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Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe 2+ chelating activity

Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe 2+ chelating activity Acta Pharm. 60 (2010) 325­337 10.2478/v10007-010-0024-9 Original research paper ZRINKA RAJI]1 MARIJANA ZOVKO KON^I]1 KRISTINA MILOLO@A1 IVANA PERKOVI]1 IVAN BUTULA1 FRANZ BUCAR2 BRANKA ZORC1,* 1 Faculty of Pharmacy and Biochemistry University of Zagreb, Zagreb, Croatia 2 Institut für Pharmazeutische Wissenschaften, Bereich Pharmakognosie Graz, Austria Novel primaquine conjugates witon-steroidal anti-inlammatory drugs (PQ-NSAIDs, 4a-h) were prepared, fully chemically characterized and screened for radical scavenging and antioxidant activities. The synthetic procedure leading to twin drugs 4a-h involved two steps: i) preparation of NSAID benzotriazolides 3a-h from the corresponding NSAID (ibuprofen, ketoprofen, fenoprofen, ketoprofen hydroxy and methylene analogues, diofenac or indomethacin) and benzotriazole carboxylic acid chloride (BtCO, 1), ii) reaction of intermediates 3a-h with PQ. The prepared PQ-NSAIDs exerted moderate activities in the DPPH free radical test and b-carotene-linoleic acid assay. Moreover, ketoprofen derivatives 4d and 4b demonstrated a notable Fe2+ chelating ability as well. On the other hand, negligible antiproliferative and antituberculotic effects of conjugates 4a-h were observed. Kewords: primaquine, NSAID, twin drug, conjugate, radical scavenging, antioxidant activity, chelating ability Accepted August 26, 2010 Excessive production of reactive oxygen species (ROS), associated with inflammation, leads to the condition of oxidative stress. Oxidative stress is a major contributing factor to the high mortality rates associated with several diseases, inuding malaria, a widespread parasitic disease in the tropical parts of the world. It seems that oxidative stress in malaria plays a dual role. Some authors suggest that oxidative stress seems to contribute to host organism defenses (1). Namely, immune cells use ROS in order to support their functions and therefore need adequate levels of antioxidant defenses in order to avoid the harmful effect of an excessive production of ROS (2). On the other hand, some authors point to deleterious effects of oxidative stress in malaria (3). In addition, some antimalarial agents have oxidative stress-inducing effects that might contribute to the development of side effects such as methemoglobinemia, hemolysis and liver damage (4). Primaquine (PQ) is the only available drug that is active against both the latent liver forms of relapsing malaria caused by Plasmodium vivax and P. ovale and the gameto- * Correspondence; e-mail: bzbz@pharma.hr Z. Raji} et al.: Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe2+ chelating activity, Acta Pharm. 60 (2010) 325­337. cytes from all species of the parasites (5). Modifications of the primary amino group protect PQ against metabolic degradation and lead to an increase in the antimalarial activity (6). In our previous paper (7) we have shown that primaquine urea derivatives possess significant antiradical and antioxidant activities. On the other hand, non-steroidal anti-inflammatory drugs (NSAIDs) and their derivatives have also exerted antioxidant activities (8). We therefore found it worth preparing a series of PQ-NSAID conjugates, twin drugs that combine both drugs in a single drug. In this study, novel primaquine conjugates with ibuprofen, ketoprofen, fenoprofen, diofenac and indomethacin (PQ-NSAIDs, 4a-h) were prepared, fully chemically characterized and screened for radical scavenging and antioxidant activity. A considerable number of iron(III) chelators, as well as certain iron(II) chelators, designed for purposes other than treating malaria have antimalarial activity in vitro (9). These facts directed us to check a potential of PQ-NSAIDs conjugates as iron chelators. A series of urea and carbamate PQ derivatives previously synthesized by our group showed antiproliferative activity (7). On the other hand, NSAIDs are potential anticancer drugs and effective chemopreventive agents (see, for example, ref. 10). Therefore, screening of cytostatic activity on a series of tumour cell lines of PQ-NSAID conjugates was performed as well. EXPERIMENTAL Melting points were determined on a Stuart Melting Point Apparatus SMP3 (Barworld Scientific, UK) and were uncorrected. IR spectra were recorded on a FTIR Perkin Elmer Paragon 500 spectrometer (Perkin Elmer, USA). 1H and NMR spectra were recorded on a Varian Gemini 300 spectrometer (Varian, USA), operating at 300 and 75.5 MHz for the 1H and nuei, respectively. Samples were measured in DMSO-d6 solutions at 20 °C in 5-mm NMR tubes. Chemical shifts (d) in ppm were referred to TMS. Coupling constants (J) are given in Hz. A Perkin Elmer Lambda 25 spectrophotometer (Perkin Elmer, USA) and Stat Fax 3200 (Awareness Technologies, USA) were used for absorbance measurements. Elemental composition of the compounds agreed to within ±0.4 % (CHN-LECO-932, LECO Corporation, USA). For thin-layer chromatography, precoated Merck silica gel 60 F254 plates (Merck, Germany) and solvent systems cyohexane/ethyl acetate/methanol (3:1:0.5) and dichloromethane/methanol (9:1) were used. Spots were visualized by short-wave UV light and iodine vapour. Column chromatography was performed on silica gel of 0.063­0.200 mm (Merck), with cyohexane/ethyl acetate/methanol (3:1:0.5) or dichloromethane/methanol (9:1) as eluents. A gradual increase of eluent polarity was also applied: first eluent was lipophylic (dichloromethane), while the second (dichloromethane/methanol 99.5:0.5) and the third one (dichloromethane/methanol 99:1) were more polar. Benzotriazole, triphosgene, triethylamine, ethylenediamine, primaquine diphosphate, butylated hydroxyanisol (BHA), 2,2-diphenyl-1-picrylhydrazyl (DPPH), b-carotene, Folin-Ciocalteu reagent, linoleic acid, Tween-40 (polyoxyethylene sorbitan monopalmitate), quercetin and 10 % Pd/C were purchased from Sigma-Aldrich (USA). NSAIDs (diofenac, ketoprofen, fenoprofen, ibuprofen and indomethacin) were obtained as gift samples from Pliva and Belupo (Croatia) and the Z. Raji} et al.: Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe2+ chelating activity, Acta Pharm. 60 (2010) 325­337. University of Potchefstroom (Republic of South Africa). Primaquine diphosphate and NSAIDs were used as racemates. Other chemicals and solvents used were of analytical grade. 1-Benzotriazole carboxylic acid chloride (1), 2-(3-benzylphenyl)propanoic acid (2a), 2-(3-(hydroxy(phenyl)methyl)phenyl)propanoic acid (2b) and NSAID benzotriazolides 3a-h were prepared according to our published procedures (11, 12). Primaquine base was prepared from primaquine diphosphate prior to use. Primaquine solution was protected against light during the whole procedure. Synthesis of PQ-NSAID twin drugs (4a-h). General procedure A light-protected solution of primaquine (0.156 g, 0.6 mmol), appropriate benzotriazolide 3 (0.5 mmol) and triethylamine (TEA) (0.209 mL, 1.5 mmol) in toluene (5 mL) was stirred at room temperature for 0.5 h. Synthesis of was performed with a double amount of primaquine and TEA, while for synthesis of 4g only half the amount of TEA was used. The reaction mixture was extracted with diluted NaOH solution pH 9 (5 ´ 10 mL). The organic layer was washed with water, dried over anhydrous sodium sulphate, filtrated and evaporated to yield a crude product. N-(4-(6-methoxyquinolin-8-ylamino)pentyl)-2-(4-isobutylphenyl)propanamide (4a). ­ Reactant: 1-(1H-benzo[d][1,2,3]triazol-1-yl)-2-(4-isobutylphenyl)propan-1-one (benzotriazolide 3a) (0.154 g, 0.5 mmol). The crude product was purified by column chromatography (eluent: dichloromethane/methanol 9:1). N-(4-(6-methoxyquinolin-8-ylamino)pentyl)-2-(3-benzylphenyl)propanamide (4b). ­ Reactant: 1-(1H-benzo[d][1,2,3]triazol-1-yl)-2-(3-benzylphenyl)propan-1-one (benzotriazolide 3b) (0.171 g, 0.5 mmol). The crude product was purified by column chromatography (eluent: cyohexane/ethyl acetate/methanol 3:1:0.5). N-(4-(6-methoxyquinolin-8-ylamino)pentyl)-2-(3-phenoxyphenyl)propanamide (4c). ­ Reactant: 1-(1H-benzo[d][1,2,3]triazol-1-yl)-2-(3-phenoxyphenyl)propan-1-one (benzotriazolide 3c) (0.174 g, 0.5 mmol). The crude product was purified by column chromatography (eluent: cyohexane/ethyl acetate/methanol 3:1:0.5). N-(4-(6-methoxyquinolin-8-ylamino)pentyl)-2-(3-benzoylphenyl)propanamide (4d). ­ Reactant: 1-(1H-benzo[d][1,2,3]triazol-1-yl)-2-(3-benzoylphenyl)propan-1-one (benzotriazolide 3d) (0.178 g, 0.5 mmol). The crude product was purified by column chromatography (eluent: cyohexane/ethyl acetate/methanol 3:1:0.5). N-(4-(6-methoxyquinolin-8-ylamino)pentyl)-2-(3-(hydroxy(phenyl)methyl)-phenyl)propan amide (4e). ­ Reactant: 1-(1H-benzo[d][1,2,3]triazol-1-yl)-2-(3-(hydroxy(phenyl)methyl) phenyl)propan-1-one (benzotriazolide 3e) (0.179 g, 0.5 mmol). The crude product was purified by column chromatography (eluent: dichloromethane®dichloromethane/methanol 99.5:0.5®dichloromethane/methanol 99:1). 2-(2-(2,6-Dichlorophenylamino)phenyl)-N-(4-(6-methoxyquinolin-8-ylamino)pentyl)acet amide (4f). ­ Reactant: 2-(2-(2,6-dichlorophenylamino)phenyl)-1-(1H-benzo[d][1,2,3]triazol-1-yl)ethanone (benzotriazolide 3f) (0.199 g, 0.5 mmol). The crude product was purified by column chromatography (eluent: cyohexane/ethyl acetate/methanol 3:1:0.5) and triturated with ether. Z. Raji} et al.: Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe2+ chelating activity, Acta Pharm. 60 (2010) 325­337. 1-(4-Chlorobenzoyl)-2-methyl-5-methoxy-N-(4-(6-methoxyquinolin-8-ylamino)pentyl)-1Hindol-3-acetamide (4g). ­ Reactants: 1-(4-chlorobenzoyl)-2-methyl-5-methoxy-1H-indol-3-(ethan-2-one-2-(1H-benzo[d][1,2,3]triazol-1-yl)) (benzotriazolide 3g) (0.229 g, 0.5 mmol), triethylamine (0.091 mL, 0.65 mmol). The crude product was purified by column chromatography (eluent: cyohexane/ethyl acetate/methanol 3:1:0.5) and triturated with ether. (3-(1-(4-(6-Methoxyquinolin-8-ylamino)pentylcarbamoyl)ethyl)phenyl)-(phenyl)methyl 4-(6methoxyquinolin-8-ylamino)pentylcarbamate (). ­ Reactant: (3-(1-(1H-benzo[d][1,2,3]triazol-1-yl)-1-oxopropan-2-yl)phenyl)(phenyl)methyl 1H-benzo[d][1,2,3]triazole-1-carboxylate (benzotriazolide 3h) (0.251 g, 0.5 mmol). The crude product was purified by column chromatography (eluent: dichloromethane/methanol 9:1) and triturated with acetone/ petrolether. DPPH radical-scavenging activity Free radical scavenging activity (RSA) was evaluated by the scavenging of a,a-diphenyl-b-picryl hydrazyl (DPPH) radicals according to the method of Yen and Chen (13). Ethanolic solution of DPPH (V = 1.0 mL, c = 0.16 mmol L­1) was added to 1.0 mL of either ethanolic solution of the test sample (g = 0.1­0.5 g L­1) or ethanol (negative control). The mixture was vortexed for 1 min and then left to stand at room temperature in the dark. After 30 min absorbance was read at 517 nm. Bleached DPPH solution, prepared by adding 1.0 mL of 0.16 mmol L­1 DPPH solution to 1.0 mL of butylated hydroxyanisol (BHA) solution (g = 1 g L­1) was used as a positive control. RSA was calculated using Acont. (absorbance of the ethanol control) and Asample (absorbance of the sample). RSA was expressed as the concentration that scavenges 50 % of DPPH free radicals (EC50). b-Carotene-linoleic acid assay The antioxidant activity (ANT) of the selected twin drugs was evaluated using the b-carotene-linoleic acid system according to modified literature procedures (14). Tween 40 (200 mg) and 1.0 mL of b-carotene solution in chloroform (g = 0.2 mg L­1) were mixed. After removing chloroform in a rotary evaporator, 20 mg of linoleic acid and 30 mL of aerated distilled water was added to the y residue with vigorous stirring. Aliquots (200 mL) of thus obtained emulsion were added to 50 mg of the test conjugate dissolved in 50 mL of methanol (final concentration of the test compound 0.2 g L­1). A reaction mixture containing 50 mL of methanol instead of sample solution served as a control. BHA was used as an antioxidant standard. After adding the emulsion to the tubes, the plate was incubated at 50 °C for 2 h. During that period, the absorbance was measured at 450 nm at 15-minute intervals, starting immediately after sample preparation (t = 0 min) until the end of the experiment (t = 120 min). The percent of antioxidant activity was calculated as described by Al-Saikhan et al. (15) using Rcont. and Rsample, average bleaching rates of the water control and antioxidant (test compound or BHA), respectively. In addition, antioxidant activity was calculated from the absolute changes in absorbance at t = 60 and 120 min (AA-60 and AA-120, Z. Raji} et al.: Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe2+ chelating activity, Acta Pharm. 60 (2010) 325­337. respectively) (14). The results were normalized using two controls: a negative control wito protection (water) and a positive control with maximum protection (BHA). Accordingly, the antioxidant activity of the test compounds was expressed as: AA 1t t AE 0 - AE t t t t 0 t t - A W ) (ABHA - ABHA ) (A t 0 W where AEt=0 is the absorbance of the conjugate at 0 min, AEt=t is the absorbance of the conjugate at t = 60 or 120 min, AWt=0 is the absorbance of the water control at 0 min, AWt=t is the absorbance of the water control at t = 60 or 120 min, ABHAt=0 is the absorbance of BHA at 0 min and ABHAt=t is the absorbance of the BHA sample at t = 60 or 120 min. Fe2+ chelating activity The chelating activity (ChA) of PQ-NSAID conjugates toward ferrous ions was studied as described by Decker and Welch with some modification (16). To an aliquot of the methanolic solution of the test conjugate (V = 150 mL, g = 0.1­0.6 g L­1), 50 mL of Fe2 solution (c = 0.25 mmol L-1) was added. After 5 minutes, the reaction was initiated by adding 100 mL of 1.0 mmol L­1 ferrozine solution. Absorbance at 545 nm was recorded after 10 min of incubation at room temperature. A reaction mixture containing 150 mL of methanol instead of conjugate solution served as a control. Quercetin was used as the chelating standard. ChA was calculated using Acont. (absorbance of the negative control, e.g., blank solution without test compound) and Asample (absorbance of the conjugate solution). Chelating activity was expressed as ChEC50, the concentration that chelates 50 % of Fe2+ ions. Statistical analysis All assays described in the paper were performed in triplicate. The results were expressed as mean ± SD. Statistical comparisons were made using Student's t-test or one-way ANOVA, followed by Dunnett's post-hoc test for multiple comparisons with the control. Statistical analyses were performed using the JMP V6 from SAS software (SAS Institute, Cary, NC, USA). RESULTS AND DISCUSSION Chemistry The synthetic procedure leading to PQ-NSAID conjugates 4a-h is presented in the Scheme 1. The first synthetic step involves preparation of NSAID benzotriazolides 3a-h from the corresponding NSAID (ibuprofen, ketoprofen, fenoprofen, ketoprofen hydroxy and methylene analogues, diofenac or indomethacin) and benzotriazole carboxylic acid chloride (BtCO, 1). Benzotriazolides 3a-h readily reacted with the primaquine Z. Raji} et al.: Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe2+ chelating activity, Acta Pharm. 