Development of novel aroylhydrazone ligands for iron chelation therapy: 2-Pyridylcarboxaldehyde isonicotinoyl hydrazone analogs

Development of novel aroylhydrazone ligands for iron chelation therapy: 2-Pyridylcarboxaldehyde isonicotinoyl hydrazone analogs ERIKA BECKER and DES R. RICHARDSON BRISBANE,QUEENSLAND, AUSTRALIA

Previous studies have demonstrated that aroylhydrazone iron (Fe) chelators of the pyridoxal isonicotinoyl hydrazone (PIH) class have high Fe chelation efficacy both in vitro and in viva. Depending on their design, these drugs may have potential as agents for the treatment of Fe overload disease or cancer. Considering the high potential of this class of ligands, we have synthesized seven novel aroylhydrazones in an attempt to identify Fe chelators more efficient than desferrioxamine (DFO) and more soluble than those of the PIH class. These compounds belong to a new series of tridentate chelators known as the 2-pyridylcarboxaldehyde isonicotinoyl hydrazones (PCIH). In this study we have examined the Fe chelation efficacy and antiproliferative activity of these chelators including their effects on the expression of genes (WAFI and GADD45) known to be important in mediating cell cycle arrest at GI/S. From seven chelators synthesized, three ligands, namely 2-pyridylcarboxaldehyde benzoyl hydrazone (PCBH), 2-pyridylcarboxaldehyde m-bromobenzoyl hydrazone (PCBBH), and 2-pyridylcarboxaldehyde 2-thiophenecarboxyl hydrazone (PCTH), showed greater Fe chelation activity than DFO and comparable or greater efficiency than PIH. These ligands were highly effective at both mobilizing 59Fe from cells and preventing 59Fe uptake from 59Fe-transferrin and caused a marked increase in the RNA-binding activity of the iron-regulatory proteins (IRP). Our studies have also demonstrated that compared with the cytotoxic Fe chelator, 2-hydroxy-I-naphthylaldehyde isonicotinoyl hydrazone (31 I), these ligands have far less effect on cellular growth and 3H-thymidine, 3H-leucine, or 3H-uridine incorporation. In addition, in contrast to 31 I, which markedly increased WAFI and GADD45 mRNA expression, PCBH and PCTH did not have any effect, whereas PCBBH increased the expression of GADD45 mRNA. Collectively, these results demonstrate the potential of several of these ligands as agents for the management of Fe overload disease. (J Lab Clin Med 1999;134:510-21)

Abbreviations: DFO = desferrioxamine; EDTA= ethylenediaminetetraacetic acid; Fe = iron; GADD45 = growth arrest and DNA damage 45 gene; MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium; PCAH : 2-pyridylcarboxaldehyde p-aminobenzoyl hydrazene; PCBH = 2-pyridylcarboxaldehyde benzoyl hydrazone; PCBBH : 2-pyridylcarboxaldehyde m-bromobenzeyl hydrazone; PCHH = 2-pyridylcarboxaldehyde p-hydroxybenzoyl hydrazone; PCIH = 2-pyridylcarboxaldehyde isonicotinoyl hydrazone; PCTH= 2-pyridylcarboxaldehyde 2-thiophenecarboxyl hydrazone; PIH = pyridoxal isonicotinoyl hydrazone; TCA = trichloroacetic acid; Tf = transferrin; WAF1= wild-type p53 activating fragment 1 gene

From the Department of Medicine, University of Queensland, Royal Brisbane Hospital. Supported by the Friedreich's Ataxia Support Group of Queensland in the form of a PhD Scholarship (E:B.) and by a grant from the National Health and Medical Research Council of Australia (D.R.R.). D.R.R was supported by a Research Fellowship and Senior Research Fellowship from the Department of Medicine, University of Queensland.

510

Submitted for publication March 1, 1999; revision submitted May i3, 1999; accepted May 21, 1999. Reprint requests: Des R. Richardson, PhD, Department of Medicine, Clinical Sciences Building Floor C, Royal Brisbane Hospital, Brisbane, Queensland, Australia 4029. Copyright © 1999 by Mosby, Inc. 0022-2143/99 $8.00 + 0

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The development of novel iron chelators for the management of Fe-overload diseases remains an important goal. This is due to the fact that the drug in current clinical use, desferrioxamine (Fig 1), is very expensive, orally ineffective, and requires long subcutaneous infusion (12 to 24 h/d, 5 to 7 d/wk) to effect significant Fe mobilization. 1,2 The need for an orally effective and economical Fe chelator has recently been emphasized by the failure of deferiprone (also known as L1 or 1,2-dimethyl-3-hydroxypyrid-4-one) to successfully chelate Fe from patients with Fe overload. 1,3 In fact, treatment of patients with this latter drug resulted in hepatic fibrosis and an increase in liver Fe levels. 1,3 One important group of chelators that have shown high Fe chelation efficacy both in vitro and in vivo are those ligands of the pyridoxal isonicotinoyl hydrazone class. 4-s These chelators have a very high affinity and specificity for Fe(III) that is similar to that found for DFO and much greater than that of ethylenediaminetetraacetic acid. 9,1° In addition, these ligands are synthesized by a simple one-step Schiff base condensation, are economical, and are orally effective, s It is interesting that PIH can chelate Fe from the mitochondrion, 4,5,8,11,12 a site that may become loaded with Fe in the neurodegenerative disease Friedreich's ataxia. 13-15 Hence, it is possible that chelators of the PIH class may have potential as agents for the treatment of a number of disease states associated with Fe overload. Previous studies have characterized the biologic and chemical properties of analogs of PIH, some of which show higher activity on a molar basis than the parent compound itself. 16-19 These compounds were derived from three groups of aromatic aldehydes, namely, pyridoxal, salicylaldehyde, and 2-hydroxy-1-naphthylaldehyde. In general, chelators derived from pyridoxal were shown to possess high chelation efficacy but low antiproliferative activity, whereas ligands derived from 2-hydroxy-1-naphthylaldehyde had high Fe chelation efficacy and potent antiproliferative activity. 17 Hence, aroylhydrazones derived from pyridoxal may be useful as agents to manage Fe overload disease, whereas chelators derived from 2-hydroxy-l-naphthylaldehyde may have potential for the treatment of cancer. 17,18,20 It should be noted that many other Fe chelators have also demonstrated antiproliferative activity including DFO. 21 In fact, some of the most potent effects of DFO have been reported when this drug was used against the pediatric tumor neuroblastoma. 22-25 When we considered the possible use of certain PIH analogs as potential antiproliferative agents, we were intrigued to find that a closely related group of chelators derived from 2-pyridylcarboxaldehyde and thiosemicarbazide (eg, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone) are among the most effec-

