Synthesis and Iron (III)-Chelating Properties of Novel 3-Hydroxypyridin

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Biomacromolecules 2008, 9, 1372–1380

Synthesis and Iron(III)-Chelating Properties of Novel 3-Hydroxypyridin-4-one Hexadentate Ligand-Containing Copolymers Tao Zhou,†,‡ Xiao Le Kong,‡ Zu Dong Liu,‡ Ding Yong Liu,‡ and Robert C. Hider*,‡ College of Food Science and Biotechnology Engineering, Zhejiang Gongshang University, Hangzhou, Zhejiang, 310035, People’s Republic of China, and Division of Pharmaceutical Science, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, United Kingdom Received October 9, 2007; Revised Manuscript Received February 12, 2008

Iron overload is a critical clinical problem that can be prevented by the use of iron-specific chelating agents. An alternative method of relieving iron overload is to reduce the efficiency of iron absorption from the intestine by administering iron chelators, which can bind iron irreversibly to form nontoxic, kinetically inert complexes that are not absorbed and are therefore excreted in the feces. A series of polymeric chelators with various iron binding capacities were therefore prepared as nonabsorbable iron-selective additives. A novel 3-hydroxypyridin-4-one hexadentate ligand CP254 has been synthesized and incorporated into polymers by copolymerisation with N,Ndimethylacrylamide (DMAA), and N,N′-ethylene-bis-acrylamide (EBAA) using (NH4)2S2O8 as the initiator. The physicochemical properties of CP254 were determined, namely, log K ) 33.2 and pFe3+ ) 27.24. The chelating capacity of the CP254-DMAA copolymers was determined at physiological pH. The iron(III) chelation was found to achieve 80% capacity after 1 h and was virtually complete after 5 h, which is much quicker than that of the commercially available chelating resin Chelex100. The chelating copolymers were found to be readily regenerated and reusable. The copolymers possess a high selectivity for iron(III). The conditional affinity (log K′) for iron(III) at pH 7.46 was determined to be 26.55, which is not significantly different to that of the hexadentate ligand CP254 (log K′ ) 26.47). In vitro perfusion studies indicate that the polymeric chelators described in this study can reduce iron absorption from the intestine.

Introduction Metal chelating polymers have been used for water treatment, recovery of metals, and as reagents for analytical chemistry.1–3 Iron chelating polymers have potential application in the biomedical field and in particular in the therapy of iron overload. Iron is an essential element for all living processes and, as such, the absorption of iron is normally under close physiological control. Iron is absorbed from the mammalian gastrointestinal tract by two protein-mediated mechanisms, one absorbing hexaquo iron(II) and the other iron as haem.4 Primates have evolved not to be able to excrete iron, and therefore, body iron levels are totally controlled by the absorption process.5 Body iron levels are tightly controlled because excess iron is extremely toxic due to its facile ability to redox cycle between the iron(III) and iron(II) states, thereby generating relatively high levels of the toxic hydroxyl radical.6 Iron overload can result from regular blood transfusions, as experienced by thalassaemia major and sickle cell patients or by the hyperabsorption from the gastrointestinal tract, when the normal control mechanisms are not fully functional.7 Examples of this latter situation arise with thalassaemia intermedia8 and hemochromatosis.9 The most common method of treating iron overload is to remove the excess iron by the use of iron-selective chelators such as desferrioxamine and deferiprone.10 These chelators are absorbed from the intestine and scavenge iron from the blood and various tissues. However, the method is not ideal as inevitably iron * To whom correspondence should be addressed. Tel.: (+)44 207 848 4882. Fax: (+)44 207 848 6394. E-mail: [email protected]. † Zhejiang Gongshang University. ‡ King’s College London.

chelators possess many toxic side effects. One possible method of avoiding the use of systemic iron chelators is to inhibit iron absorption from the gastrointestinal tract, this method being particularly relevant to thalassaemia intermedia and hemochromatosis. Iron binding polymers have considerable potential in this therapeutic approach as they can effectively bind iron irreversibly to form nontoxic, kinetically inert complexes that are not absorbed by the gastrointestinal tract, thereby reducing the absorption of iron from the intestine. Design of iron chelating polymers presently falls into two strategies, one involves the immobilization of natural chelators (e.g., desferrioxamine B) onto activated supports11–13 and a second involves the conjugation of specific bidentate ligands with activated polymers14–17 or the copolymerisation of 1-(βacrylamidoethyl)-3-hydroxy-2-methyl-4(1H)-pyridinone (AHMP) with other cross-linking agents.18,19 There are disadvantages with both of these strategies. The former structures are limited to carbohydrate matrices and, as a consequence, are expensive to prepare, and high binding capacities are difficult to achieve. The latter group of polymers lack a uniform high affinity for iron(III), and with bidentate ligand-containing polymeric chelators, it is difficult for each bidentate ligand to form part of an ideal octahedral iron(III) coordination site. Thus, the complexation of three bidentate ligands with iron will not be consistently strong, with partial chelation of iron being likely, for instance, where only two bidentate ligands bind an iron atom (Figure 1a). Such structures possess a markedly lower affinity for iron(III).20 One approach that avoids the disadvantage of incorporating bidentate ligands is to use similar technology but with hexadentate ligands. With hexadentate ligand-containing polymeric chelators, all the hexadentate moieties possess the

10.1021/bm701122u CCC: $40.75  2008 American Chemical Society Published on Web 03/29/2008