60 (2010) 325­337. base. In that reaction, the primaquine terminal amino group formed an amide bond with the carboxylic group present in the title NSAIDs. The reaction was performed in toluene, at room temperature for 0.5 h. In general, 3-fold excess of TEA was used. Triethylamine formed a water soluble salt with benzotriazole, a by-product of the reaction, which was readily extracted with water. Synthesis of the PQ-indomethacin derivative 4g was performed with benzotriazolide 3g : TEA molar ratio 1 : 1.3 to avoid indomethacin decomposition. Compound consists of one reduced ketoprofen moiety and two primaquine units (one bound by an amide and the other by the carbamate bond), and its preparation required a double amount of primaquine, e.g., PQ to benzotriazolide 3h molar ratio 2 : 1. O O R OH BtCO/TEA toluene O R 3a-h PQ/TEA Bt toluene R O 4a-h HN N NSAID O N HN H2N N PQ 3b, 4b O Bt 3a, 4a R H3C 3c, 4c 3d, 4d O 3e, 3e OH 3f, 4f 3g, 4g 3h O O Bt O O O N H3C N O HN Scheme 1 Structures of compounds 4a-h were deduced from the analysis of their IR, 1H and NMR spectra and confirmed by elemental analysis. The chemical shifts were consistent with the proposed structures of the novel compounds. In 1MR spectrum of amidocarbamate derivative each atom of two primaquine residues always appeared as one signal with a double integral, except for NH group at position 2: carbamate NH (') appeared at d 7.47­7.39 ppm, while amide NH (2) had a signal at d 7.95­7.91 ppm. Physicochemical and spectroscopic data are presented in Tables I and II. Radical scavenging, antioxidant activity and Fe2+ chelating ability The investigated conjugates demonstrated moderate antiradical activities, with EC50 between 269.5 ± 10.7 and 379.3 ± 59.1 mg L­1, with the exception of conjugate 4e Z. Raji} et al.: Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe2+ chelating activity, Acta Pharm. 60 (2010) 325­337. Table I. Physicochemical and IR spectroscopic data for PQ-NSAID conjugates 4a-h Elemental analysis calcd./found C 75.13 75.49 77.31 77.48 74.51 74.39 H 8.33 8.40 7.32 7.44 6.88 6.71 N 9.39 9.20 8.72 8.60 8.69 8.95 Compd. Yield (%) M.p. (ºC) IR (nmax, KBr or Na, cm­1) 3384, 3302, 3052, 2957, 2930, 1868, 1645, 1616, 1577, 1520, 1456, 1387, 1220, 1203, 1166, 1052, 822, 791 3386, 3301, 3060, 3027, 2965, 2930, 1647, 1616, 1577, 1520, 1455, 1388, 1220, 1202, 1167, 1159, 822, 791 3385, 3300, 3069, 2964, 2931, 1646, 1616, 1581, 1520, 1488, 1456, 1388, 1245, 1222, 1206, 1161, 1051, 932, 821, 791, 693 3380, 3310, 3059, 2965, 2932, 1655, 1616, 1596, 1578, 1520, 1455, 1388, 1284, 1221, 1202, 1159, 1052, 822, 791, 722, 705 3391, 3310, 3061, 2965, 1932, 2870, 1650, 1616, 1578, 1520, 1454, 1388, 1221, 1202, 1159, 823, 791, 703 Molecular formula (Mr) C28H37N3O2 (447.61) C31H35N3O2 (481.63) C30H33N3O3 (483.60) 4a 4b 4c 4d 34­38 C31H33N3O3 (495.61) C31H35N3O3 (497.63) 4e 4f 3393, 3298, 3070, 2964, 2941, 1661, C H N O 64.80 128­130 1633, 1616, 1591, 1578, 1521, 1454, 29 30 2 4 2 (536.17) 64.46 1389, 1205, 1158, 820, 768, 752 113­116 3383, 3302, 3083, 2960, 2929, 1669, 1641, 1617, 1596, 1520, 1478, 1458, C335N4O4 68.16 1388, 1364, 1332, 1222, 1154, 1090, (599.12) 67.83 823, 792, 754 3384, 3055, 2962, 2932, 1716, 1660, 1616, 1588, 1520, 1455, 1424, 1388, 1238, 1221, 1203, 1159, 1051, 1031, 822, 792 C47H54N6O5 (782.97) 72.10 72.44 4g 82­86 EC 50 (g L ) ­1 Fig. 1. DPPH radical scavenging activities of the PQ-NSAID conjugates and butylated hydroxyanisol (BHA). Mean ± SD, n = 3. 0 BHA 4b 4d 4e 4f 4g Z. Raji} et al.: Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe2+ chelating activity, Acta Pharm. 60 (2010) 325­337. Table II. 1H and NMR data for PQ-NSAID conjugates 4a-h O HN R N Compd. 1H NMR (DMSO-d6, d/ppm, J/Hz) NMR (DMSO-d6, d/ppm) 1 7' 8' 3' 4' 4a 1 8.53 (d, 1H, 17, J = 3.79), 8.07 (dd, 1H, 15, J = 1.61, J = 6.65), 7.92­7.87 (m, 1H, 2), 7.42 (dd, 1H, 16, J = 3.96, J = 4.30), 7.19 (dd, 2H, 4', 8', J = 1.81, J = 6.25), 7.02 (dd, 2H, , 7', J = 2.02, J = 6.25), 6.47 (d, 1H, 10, J = 2.42), 6.25 (d, 1H, 12, J = 1.61), 6.07 (dd, 1H, 8, J = 2.42, J = 6.25), 3.82 (s, 3H, 18), 3.60­3.48 (m, 2H, 6, ), 2.96­2.87 (m, 2H, 3), 2.35 (d, 2H, , J = 6.65), 1.82­1.69 (m, 1H, ), 1.60­1.38 (m, , 4, 5), 1.28 (dd, 3H, , J = 3.02, J = 3.83), 1.14 (dd, 3H, 7, J = 2.22, J = 3.93), 0.82 (d, 6H, 1, 1, J = 6.45) 173.68 (1), 159.49 (11), 145.09 (9), 144.67 (17), 140.15, 139.55 (3', ), 135.25 (15), 135.01 (14), 130.05 (13), 129.15, 127.34 (4', 8', , 7'), 122.55 (16), 96.55 (10), 92.06 (12), 55.44 (18), 47.43 (6), 45.26 (), 44.69 (), 38.91 (3), 33.77 (5), 30.06 (), 26.36 (4), 22.61 (1, 1), 20.61 (7), 18.84 () 8.53 (dd, 1H, 17, J = 1.55, J = 2.62), 8.08 (dd, 1H, 15, J = 1.54, J = 6.73), 7.91 (t, 1H, 2, J = 5.32), 7.44­7.40 (m, 1H, 16), 7.28­7.01 (m, 9H, 4'­, 8', 1­1), 6.48 (d, 1H, 10, J = 2.53), 6.25 (s, 1H, 12), 6.09 (dd, 1H, 8, J = 1.61, J = 7.01), 3.88 (s, 2H, ), 3.82 (s, 3H, 18), 3.45­3.34 (m, 2H, 6, ), 2.95­2.87 (m, 2H, 3), 1.61­1.40 (m, , 4, 5), 1.28 (dd, 3H, , J = 3.52), 1.14 (dd, 3H, 7, J = 1.55, J = 4.58) 173.47 (1), 159.49 (11), 145.09 (9), 144.69 (17), 143.03, 141.64, 141.43 (3', 7', ), 135.26 (15), 135.01 (14), 130.05 (13), 129.09, 128.82, 128.69, 128.08, 127.26, 126.37, 125.30 (4'­, 8', 1­1), 122.57 (16), 96.59 (10), 92.07 (12), 55.45 (18), 47.36 (6), 45.57 (), 41.63 (), 38.98 (3), 33.79 (4), 26.37 (5), 20.63 (7), 18.99 () 8.53 (dd, 1H, J = 1.51, 17, J = 2.65), 8.08 (dd, 1H, 15, J = 1.51, J = 6.81), 7.96 (t, 1H, 2, J = 5.39), 7.44­6.82 (m, 10H, 16, 4'­, 8', ­14'), 6.48 (d, 1H, 10, J = 2.44), 6.26 (s, 1H, 12), 6.09 (dd, 1H, 8, J = 1.75, J = 6.90), 3.82 (s, 3H, 18), 3.61­3.52 (m, 2H, 6, ), 3.08­3.00 (m, 2H, 3), 1.62­1.39 (m, , 4, 5), 1.29 (dd, 3H, , J = 3.50), 1.15 (dd, 3H, 7, J = 1.06, J = 4.94) 173.18 (1), 159.48 (11), 157.05, 156.89 (3', 7', ), 145.09 (9), 144.70 (17), 135.26 (15), 135.01 (14), 130.05 (13), 122.56 (16), 130.44, 130.15, 123.79, 122.79, 118.96, 117.99, 116.99 (4'­, 8', ­14'), 96.58 (10), 92.08 (12), 55.44 (18), 47.35 (6), 45.44 (), 39.01 (3), 33.84 (4), 26.37 (5), 20.63 (7), 18.85 () 1 14' 1 1 7' 8' 3' 4' 4b 13' 14' 4c 13' 1 1 7' 8' 3' 4' Z. Raji} et al.: Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe2+ chelating activity, Acta Pharm. 60 (2010) 325­337. 1 7' 8' 3' 4' 4d 14' 13' 1 1 8.52 (d, 1H, 17, J = 4.18), 8.07 (dd, 1H, 15, J = 1.53, J = 6.64), 8.04 (t, 1H, 2, J = 5.49, 2), 7.72­7.46 (m, 9H, 4'­, 8', 1­1), 7.41 (dd, 1H, 16, J = 4.09, J = 4.18), 6.47 (d, 1H, 10, J = 2.17), 6.24 (t, 1H, 12, J = 2.63), 6.08 (dd, 1H, 8, J = 4.03, J = 4.61), 3.82 (d, 3H, 18, J = 2.02), 3.68 (q, 1H, , J = 7.03), 3.61­3.54 (m, 1H, 6), 3.10­3.00 (m, 2H, 3), 1.62­1.41 (m, , 4, 5), 1.34 (t, 3H, , J = 6.78), 1.14 (dd, 3H, 7, J = 5.76, J = 6.20) 196.16 (), 173.16 (1), 159.47 (11), 145.08 (9), 144.68 (17), 143.33, 137.51, 137.29 (3', 7', ), 135.25 (15), 135.00 (14, 13), 133.09, 132.04, 130.05, 128.99, 128.94, 128.50 (4'­, 8', 1­1), 122.55 (16), 96.56 (10), 92.07 (12), 55.44 (18), 47.35 (6), 45.39 (), 38.94 (3), 33.84 (4), 26.31 (5), 20.63 (7), 18.87 () 8.53 (d, 1H, 17, J = 4.13), 8.08 (dd, 1H, 15, J = 1.45, J = 6.77), 7.93­7.91 (m, 1H, 2), 7.42 (dd, 1H, 16 J = 4.05, J = 4.20), 7.35­7.13 (m, 9H, 4'­, 8', 1­1), 6.48 (d, 1H, 10, J = 2.42), 6.26 (t, 1H, 12, J = 2.20), 6.09 (dd, 1H, 8, J = 3.13, J = 5.44), 5.85 (d, 1H, , J = 3.91), 5.65­5.64 (m, 1H, ), 3.82 (s, 3H, 18), 3.61­3.56 (m, 1H, 6), 3.53 (q, 1H, , J = 7.16), 3.08­2.95 (m, 2H, 3), 1.61­1.58, 1.52­1.41 (2m, , 4, 5), 1.29­1.26 (m, 3H, ), 1.17­1.14 (m, 3H, 7) 173.47 (1), 159.49 (11), 146.12, 145.98, 142.66 (3', 7', ), 145.11 (9), 144.71 (17), 135.27 (15), 135.02 (14), 130.06 (13), 128.46, 128.30, 127.10, 126.69, 126.03, 125.60, 124.89 (4'­, 8', 1­1), 122.57 (16), 96.60 (10), 92.11 (12), 74.82 (), 55.46 (18), 47.46 (6), 45.64 (), 39.03 (3), 33.83 (4), 26.33 (5), 20.64 (7), 19.06 () 8.53 (dd, 1H, 17, J = 1.56, J = 2.66, 17), 8.41 (s, 1H, 8'), 8.36 (t, 1H, 2, J = 5.42), 8.07 (dd, 1H, 15, J = 1.52, J = 6.79), 7.50 (d, 2H, 1, 13', J = 8.08), 7.42 (dd, 1H, 16, J = 4.14), 7.20­7.12 (m, 2H, 3', ), 7.05­7.00 (m, 1H, 1), 6.85­6.80 (m, 1H, 4'), 6.47 (d, 1H, 10, J = 2.41), 6.30­6.27 (m, 2H, 12, ), 6.12 (d, 1H, 8, J = 8.80), 3.82 (s, 3H, 18), 3.67­3.56 (m, 1H, 6), 3.56 (s, 