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tive inhibitors of ribonucleotide reductase yet identified. 26,27 In an attempt to improve on the Fe chelation efficacy of the aroylhydrazone ligands already screened in our laboratory,17, is we attempted to examine the activity of "hybrid" chelators derived from 2-pyridylcarboxaldehyde and a number of acid hydrazides previously used for synthesis of the PIH analogs. 16-18 These new chelators are called the analogs of 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (Fig 1). The results reported here demonstrate that several PCIH analogs show high Fe chelation efficacy and in contrast to expectations, low antiproliferative activity. Hence, these latter characteristics suggest that some of these ligands may have potential as agents for the management of Fe overload disease. We suggest that further investigation of these chelators is warranted. METHODS Synthesis of iron chelators and their preparation for screening in culture. The seven PCIH analogs were synthe-

sized by Schiff base condensation between 2-pyridinecarboxaldehyde and the respective acid hydrazides with standard procedures. 2s The chelators were characterized by a combination of elemental analysis, infrared spectroscopy, 1H-NMR spectroscopy, and x-ray crystallography. The chemical characterization of these chelators will be reported in a subsequent publication. Both PIH and the PIH analog 2-hydroxy-l-naphthylaldehyde isonicotinoyl hydrazone (311) were synthesized and characterized as described previously.17 Desferrioxamine (desferrioxamine mesylate) was purchased from Ciba-Geigy Pharmaceutical Co (Summit, NJ). All of the aroylhydrazone chelators were dissolved in dimetbyl sulfoxide as 10 mmol/L stock solutions immediately before an experiment and then diluted in 10% fetal calf serum (Commonwealth Serum Laboratories, Melbourne, Australia) so that the final concentration of dimethyl sulfoxide was equal to or less than 0.5% (vol/vol). After dilution was performed, the solutions were mixed vigorously to ensure total solubilization. Previous studies by the authors have demonstrated that dimethyl sulfoxide at this concentration has no effect on either cellular proliferation, 59Fe release from prelabeled cells, or the ability of the cells to remove 59Fe from Tf. 17 Cell culture. The human SK-N-MC neuroepithelioma and SK-Mel-28 melanoma cell lines were from the American Type Culture Collection (Rockville, MD). The SK-N-MC cell line was originally classified as a neuroblastoma but has been recently reclassified as a neuroepithelioma, a closely related neuroectodermal malignancy. The BE-2 neuroblastoma cell line was a gift from Dr Greg Anderson, Queensland Institute of Medical Research, Queensland. The SK-N-MC and SKMel-28 cell lines were grown in Eagle's modified minimum essential medium (Gibco BRL, Sydney, Australia) containing 10% fetal calf serum, 1% (vol/vol) nonessential amino acids (Gibco), 2 mmol/L L-glutamine (Sigma Chemical Co, St Louis, MO), 100 gg/mL streptomycin (Gibco), 100 U/mL penicillin (Gibco), and 0.28 gg/mL fungizone (Squibb Pharmaceuticals, Montreal, Canada). This growth medium will be

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Fig 1. Structures of the iron chelators examined in this study: desferrioxamine (DFO), 2hydroxy-1-naphthylaldehydeisonicotinoylhydrazone (311), pyridoxal isonicotinoylhydrazone (PIH), 2-pyridylcarboxaldehydeisonicotinoyl hydrazone (PCIH), 2-pyridylcarboxaldehyde thiophenecarboxylhydrazone (PCTH), 2-pyridylcarboxaldehydebenzoyl hydrazone (PCBH), 2-pyridylcarboxaldehydem-bromobenzoylhydrazone(PCBBH), 2-furoylcarboxaldehydeisonicotinoyl hydrazone (FIH), 2-pyridylcarboxaldehydep-aminobenzoylhydrazone (PCAH), 2pyridylcarboxaldehydep-hydroxybenzoylhydrazone (PCHH).

subsequently referred to as complete medium. The BE-2 cell line was grown in Rosewall Park Memorial Institute medium with all of the supplements described previously for Eagle's modified minimum essential medium. Cells were grown in an incubator (Forma Scientific, Ohio) at 37°C in a humidified atmosphere of 5% CO2/95% air and subcultured as described previously.29 Cellular growth and viability were

monitored with phase-contrast microscopy and trypan blue staining. Effect of chelators on cellular proliferation. The effect of the chelators on the proliferation of SK-N-MC neuroepithelioma cells were examined with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium) assay by essentially the same method as described previously) 7 Cellular