3-Hydroxypyridin-4-one Hexadentate Copolymers

Biomacromolecules, Vol. 9, No. 5, 2008

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Figure 1. (a) Iron chelation by a bidentate ligand-containing polymeric chelator: three bidentate moieties bind one iron with ideal stereochemistry (left), only two bidentate moieties bind one iron and two orbits are occupied by water molecules (middle), three bidentate moieties bind one iron in a nonideal geometry (right); (b) Iron chelation by a hexadentate ligand-containing polymeric chelator: all the hexadentate moieties bind iron with an ideal stereochemistry.

ideal geometry to provide octahedral coordination sites for iron chelation and so form consistently stable iron complexes (Figure 1b). Such chelation will optimize the iron(III) affinity of the polymer. These considerations prompted us to explore the design and synthesis of high affinity iron(III)-selective hexadentate ligand-containing polymeric chelators. These polymers are novel and have not been previously reported. 3-Hydroxypyridin-4ones (HPOs) are one of the main candidates for the development of orally active iron chelators,21 and 1,2-dimethyl-3-hydroxypyridin-4-one (deferiprone) is currently available for clinical use (Apotex Inc., Toronto, Canada as Ferriprox). In this work, the synthesis of a novel 3-hydroxypyridin-4-one hexadentate ligand and its incorporation into copolymer structures is reported. An investigation of the physicochemical characterization of the monomer hexadentate ligand and the iron-chelating properties of this class of polymer has been undertaken. The effects of one of these polymeric chelators on iron absorption from rat intestine has been investigated using an in vitro perfusion model.

Experimental Section General. All chemicals were purchased from Aldrich or Merck Chemical Company and used without any further purification. Di-tertbutyl 4-amino-4-[2-(tert-butoxycarbonyl)ethyl]-heptanedioate (2) was prepared as reported.22 58Fe was purchased from Chemgas (Boulogne, France). This product contains 56Fe 0.35%, 57Fe 6.50%, and 58Fe 93.13%. Male Wistar rats (230-250 g) were purchased from Harlan U.K. Ltd. (Oxon, England) and housed in the Biological Service Unit, King’s College London, for at least three days before the investigation. The rats were maintained at a temperature between 20 to 23 °C with food and water ad libitum in accordance with Animals (Scientific Procedures) Act 1986 Home Office regulations. Melting points were determined using an Electrothermal IA 9100 Digital Melting Point Apparatus and were uncorrected. 1H NMR and 13 C NMR spectra were recorded on a Bruker Avance 360 spectrometer with TMS as an internal standard. Matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectra were recorded on Autoflex MALDI-TOF mass spectrometer (Brukes Daltonics, Coventry, U.K.). Electrospray ionization (ESI) mass spectra were obtained by infusing samples into an LCQ Deca XP ion trap instrument. High resolution mass spectra (HRMS) were determined at the Strand

campus, King’s College London. Metal ion concentrations were determined by inductively coupled plasma mass spectroscopy (ICPMS). A quadrupole-based ICP-MS spectrometer (Perkin-Elmer SCIEX ELAN 6100 DRC) with a dynamic reaction cell (DRC) was used in the present study (PerkinElmer SCIEX, Concord, Ontario, Canada). The size of copolymer particles were determined on a Nikon Labophot microscope. Synthesis of Hexadentate Monomer CP254 Hydrochloride (11). The synthetic route is summarized in Scheme 1. Di-tert-butyl 4-(Acryloylamino)-4-(3-tert-butoxy-3-oxopropyl)heptanedioate (3). Acryloyl chloride (12 mmol) was added to a solution of amine 2 (10 mmol) and triethylamine (12 mmol) in 50 mL of dichloromethane cooled with an ice bath. The mixture was stirred for 4 h at room temperature. The reaction mixture was washed with 10% NaHCO3, brine, cold 10% HCl, and brine. The organic layer was dried in anhydrous Na2SO4. After the removal of solvent, the residue was chromatographed on silica gel using CHCl3/MeOH (20:1) as an eluent. The product was obtained in 76% yield. Mp 132–133 °C. 1H NMR (CDCl3) δ 1.44 (s, CH3, 27H), 2.03 (t, J ) 7.3 Hz, CH2, 6H), 2.25 (t, J ) 7.3 Hz, CH2, 6H), 5.59 (m, 1H), 6.03 (m, 1H), 6.24 (m, 1H); 13C NMR δ 28.07 (CH3), 29.84 (CH2), 30.08 (CH2), 57.57 (NHC), 80.04 (OCMe3), 125.85 (CH2dCH), 131.76 (CH2dCH), 164.82 (COO), 173.01 (CONH). HRMS (ESI) calcd for C25H43NO7Na, 492.2938; found, 492.2935 ([M + Na]+). 4-(Acryloylamino)-4-(2-carboxyethyl)heptanedioic Acid (4). A solution of 3 (3 g, 6.39 mmol) in formic acid (15 mL) was stirred at room temperature overnight. After concentration and removal of residual formic acid by the addition of toluene (3 × 15 mL), the product was obtained in quantitative yield. Mp. 148–150 °C; 1H NMR (DMSO-d6) δ 1.87 (m, 6H, CH2), 2.11 (m, 6H, CH2), 5.53 (m, 1H), 6.03 (m, 1H), 6.30 (m, 1H), 7.44 (s, NH, 1H). 13C NMR δ 27.92, 28.89 (CH2), 56.54 (NHC), 124.75 (CH2dCH), 132.17 (CH2)CH), 163.90 (COOH), 174.24 (CO). HRMS (ESI) calcd for C13H20NO7, 302.1239; found, 302.1234 ([M + H]+). 3-Methoxy-2-methyl-4H-pyran-4-one (6). To a solution of methyl maltol (5; 20 g, 158.6 mmol) in methanol (30 mL) was added aqueous sodium hydroxide (6.98 g in 60 mL of water, 174.4 mmol). The solution was cooled with ice-bath. Dimethyl sulfate (22 g, 174.4 mmol) was added dropwise over a period of 1 h. Stirring was continued at room temperature for one hour and then at 60 °C for an additional 4 h. After the removal of methanol, the solution was extracted with dichloromethane (3 × 100 mL). The combined organic layers were washed