2H, ), 3.15­3.07 (m, 2H, 3), 1.69­1.49 (m, , 4, 5), 1.17 (d, 3H, 7, J = 6.42) 171.89 (1), 159.49 (11), 145.12 (9), 144.69 (17), 143.42, 137.66, 129.84, 126.05 (, 7', , , 14'), 135.26 (15), 135.01 (14), 130.05 (13), 122.55 (16), 130.80, 129.63, 127.60, 125.41, 121.10, 116.40 (3'­, 1­13'), 96.59 (10), 92.08 (12), 55.45 (18), 47.42 (6), 40.07 (), 39.18 (3), 33.83 (4), 26.24 (5), 20.68 (7) 8.52 (dd, 1H, 17, J = 1.52, J = 2.64), 8.07 (dd, 1H, 15, J = 1.45, J = 6.86), 8.04 (t, 1H, 2, J = 5.59), 7.65 (q, , 1, 1, 13', 1, J = 8.64,), 7.42 (dd, 1H, 16, J = 4.03, J = 4.21), 7.11 (d, 1H, , J = 2.38), 6.94 (d, 1H, , J = 8.97), 6.69 (dd, 1H, 8', J = 2.44, J = 6.60), 6.47 (d, 1H, 10, J = 2.38), 6.25 (d, 1H, 12, J = 2.38), 6.10 (d, 1H, 8, J = 8.71), 3.81 (s, 3H, 18), 3.72 (s, 3H, '), 3.65­3.56 (m, 1H, 6), 3.48 (s, 2H, ), 3.12­3.05 (m, 2H, 3), 2.21 (s, 3H, '), 1.66­1.46 (m, , 4, 5), 1.16 (d, 3H, 7, J = 6.20) 169.66 (1), 168.29 (), 159.47 (11), 156.00 (7'), 145.09 (9), 144.69 (17), 137.98 (14'), 135.53 (3'), 135.26 (15), 135.00 (14), 134.74 (4'), 131.58 (1, 1), 131.35 (1), 130.75 (), 130.04 (13), 129.47 (13', 1), 122.57 (16), 115.01 (), 114.92 (), 111.67 (), 102.34 (8'), 96.57 (10), 92.07 (12), 55.83 ('), 55.44 (18),47.46 (6), 39.14 (3), 33.93 (4), 31.70 (), 26.50 (5), 20.63 (7), 13.82 (') OH 1 7' 8' 3' 4' 4e 1 14' 13' 1 1 14' 4f 13' 1 1 8' 7' 3' 4' ' 7' 8' 3' 4g 1 13' 14' 1 ' 4' 1 1 Z. Raji} et al.: Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe2+ chelating activity, Acta Pharm. 60 (2010) 325­337. O 1' ' 18'' 17'' 13'' ' 1' 4'' ' 8' 7'' ' ' NH 8'' 1 14' 13' 1 1 ' 7' N H 3'' 3' 4' 8.53 (d, 2H, 17, 17'', J = 3.67), 8.07 (dd, 2H, 15, 1', J = 0.97, J = 7.15), 7.95­7.91 (m, 1H, 2), 7.47­7.39 (m, 3H, 16, ', 1'), 14'' 7.30­7.17 (m, 9H, 4'­, 8', 1­1), 6.62 (s, 1H, ), 6.47 (d, 2H, 10, ', J = 1.31), 6.25 (s, 2H, 12, ), 6.10 (t, 2H, 8, 8'', J = 1' 8.28), 3.81 (s, 6H, 18, 18''), 3.64­3.50 (m, 3H, 6, , '), 3.07­2.95 (m, , 3, 3''), 1.65­1.39 (m, 8H, 4, 5, 4'', '), 1.27 (dd, 3H, , J = 2.28, J = 3.63), 1.18­1.11 (m, 6H, 7, 7'') 173.31 (1), 159.48 (11), 155.79 (1), 145.10 (9, '), 144.69 (17, 17''), 143.02, 141.76, 141.57 (3', 7', ), 135.25 (15, 1'), 135.02 (14, 14''), 130.05 (13, 13''), 128.80, 127.92, 126.99, 126.95, 125.94, 125.10 (4'­, 8', 1­1), 122.56 (16, 1'), 96.58 (10, '), 92.07 (112, ), 76.53 (), 55.44 (18, 18''), 47.44 (6, '), 45.54 (), 39.02 (3, 3''), 33.77 (4, 4''), 26.70 (5), 26.30 ('), 20.62 (7, 7''), 19.02 () with EC50 = 528.8 mg L­1. BHA, investigated under the same conditions, had the EC50 value of 5.9 mg L­1. The results are presented in Fig. 1. Reduction of the absorbance of the b-carotene-linoleate emulsion in the presence of PQ-NSAID conjugates is shown in Fig. 2. All the investigated derivatives significantly inhibited b-carotene bleaching in comparison with the control. According to Amarowicz et al. (17), the normalized antioxidant activity at 60 and 120 min of incubation (AA-60 and AA-120) probably reflects the antioxidant activity of the test compound more accurately than the ANT value (Table III). Differences among the PQ-NSAID conjugates were minor. The most active conjugate, according both to the ANT value (69.4 ± 0.9 %) or AA-60 (58.4 ± 3.1 %) and AA-120 (59.3 ± 1.8 %), was conjugate with two primaquine units. The activity of diofenac derivative 4f was the lowest; however, activities of both and 4f were not statistically different from the activity of the other conjugates. Small differences in antioxidant, as well as in antiradical activities, could indicate that the primaquine moiety, which all the investigated substances have in common, might be responsible for both activities. Under the conditions applied in our experiments only ketoprofen derivatives 4d and 4e demonstrated chelating activities with ChEC50 of 1074.7 ± 23.2 and 861.9 ± 50.4 mg L­1, respectively. ChEC50 of standard quercetin was 537.6 ± 13.5 mg L­1. Other more Fig. 2. Reduction of absorbance in the b-carotene-linoleate assay in the presence of the PQ-NSAID conjugates and butylated hydroxyanisol (BHA). Mean ± SD, n = 3. BHA control 4b 4d 4e 4f 4g Absorbance 0.0 0 30 60 Time (min) 90 120 Z. Raji} et al.: Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe2+ chelating activity, Acta Pharm. 60 (2010) 325­337. Table III. Antioxidant activity in the b-carotene-linoleate assay (ANT, AA-60, AA-120) and metal chelating activity (ChEC50) of PQ-NSAID conjugates Compd. 4b 4d 4e 4f 4g Standarda,b ANT (%)a 60.8 ± 1.8c,d,e 6 3.2 ± 55.8 ± 1.8c,f,g 1.7c,d,f,h,j,k 62.8 ± 1.7c,h,i 61.4 ± 2.3c,j,l 69.4 ± 0.9c,e,g,i,k,l 96.6 ± 1.1 AA-60 (%)a 44.8 ± 8.2c 50.2 ± 39.8 ± 8.9c 4.9c,d 49.1 ± 5.9c 46.6 ± 6.3c 58.4 ± 3.1c,d 95.7 ± 5.2 AA-120 (%)a 48.1 ± 5.2c,d 50.5 ± 2.3c 52.0 ± 3.4c 44.5 ± 3.1c,e 50.1 ± 4.0c 59.3 ± 1.8c,d,e 95.0 ± 3.3 ChEC50 (mg L-1)b ND 1074.7 ± 23.2c,d 861.9 ± 50.4c,d ND ND ND 537.6 ± 13.5 ND - activity could not be determined due to precipitation. ANT ­ Antioxidant activity relative to water control (determined at test compounds concentration of 0.2 g L­1). AA-60 and AA-120 ­ absolute changes in absorbance at t = 60 and 120 min (compounds concentration of 0.2 g L­1). Standards: a BHA (0.2 g L­1), b quercetin. Statistically significant difference (p < 0.05) within columns: c vs. standard, d­l between test compounds (the same letter indicates significant difference). lipophilic derivatives precipitated upon addition of aqueous reagent solutions, so it was not possible to determine their chelating abilities. Antiproliferative and antituberculotic activity Antiproliferative activity of PQ-NSAID conjugates was screened in vitro on 4 human cell lines, which are derived from 4 cancer types. The following cell lines were used: HCT 116 (colon carcinoma), SW 620 (colon carcinoma), MCF-7 (breast carcinoma), and H 460 (lung carcinoma). Unfortunately, activities of all conjugates were too weak (concentration that causes 50 % growth inhibition was ³ 100 µmol L­1). Preliminary antituberculotic activity was checked as well. The minimal inhibitory concentration determined on the strain Mycobacterium smegmatis ATCC 14468 was too high (³ 128 mg L­1), so no further tests were done. CONUSIONS All the tested PQ-NSAID conjugates were found to possess moderate antiradical acivity, with EC50 between 269.5 ± 10.7 and 379.3 ± 59.1 mg L­1, with the exception of conjugate 4e with EC50 = 528.8 mg L­1. Conjugate exerted the strongest antioxidant activity, as determined by the b-carotene-linoleic acid assay (ANT = 69.4 ± 0.9 %; AA-60 and AA-120 were approximately 59 %). Moreover, primaquine derivatives with the ketoprofen moiety demonstrated notable Fe2+ chelating ability. On the other hand, negligible antiproliferative and antituberculotic effects of conjugates 4a-h were observed. Screening of antimalarial activity is in progress and the results will be published elsewhere. Z. Raji} et al.: Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe2+ chelating activity, Acta Pharm. 60 (2010) 325­337. Acknowledgements. ­ Support for this study was provided by the Ministry of Science, Education and Sports of the Republic of Croatia (Projects 006-0000000-3216 and 0006-0061246-1251). We are grateful to Marijeta Kralj, PhD for antiproliferative screening. Acronyms. ­ AA-60 and AA-120 ­ absolute changes in absorbance at t = 60 and 120 min, respectively; ANT ­ antioxidant activity; BHA ­ butylated hydroxyanisol; Bt ­ benzotriazolyl; ChA ­ metal chelating activity; ChEC50 ­ concentration that chelates 50 % of Fe2+ ions); DPPH ­ a,a-diphenyl-b-picryl hydrazyl; EC50 ­ concentration that scavenges 50 % of DPPH free radicals; NSAID ­ non-steroidal anti-inflammatory drug; PQ ­ primaquine; ROS ­ reactive oxygen species; RSA ­ radical scavenging activity; TEA ­ triethylamine. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Pharmaceutica de Gruyter

Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe 2+ chelating activity