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proliferation was examined by seeding cells in 96-well microtiter plates at 15,000 cells/well in 0.1 mL of Complete medium containing human diferric Tf (1.25 gmol/L). This seeding density resulted in exponential growth of the cells for the duration of the assay. The cells were allowed to grow overnight, and the chelators were then added in 0.1 mL of complete medium containing diferric transferrin (1.25 gmol/L). The final concentrations of the chelators were 0.39 to 50 gmol/L. Control samples contained complete medium and diferric Tf (1.25 gmol/L). Cells were incubated with the chelators for 90 hours at 37°C in a humidified atmosphere containing 95% air and 5% CO 2. After this incubation was performed, 0.01 mL MTT was added to each well, and the plates were incubated for 2 hours at 37°C. The cells were then solubilized by adding 0.1 mL of 10% SDS-50% isobutanol in 0.01 mol/L HC1, and the plates were then read at 570 nm on a scanning multiwell spectrophotometer (Titertek Multiscan; Beckman Instruments Inc, CA). As shown previously for the SK-N-MC cell line, MTT color formation was directly proportional to the number of viable cells. 17 The results of the MTT assays are expressed as a percentage of the control value. Preparation of 59Fe-transferrin. Apotransferrin (Sigma Chemical Co, St Louis, MO) was prepared and labeled with 59Fe (as ferric chloride in 0.1 mol/L HC1, Dupont NEN, MA) to produce 59Fe2-transferrin (59Fe-Tf) with standard procedures described previously.29 The saturation of Tf with Fe was monitored by ultraviolet visible spectrophotometry with the absorbance at 280 nm (protein) compared with that at 465 nm (Fe-binding site). In all experiments fully saturated diferric Tf was used. Effect of the chelators on 3H-thymidine, 3H-leucine, and 3H-uridine incorporation. Labeling of ceils with 3H-thymidine (20 Ci/mmol; Dupont NEN, MA), 3H-uridine (42.7 Ci/mmol; Dupont NEN), and 3H-leucine (52 Ci/mmol; Dupont NEN) was estimated after precipitation with TCA. After a 20-hour incubation with the chelators, 3H-leucine, 3Huridine, or 3H-thymidine (1 gCi/mL) were then added for 2 hours at 37°C. Subsequently, the petri dishes were placed on ice and washed four times with ice-cold Hanks' balanced salt solution, and the cells were detached from the plate with 1 mmol/L EDTA in Ca/Mg-free saline solution. The cells were then pelleted by centrifugation, the supernatant removed, and the pellet frozen at -70°C. After the cells were thawed on ice, 1 mL of ice-cold 20% TCA was added, and the solution was then vortexed and kept on ice for 1 hour with periodic mixing. This solution was then centrifuged at 15,000 rpm for 15 minutes at 4°C, and the supernatant was removed. The pellet was then washed twice with 1 mL of ice-cold 10% TCA. The pellet was dissolved in 0.5 mL of 1 mol/L NaOH and transferred to scintillation tubes with 3 mL of scintillant. Radioactivity was measured on a B-scintillation counter (LKB Wallace, Finland). Iron uptake and iron efflux experiments. The effect of chelators on 59Fe uptake from 59FeTf and 59Fe release from prelabeled cells was studied with standard procedures reported previously. I7,30 The amount of 59Fe internalized by the cells was measured by incubation with the general protease

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pronase (1 mg/mL) for 30 minutes at 4°C to remove membrane-bound 59Fe and Tf. 2931 Control experiments reported in previous studies have found that this technique is valid for estimation of 59Fe internalization by cells. 29,31 Iron regulatory protein gel-retardation assay. The gelretardation assay was used to measure the interaction between the iron regulatory proteins and iron-responsive element with established techniques.32,33 In brief, after incubation with medium alone (control) or medium containing ferric ammonium citrate (100 gg/mL; Aldrich Ltd, Sydney, Australia) or the chelators, 2-5 x 106 cells were washed with ice-cold phosphate-buffered saline solution and lysed at 4°C in 40 gL of ice-cold Munro extraction buffer (10 mmol/L N-2-hydroxyethylpiperazine-N-2-ethanesulfonicacid, pH 7.6, 3 mmol/L MgC12, 40 mmol/L KC1, 5% glycerol, 1 mmol/L dithiothreitol, and 0.5% Nonidet P-40). After lysis was performed, the samples were then centrifuged at 10,000g for 3 minutes at 4°C to remove nuclei, and the supernatant was stored at -70°C. Frozen cytoplasmic extracts were thawed on ice and then centrifuged at 15,000 rpm for 10 minutes at 4°C. The protein concentration of the soluble supernatant was determined with the BioRad protein assay (BioRad Ltd). Samples of cytoplasmic extracts were diluted to a protein concentration of 100 gg/mL in Munro buffer without Nonidet P-40, and 1 gg aliquots were analyzed for IRP by incubation with 0.1 ng (approximately 1 x 105 cpm) of 32p-labeled pGL66 RNA transcript. 32 The riboprobe was transcribed in vitro from linearized plasmid templates with SP6 RNA polymerase in the presence of c~32p UTP (Dupont, NEN). This latter reaction was performed with the Promega Riboprobe In Vitro Transcription Kit (Promega, Madison, WI). The probe was subsequently purified on a 6% urea/PAGE gel. To form RNA-protein complexes, cytoplasmic extracts containing 1 gg protein were incubated for 10 minutes at room temperature with the 32p_ labeled riboprobe. Unprotected probe was degraded by incubation with 1 U of RNAse T1 for 10 minutes at room temperature. Heparin (Sigma) at a final concentration of 5 mg/mL was then added and incubated with the extract for another 10 minutes at room temperature to exclude nonspecific binding. RNA protein complexes were analyzed in 6% nondenaturing polyacrylamide gels at 4°C as described by Konarska and Sharp. 34 Gels were dried, covered in plastic film, and exposed to Kodak XAR films at -70°C with an intensifying screen. Northern blot analysis. Northern blot analysis was performed by isolating total RNA with the Total RNA Isolation Reagent from Advanced Biotechnologies Ltd (Surrey, United Kingdom). The RNA (15 gg) was heat denatured at 90°C for 2 minutes in RNA-loading buffer and then loaded onto a 1.2% agarose-formaldehyde gel. After electrophoresis was performed, RNA was transferred to a nylon membrane (GeneScreen, New England Nuclear, Boston) in 10 x SSC with the capillary blotting method. The RNA was then cross-linked to the membrane with a UV-crosslinker (UV Stratalinker 1800, Stratagene Ltd). The membranes were hybridized with probes specific for human WAF1, GADD-45, and ~-actin. The WAF1 probe con-

