stirred at 50ᴼC for one hour and then evaporated to remove THF, water and acetic acid under reduced pressure. Ethyl acetate (50 ml) was added to the residue, which was washed with 5 % NaHCO3 (2 × 25 ml) and water (2 × 25 ml). The ethyl acetate layer was dried over Na2SO4, filtered and evaporated. The desired product was then crystallized from ethanol. (Z)-2- ( 3-oxopropenyl)phenyl dichloroacetate (6) A suspension of 5 (2.09 g, 8 mmol) and MnO2 (3.48 g, 40 mmol) in CHCl3 (50 ml) was heated at reflux for 20 hours. The hot mixture was filtered, washed with warm CHCl3 (2×25 ml) and the solvent was evaporated under reduced pressure. The residue was re-dissolved in 30 ml acetone, filtered and the filtrate was evaporated to afford the desired product. (Z)- 3- [2- (dichloroacetyloxy) phenyl ] acrylic acid (7) A solution of sodium chlorite (660 mg, 7.33 mmol) in water (6 ml) was added dropwise very slowly to a stirred mixture of compound 6 (1.04 g, 4 mmol) in ACN (15 ml), sodium dihydrogen phosphate (102 mg, 0.85 mmol) in water (1.65 ml), and 30 % hydrogen peroxide (0.5 ml, 4.17 mmol). During the addition, the reaction temperature was kept at 10ᴼC in a cold water bath and oxygen evolution from the solution was observed visually until the end of the reaction. When oxygen evolution was ended (about 155 minutes from the first addition of sodium chlorite), a small amount of sodium sulfite (0.05 g) was added to destroy the unreacted HOCl and H2O2. The solution was acidified with 1 M HCl to pH 2. The mixture was then extracted with ethyl acetate (2×50 ml). The combined ethyl acetate layer was washed with saturated sodium chloride solution (2 × 25 ml), dried over anhydrous Na2SO4, filtered and evaporated. The residue was re-dissolved in ACN, treated with aqueous solution of 10% NaHCO3 to pH 6.5 and filtered. The filtrate was acidified with 1M HCl to pH 2.5 and the desired product obtained by filtration. (Z)- 2 [ 3- ( 5 – fluorouricyl )-3-oxopropenyl] phenyl dichloroacetate (prodrug) To a cold solution of 7 (1.1 g, 4 mmol) in 50 ml freshly distilled DMSO, 5-FU (0.52 g, 4 mmol), DCC (1 g, 4.8 mmol), DMAP (40 mg, 0.33 mmol) and triethylamine (0.6 ml , 4 mmol) were sequentially added to the reaction mixture. The solution was stirred overnight at room temperature. After addition of methanol (10 ml) and acetic acid (0.75 ml), the mixture was stirred for one hour and then neutralized with 10% aqueous NaHCO3 solution. The resulting solid was filtered and the filtrate was evaporated by rotary evaporator. The resulted residue was washed with (20 ml×3) distilled water and then dried. The desired product was crystallized from a mixture of chloroform and petroleum ether. Kinetic study The synthesized prodrug was subjected to chemical hydrolysis in buffers of physiological pH values, and enzymatic hydrolysis in human serum. These hydrolytic reactions were monitored by double beam UV/Visible spectrophotometer for decreasing in the concentration of prodrug with the time by applying a Beer’s law equation, that is: Iraqi J Pharm Sci, Vol.25(1) 2016 Coumarin- based mutual prodrug of 5-fluorouracil 10 Absorbance = Ԑ × L × C Where Ԑ is the molar extinction coefficient or called absorbance coefficient. L is the path length of cell holder (2cm). C is the concentration. Stability study in acidic buffer (pH 1.2) and in basic buffer (pH 7.4). The in vitro chemical hydrolysis of the prodrug was studied in (0.1 M) hydrochloric acid buffer (pH 1.2) and in (0.1 M) phosphate buffered saline (pH 7.4). A sample (5μmol) of prodrug was dissolved in 2 ml anhydrous DMSO in a 100ml beaker. To this solution, 48 ml preheated buffer solution was added with gentle stirring to achieve a final concentration of 100 μM. At the end of addition, the time was detected and the resulted solution was kept at a constant temperature (37 ± 1°C) using a water bath. Then, the solution was divided into a set of ten test tubes; each one would contain 5 ml. At selected time intervals of 30, 60, 90, 120, 150, 180, 210 and 240 minutes, a test tube was removed from a water bath and its content extracted with 2 ml CH2Cl2. Aliquot (2 ml) was withdrawn from aqueous layer and estimated at defined λmax on UV/Visible spectrophotometer to determine the remaining concentration of prodrug. Release