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10.2478/v10007-010-0024-9
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Abstract

Acta Pharm. 60 (2010) 325­337 10.2478/v10007-010-0024-9 Original research paper ZRINKA RAJI]1 MARIJANA ZOVKO KON^I]1 KRISTINA MILOLO@A1 IVANA PERKOVI]1 IVAN BUTULA1 FRANZ BUCAR2 BRANKA ZORC1,* 1 Faculty of Pharmacy and Biochemistry University of Zagreb, Zagreb, Croatia 2 Institut für Pharmazeutische Wissenschaften, Bereich Pharmakognosie Graz, Austria Novel primaquine conjugates witon-steroidal anti-inlammatory drugs (PQ-NSAIDs, 4a-h) were prepared, fully chemically characterized and screened for radical scavenging and antioxidant activities. The synthetic procedure leading to twin drugs 4a-h involved two steps: i) preparation of NSAID benzotriazolides 3a-h from the corresponding NSAID (ibuprofen, ketoprofen, fenoprofen, ketoprofen hydroxy and methylene analogues, diofenac or indomethacin) and benzotriazole carboxylic acid chloride (BtCO, 1), ii) reaction of intermediates 3a-h with PQ. The prepared PQ-NSAIDs exerted moderate activities in the DPPH free radical test and b-carotene-linoleic acid assay. Moreover, ketoprofen derivatives 4d and 4b demonstrated a notable Fe2+ chelating ability as well. On the other hand, negligible antiproliferative and antituberculotic effects of conjugates 4a-h were observed. Kewords: primaquine, NSAID, twin drug, conjugate, radical scavenging, antioxidant activity, chelating ability Accepted August 26, 2010 Excessive production of reactive oxygen species (ROS), associated with inflammation, leads to the condition of oxidative stress. Oxidative stress is a major contributing factor to the high mortality rates associated with several diseases, inuding malaria, a widespread parasitic disease in the tropical parts of the world. It seems that oxidative stress in malaria plays a dual role. Some authors suggest that oxidative stress seems to contribute to host organism defenses (1). Namely, immune cells use ROS in order to support their functions and therefore need adequate levels of antioxidant defenses in order to avoid the harmful effect of an excessive production of ROS (2). On the other hand, some authors point to deleterious effects of oxidative stress in malaria (3). In addition, some antimalarial agents have oxidative stress-inducing effects that might contribute to the development of side effects such as methemoglobinemia, hemolysis and liver damage (4). Primaquine (PQ) is the only available drug that is active against both the latent liver forms of relapsing malaria caused by Plasmodium vivax and P. ovale and the gameto- * Correspondence; e-mail: bzbz@pharma.hr Z. Raji} et al.: Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe2+ chelating activity, Acta Pharm. 60 (2010) 325­337. cytes from all species of the parasites (5). Modifications of the primary amino group protect PQ against metabolic degradation and lead to an increase in the antimalarial activity (6). In our previous paper (7) we have shown that primaquine urea derivatives possess significant antiradical and antioxidant activities. On the other hand, non-steroidal anti-inflammatory drugs (NSAIDs) and their derivatives have also exerted antioxidant activities (8). We therefore found it worth preparing a series of PQ-NSAID conjugates, twin drugs that combine both drugs in a single drug. In this study, novel primaquine conjugates with ibuprofen, ketoprofen, fenoprofen, diofenac and indomethacin (PQ-NSAIDs, 4a-h) were prepared, fully chemically characterized and screened for radical scavenging and antioxidant activity. A considerable number of iron(III) chelators, as well as certain iron(II) chelators, designed for purposes other than treating malaria have antimalarial activity in vitro (9). These facts directed us to check a potential of PQ-NSAIDs conjugates as iron chelators. A series of urea and carbamate PQ derivatives previously synthesized by our group showed antiproliferative activity (7). On the other hand, NSAIDs are potential anticancer drugs and effective chemopreventive agents (see, for example, ref. 10). Therefore, screening of cytostatic activity on a series of tumour cell lines of PQ-NSAID conjugates was performed as well. EXPERIMENTAL Melting points were determined on a Stuart Melting Point Apparatus SMP3 (Barworld Scientific, UK) and were uncorrected. IR spectra were recorded on a FTIR Perkin Elmer Paragon 500 spectrometer (Perkin Elmer, USA). 1H and NMR spectra were recorded on a Varian Gemini 300 spectrometer (Varian, USA), operating at 300 and 75.5 MHz for the 1H and nuei, respectively. Samples were measured in DMSO-d6 solutions at 20 °C in 5-mm NMR tubes. Chemical shifts (d) in ppm were referred to TMS. Coupling constants (J) are given in Hz. A Perkin Elmer Lambda 25 spectrophotometer (Perkin Elmer, USA) and Stat Fax 3200 (Awareness Technologies, USA) were used for absorbance measurements. Elemental composition of the compounds agreed to within ±0.4 % (CHN-LECO-932, LECO Corporation, USA). For thin-layer chromatography, precoated Merck silica gel 60 F254 plates (Merck, Germany) and solvent systems cyohexane/ethyl acetate/methanol (3:1:0.5) and dichloromethane/methanol (9:1) were used. Spots were visualized by short-wave UV light and iodine vapour. Column chromatography was performed on silica gel of 0.063­0.200 mm (Merck), with cyohexane/ethyl acetate/methanol (3:1:0.5) or dichloromethane/methanol (9:1) as eluents. A gradual increase of eluent polarity was also applied: first eluent was lipophylic (dichloromethane), while the second (dichloromethane/methanol 99.5:0.5) and the third one (dichloromethane/methanol 99:1) were more polar. Benzotriazole, triphosgene, triethylamine, ethylenediamine, primaquine diphosphate, butylated hydroxyanisol (BHA), 2,2-diphenyl-1-picrylhydrazyl (DPPH), b-carotene, Folin-Ciocalteu reagent, linoleic acid, Tween-40 (polyoxyethylene sorbitan monopalmitate), quercetin and 10 % Pd/C were purchased from Sigma-Aldrich (USA). NSAIDs (diofenac, ketoprofen, fenoprofen, ibuprofen and indomethacin) were obtained as gift samples from Pliva and Belupo (Croatia) and the Z. Raji} et al.: Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe2+ chelating activity, Acta Pharm. 60 (2010) 325­337. University of Potchefstroom (Republic of South Africa). Primaquine diphosphate and NSAIDs were used as racemates. Other chemicals and solvents used were of analytical grade. 1-Benzotriazole carboxylic acid chloride (1), 2-(3-benzylphenyl)propanoic acid (2a), 2-(3-(hydroxy(phenyl)methyl)phenyl)propanoic acid (2b) and NSAID benzotriazolides 3a-h were prepared according to our published procedures (11, 12). Primaquine base was prepared from primaquine diphosphate prior to use. Primaquine solution was protected against light during the whole procedure. Synthesis of PQ-NSAID twin drugs (4a-h). General procedure A light-protected solution of primaquine (0.156 g, 0.6 mmol), appropriate benzotriazolide 3 (0.5 mmol) and triethylamine (TEA) (0.209 mL, 1.5 mmol) in toluene (5 mL) was stirred at room temperature for 0.5 h. Synthesis of was performed with a double amount of primaquine and TEA, while for synthesis of 4g only half the amount of TEA was used. The reaction mixture was extracted with diluted NaOH solution pH 9 (5 ´ 10 mL). The organic layer was washed with water, dried over anhydrous sodium sulphate, filtrated and evaporated to yield a crude product. N-(4-(6-methoxyquinolin-8-ylamino)pentyl)-2-(4-isobutylphenyl)propanamide (4a). ­ Reactant: 1-(1H-benzo[d][1,2,3]triazol-1-yl)-2-(4-isobutylphenyl)propan-1-one (benzotriazolide 3a) (0.154 g, 0.5 mmol). The crude product was purified by column chromatography (eluent: dichloromethane/methanol 9:1). N-(4-(6-methoxyquinolin-8-ylamino)pentyl)-2-(3-benzylphenyl)propanamide (4b). ­ Reactant: 1-(1H-benzo[d][1,2,3]triazol-1-yl)-2-(3-benzylphenyl)propan-1-one (benzotriazolide 3b) (0.171 g, 0.5 mmol). The crude product was purified by column chromatography (eluent: cyohexane/ethyl acetate/methanol 3:1:0.5). N-(4-(6-methoxyquinolin-8-ylamino)pentyl)-2-(3-phenoxyphenyl)propanamide (4c). ­ Reactant: 1-(1H-benzo[d][1,2,3]triazol-1-yl)-2-(3-phenoxyphenyl)propan-1-one (benzotriazolide 3c) (0.174 g, 0.5 mmol). The crude product was purified by column chromatography (eluent: cyohexane/ethyl acetate/methanol 3:1:0.5). N-(4-(6-methoxyquinolin-8-ylamino)pentyl)-2-(3-benzoylphenyl)propanamide (4d). ­ Reactant: 1-(1H-benzo[d][1,2,3]triazol-1-yl)-2-(3-benzoylphenyl)propan-1-one (benzotriazolide 3d) (0.178 g, 0.5 mmol). The crude product was purified by column chromatography (eluent: cyohexane/ethyl acetate/methanol 3:1:0.5). N-(4-(6-methoxyquinolin-8-ylamino)pentyl)-2-(3-(hydroxy(phenyl)methyl)-phenyl)propan amide (4e). ­ Reactant: 1-(1H-benzo[d][1,2,3]triazol-1-yl)-2-(3-(hydroxy(phenyl)methyl) phenyl)propan-1-one (benzotriazolide 3e) (0.179 g, 0.5 mmol). The crude product was purified by column chromatography (eluent: dichloromethane®dichloromethane/methanol 99.5:0.5®dichloromethane/methanol 99:1). 2-(2-(2,6-Dichlorophenylamino)phenyl)-N-(4-(6-methoxyquinolin-8-ylamino)pentyl)acet amide (4f). ­ Reactant: 2-(2-(2,6-dichlorophenylamino)phenyl)-1-(1H-benzo[d][1,2,3]triazol-1-yl)ethanone (benzotriazolide 3f) (0.199 g, 0.5 mmol). The crude product was purified by column chromatography (eluent: cyohexane/ethyl acetate/methanol 3:1:0.5) and triturated with ether. Z. Raji} et al.: Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe2+ chelating activity, Acta Pharm. 60 (2010) 325­337. 1-(4-Chlorobenzoyl)-2-methyl-5-methoxy-N-(4-(6-methoxyquinolin-8-ylamino)pentyl)-1Hindol-3-acetamide (4g). ­ Reactants: 1-(4-chlorobenzoyl)-2-methyl-5-methoxy-1H-indol-3-(ethan-2-one-2-(1H-benzo[d][1,2,3]triazol-1-yl)) (benzotriazolide 3g) (0.229 g, 0.5 mmol), triethylamine (0.091 mL, 0.65 mmol). The crude product was purified by column chromatography (eluent: cyohexane/ethyl acetate/methanol 3:1:0.5) and triturated with ether. (3-(1-(4-(6-Methoxyquinolin-8-ylamino)pentylcarbamoyl)ethyl)phenyl)-(phenyl)methyl 4-(6methoxyquinolin-8-ylamino)pentylcarbamate (). ­ Reactant: (3-(1-(1H-benzo[d][1,2,3]triazol-1-yl)-1-oxopropan-2-yl)phenyl)(phenyl)methyl 1H-benzo[d][1,2,3]triazole-1-carboxylate (benzotriazolide 3h) (0.251 g, 0.5 mmol). The crude product was purified by column chromatography (eluent: dichloromethane/methanol 9:1) and triturated with acetone/ petrolether. DPPH radical-scavenging activity Free radical scavenging activity (RSA) was evaluated by the scavenging of a,a-diphenyl-b-picryl hydrazyl (DPPH) radicals according to the method of Yen and Chen (13). Ethanolic solution of DPPH (V = 1.0 mL, c = 0.16 mmol L­1) was added to 1.0 mL of either ethanolic solution of the test sample (g = 0.1­0.5 g L­1) or ethanol (negative control). The mixture was vortexed for 1 min and then left to stand at room temperature in the dark. After 30 min absorbance was read at 517 nm. Bleached DPPH solution, prepared by adding 1.0 mL of 0.16 mmol L­1 DPPH solution to 1.0 mL of butylated hydroxyanisol (BHA) solution (g = 1 g L­1) was used as a positive control. RSA was calculated using Acont. (absorbance of the ethanol control) and Asample (absorbance of the sample). RSA was expressed as the concentration that scavenges 50 % of DPPH free radicals (EC50). b-Carotene-linoleic acid assay The antioxidant activity (ANT) of the selected twin drugs was evaluated using the b-carotene-linoleic acid system according to modified literature procedures (14). Tween 40 (200 mg) and 1.0 mL of b-carotene solution in chloroform (g = 0.2 mg L­1) were mixed. After removing chloroform in a rotary evaporator, 20 mg of linoleic acid and 30 mL of aerated distilled water was added to the y residue with vigorous stirring. Aliquots (200 mL) of thus obtained emulsion were added to 50 mg of the test conjugate dissolved in 50 mL of methanol (final concentration of the test compound 0.2 g L­1). A reaction mixture containing 50 mL of methanol instead of sample solution served as a control. BHA was used as an antioxidant standard. After adding the emulsion to the tubes, the plate was incubated at 50 °C for 2 h. During that period, the absorbance was measured at 450 nm at 15-minute intervals, starting immediately after sample preparation (t = 0 min) until the end of the experiment (t = 120 min). The percent of antioxidant activity was calculated as described by Al-Saikhan et al. (15) using Rcont. and Rsample, average bleaching rates of the water control and antioxidant (test compound or BHA), respectively. In addition, antioxidant activity was calculated from the absolute changes in absorbance at t = 60 and 120 min (AA-60 and AA-120, Z. Raji} et al.: Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe2+ chelating activity, Acta Pharm. 60 (2010) 325­337. respectively) (14). The results were normalized using two controls: a negative control wito protection (water) and a positive control with maximum protection (BHA). Accordingly, the antioxidant activity of the test compounds was expressed as: AA 1t t AE 0 - AE t t t t 0 t t - A W ) (ABHA - ABHA ) (A t 0 W where AEt=0 is the absorbance of the conjugate at 0 min, AEt=t is the absorbance of the conjugate at t = 60 or 120 min, AWt=0 is the absorbance of the water control at 0 min, AWt=t is the absorbance of the water control at t = 60 or 120 min, ABHAt=0 is the absorbance of BHA at 0 min and ABHAt=t is the absorbance of the BHA sample at t = 60 or 120 min. Fe2+ chelating activity The chelating activity (ChA) of PQ-NSAID conjugates toward ferrous ions was studied as described by Decker and Welch with some modification (16). To an aliquot of the methanolic solution of the test conjugate (V = 150 mL, g = 0.1­0.6 g L­1), 50 mL of Fe2 solution (c = 0.25 mmol L-1) was added. After 5 minutes, the reaction was initiated by adding 100 mL of 1.0 mmol L­1 ferrozine solution. Absorbance at 545 nm was recorded after 10 min of incubation at room temperature. A reaction mixture containing 150 mL of methanol instead of conjugate solution served as a control. Quercetin was used as the chelating standard. ChA was calculated using Acont. (absorbance of the negative control, e.g., blank solution without test compound) and Asample (absorbance of the conjugate solution). Chelating activity was expressed as ChEC50, the concentration that chelates 50 % of Fe2+ ions. Statistical analysis All assays described in the paper were performed in triplicate. The results were expressed as mean ± SD. Statistical comparisons were made using Student's t-test or one-way ANOVA, followed by Dunnett's post-hoc test for multiple comparisons with the control. Statistical analyses were performed using the JMP V6 from SAS software (SAS Institute, Cary, NC, USA). RESULTS AND DISCUSSION Chemistry The synthetic procedure leading to PQ-NSAID conjugates 4a-h is presented in the Scheme 1. The first synthetic step involves preparation of NSAID benzotriazolides 3a-h from the corresponding NSAID (ibuprofen, ketoprofen, fenoprofen, ketoprofen hydroxy and methylene analogues, diofenac or indomethacin) and benzotriazole carboxylic acid chloride (BtCO, 1). Benzotriazolides 3a-h readily reacted with the primaquine Z. Raji} et al.: Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe2+ chelating activity, Acta Pharm. 