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Scheme 1. Synthesis of Hydroxypyridinone Hexadentate Ligand (CP254)a

a Reagents and conditions: (a) CH2Cl2, Et3N, 0 °C to rt, 4 h, 76% yield; (b) HCOOH, rt, 24 h, 96% yield; (c) (CH3)2SO4; (d) 40% NH3 aqueous, reflux overnight; (e) BnNHCH3, HCHO, EtOH, reflux 2 d, 89% yield; (f) Pd/C, 40 psi H2, 24 h, 95% yield; (g) 4, 1-hydroxybenzotriazole, DCCI, DMF, rt, 2 d, 74% yield; (h) BCl3, CH2Cl2, 0 °C to rt, 1 d, 73% yield.

with 5% aqueous sodium hydroxide (2 × 200 mL), followed by water (3 × 200 mL), and then dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to yield an pale brown oil (15.2 g, 68%). 1H NMR (CDCl3) δ 2.33 (s, 3H, CH3), 3.86 (s, 3H, CH3), 6.36 (d, J ) 5.6 Hz, 1H, C-5H), 7.63 (d, J ) 5.6 Hz, 1H, C-6H). 3-Methoxy-2-methylpyridin-4(1H)-one (7). To a solution of 6 (10 g, 71.4 mmol) in ethanol (25 mL) was added 35% ammonium aqueous (70 mL). The mixture was refluxed overnight. The reactant was concentrated under vacuum to dryness, and the resulting residue was solidified in acetone to obtain the white crystals (8.75 g, 88%). Mp. 153–155 °C (lit23 155–156.5 °C; 1H NMR (DMSO-d6) δ 2.19 (s, 3H, CH3), 3.69 (s, CH3, 3H), 6.13 (d, J ) 7.0 Hz, 1H, C-5H), 7.47 (d, J ) 7.0 Hz, 1H, C-6H). 5-{[Benzyl(methyl)amino]methyl}-3-methoxy-2-methylpyridin4(1H)-one (8). A solution of 40% HCHO (100 mmol) and Nbenzylmethylamine (200 mmol) in ethanol (100 mL) was stirred for 0.5 h before adding to a solution of 7 (50 mmol) in ethanol (50 mL). The mixture was refluxed for 2 days. After removing the solvent, chloroform (200 mL) was added and the organic layer was washed with water (20 mL) and then dried over anhydrous sodium sulfate. After removal of the solvent, the residue was recrystallized from ethanol/acetone to afford pure product 8 in 89% yield. Mp. 137–139 °C; 1H NMR (CDCl3) δ 2.26 (s, 3H, CH3), 2.44 (s, 3H, CH3), 3.63 (s, 2H, CH2), 3.75 (s, 2H, CH2), 3.88 (s, 3H, CH3), 7.29–7.35 (m, 5H, ArH), 7.79 (s, 1H, C6-H), 10.7 (br, 1H, NH). MS m/z 272 (M+, 100%), 181 (10%), 152 (35%), 150 (15%), 120 (33%); HRMS (ESI) calcd for C16H21N2O2, 273.1603; found, 273.1598 ([M + H]+). 3-Methoxy-2-methyl-5-[(methylamino)methyl]pyridin-4(1H)one (9). A solution of 8 (10 mmol) in ethanol (60 mL) was subjected to hydrogenation under 40 psi H2 in the presence of 5% Pd/C catalyst (0.5 g) for 24 h. Following filtration, the filtrate was concentrated in vacuo and the crude material was recrystallized from dichloromethane to give white solid (95% yield). 1H NMR (DMSO-d6) δ 2.22 (s, 3H, CH3), 2.36 (s, 3H, CH3), 3.64 (s, 2H, CH2), 3.73 (s, 3H, CH3), 7.62 (s, 1H, C6-H); 13C NMR δ 13.52 (CH3), 33.53 (NCH3), 46.91 (CH2), 58.45 (OCH3), 121.65 (C-5 in pyridinone), 134.08 (HC-6 in pyridinone), 139.04 (C-2 in pyridinone), 144.74 (C-3 in pyridinone), 171.77 (CO).