study in serum. The in vitro enzymatic hydrolysis of the prodrug was studied in serum; a sample (2.5μmol) of prodrug was dissolved in 2 ml phosphate buffered saline in a 50ml beaker. To this solution, 23 ml preheated serum was added with gentle stirring to achieve a final concentration of 100 μM. At the end of addition, the time was detected and the resulted solution was kept at a constant temperature (37 ± 1°C) using a water bath. Then, the solution was divided into a set of ten test tubes; each one would contain 2.5 ml. At selected time intervals of 30, 60, 90, 120, 150, 180, 210 and 240 minutes, a test tube was removed from a water bath and its content extracted with 2 ml CH2Cl2. Aliquot (2 ml) was withdrawn from aqueous layer and estimated at defined λmax on UV/Visible spectrophotometer to determine the remaining concentration of prodrug. Results and Discussion The oral use of 5-FU was forsaken during the past decades because of its irregular absorption (24) and unpredictable plasma level with high intra- and inter-individual variations due to the fickle activity of DPD in the gastrointestinal mucosa [25] . Although the oral use of DCA is well tolerated, but it may cause many gastrointestinal (e.g. nausea, vomiting and heartburn) [26] and central nervous system (e.g. neuropathy, confusion and twitching) related side effects that are highly dose dependent [27] . In an attempt to optimize the drug-like properties of 5-FU and DCA, a novel coumarin-based esterase-sensitive mutual prodrug was designed, synthesized and evaluated depending on in vitro stability study in HCl buffer (pH 1.2) and in phosphate buffer (pH 7.4), as well as on in vitro release study in human serum. Synthetic part The prodrug was synthesized through a sequence of 7 linear steps starting from coumarin; this synthetic pathway (scheme 2) can be considered as a modification to that described by Wang et al [28] . The first step involved reduction of coumarin to an open ring diol with lithium aluminum hydride (LiAlH4). Higher temperature than 0°C and/or longer reaction time may lead to reduce the exocyclic double bond whereas the use of commercially available LiAlH4 may lead to lower the yield to high extend. The following steps involved selective protection of allylic hydroxyl group resulted from the previous step with TBDMS-chloride, and acylation of free phenolic hydroxyl group with dichloroacetyl chloride. When aromatic ester is formed, the TBDMS ether was cleaved under acidic condition to afford allylic hydroxyl group that converted to carboxylic acid group in two steps. Coupling of the free carboxylic acid with 5-FU was carried out by using dicyclohexyl carbodiimide (DCC) as an activating agent. Iraqi J Pharm Sci, Vol.25(1) 2016 Coumarin- based mutual prodrug of 5-fluorouracil 11 Table( 1): The physicochemical properties of compounds (1-8). Compound number Physical appearance Yield % M.p. (ᴼC) Rf chloroform: acetone (4:1) λmax (nm)e 1 White needle like crystals ------- 68-70 0.696 315 2 White crystals 41 148-151 0.337 286 3 White crystals 76 122-124 0.556 281 4 White needle like crystals 79 86-88 0.601 307 5 White crystals 92 102-105 0.478 304 6 White powder 68 119-122 0.576 316 7 White crystals 89 154-156 0.489 311 prodrug White needle like crystals 82 136-138 0.589 326 Table (2): The calculated molecular formula, molecular weight and elemental analysis of compounds (1-8). Comp. No. Molecular formula M. Wt. Elemental analysis %C %H %Cl %F %N %O %Si 1 C9H6O2 146.14 73.96 4.14 ----- ----- ----- 21.90 ----- 2 C9H10O2 150.17 71.98 6.71 ----- ----- ----- 21.31 ----- 3 C15H24O2Si 264.44 68.13 9.15 ----- ----- ----- 12.10 10.62 4 C17H24Cl2O3Si 375.36 54.40 6.44 18.89 ----- ----- 12.79 7.48 5 C11H10Cl2O3 216.10 50.60 3.86 27.16 ----- ----- 18.38 ----- 6 C11H8Cl2O3 259.08 50.99 3.11 27.37 ----- ----- 18.53 ----- 7 C11H8Cl2O4 275.08 48.03 2.93 25.78 ----- ----- 23.26 ----- Prodrug C15H9Cl2FN2O5 387.15 46.54 2.34 18.31 4.91 7.24 20.66 ----- The structure of compounds (2-7) was characterized by monitoring the presence and/or absence of specific functional groups using the FTIR spectrophotometer, whereas the prodrug structure was established by analyzing its FTIR, 1 H NMR, 13 C NMR and mass spectra. 