60 (2010) 325­337. base. In that reaction, the primaquine terminal amino group formed an amide bond with the carboxylic group present in the title NSAIDs. The reaction was performed in toluene, at room temperature for 0.5 h. In general, 3-fold excess of TEA was used. Triethylamine formed a water soluble salt with benzotriazole, a by-product of the reaction, which was readily extracted with water. Synthesis of the PQ-indomethacin derivative 4g was performed with benzotriazolide 3g : TEA molar ratio 1 : 1.3 to avoid indomethacin decomposition. Compound consists of one reduced ketoprofen moiety and two primaquine units (one bound by an amide and the other by the carbamate bond), and its preparation required a double amount of primaquine, e.g., PQ to benzotriazolide 3h molar ratio 2 : 1. O O R OH BtCO/TEA toluene O R 3a-h PQ/TEA Bt toluene R O 4a-h HN N NSAID O N HN H2N N PQ 3b, 4b O Bt 3a, 4a R H3C 3c, 4c 3d, 4d O 3e, 3e OH 3f, 4f 3g, 4g 3h O O Bt O O O N H3C N O HN Scheme 1 Structures of compounds 4a-h were deduced from the analysis of their IR, 1H and NMR spectra and confirmed by elemental analysis. The chemical shifts were consistent with the proposed structures of the novel compounds. In 1MR spectrum of amidocarbamate derivative each atom of two primaquine residues always appeared as one signal with a double integral, except for NH group at position 2: carbamate NH (') appeared at d 7.47­7.39 ppm, while amide NH (2) had a signal at d 7.95­7.91 ppm. Physicochemical and spectroscopic data are presented in Tables I and II. Radical scavenging, antioxidant activity and Fe2+ chelating ability The investigated conjugates demonstrated moderate antiradical activities, with EC50 between 269.5 ± 10.7 and 379.3 ± 59.1 mg L­1, with the exception of conjugate 4e Z. Raji} et al.: Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe2+ chelating activity, Acta Pharm. 60 (2010) 325­337. Table I. Physicochemical and IR spectroscopic data for PQ-NSAID conjugates 4a-h Elemental analysis calcd./found C 75.13 75.49 77.31 77.48 74.51 74.39 H 8.33 8.40 7.32 7.44 6.88 6.71 N 9.39 9.20 8.72 8.60 8.69 8.95 Compd. Yield (%) M.p. (ºC) IR (nmax, KBr or Na, cm­1) 3384, 3302, 3052, 2957, 2930, 1868, 1645, 1616, 1577, 1520, 1456, 1387, 1220, 1203, 1166, 1052, 822, 791 3386, 3301, 3060, 3027, 2965, 2930, 1647, 1616, 1577, 1520, 1455, 1388, 1220, 1202, 1167, 1159, 822, 791 3385, 3300, 3069, 2964, 2931, 1646, 1616, 1581, 1520, 1488, 1456, 1388, 1245, 1222, 1206, 1161, 1051, 932, 821, 791, 693 3380, 3310, 3059, 2965, 2932, 1655, 1616, 1596, 1578, 1520, 1455, 1388, 1284, 1221, 1202, 1159, 1052, 822, 791, 722, 705 3391, 3310, 3061, 2965, 1932, 2870, 1650, 1616, 1578, 1520, 1454, 1388, 1221, 1202, 1159, 823, 791, 703 Molecular formula (Mr) C28H37N3O2 (447.61) C31H35N3O2 (481.63) C30H33N3O3 (483.60) 4a 4b 4c 4d 34­38 C31H33N3O3 (495.61) C31H35N3O3 (497.63) 4e 4f 3393, 3298, 3070, 2964, 2941, 1661, C H N O 64.80 128­130 1633, 1616, 1591, 1578, 1521, 1454, 29 30 2 4 2 (536.17) 64.46 1389, 1205, 1158, 820, 768, 752 113­116 3383, 3302, 3083, 2960, 2929, 1669, 1641, 1617, 1596, 1520, 1478, 1458, C335N4O4 68.16 1388, 1364, 1332, 1222, 1154, 1090, (599.12) 67.83 823, 792, 754 3384, 3055, 2962, 2932, 1716, 1660, 1616, 1588, 1520, 1455, 1424, 1388, 1238, 1221, 1203, 1159, 1051, 1031, 822, 792 C47H54N6O5 (782.97) 72.10 72.44 4g 82­86 EC 50 (g L ) ­1 Fig. 1. DPPH radical scavenging activities of the PQ-NSAID conjugates and butylated hydroxyanisol (BHA). Mean ± SD, n = 3. 0 BHA 4b 4d 4e 4f 4g Z. Raji} et al.: Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe2+ chelating activity, Acta Pharm. 60 (2010) 325­337. Table II. 1H and NMR data for PQ-NSAID conjugates 4a-h O HN R N Compd. 1H NMR (DMSO-d6, d/ppm, J/Hz) NMR (DMSO-d6, d/ppm) 1 7' 8' 3' 4' 4a 1 8.53 (d, 1H, 17, J = 3.79), 8.07 (dd, 1H, 15, J = 1.61, J = 6.65), 7.92­7.87 (m, 1H, 2), 7.42 (dd, 1H, 16, J = 3.96, J = 4.30), 7.19 (dd, 2H, 4', 8', J = 1.81, J = 6.25), 7.02 (dd, 2H, , 7', J = 2.02, J = 6.25), 6.47 (d, 1H, 10, J = 2.42), 6.25 (d, 1H, 12, J = 1.61), 6.07 (dd, 1H, 8, J = 2.42, J = 6.25), 3.82 (s, 3H, 18), 3.60­3.48 (m, 2H, 6, ), 2.96­2.87 (m, 2H, 3), 2.35 (d, 2H, , J = 6.65), 1.82­1.69 (m, 1H, ), 1.60­1.38 (m, , 4, 5), 1.28 (dd, 3H, , J = 3.02, J = 3.83), 1.14 (dd, 3H, 7, J = 2.22, J = 3.93), 0.82 (d, 6H, 1, 1, J = 6.45) 173.68 (1), 159.49 (11), 145.09 (9), 144.67 (17), 140.15, 139.55 (3', ), 135.25 (15), 135.01 (14), 130.05 (13), 129.15, 127.34 (4', 8', , 7'), 122.55 (16), 96.55 (10), 92.06 (12), 55.44 (18), 47.43 (6), 45.26 (), 44.69 (), 38.91 (3), 33.77 (5), 30.06 (), 26.36 (4), 22.61 (1, 1), 20.61 (7), 18.84 () 8.53 (dd, 1H, 17, J = 1.55, J = 2.62), 8.08 (dd, 1H, 15, J = 1.54, J = 6.73), 7.91 (t, 1H, 2, J = 5.32), 7.44­7.40 (m, 1H, 16), 7.28­7.01 (m, 9H, 4'­, 8', 1­1), 6.48 (d, 1H, 10, J = 2.53), 6.25 (s, 1H, 12), 6.09 (dd, 1H, 8, J = 1.61, J = 7.01), 3.88 (s, 2H, ), 3.82 (s, 3H, 18), 3.45­3.34 (m, 2H, 6, ), 2.95­2.87 (m, 2H, 3), 1.61­1.40 (m, , 4, 5), 1.28 (dd, 3H, , J = 3.52), 1.14 (dd, 3H, 7, J = 1.55, J = 4.58) 173.47 (1), 159.49 (11), 145.09 (9), 144.69 (17), 143.03, 141.64, 141.43 (3', 7', ), 135.26 (15), 135.01 (14), 130.05 (13), 129.09, 128.82, 128.69, 128.08, 127.26, 126.37, 125.30 (4'­, 8', 1­1), 122.57 (16), 96.59 (10), 92.07 (12), 55.45 (18), 47.36 (6), 45.57 (), 41.63 (), 38.98 (3), 33.79 (4), 26.37 (5), 20.63 (7), 18.99 () 8.53 (dd, 1H, J = 1.51, 17, J = 2.65), 8.08 (dd, 1H, 15, J = 1.51, J = 6.81), 7.96 (t, 1H, 2, J = 5.39), 7.44­6.82 (m, 10H, 16, 4'­, 8', ­14'), 6.48 (d, 1H, 10, J = 2.44), 6.26 (s, 1H, 12), 6.09 (dd, 1H, 8, J = 1.75, J = 6.90), 3.82 (s, 3H, 18), 3.61­3.52 (m, 2H, 6, ), 3.08­3.00 (m, 2H, 3), 1.62­1.39 (m, , 4, 5), 1.29 (dd, 3H, , J = 3.50), 1.15 (dd, 3H, 7, J = 1.06, J = 4.94) 173.18 (1), 159.48 (11), 157.05, 156.89 (3', 7', ), 145.09 (9), 144.70 (17), 135.26 (15), 135.01 (14), 130.05 (13), 122.56 (16), 130.44, 130.15, 123.79, 122.79, 118.96, 117.99, 116.99 (4'­, 8', ­14'), 96.58 (10), 92.08 (12), 55.44 (18), 47.35 (6), 45.44 (), 39.01 (3), 33.84 (4), 26.37 (5), 20.63 (7), 18.85 () 1 14' 1 1 7' 8' 3' 4' 4b 13' 14' 4c 13' 1 1 7' 8' 3' 4' Z. Raji} et al.: Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe2+ chelating activity, Acta Pharm. 60 (2010) 325­337. 1 7' 8' 3' 4' 4d 14' 13' 1 1 8.52 (d, 1H, 17, J = 4.18), 8.07 (dd, 1H, 15, J = 1.53, J = 6.64), 8.04 (t, 1H, 2, J = 5.49, 2), 7.72­7.46 (m, 9H, 4'­, 8', 1­1), 7.41 (dd, 1H, 16, J = 4.09, J = 4.18), 6.47 (d, 1H, 10, J = 2.17), 6.24 (t, 1H, 12, J = 2.63), 6.08 (dd, 1H, 8, J = 4.03, J = 4.61), 3.82 (d, 3H, 18, J = 2.02), 3.68 (q, 1H, , J = 7.03), 3.61­3.54 (m, 1H, 6), 3.10­3.00 (m, 2H, 3), 1.62­1.41 (m, , 4, 5), 1.34 (t, 3H, , J = 6.78), 1.14 (dd, 3H, 7, J = 5.76, J = 6.20) 196.16 (), 173.16 (1), 159.47 (11), 145.08 (9), 144.68 (17), 143.33, 137.51, 137.29 (3', 7', ), 135.25 (15), 135.00 (14, 13), 133.09, 132.04, 130.05, 128.99, 128.94, 128.50 (4'­, 8', 1­1), 122.55 (16), 96.56 (10), 92.07 (12), 55.44 (18), 47.35 (6), 45.39 (), 38.94 (3), 33.84 (4), 26.31 (5), 20.63 (7), 18.87 () 8.53 (d, 1H, 17, J = 4.13), 8.08 (dd, 1H, 15, J = 1.45, J = 6.77), 7.93­7.91 (m, 1H, 2), 7.42 (dd, 1H, 16 J = 4.05, J = 4.20), 7.35­7.13 (m, 9H, 4'­, 8', 1­1), 6.48 (d, 1H, 10, J = 2.42), 6.26 (t, 1H, 12, J = 2.20), 6.09 (dd, 1H, 8, J = 3.13, J = 5.44), 5.85 (d, 1H, , J = 3.91), 5.65­5.64 (m, 1H, ), 3.82 (s, 3H, 18), 3.61­3.56 (m, 1H, 6), 3.53 (q, 1H, , J = 7.16), 3.08­2.95 (m, 2H, 3), 1.61­1.58, 1.52­1.41 (2m, , 4, 5), 1.29­1.26 (m, 3H, ), 1.17­1.14 (m, 3H, 7) 173.47 (1), 159.49 (11), 146.12, 145.98, 142.66 (3', 7', ), 145.11 (9), 144.71 (17), 135.27 (15), 135.02 (14), 130.06 (13), 128.46, 128.30, 127.10, 126.69, 126.03, 125.60, 124.89 (4'­, 8', 1­1), 122.57 (16), 96.60 (10), 92.11 (12), 74.82 (), 55.46 (18), 47.46 (6), 45.64 (), 39.03 (3), 33.83 (4), 26.33 (5), 20.64 (7), 19.06 () 8.53 (dd, 1H, 17, J = 1.56, J = 2.66, 17), 8.41 (s, 1H, 8'), 8.36 (t, 1H, 2, J = 5.42), 8.07 (dd, 1H, 15, J = 1.52, J = 6.79), 7.50 (d, 2H, 1, 13', J = 8.08), 7.42 (dd, 1H, 16, J = 4.14), 7.20­7.12 (m, 2H, 3', ), 7.05­7.00 (m, 1H, 1), 6.85­6.80 (m, 1H, 4'), 6.47 (d, 1H, 10, J = 2.41), 6.30­6.27 (m, 2H, 12, ), 6.12 (d, 1H, 8, J = 8.80), 3.82 (s, 3H, 18), 3.67­3.56 (m, 1H, 6), 3.56 (s, 2H, ), 3.15­3.07 (m, 2H, 3), 