MS m/z 182 (M+, 95%), 150 (100%), 149 (67%); HRMS (ESI) calcd for C9H15N2O2, 183.1133; found, 183.1128 ([M + H]+). Protected Hexadentate Ligand 10. A mixture of 4 (3 mmol), 9 (10.8 mmol), N,N-dicyclohexylcarbodiimide (DCCI; 10.8 mmol), 1-hydroxybenzotriazole (HOBt; 10.8 mmol), and N,N dimethylformamide (DMF; 30 mL) was stirred for 2 days. After filtration and removal of the solvent under reduced pressure, the residue was chromatographed on silica gel using MeOH/CHCl3 (1:2) as an eluent to afford pure 10 (74% yield). Mp. 158–161 °C; 1H NMR (DMSO-d6) δ 1.93 (m, 6H, CH2), 2.20 (s, 9H, CH3), 2.25–2.39 (m, 6H, CH2), 2.71 and 2.95 (m, 9H, CH3), 3.69 (m, 9H, CH3), 4.19 and 4.23 (s, 6H, CH2), 5.48 (m, 1H, vinyl), 6.01 (m, 1H, vinyl), 6.27 (m, 1H, vinyl), 7.24 and 7.30 (s, 3H, pyridinone C6-H), 7.49 (m, 1H, NH), 11.74 (br, 3H, NH); 13C NMR δ 13.7 (CH3), 26.8 (CCH2CH2CO), 30.0 (CCH2CH2CO), 33.2 (NCH3), 44.3 (NCH2), 57.6 (NHC), 58.8 (OCH3), 123.2 (C-5 in pyridinone), 124.3 (CH2dCH), 132.4 (HC-6 in pyridinone), 132.8 CH2dCH), 139.0 (C-2 in pyridinone), 145.3 (C-3 in pyridinone), 164.5 (CONH), 172.5 (CH2CH2CO), 172.7 (C-4 in pyridinone). Calcd for C40H56N7O10: [M + 1]+ m/z ) 794.4048. Found: MALDI-TOF MS (R-cyano-4-hydroxycinnamic acid (CHCA) as a matrix): 794.26 ([M + H]+), 816.25 ([M + Na]+), 838.25 ([M + K]+). HRMS (ESI): 794.4059 ([M + H]+). Hexadentate Ligand CP254 Hydrochloride Salt (11). Under an atmosphere of nitrogen, 1 M boron trichloride in dichloromethane (18 mL, 18 mmol) was dropped slowly onto an ice-bath cooled solution of 10 (2 mmol) in dichloromethane (15 mL). The mixture was stirred at room temperature for 1 day. Methanol (20 mL) was added to quench the reaction. After removal of the solvent, the residue was recrystallized with methanol/acetone to afford the hydrochloric acid salt of CP254 as a white powder (73% yield). 1H NMR (DMSO-d6) δ 1.95 (m, 6H, CH2), 2.33 (m, 6H, CH2), 2.50 (s, 9H, CH3), 2.74 and 3.05 (m, 9H, CH3), 4.47 (s, 6H, CH2), 5.50 (m, 1H, vinyl), 5.99 (m, 1H, vinyl), 6.32 (m, 1H, vinyl), 7.76 (s, CONH, 1H), 8.02 (s, pyridinone C6-H, 3H); 13C NMR δ 14.4 (CH3), 27.2 (CCH2CH2CO), 29.5 (CCH2CH2CO), 36.2 (NCH3), 44.8 (NCH2), 121.5 (C5 in pyridinone), 133.1 (C6 in pyridinone), 139.9 (C2 in pyridinone), 142.3 (C3 in pyridinone), 158.8 (C4 in pyridinone), 174.9 (CONCH3). MALDI-TOF MS (CHCA



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Table 1. Preparation of Cross-Linked CP254-DMAA Copolymers copolymer code 1 2 3 4 5

monomer composition of reaction mixture (mol %) CP254

DMAA

EBAA

weight recovery (%)

iron binding capacity (µmol/1 g)

monomera recovery (%)

0.00 1.25 2.50 5.00 7.50

95.00 93.75 92.50 90.00 87.50

5.00 5.00 5.00 5.00 5.00

98 95 85 77 73

0 106 175 234 271

89 71 49 40

a Monomer recovery means the percentage of monomer incorporated in the copolymer in relation to the amount of monomer used in the polymerization reaction. It was calculated from the capacity of the copolymer based on the assumption that all the hexadentated ligand units bind one iron atom.

Scheme 2. Synthesis of Crosslinked Chelating Copolymer

as a matrix): 752.22 ([M + H]+), 774.21 ([M + Na]+), 790.17 ([M + K]+). HRMS (ESI) calcd for C37H50N7O10 [M + H]+ m/z ) 752.3614, calcd for C37H49N7O10Na [M + Na]+ m/z ) 774.3433. Found: 752.3601 ([M + H]+), 774.3432 ([M + Na]+). Preparation of Cross-Linked CP254-DMAA Copolymers. The iron chelating copolymers were prepared by the reverse-phase suspension polymerization of CP254 hydrochloride salt, DMAA, and EBAA (Table 1, Scheme 2). In a typical procedure, to a stirring mixture of CP254 hydrochloride salt (0.861 g, 1 mmol), DMAA (12; 7.43 g, 75 mmol), EBAA (13; 0.673 g, 4 mmol), (NH4)2S2O8 (0.183 g, 0.8 mmol), H2O (55 mL), hexane (140 mL), and CCl4 (8 mL), sorbitan monostearate (140 mg) was added, and the mixture was flushed with N2 for 20 min. N,N,N′,N′-Tetramethylethylenediamine (0.5 mL) was added and stirring was continued for 3 h at 40 °C. After filtration, the solid product was washed with 2-propanol, methanol, water, ethanol, carbon tetrachloride, and ethyl acetate and then dried in vacuo at 60 °C for 2 days, and copolymer 2 was obtained in 95% yield (8.410 g). The average diameter of the final polymer particles was determined by microscopy to be 80 µm. pKa Determination of CP254. Equilibrium constants of protonated ligands were determined using an automated computerized system, consisting of a UV/vis spectrophotometer (Perkin-Elmer Lambda 5), an autoburette (Metrohm Dosimat 665), a pH meter (Corning Delta 225), and a peristaltic pump (Watson-M arlow 101U/R M2) all interfaced to a computer. A blank titration of 0.1 M KCl (25 mL) was carried out to determine the electrode zero and slope using GLEE.24 The solution (0.1 M KCl, 25 mL), contained in a jacketed titration cell, was acidified by 0.15 mL of 0.2 M HCl. Titration was carried out against 0.3 mL of 0.2 M KOH using 0.01 mL increments dispensed from the dosimat. All solutions were maintained at 25 ( 0.5 °C under an argon atmosphere. The above titration was repeated in the presence of CP254. The data obtained from titration were analyzed using the pHab software.25