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sisted of a 1-kb fragment from pSXV (ATCC; category number 79928). The GADD45 probe consisted of a 760 bp fragment from human GADD45 cDNA cloned into pHu145B2 (supplied by Dr Albert Fornace, National Cancer Institute, National Institutes of Health, Maryland). The ~-actin probe consisted of a 1.4-kb fragment from human ~-actin cDNA cloned into pBluescript SK- (ATCC; category number 37997). Hybridization of probes to the membranes and their subsequent washing were performed as described by Mahmoudi and Lin 35 with a Hybaid Shake and Stack Hybridization oven (Hybaid Ltd, Middlesex, United Kingdom). The membranes were then exposed to Kodak XAR films at -70°C with an intensifying screen. The probes were stripped from the nylon membrane by boiling in a solution containing 10 mmol/L Tris-HC1 (pH 7.0), 1 mmol/L EDTA (pH 8.0), and 1% SDS for 15 to 30 minutes, as described by the membrane manufacturer. Densitometric data were collected with a Laser Densitometer and analyzed by Kodak Biomax I Software (Kodak Ltd).

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Fig 2. The effect of DFO, 311, PIH, or the PCIH analogs on (A) 59Fe release from prelabeled SK-N-MC cells and (B) 59Fe uptake from 59Fe-transferrin (59Fe-Tf) by SK-N-MC cells. A, SK-N-MC neuroepithelioma cells were labeled with 59Fe-Tf(0.75 gmol/L) for 3 hours at 37°C, washed, and then reincubated for 3 hours at 37°C in the presence of medium alone (control) or medium containing DFO (100 gmol/L) or the other chelators (50 gmol/L). B, SK-NMC cells were incubated for 3 hours in media containing 59Fe-Tf (0.75 gmol/L) and either DFO (100 gmol/L) or the other chelators (50 gmol/L), washed, and then incubated with pronase (1 mg/mL) for 30 minutes at 4°C. Results are expressed as the mean _+SD of three replicates in a typical experiment of two experiments performed.

seven PCIH analogs to increase 59Fe release from SKN-MC cells was compared with that of"standard" chelators (DFO, PIH, and 311) whose activity has been documented previously in this cell line. 17,30 The efflux of 59Fe from S K - N - M C cells was examined after a 3-hour labeling period with 59Fe-Tf (0.75 ~tmol/L) followed by a 3-hour reincubation in the presence and absence of D F O (100 g m o l / L ) or the r e m a i n d e r o f the ligands at 50 g m o l / L (Fig 2, A). It should be noted that D F O was screened at 100 ~tmol/L in all experiments because o f its low Fe chelation efficacy in S K - N - M C cells. 17,1s Chelators 311, PIH, PCTH, PCBH, and PCBBH showed similar activity resulting in the release o f 40% to 42% of cellular 59Fe. The mobilization of 59Fe by PCIH was similar to that of DFO, which released 19% of cellular 59Fe. In contrast, FIH, PCAH, and PCHH did not appreciably increase 59Fe mobilization over that observed for the control medium (Fig 2, A). To determine the ability of chelators to inhibit 59Fe uptake from 59Fe-Tf (0.75 g m o l / L ) , S K - N - M C cells were incubated for 3 hours at 37°C with 59Fe-Tf (0.75 g m o l / L ) and either D F O (100 g m o l / L ) or the other chelators (50 g m o l / L ) (Fig 2, B). L i g a n d s 311, PIH, PCTH, PCBH, and P C B B H showed much greater efficacy than D F O at preventing 59Fe uptake from 59Fe-Tf (Fig 2, B), decreasing it to 13% to 30% of the control value, respectively, whereas D F O reduced it to 91% of the control (Fig 2, B). In terms of the other chelators, P C I H was slightly m o r e effective than DFO, whereas FIH, P C A H , and P C H H had no effect on 59Fe uptake from 59Fe-Tf. Considering these data, in both 59Fe uptake and 59Fe efflux studies, 3 of the PCIH analogs,

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Table I. T h e e f f e c t o f DFO, 311, PCTH, PCBH, o r PCBBH o n 59Fe r e l e a s e f r o m BE-2 n e u r o b i a s t o m a cells, S K - N - M C n e u r o e p i t h e l i o m a 28 m e l a n o m a

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Control DFO 311 PCTH PCBH PCBBH