1.69­1.49 (m, , 4, 5), 1.17 (d, 3H, 7, J = 6.42) 171.89 (1), 159.49 (11), 145.12 (9), 144.69 (17), 143.42, 137.66, 129.84, 126.05 (, 7', , , 14'), 135.26 (15), 135.01 (14), 130.05 (13), 122.55 (16), 130.80, 129.63, 127.60, 125.41, 121.10, 116.40 (3'­, 1­13'), 96.59 (10), 92.08 (12), 55.45 (18), 47.42 (6), 40.07 (), 39.18 (3), 33.83 (4), 26.24 (5), 20.68 (7) 8.52 (dd, 1H, 17, J = 1.52, J = 2.64), 8.07 (dd, 1H, 15, J = 1.45, J = 6.86), 8.04 (t, 1H, 2, J = 5.59), 7.65 (q, , 1, 1, 13', 1, J = 8.64,), 7.42 (dd, 1H, 16, J = 4.03, J = 4.21), 7.11 (d, 1H, , J = 2.38), 6.94 (d, 1H, , J = 8.97), 6.69 (dd, 1H, 8', J = 2.44, J = 6.60), 6.47 (d, 1H, 10, J = 2.38), 6.25 (d, 1H, 12, J = 2.38), 6.10 (d, 1H, 8, J = 8.71), 3.81 (s, 3H, 18), 3.72 (s, 3H, '), 3.65­3.56 (m, 1H, 6), 3.48 (s, 2H, ), 3.12­3.05 (m, 2H, 3), 2.21 (s, 3H, '), 1.66­1.46 (m, , 4, 5), 1.16 (d, 3H, 7, J = 6.20) 169.66 (1), 168.29 (), 159.47 (11), 156.00 (7'), 145.09 (9), 144.69 (17), 137.98 (14'), 135.53 (3'), 135.26 (15), 135.00 (14), 134.74 (4'), 131.58 (1, 1), 131.35 (1), 130.75 (), 130.04 (13), 129.47 (13', 1), 122.57 (16), 115.01 (), 114.92 (), 111.67 (), 102.34 (8'), 96.57 (10), 92.07 (12), 55.83 ('), 55.44 (18),47.46 (6), 39.14 (3), 33.93 (4), 31.70 (), 26.50 (5), 20.63 (7), 13.82 (') OH 1 7' 8' 3' 4' 4e 1 14' 13' 1 1 14' 4f 13' 1 1 8' 7' 3' 4' ' 7' 8' 3' 4g 1 13' 14' 1 ' 4' 1 1 Z. Raji} et al.: Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe2+ chelating activity, Acta Pharm. 60 (2010) 325­337. O 1' ' 18'' 17'' 13'' ' 1' 4'' ' 8' 7'' ' ' NH 8'' 1 14' 13' 1 1 ' 7' N H 3'' 3' 4' 8.53 (d, 2H, 17, 17'', J = 3.67), 8.07 (dd, 2H, 15, 1', J = 0.97, J = 7.15), 7.95­7.91 (m, 1H, 2), 7.47­7.39 (m, 3H, 16, ', 1'), 14'' 7.30­7.17 (m, 9H, 4'­, 8', 1­1), 6.62 (s, 1H, ), 6.47 (d, 2H, 10, ', J = 1.31), 6.25 (s, 2H, 12, ), 6.10 (t, 2H, 8, 8'', J = 1' 8.28), 3.81 (s, 6H, 18, 18''), 3.64­3.50 (m, 3H, 6, , '), 3.07­2.95 (m, , 3, 3''), 1.65­1.39 (m, 8H, 4, 5, 4'', '), 1.27 (dd, 3H, , J = 2.28, J = 3.63), 1.18­1.11 (m, 6H, 7, 7'') 173.31 (1), 159.48 (11), 155.79 (1), 145.10 (9, '), 144.69 (17, 17''), 143.02, 141.76, 141.57 (3', 7', ), 135.25 (15, 1'), 135.02 (14, 14''), 130.05 (13, 13''), 128.80, 127.92, 126.99, 126.95, 125.94, 125.10 (4'­, 8', 1­1), 122.56 (16, 1'), 96.58 (10, '), 92.07 (112, ), 76.53 (), 55.44 (18, 18''), 47.44 (6, '), 45.54 (), 39.02 (3, 3''), 33.77 (4, 4''), 26.70 (5), 26.30 ('), 20.62 (7, 7''), 19.02 () with EC50 = 528.8 mg L­1. BHA, investigated under the same conditions, had the EC50 value of 5.9 mg L­1. The results are presented in Fig. 1. Reduction of the absorbance of the b-carotene-linoleate emulsion in the presence of PQ-NSAID conjugates is shown in Fig. 2. All the investigated derivatives significantly inhibited b-carotene bleaching in comparison with the control. According to Amarowicz et al. (17), the normalized antioxidant activity at 60 and 120 min of incubation (AA-60 and AA-120) probably reflects the antioxidant activity of the test compound more accurately than the ANT value (Table III). Differences among the PQ-NSAID conjugates were minor. The most active conjugate, according both to the ANT value (69.4 ± 0.9 %) or AA-60 (58.4 ± 3.1 %) and AA-120 (59.3 ± 1.8 %), was conjugate with two primaquine units. The activity of diofenac derivative 4f was the lowest; however, activities of both and 4f were not statistically different from the activity of the other conjugates. Small differences in antioxidant, as well as in antiradical activities, could indicate that the primaquine moiety, which all the investigated substances have in common, might be responsible for both activities. Under the conditions applied in our experiments only ketoprofen derivatives 4d and 4e demonstrated chelating activities with ChEC50 of 1074.7 ± 23.2 and 861.9 ± 50.4 mg L­1, respectively. ChEC50 of standard quercetin was 537.6 ± 13.5 mg L­1. Other more Fig. 2. Reduction of absorbance in the b-carotene-linoleate assay in the presence of the PQ-NSAID conjugates and butylated hydroxyanisol (BHA). Mean ± SD, n = 3. BHA control 4b 4d 4e 4f 4g Absorbance 0.0 0 30 60 Time (min) 90 120 Z. Raji} et al.: Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe2+ chelating activity, Acta Pharm. 60 (2010) 325­337. Table III. Antioxidant activity in the b-carotene-linoleate assay (ANT, AA-60, AA-120) and metal chelating activity (ChEC50) of PQ-NSAID conjugates Compd. 4b 4d 4e 4f 4g Standarda,b ANT (%)a 60.8 ± 1.8c,d,e 6 3.2 ± 55.8 ± 1.8c,f,g 1.7c,d,f,h,j,k 62.8 ± 1.7c,h,i 61.4 ± 2.3c,j,l 69.4 ± 0.9c,e,g,i,k,l 96.6 ± 1.1 AA-60 (%)a 44.8 ± 8.2c 50.2 ± 39.8 ± 8.9c 4.9c,d 49.1 ± 5.9c 46.6 ± 6.3c 58.4 ± 3.1c,d 95.7 ± 5.2 AA-120 (%)a 48.1 ± 5.2c,d 50.5 ± 2.3c 52.0 ± 3.4c 44.5 ± 3.1c,e 50.1 ± 4.0c 59.3 ± 1.8c,d,e 95.0 ± 3.3 ChEC50 (mg L-1)b ND 1074.7 ± 23.2c,d 861.9 ± 50.4c,d ND ND ND 537.6 ± 13.5 ND - activity could not be determined due to precipitation. ANT ­ Antioxidant activity relative to water control (determined at test compounds concentration of 0.2 g L­1). AA-60 and AA-120 ­ absolute changes in absorbance at t = 60 and 120 min (compounds concentration of 0.2 g L­1). Standards: a BHA (0.2 g L­1), b quercetin. Statistically significant difference (p < 0.05) within columns: c vs. standard, d­l between test compounds (the same letter indicates significant difference). lipophilic derivatives precipitated upon addition of aqueous reagent solutions, so it was not possible to determine their chelating abilities. Antiproliferative and antituberculotic activity Antiproliferative activity of PQ-NSAID conjugates was screened in vitro on 4 human cell lines, which are derived from 4 cancer types. The following cell lines were used: HCT 116 (colon carcinoma), SW 620 (colon carcinoma), MCF-7 (breast carcinoma), and H 460 (lung carcinoma). Unfortunately, activities of all conjugates were too weak (concentration that causes 50 % growth inhibition was ³ 100 µmol L­1). Preliminary antituberculotic activity was checked as well. The minimal inhibitory concentration determined on the strain Mycobacterium smegmatis ATCC 14468 was too high (³ 128 mg L­1), so no further tests were done. CONUSIONS All the tested PQ-NSAID conjugates were found to possess moderate antiradical acivity, with EC50 between 269.5 ± 10.7 and 379.3 ± 59.1 mg L­1, with the exception of conjugate 4e with EC50 = 528.8 mg L­1. Conjugate exerted the strongest antioxidant activity, as determined by the b-carotene-linoleic acid assay (ANT = 69.4 ± 0.9 %; AA-60 and AA-120 were approximately 59 %). Moreover, primaquine derivatives with the ketoprofen moiety demonstrated notable Fe2+ chelating ability. On the other hand, negligible antiproliferative and antituberculotic effects of conjugates 4a-h were observed. Screening of antimalarial activity is in progress and the results will be published elsewhere. Z. Raji} et al.: Primaquine-NSAID twin drugs: Synthesis, radical scavenging, antioxidant and Fe2+ chelating activity, Acta Pharm. 60 (2010) 325­337. Acknowledgements. ­ Support for this study was provided by the Ministry of Science, Education and Sports of the Republic of Croatia (Projects 006-0000000-3216 and 0006-0061246-1251). We are grateful to Marijeta Kralj, PhD for antiproliferative screening. Acronyms. ­ AA-60 and AA-120 ­ absolute changes in absorbance at t = 60 and 120 min, respectively; ANT ­ antioxidant activity; BHA ­ butylated hydroxyanisol; Bt ­ benzotriazolyl; ChA ­ metal chelating activity; ChEC50 ­ concentration that chelates 50 % of Fe2+ ions); DPPH ­ a,a-diphenyl-b-picryl hydrazyl; EC50 ­ concentration that scavenges 50 % of DPPH free radicals; NSAID ­ non-steroidal anti-inflammatory drug; PQ ­ primaquine; ROS ­ reactive oxygen species; RSA ­ radical scavenging activity; TEA ­ triethylamine.

Journal

Acta Pharmaceutica – de Gruyter

Published: Sep 1, 2010

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