Determination of Stability Constant of Iron(III)-CP254 Complex. Iron(III) (51.9 µM), CP254 (52.6 µM), and EDTA (95.3 mM) were added to a 20.33 mL of KCl (0.1 M) solution and alkalimetrically titrated from pH 6.14 to 9.36. Each pH observation was taken after standing for 30 min to achieve equilibrium. The data obtained were analyzed using pHab software.25 Measurement of Iron(III) Chelation of the Copolymers. Determination of iron(III) chelation was carried out with ferric ammonium sulfate-nitrilotriacetic acid (NTA; 1:2 mol ratio) at pH 7.4 in 3-(Nmorpholine)-propanesulfonic acid (MOPS) buffer solution. Chelating Capacity. A total of 30 mg of dry chelating copolymer and 5 mL iron(III) solution (1.6 mM) were placed in a stoppered vessel. The mixture was rotated for 24 h at 25 °C. The amount of iron(III) chelated by the copolymer was calculated from the change in the solution iron(III) concentration. For comparison, the procedure was carried out using a blank control (in the absence of copolymer). Rate of Iron(III) Chelation. Copolymer 5 or Chelex 100 (30 mg) was added to an iron(III) solution (0.4 mM, 30 mL) and rotated at 25 °C. The iron(III) concentration of the supernatant was measured at regular intervals, and the amount of the iron(III) bound to the polymer was calculated from the change in the solution iron(III) concentration. The supernatant was obtained by filtration through a syringe filter (0.2 µm pore size, Whatman Inc.). Recycling InVestigation. Copolymer 5 (30 mg) was added to an iron(III) solution (0.17 mM, 5 mL), and after a 30 min rotation at 25 °C, the supernatant of the mixture was analyzed for iron(III) concentration. The resulting copolymer (containing bound iron(III)) was added to an excess of HCl (1 M) and rotated overnight at 25 °C. Before reuse, the copolymer was filtered and washed with HCl (1 M), EDTA (0.2 mM, pH 3), and MOPS buffer (pH 7.4). SelectiVity of the Copolymer for Iron(III). NH4Fe(SO4)2 solution and solutions of bivalent metal chlorides or sulfates (approximate 0.8 mM) were prepared in citrate buffer (10 mM) at pH 5.6. Two test systems

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were investigated. The single metal chelation test involved the incubation of copolymer 2 (60 mg) in a metal solution (10 mL), which was rotated at room temperature for 24 h. A competition test was also designed, which involved the incubation of copolymer 2 (60 mg) with Fe3+ in the presence of a second metal ion. To a mixture of an Fe3+ solution (10 mL) and a M2+ solution (10 mL) was added copolymer 2 (60 mg), and the mixture was rotated at room temperature for 24 h. The concentrations of Fe3+ and M2+ remaining in the solution were determined by ICP-MS. The amount of the metal ion scavenged by the copolymer was calculated from the change of the metal ion concentration in the solution. Determination of the Conditional Stability Constant of the Iron(III)Copolymer 2 Complex. Deferiprone solution (5.48 mM) was prepared by dissolving 35 mg of deferiprone in 36.3 mL of MOPS buffer (100 mM, pH 7.46). An iron(III)-deferiprone complex was prepared from this solution by the addition of 90 µL of standard atomic absorbance iron solution (0.01771131 M). The competition experiment was performed at room temperature for 4 days by rotating known amounts of copolymer 2 (1.47 mg, 0.1557 µmol; 4.50 mg, 0.4766 µmol; 9.70 mg, 1.0275 µmol; 14.32 mg, 1.5169 µmol; 19.64 mg, 2.0805 µmol) in a solution of the deferiprone-iron(III) complex (5.3 mL). The concentration of (deferiprone)3-Fe complex at equilibrium was determined by UV–visible spectrophotometry in a 5 cm cuvette. CP254 forms a 1:1 iron(III)-ligand complex. Assuming that a 1:1 iron(III)-ligand complex is also formed on copolymer 2, competition with deferiprone will give rise to two chelation reactions:

Fe + 3L h FeL3 ; K )

[FeL3]

[Fe][L]3 [FeL ′ ] Fe + L ′ h FeL ′ ; K′ ) [Fe][L ′ ]

(1) (2)

where L is the soluble ligand (deferiprone) and L′ is the immobilized hexadentate ligand on the copolymer. For the sake of brevity, all charges have been omitted. In the equilibrium situation, the system can be represented in the following way:

FeL3 + L ′ h FeL ′ + 3L; Kq )

[FeL ′ ][L]3 K′ ) [FeL3][L ′ ] K

(3)

The conditional stability constant K′ at pH 7.46 was calculated from the stability constant of deferiprone (log K ) 36.7) and Kq.24 In Vitro Intestinal Perfusion. 58Fe was dissolved in nitric acid (1 M) to yield 170 mM 58Fe and diluted to 25 mM or 5 mM 58Fe using NTA solution (pH 7.0). The molecular ratio of 58Fe/NTA in these two 58 Fe solutions was maintained at 1:5. The experiments were undertaken using an in vitro intestinal perfusion model described by Schümann et al.26–28 The animals were terminally anaesthetised by a combined i.p. application of Hypnorm and Hypnovel. A total of 10–15 cm of the jejunal section was removed from the body and perfused for periods up to 2 h using 50 mL of Tyrode solution at 37 °C, pH 7.0, saturated with carbogen (95% O2 and 5% CO2). Tyrode solution contains NaCl (137 mM), KCl (2.7 mM), NaHCO3 (11.9 mM), NaH2PO4 (0.4 mM), CaCl2 · 2H2O (0.14 mM), MgCl2 · 6H2O (0.05 mM), and D-glucose (15 mM). For the control group, 10 min after the start of perfusion, 100 µL of 58 Fe solution (25 mM or 5 mM) was added to the perfusate (50 mL; final concentration of 58Fe in the solution is 50 µM and 10 µM, respectively). The perfusion time was reset and the samples (absorbates) were collected over the periods 0–30 min, 30–60 min, and 60–120 min. For the test groups, 5 min after the start of perfusion, copolymer 2 (iron binding capacity, 106 mmol/g) was added to the perfusate. After 10 min of perfusion, 100 µL of 58Fe solution (25 mM or 5 mM) was added to perfusate (50 mL). The ratios of the calculated iron binding site numbers of copolymer 2 to 58Fe were adjusted to 1:1, 2:1, or 5:1. Thus, in the case of 50 µM iron concentration, the amount of copolymer