9 _+ 1 44_+ 2 52 _+ 1 50_+1 44 _+ 1 48 _+3

5 _+ 1 18_+ 3 49_+ 1 51+5 47 _+ 3 47 _+2

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namely PCTH, PCBH, and PCBBH, showed activity that was greater than DFO and comparable to that of PIH and 311 (Fig 2, A and B). To further investigate the efficacy of the most effective PCIH analogs identified from the screening studies above, we compared the efficacy of PIH, PCIH, PCTH, PCBH, and P C B B H at mobilizing 59Fe from SK-N-MC cells at a range of ligand concentrations (0.5 to 50 gmol/L; Fig 3, A). The mobilization of 59Fe from S K - N - M C cells was examined after a 3-hour labeling period with 59Fe-Tf (0.75 gmol/L) followed by a 3-hour reincubation in the presence and absence of chelators (0.5 to 50 gmol/L; Fig 3, A). The 59Fe release mediated by all chelators was biphasic as a function of chelator concentration, with 59Fe release beginning to plateau at a ligand concentration of 25 gmol/L. It is apparent that at chelator concentrations up to 10 g m o l / L , the m o s t effective chelators at m o b i l i z i n g 59Fe were P C T H and P C B B H (Fig 3, A). However, as the ligand c o n c e n t r a t i o n was increased up to 25 and 50 g m o l / L , the activity of P C T H and P C B B H b e c a m e similar to that o f P I H and P C B H . P C I H was the least effective chelator at all concentrations (Fig 3, A). Further studies examined the effect of chelator concentration (0.5 to 50 gmol/L) on the uptake of 59Fe from 59Fe-Tf (0.75 ~tmol/L) during a 3-hour incubation (Fig 3, B). Similar to the results found in the efflux studies described previously, PCTH and P C B B H were the most effective chelators at preventing 59Fe uptake from 59Fe-Tf at ligand concentrations up to 10 gmol/L, whereas at concentrations from 25 to 50 gmol/L, the activity of PIH, PCIH, PCTH, and PCBH was similar

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Fig 3. The effect of chelator concentration on (A) iron release from prelabeled SK-N-MC cells and (B) 59Feuptake from 59Fe-transferrin (59Fe-Tf) by SK-N-MC cells. A, SK-N-MC neuroepithelioma cells were labeled with 59Fe-Tf(0.75 gmol/L) for 3 hours at 37°C, washed, and then reincubated for 3 hours at 37°C in the presence of the chelators (0.5 to 50 pmol/L). B, SK-N-MC cells were incubated for 3 hours in media containing59Fe-Tf(0.75 gmol/L) and the chelators (0.5 to 50 gmol/L), washed, and then incubated with pronase (1 mg/mL) for 30 minutes at 4°C. Results are expressed as the mean of three replicates in a typical experiment of two experiments performed. (Fig 3, B). Again, PCIH was the least effective chelator at all concentrations examined. To determine whether there were any differences in Fe chelation efficacy of the ligands between different cell types, the activity of the three most effective PCIH

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1"able II. The effect of DFO, 31 1, PCTH, PCBH, or PCBBH on internalized 59Fe uptake from 59Fe-transferrin by BE-2 neuroblastoma cells, SK-N-MC neuroepithelioma cells, and SK-Mel-28 melanoma cells

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analogs (PCTH, PCBH, and PCBBH) was compared with that of DFO and 311 in BE-2 neuroblastoma cells, SK-N-MC neuroepithelioma cells, and SK-Mel-28 melanoma cells (Tables I and II). When the ability of the chelators to mobilize 59Fe from cells prelabeled for 3 hours with 59Fe-Tf (0.75 gmol/L) and then reincubated for 3 hours was examined, DFO (100 gmol/L) had comparable activity to 311 and the three PCIH analogs (50 pmollL) at mobilizing 59Fe from BE-2 cells (Table I). In contrast, DFO was far less effective than either 311 or the PCIH analogs at mobilizing 59Fe from SKN-MC or SK-Mel-28 cells (Table I). The effect of the chelators at preventing 59Fe uptake from 59Fe-Tf (0.75 gmol/L) after a 3-hour incubation was also examined in the same cell lines (Table II). The most effective chelator at inhibiting 59Fe uptake from 59Fe-Tf was 311, which reduced 59Fe uptake similarly in all three cell lines to 8% to 10% of the control value. The three PCIH analogs showed less activity than 311 but were far more effective than DFO (Table II). It is interesting to note that DFO had little effect at inhibit-

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100 + 10 98 + 7 9_+1 38 _+ 7 36 + 6 54_+ 1

Cells were incubated with the chelators and 59Fe-transferrin (0.75 gmol/L) for 3 hours a t 37°C, washed, a n d i n c u b a t e d for 30 minutes at 4°C with pronase (1 mg/mL) to separate the internalized from the m e m b r a n e - b o u n d 59Fe. All chelators were screened at a concentration of 50 l~mol/L e x c e p t for DFO, which was examined at 100 gmol/L. Results are mean _+SD (three determinations) from a typical experiment,

ing 59Fe uptake in both SK-N-MC neuroepithelioma cells and SK-Mel-28 melanoma cells (100% and 98% of the control, respectively), whereas it reduced internalized 59Fe uptake from 59Fe-Tf to 42% of the control in the BE-2 cell line (Table II). In contrast, the activity of the three PCIH analogs was somewhat similar in all three cell lines, reducing 59Fe uptake from 59Fe-Tf to 15% to 38% of the control. The only exception to this was PCBBH, which was much less effective at reducing 59Fe uptake in SK-Mel-28 cells than either SK-NMC or BE-2 cells (Table II). Effect of the PCIH a n a l o g s o n c e l l u l a r p r o l i f e r a t i o n . The studies described above have clearly demonstrated that PCBH, PCBBH, and PCTH have Fe chelation efficacy that is comparable to that of PIH or 311 and greater than that found for DFO. Hence, it was important to determine the antiproliferative effects of the PCIH analogs compared with those of DFO, PIH, and 311, whose activity has been characterized previously.17J 8 From Fig 4, it is clear that all of the PCIH analogs have much less effect on proliferation than chelator 311, which has been shown in previous studies to be potent at inhibiting the growth of a wide range of neoplastic cell lines.17,18 As shown previously, the ability of DFO to inhibit growth of SK-N-MC cells was far less than that of 311 (ICs0 DFO = 47 gmol/L; ICs0 311 = 2 gmol/L). Of the PCIH analogs, PCBBH and PCBH had antiproliferative activity comparable to that of DFO (ICs0 PCBBH = 42 gmol/L; ICs0 PCBH = 50 gmol/L). The remaining PCIH analogs had little effect at inhibiting growth. It is interesting to note that although PIH, PCTH, PCBH, and PCBBH had comparable Fe chela-