1 H-NMR (DMSO-d6) spectrum of the prodrug (Figure 1) showed the chemical shift of the following protons: δ 11.95 ppm (s, 1H, NH), δ 7.97 ppm (d, 1H, -FC=CH-N), δ 7.72 ppm (d, 1H, Ar-CH), δ 7.30-7.45 ppm (m, 4H, aromatic), δ 6.45 ppm (d, 1H, CH=CH-CO), and δ 6.25 ppm (s, 1H, CHCl2). 13 C-NMR (DMSO-d6) spectrum of the prodrug (Figure 2) reported the chemical shift of the following carbons: δ 164.75 ppm (O-CO-), δ 160.90 ppm (=CH-CO-N), δ 158.08 ppm (CF-CO-), δ 154.03 ppm (-C-O), δ 150.05 ppm (N-CO- NH), δ 143.55 ppm (Ar-CH), δ 141.39 ppm (- CF), δ 131.88, 127.91, 124.48, 116.93, 116.68 ppm (aromatic), δ 126.09 ppm (FC=CH), δ 118.87 ppm (-CH-CO-N) and δ 73.53 ppm (- CHCl2). Iraqi J Pharm Sci, Vol.25(1) 2016 Coumarin- based mutual prodrug of 5-fluorouracil 12 Figure (1): 1 H-NMR (DMSO-d6) spectrum of the prodrug. Figure (2): 13 C-NMR (DMSO-d6) spectrum of the prodrug. Iraqi J Pharm Sci, Vol.25(1) 2016 Coumarin- based mutual prodrug of 5-fluorouracil 13 FTIR spectrum of the prodrug revealed the characteristic absorption band for the following functional groups: ν 797 cm -1 (C- Cl), ν 1167 cm -1 (C-F), ν 1648 cm -1 (C=O, 3ᴼ amide), ν 1668 cm -1 (C=O, 2ᴼ amide), ν 1732 cm -1 (C=O, ester), ν 2822 cm -1 (-CH), ν 3037 cm -1 (=CH) and multiple bands for N-H at ν 3114.33, 3153, 3186 cm -1 . MS-ESI spectrum (m/z) of the prodrug operated in a positive mode ( Figure 3) characterized the mass of the following products: 388 [M+H] + , 402 [M+Nebulizer gas,CH3], 410 [M+Na] + , 276 [M- O=C=Cl2], 248 [M-(O=C=Cl2 & CO)]. Figure (3): MS-ESI spectrum (m/z) of the prodrug operated in a positive mode. Kinetic part Under experimental conditions, the stability study in HCl buffer (pH 1.2) and in phosphate buffer (pH 7.4) showed a significant stability of prodrug with half-lives of about 33hr and 18hr respectively. In human serum, the prodrug was liberated 5-FU and DCA followed pseudo first order kinetics (Figure 4) with half-life of about 7hr. Data obtained from the kinetic study (average of three trials) were listed in Table 3, while the kinetic parameters listed in Table 4. Iraqi J Pharm Sci, Vol.25(1) 2016 Coumarin- based mutual prodrug of 5-fluorouracil 14 Table (3): Data obtained from kinetic study. Absorbance Medium Time (min.) x (M×10 6 ) a-x (M×10 6 ) ln a/a-x 0.0568 pH 1.2 0 0 100.000 0 0.0596 pH 7.4 0 100.000 0 0.0531 Serum 0 100.000 0 0.0562 pH 1.2 30 1.0391 98.9609 0.0104 0.0584 pH 7.4 1.8940 98.0160 0.0200 0.0505 Serum 4.8206 95.1794 0.0494 0.0556 pH 1.2 60 2.0722 97.9278 0.0209 0.0574 pH 7.4 3.7521 96.2479 0.0382 0.0484 Serum 8.8512 91.1488 0.0927 0.0551 pH 1.2 90 3.0282 96.9718 0.0308 0.0561 pH 7.4 5.8725 94.1275 0.0605 0.0458 Serum 13.7848 86.2152 0.1483 0.0544 pH 1.2 120 4.2077 95.7923 0.0430 0.0553 pH 7.4 7.2148 92.7852 0.0749 0.0436 Serum 17.9397 82.0603 0.1977 0.0539 pH 1.2 150 5.1002 94.8998 0.0523 0.0542 pH 7.4 9.1189 90.8811 0.0956 0.0419 Serum 21.0923 78.9077 0.2369 0.0533 pH 1.2 180 6.1620 93.8380 0.0636 0.0533 pH 7.4 10.5705 89.4295 0.1117 0.0391 Serum 26.3653 73.6347 0.3061 0.0528 pH 1.2 210 7.0246 92.9754 0.0728 0.0519 pH 7.4 12.9195 87.0805 0.1383 0.0376 Serum 29.2399 70.7601 0.3459 0.0522 pH 1.2 240 8.0371 91.9629 0.0838 0.0511 pH 7.4 14.1847 85.8153 0.1529 0.0355 Serum 33.1450 66.8550 0.4026 a = conc. of prodrug at zero time and (a-x) = conc. of prodrug remaining for any time. Table (4): Parameters obtained from kinetic study. pH1.2 pH 7.4 Serum Ԑ = 284 Ԑ = 298 Ԑ = 265.5 λmax = 313 nm λmax = 338 nm λmax = 332 nm t1/2 = 1991.38 min t1/2 = 1087.91 min t1/2 = 420.80 min kobs = 0.000348 min -1 kobs = 0.000637 min -1 kobs = 0.001647 min -1 ε = absorbance coefficient and Kobs = observed rate constants of hydrolysis. Iraqi J Pharm Sci, Vol.25(1) 2016 Coumarin- based mutual prodrug of 5-fluorouracil 15 Figure (4): Pseudo first order slope of the in vitro hydrolysis of prodrug in human serum. Conclusion This work reported the first attempt to utilize a coumarin-based prodrug system to deliver two active moieties which are 5-FU and DCA. Thus, it is believed that the synthesized compound is a first agent belongs to a new prodrug strategy, the coumarin-based mutual prodrug system, and is a promising oral chemotherapeutic agent. Further studies are recommended to establish the result obtained from this work. References 1. 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