2 in perfusate was 23.6, 47.2, and 118 mg, respectively, because iron binding capacity of copolymer 2 is 106 mmol/g. Similarly, in the case of 10 µM 58Fe concentration, the amount of copolymer 2 in perfusate was 4.7, 9.4, and 23.5 mg, respectively. The samples were collected in the same periods as the control groups. The 58Fe content in the samples was determined by ICP-MS. 58 Fe analysis: A quartz cyclonic spray chamber with Meinhard nebulizer was used for sample introduction. The dynamic reaction cell gas used was 5% hydrogen in argon (BOC Special Gases, Guildford, Surrey). Samples (200 µL) were diluted to 1.5 mL using 0.5% HNO3 containing indium (10 mg/L as internal standard) before being subjected to ICP-MS analysis.

Results and Discussion Chemistry. Hexadentate ligands can be constructed by attaching three bidentate units to suitable molecular backbones. Theoretically, three 3-hydroxypyridin-4-one ligands with either the 2- or 5-substituents attached to a suitable tripodal molecule can form a high affinity hexadentate ligand. The readily accessible amine 9 and triacid 4 were chosen as starting materials. The synthetic procedure for the hydroxypyridinone hexadentate monomer 11 is presented in Scheme 1. Treatment of acryloyl chloride with amine 2 furnished the desired triester 3, the structure of which was confirmed by the 13C NMR peaks at 164.82 (COO), 173.01 (CONH), and 57.57 (NHC). Hydrolysis of triester 3 was accomplished by treatment with formic acid to give the triacid 4. 3-Methoxy-2-methyl pyridin-4(1H)-one 7 was readily prepared from commercially available methyl maltol 5 in a two-step reaction. The introduction of a [benzyl(methyl)amino]methyl group to the 5-position of 7 was achieved by the use of the Mannich reaction. The desired amine 9 was then prepared by hydrogenation of 8 to remove the benzyl group in the presence of Pd/C catalyst. Application of peptide coupling procedures to the condensation of acid 4 with amine 9 was undertaken in the presence of DCCI and HOBt in DMF to afford methyl-protected hexadentate 10. The desired hexadentate monomer 11 (CP254) was obtained after removal of methyl groups by treatment of 10 with boron trichloride. The synthesis of cross-linked copolymeric chelators and a schematic structure of the resins are presented in Scheme 2. CP254-DMAA copolymers were prepared by varying the initial mole percentage of 11 from 0 to 7.5%, while keeping the content of the cross-linking agent at 5 mol% (Table 1). Cross-linked poly(DMAA) (copolymer 1) was obtained in almost quantitative yield, whereas the weight recovery of the CP254-DMAA copolymers (copolymer 2–5) decreased with increasing the percentage of the hexadentate ligand monomer, indicating that the bulky and polar CP254 group renders the CP254 polymerization more difficult. Characterization of hexadentate monomer CP254. To demonstrate the ligand affinity, both pKa values of CP254 and the stability constant of its iron(III) complex were evaluated using an automated titration system.29–31 pKa Value Determination. The pH dependence UV spectra of CP254 (Figure 2) was recorded between 200 and 340 nm over the pH range 1.63–12.16. The speciation spectra demonstrate a clear shift in λmax from 260 to 288 nm, which displays the pH dependence of the ligand ionization equilibrium. The pKa values obtained from nonlinear least-squares regression analysis are 1.69, 2.69, 3.29, 9.45, 9.73, and 10.16. CP254 possesses six pKa values, with the lower three values corresponding to the 4-hydroxyl functions and the high three corresponding to the 3-hydroxyl functions. Iron Chelation Ability. Iron chelating ability of CP254 was investigated using matrix-assisted laser desorption/ionization



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Figure 2. UV spectra of CP254. The spectra were recorded between 200 and 340 nm over the pH range 1.63–12.16.

Figure 4. An energy-minimized structure of Fe(III)-CP254 complex. Atoms are colored as follows: hydrogen, white; carbon, green; oxygen, red; nitrogen, blue; and iron, yellow.