J L a b Clin M e d V o l u m e 134, N u m b e r 5

Becker a n d Richardson

-I-

Table III. T h e e f f e c t o f t h e c h e l a t o r s o n 3 H - t h y m i dine, 3H-teucine, or 3H-uridine incorporation SK-N-MC neuroepithelioma

into

cells % Control

Chelator Control DFO 311 PIH PCIH PCTH PCBH PCBBH FIH PCAH PCHH

3H-Thymidine 100 29 0.1 52 43 64 72 33 17 12 11

_+ 7 _+ 13 +0,1 + 9 + 0.5 -+ 14 + 16 -+ 8 -+ 4 -+ 1 -+ 2

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100 + 7 9 _ 0.7 2+0,3 84 + 5 74 + 7 21 -+ 6 30 -+ 13 16 -+ 5 78 + 5 76 + 2 80 _+ 10

100 _+ 2 34_+ 5 5-+2 56 -+ 9 50 -+ 8 43 + 2 31 + 9 47 _+2 48 _ 10 40 _ 9 41 + 11

Cells were i n c u b a t e d for 20 hours a t 37oC with either DFO (100 gmol/L) or the other chelators (50 gmol/L). After this, either 3Hthymidine, 3H-leucine, or 3H-uridine (1 gCi/mL) were a d d e d , and the cells were i n c u b a t e d for an additional 2 hours at 37°C (see Methods for details), Results are mean + SD (four to five determinations) from a typical experiment of two to four experiments performed,

tion activity to 311 in SK-N-MC cells (Fig 21 Tables I and II), their ability to inhibit proliferation was much less. These results concur with our previous studies, which demonstrated that Fe chelation efficacy is not always well correlated to the ability of a ligand to inhibit proliferation.17,3° Effect of the chelators on 3H-thymidine, 3H-leucine, or 3H-uridine incorporation. T h e e f f e c t o f t h e c h e l a t o r s o n

3H-thymidine, 3H-leucine, or 3H-uridine incorporation into SK-N-MC cells was examined to obtain further information on the possible mechanisms of action of these ligands (Table III). In these experiments the effect of the PCIH analogs has been compared to that of DFO and 311, as the antiproliferative activity of these latter compounds has been previously characterized with SKN-MC cells.17.18 The PCIH analogs with the greatest Fe chelation efficacy, namely PCBBH, PCTH, and PCBH, reduced 3H-thymidine incorporation to 33%, 64%, and 72% of the control, respectively, which was far less than the inhibition observed with 311 (0.1% of the control) and similar to that found with PIH (52% of the control). It is interesting that three chelators that showed little Fe chelation efficacy, namely FIH, PCAH, and PCHH (Fig 2), caused a considerable decrease in 3H-thymidine incorporation to 11% to 17% of the control value (Table III). When the effect of the chelators at inhibiting 3Hleucine and 3H-uridine incorporation (Table III) was examined, again the most effective chelator was 311, which reduced their incorporation to 2% and 5% of the

Fig 5. The effect of the chelators on the RNA-binding activity of the iron-regulatory proteins (IRPs) in SK-N-MC neuroepithelioma ceils. Cells were incubated for 20 hours with either medium alone (control), ferric ammonium citrate (100 gg/mL), DFO (100 gmol/L), or the other chelators (25 gmol/L). The result illustrated is a typical experiment from two experiments performed.

control value, respectively. On the other hand, PCBH, PCBBH, and PCTH were far less active than 311 at inhibiting 3H-leucine and 3H-uridine incorporation, reducing it to 16% to 47% of the control (Table III). DFO was also less effective than 311 at inhibiting 3Hleucine and 3H-uridine incorporation, reducing it to 9% and 34% of the control, respectively. The three PCIH analogs that caused a considerable decrease in 3Hthymidine incorporation (FIH, PCAH, and PCHH) did not inhibit 3H-leucine and 3H-uridine incorporation to the same extent, reducing it to between 40% to 80% of the control value (Table III). The effect of chelators on the RNA-binding activity of the iron regulatory proteins. An important effect of intra-

cellular Fe depletion with DFO is the activation of RNA-binding activity of the iron regulatory proteins. 36 Although the effect of DFO on the RNA-binding activity of this protein has been well characterized, 36 little is known concerning the effect of other Fe chelators such as the PCIH analogs. We examined the effect of a 20-hour incubation with 311, PIH, and the PCIH analogs (25 gmol/L) on IRP-RNA binding activity in SK-N-MC cells. In all experiments DFO (100 gmol/L) was used as a positive control to deplete cells of Fe and to increase IRP-RNA binding activity. In contrast, ferric ammonium citrate (100 gg/mL) was used to donate Fe to cells and to reduce IRP-RNA binding. In Fig 5 it is obvious that only one major IRP-IRE band is present, which is due to the fact that human IRPI-IRE and IRP2-IRE complexes comigrate in nondenaturing polyacrylamide gels. 37 As expected, an increase in IRPRNA binding activity occurred after a 20-hour incubation with DFO, whereas after a 20-hour incubation with the Fe donor ferric ammonium citrate, there was an appreciable decrease in IRP-RNA binding activity compared with the control (Fig 5). Compared with the control, a marked increase occurred in IRP-RNA binding activity after treatment of cells with 311, PCTH, PCBH, and PCBBH (Fig 5), which most likely reflects their high Fe chelation efficacy (Figs 2 and 3). It was sur-