Figure 3. The MALDI TOF mass spectrum of a one-to-one mixture of CP254 and iron(III) using DCTB (trans-2-[3-{4-tert-butylphenyl}-2methyl-2-propenylidene]malononitrile) as a matrix. The mixture was prepared by adding an equivalent volume of iron solution (100 µM, 1 mM NTA, 50 mM NH4HCO3) to CP254 (100 µM in water; final pH was 7.2). This solution was maintained at room temperature for 15 min prior to spectral acquisition. The peaks at m/z 805.23 and 843.24 correspond to the proton adduct of the 1:1 iron(III)-CP254 complex [M + (FeIII - 3H) + H]+ and the potassium adduct of the 1:1 iron(III)-CP254 complex [M + (FeIII - 3H) + K]+, respectively.

time-of-light mass spectrometry (MALDI-TOF MS). Direct evidence was obtained for the formation of 1:1 complex of CP254 and iron. The MALDI spectrum recorded from the oneto-one mixture of CP254 and iron shows a main signal at m/z 805.23, which corresponds to the proton adduct of one-to-one CP254-iron complex, also annotated as [M + (FeIII - 3H) + H]+; a small signal at m/z 843.24 was also found, corresponding to the potassium adduct of one-to-one CP254-iron complex ([M + (FeIII - 3H) + K]+; Figure 3). Evidently, three protons are released upon complexation of each iron(III). An energyminimized structure of Fe(III)-CP254 complex revealed that CP254 binds iron in an octahedral coordination fashion (Figure 4). The structure optimization of Fe(III)-CP254 complex was carried out using the AM1 level semiempirical algorithm by Hyperchem 5. The ball-stick structure was presented by ViewerLite 5.0. Spectrophotometric Determination of Affinity Constant for Iron(III). The stability constant of an iron-ligand complex is one of the key parameters related to the in vivo chelation efficacy of a ligand. The ligand competition for CP254 with EDTA in the presence of iron(III) is presented in Figure 5. With the increase of the pH from 6.14 to 9.36, CP254 gradually removed more iron(III) from the EDTA-iron complex, and the color of the solution changed to red. The log stability constant

Figure 5. Visible spectra for CP254 (52.6 µM); EDTA (95.3 mM) competition titration with iron(III) (51.9 µM) in a 5 cm flow cell.

of CP254 for iron(III) was determined to be 33.2. The pFe3+ value, which is defined as the negative logarithm of concentration of free iron(III) in solution under defined conditions was derived from the affinity constant. For clinically relevant conditions, pFe3+ values are typically calculated for total [ligand] ) 10-5 M and total [iron] ) 10-6 M at pH 7.45, the corresponding pFe3+ value of CP254, using these conditions, is 27.24. This value is high even for a hexadentate ligand; the value is almost 10 times higher than that corresponding to the commercially available hexadentate ligand, desferrioxamine (26.3).32 The speciation plot of CP254-iron(III) complexes demonstrated that the 1:1 ligand-iron(III) complex is the dominant species over the pH range 2–11 (Figure 6). Characterization of Hexadentate-Containing Copolymers. To facilitate the assessment of the copolymers, a comparison has been made with Chelex 100, a widely used commercially available polymeric chelator. Iron(III) Chelating Capacity. All the CP254-DMAA copolymers were found to bind iron(III) tightly, and the chelating capacity depended on the initial CP254 concentration in the polymerization reaction (Table 1), the larger the initial CP254 concentration, the greater the chelating capacity of copolymer. For instance, when the initial mole ratio of CP254 was 1.25% of the total composition, the iron chelating capacity of the

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Zhou et al. Table 2. Reutilization Test with Copolymer 5 and Chelex 100a iron remaining (µM) number of cycles

polymer 5 (30 mg)

Chelex 100 (30 mg)

1 2 3 4 5

0.17 0.04 0.08 0.10 0.16

118 111 97 115 114

a The initial iron concentration was 170 µM, the volume was 5 mL, and the rotation time was 30 min.

Table 3. Chelation of Iron and Other Metals by Copolymer 2a initial final metal metal concentration (µM) concentration (µM) scavenged (µmol) Figure 6. Speciation plot of CP254 (1 µM) in the presence of iron(III) (1 µM).

Fe3+ Cu2+ Zn2+ Ni2+ Mn2+ Ca2+

969 841 878 935 825 997

471 389 822 873 758 908

4.98 4.52 0.56 0.62 0.67 0.89

a Determined by chelation experiments at pH 5.6 for 24 h at room temperature.

Figure 7. Iron(III) chelation of copolymer 5 (black) and Chelex 100 (red): chelation was carried out by adding 30 mg of copolymer 5 or Chelex 100 to an iron(III) solution (0.4 mM, pH 7.4, 30 mL) and rotating at 25 °C.

copolymer was determined as 106 µmol/1 g of copolymer; whereas when the initial mole ratio of CP254 was 7.5% of the total composition, the iron chelating capacity of the obtained copolymer reached 271 µmol/1 g of copolymer. The chelating capacity of Chelex 100 was determined as 110 µmol/1 g. Iron(III) ScaVenging Properties. Iron(III) chelation was studied with polymer 5 and Chelex 100 to determine the rate of iron(III) chelation. Copolymer 5 was found to bind iron(III) much more quickly than Chelex 100. Iron(III) chelation by the copolymer nearly reached its maximal capacity (96%) at 5 h. In contrast, only 45% of the chelating capacity of Chelex 100 was utilized at 5 h, and 88% was achieved after 48 h (Figure 7). It is important that there is a relatively fast onset of iron(III) binding if such polymers are to be clinically useful. For polymers to be suitable for use in chemical and biomedical systems, they should be chemically stable and reusable. The recycling efficiency of this copolymer group was demonstrated by repeated exposure to an iron(III) containing solution, followed by acid wash regeneration (Table 2). The copolymer was found to be readily regenerated without damage to the structure. The copolymer continued to bind iron efficiently, after several recycling stages. During these experiments the iron concentration was reduced from 170 µM to 0.04–0.17 µM (average 0.11 µM, 5.5 × 10-4 µmol). Thus, over 99.9% of iron was bound to the copolymer under these conditions. For comparison, a similar test was carried out with Chelex 100, where the iron concentrations were only reduced from 170 µM to 96.59–117.92 µM (average 110.83 µM). Thus, only 35% of iron was bound to Chelex 100 under these conditions.