518

J Lab Clin Med November 1999

Becket and Richardson

~18S

GADD45

WAF1

~-actin Z

X~W~ ~

XZ Z

Fig 6. The effect of the chelators on m R N A levels of GADD45, WAF1, and fl-actin in SK-N-MC neuroepithelioma cells. Total RNA was extracted from cells after a 20-hour incubation with medium alone (control) or medium containing ferric ammonium citrate (100 gg/mL), DFO (100 gmol/L), or the other chelators (25 gmol/L). The isolated RNA then underwent electrophoresis on a 1.2% agarose-formaldehyde gel, was transferred to a hybridization membrane, and was probed under high stringency conditions. The result illustrated is a typical experiment from three experiments performed.

prising that PIH and PCIH had little effect on IRP-RNA binding activity, whereas FIH, PCAH, and PCHH decreased IRP-RNA binding compared with the control (Fig 5). Addition of [3-mercaptoethanol to the cell lysates demonstrated that no change occurred in the total amount of IRP-RNA binding activity after incubation with the chelators (data not shown). Effect of the PCIH analogs on the expression of genes involved in the cell cycle. Recently we have demonstrated that treatment of cells with high concentrations of

DFO (150 gmol/L) or much lower concentrations of 311 (2.5 to 5 gmol/L) resulted in an increase in the expression of WAF-1 and GADD45. 2° WAF-1 is a potent universal inhibitor of cyclin-dependent kinases 38 and can induce a G1/S arrest 39-41 and possibly a G2/M arrest. 42 GADD45 is induced on DNA damage and can arrest the cell cycle and is also involved in DNA nucleotide excision repair. 41,43 Although it is well known that DFO, 311, and other Fe chelators can cause cell cycle a r r e s t , 18,21,44,45 little is understood concerning the changes in gene expression that may play a role in inhibiting the cell cycle. In this study incubation with 311 (25 gmol/L) and to a lesser extent DFO (100 gmol/L) caused an increase in the levels of both WAF1 and GADD45 mRNA in SK-N-MC cells (Fig 6). Of the PCIH analogs, only PCBBH markedly increased the level of GADD45 mRNA but not WAF1 mRNA. This latter effect corresponds to the greater antiproliferative activity of PCBBH relative to the other PCIH analogs (Fig 4). DISCUSSION In this study we have synthesized and screened seven novel aroylhydrazone ligands based on PCIH. Three of

these chelators, namely PCBH, PCBBH, and PCTH, showed Fe chelation activity that was greater than DFO and comparable to that of PIH and 311. In addition, the antiproliferative activity of these chelators was far less than that found for analog 311, an aroylhydrazone ligand previously shown to possess high cytotoxic activity. 17,18 These properties suggest that these three PCIH analogs would be more appropriate for the management of Fe-loading diseases rather than as antiproliferative agents against cancer. In this investigation we attempted to synthesize ligands with high antiproliferative activity by condensing 2-pyridylcarboxaldehyde with a range of acid hydrazides previously used in the synthesis of the PIH analogs. I6A7 The 2-pyridylcarboxaldehyde moiety was examined because when it is condensed with thiosemicarbazide to form the relevant thiosemicarbazone, this ligand has potent antiproliferative activity.26, 27 In fact, this latter group of c~-N-heterocyclic carboxaldehyde thiosemicarbazones has been described as the most effective ribonucleotide reductase inhibitors yet identified. 26,27 Obviously, these results demonstrate that the PCIH analogs show little antiproliferative activity, being far less effective than 311. These data may indicate that in contrast to the 2-pyridylcarboxaldehyde moiety, the thiosemicarbazide component of the 2pyridylcarboxaldehyde thiosemicarbazones may be critical for antiproliferative activity. In fact, condensation of 2-hydroxy-1-naphthylaldehyde with thiosemicarbazide results in a chelator with high antiproliferative activity (Lovejoy D, Richardson DR, unpublished results). Further studies on these "hybrid" ligands derived from the PIH analogs and thiosemicarbazones are underway. All of the PCIH analogs examined in this investigation except FIH have the same potential Fe ligating sites, namely the carbonyl oxygen, aldimine nitrogen, and 2-pyridyl nitrogen46-48 (Fig 1). However, it is interesting that the biologic activity of these ligands can be influenced markedly by the nature of the substituents placed distal to the Fe-binding site. For example, both PCHH and PCAH display very poor Fe chelation activity, whereas PCTH, PCBBH, and PCBH, show very high efficacy (Fig 2, A and B). Because lipophilicity is an important criterion for the membrane permeability and Fe chelation efficacy of ligands,49, 50 it may be that the increased hydrophilicity of PCHH and PCAH (caused by the presence of a hydroxyl and amino group, respectively) could prevent the access of these chelators to intracellular Fe pools. Although PCAH, PCHH, and FItt showed little ability to mobilize 59Fe and inhibit 59Fe uptake from 59Fe-Tf, it was interesting that these chelators were more effective than the other PCIH analogs at inhibiting 3H-thymidine incorpora-