SelectiVity of the Copolymer. As CP254-DMAA copolymers possess a high affinity for iron(III), it was anticipated that the copolymers would also possess a high selectivity for Fe3+ over other metal ions. It has been previously demonstrated that hexadentate hydroxypyridinones possess a high selectivity for tribasic cations over dibasic cations.33 This renders the ligands effectively selective for iron(III) under biological conditions, as gallium(III), indium(III), aluminum(III), and chromium(III) are only present at relatively low levels and are not essential for living processes. In contrast, copper(II), zinc(II), nickel(II), manganese(II), and calcium(II) are all present in biological tissues and food stuffs at relatively high levels. As these five metals are essential for life, it is important that the polymers designed in this study possess much low affinities for this group of dibasic cations. The chelation of this range of metals by copolymer 2 was investigated. The copolymer was found to bind Cu2+ efficiently, but to possess a relatively low affinity for Zn2+, Ni2+, Mn2+, and Ca2+ (Table 3). In competition studies, iron(III) was shown to be selectively bound to the copolymer in the presence of competing metals, even copper(II) (Table 4). In all these cases, iron concentrations were decreased significantly after incubation with copolymer, whereas the concentrations of the competing bivalent metal remained almost unchanged. Conditional Stability Constant of Iron(III)-Copolymer 2 at pH 7.46. The ligand competition method is widely used for the determination of stability constants of both soluble iron(III)-ligand complexes34 and iron-polymeric chelators.16,35 In this study, the conditional stability constant (log K′) of iron(III)-ligand complexes of copolymer 2 was determined as 26.55 ( 0.07 from spectrophometric competition data (Figure 8). This conditional stability constant is slightly higher than that of CP254 (log [email protected]′ ) log KCP254 - log(acidic factor)[email protected] ) 33.2 - 6.73 ) 26.47), but this difference is within the error range, which indicates that copolymer 2 possesses almost the same affinity to iron as CP254. No appreciable statistical effects were observed. Chelating properties of a polymeric chelator can in principle be affected by steric hindrance between the ligand and the polymeric matrix,36,37 but for this copolymer series there appears to be little inference by the polymer backbone in the iron chelation process. In Vitro Intestinal Perfusion Study. To evaluate the ability of these polymeric chelators to prevent gastointestinal iron(III)



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Table 4. Selectivity of Copolymer 2 for Iron(III) in Competition with Other Metal Ionsa Fe3+ metal ions 3+

2+

Fe /Cu Fe3+/Zn2+ Fe3+/Ni2+ Fe3+/Mn2+ Fe3+/Ca2+ a

M2+

initial concentration (µM)

final concentration (µM)

scavenged (µmol)

initial concentration (µM)

final concentration (µM)

scavenged (µmol)

477 471 478 471 473

239 230 236 230 237

4.76 4.82 4.84 4.82 4.72

449 466 471 403 493

451 463 470 397 488

0 0.06 0.02 0.12 0.1

Determined by chelation experiments at pH 5.6 for 24 h at room temperature.

Figure 8. Visible spectra for varying copolymer 2 (0 µmol, 0.1557 µmol, 0.4766 µmol, 1.0275 µmol, 1.5169 µmol, 2.0805 µmol) in competition with a fixed concentration of deferiprone (5.48 mM) for binding iron(III) (43.8 µM) in a 5 cm flow cell at a fixed pH 7.46.

presented to samples, can be selectively measured by ICP-MS, thus avoiding interference from the endogenous iron in lumen. Two 58Fe concentrations (50 µM and 10 µM) were used in the perfusion studies. The ratios of the calculated iron binding site numbers of copolymer 2 to 58Fe were adjusted to 1:1, 2:1, and 5:1. In the 50 µM 58Fe concentration perfusion experiments, the accumulated iron contents in the absorbates of the 1:1, 2:1, and 5:1 groups at 120 min were 399.3, 129.9, and 42.7 pmole per centimeter tissue, respectively, significantly less than that from the control groups (1826.5 pmole per centimeter tissue; Figure 9a). In the 10 µM 58Fe concentration perfusion experiments, the accumulated iron contents in the absorbates of the 1:1, 2:1, and 5:1 groups in 120 min were 133.9, 61.9, and 34.5 pmole per centimeter tissue, respectively (Figure 9b). Compared with the control groups (247.2 pmole per centimeter tissue), the absorption of iron was again significantly reduced. Thus, iron binding copolymers, by reducing iron absorption, have clear potential in iron overload therapy.

Conclusions 3-Hydroxypyridin-4-one hexadentate functionalized iron chelating copolymers have been synthesized by copolymerisation of CP254 and DMAA in the presence of a cross-linking agent (EBAA). The copolymers showed high affinity and selectivity for iron(III) and may find application in iron overload therapy by reducing iron absorption in the gastrointestinal tract. Acknowledgment. The authors gratefully thank the financial support from EC Framework 5 project (QLK1-CT-2002-00444).

References and Notes

Figure 9. In vitro intestinal perfusion investigation with different ratios of copolymer 2 and 58Fe (9 control (black), b copolymer/58Fe ) 1:1 (red), 2 copolymer/58Fe ) 2:1 (green), 1 copolymer/58Fe ) 5:1 (blue)). (a) Initial concentration of 58Fe of 50 µM; (b) Initial concentration of 58Fe of 10 µM.

absorption, an in vitro rat intestinal perfusion method described by Schümann et al. was adopted. Copolymer 2 was used in this study. 58Fe is utilized in these experiments because 58Fe, when

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BM701122U

Synthesis and Iron (III)-Chelating Properties of Novel 3-Hydroxypyridin

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