J Lab Clin M e d Volume 134, Number 5

tion (Table III). Considering this, it is possible that FIH, PCAH, and PCHH may be relatively more efficient at inhibiting ribonucleotide reductase, a crucial Fe-containing enzyme that is involved in the conversion of ribonucleotides into deoxyribonucleotides for DNA synthesis. 51 Further studies will directly examine the effect of these chelators on ribonucleotide reductase activity to determine whether this is the target affected. One advantage of the PCIH analogs compared with the PIH analogs was their higher solubility in aqueous solutions (data not shown). Although lipophilicity is an important property for membrane permeability,49, 50 the solubility of a chelator in water is also an important factor in terms of its practical and clinical use. Hence, for a chelator to be used as a useful therapeutic agent, an appropriate balance between solubility in aqueous solutions and the ability to permeate biologic membranes must be reached. For several of the PCIH analogs this appears to have been achieved. The effect of DFO at increasing the RNA-binding activity of the IRPs has been well characterized. 36 However, little is known concerning the effect of other Fe chelators. The fact that 311, PCTH, PCBH, and PCBBH all increased IRP-RNA binding activity in a similar way to DFO (Fig 5) may suggest that these ligands act on the same or a similar intracellular pool of Fe. In contrast to expectations, PIH and PCIH had no effect at increasing IRP-RNA binding activity despite the fact that these chelators were highly effective at mobilizing 59Fe from cells and preventing 59Fe uptake from 59Fe-Tf (Figs 2 and 3). Further studies are necessary to determine why no increase in IRP-RNA binding activity occurred. However, higher concentrations of PIH did increase IRP-RNA binding activity, suggesting that a concentration effect may be involved (Richardson, unpublished results). It is also important to note that FIH, PCAH, and PCHH inhibited IRP-RNA binding activity to a level comparable to that found after incubation with ferric ammonium citrate (Fig 5). Considering these results, it can be speculated that PCAH, PCHH, and FIH may disturb the intracellular distribution of Fe, such that there is an increase in the Fe pool sensed by the IRPs. Although the ability of Fe chelators to inhibit the cell cycle at G1/S is well known, 18,21,44,45 very little is understood about the changes in gene expression that may play a role in this process. In a previous study with neuroblastoma cell lines and K562 cells, 2° we demonstrated that incubation with DFO (150 jxmol/L) or 311 (2.5 to 5 gmol/L) resulted in a marked increase in the expression of WAF1 and GADD45, two molecules that play key roles in inducing cell cycle arrest at G1/S. 41 In this study we have confirmed these results with DFO

Becker a n d Richardson

519

and 311 and have shown that of the PCIH analogs, only PCBBH increased the expression of GADD45 mRNA (Fig 6). Obviously, the ability of a chelator to increase the expression of molecules involved in inhibiting the cell cycle is not an appropriate characteristic of a compound to be used for treating Fe overload. Considering this and the fact that PCBBH was the most effective ligand at inhibiting proliferation, PCTH and PCBH appear to be preferable candidate chelators for the management of Fe-loading diseases. In this work all of the PCIH analogs showed far less antiproliferative activity than 311 (Fig 4) despite the high Fe chelation efficacy of some of these compounds such as PCBBH, PCBH, and PCTH (Figs 2 and 3). Recently, we have shown that when the Fe complex of 311 is prepared, it totally prevents its antiproliferative activity 52 and also its ability to increase the expression of GADD45 and WAF1 ml~NA, ao These results, together with its high Fe chelation efficacy,17j8,20 suggest that 311 inhibits growth by depleting intracellular Fe pools. Several studies have suggested that Friedreich's ataxia may be caused by an accumulation of Fe in the mitochondrion.13-15 If this latter disease is caused by mitochondrial Fe overload, possible treatment regimens could include Fe chelation therapy. DFO effectively depletes cytosolic Fe pools, I but it is unknown whether it can chelate mitochondrial Fe. In contrast, previous studies have shown that aroylhydrazone chelators such as PIH can remove Fe from the mitochondrion.4,5,sAM 2 Considering the structural similarities between PIH and the PCIH analogs (Fig 1), further studies will examine whether these chelators are also active at mobilizing Fe from Fe-loaded mitochondria. In summary, we have identified three PCIH analogs that show high Fe chelation efficacy and low antiproliferative activity. Considering their properties, these ligands warrant further investigation as suitable chelators for the management of Fe overload disease. Obviously, additional studies in vivo are essential to determine the toxicologic profile of these chelators and their ability to increase Fe excretion when given via the oral route. REFERENCES

1. Olivieri NF, Brittenham GM. Iron-chelating therapy and the treatment of thalassemia. Blood 1997;89:739-61. 2. Richardson DR, Ponka R The development of iron chelators to treat iron overload disease and their use as experimental tools to probe intraceilular iron metabolism. Am J Hematol 1998;58:299-305. 3. Olivieri NF, Brittenham GM, McLaren CE, Templeton DM, Cameron RG, McClelland RA, et al. Long-term safety and effectiveness of iron-chelation therapy with deferiprone for thalassemia major. N Engl J Med 1998;339:417-23. 4. Ponka R Borova J, Neuwirt J, Fuchs O. Mobilization of iron

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39. E1-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, et al. WAF1, a potent mediator of p53 tumor suppression. Cell 1993;75:817-25. 40. Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ. The p21 Cdk-interacting protein Cip 1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 1993;75:805-16. 41. Levine AJ. p53, the cellular gatekeeper for growth and division. Cell 1997;88:323-31. 42. Agarwal ML, Agarwal A, Taylor WR, Stark GR. p53 controls both the G2/M and the G1 cell cycle checkpoints and mediates reversible growth arrest in human fibroblasts. Proc Nail Acad Sci USA 1995;92:8493-7. 43. Kastan MB, Zhan Q, E1-Deiry WS, Carrier F, Jacks T, Walsh WV, et al. A mammalian cell cycle check point pathway utilizing p53 and gadd45 is defective in ataxia telangiectasia. Cell 1992;71:587-97. 44. Hoyes KP, Hider RC, Porter JB. Cell cycle synchronization and growth inhibition by 3-hydroxypyridin-4-one iron chelators in leukemic cell lines. Cancer Res 1992;52:4591-9. 45. Brodie C, Siriwardana G, Lucas J, Schleicher R, Terada N, Szepesi A, et al. Neuroblastoma sensitivity to growth inhibition by deferoxamine: evidence for a block in the G1 phase of the cell cycle. Cancer Res 1993;53:3968-75. 46. Domiano P, Musatti A, Nardelli M, Pelizzi C. Crystal structure and chemical properties of bis(N-picolinylidene-N'-salicyloylhydrazinato)nickle(II). J Chem Soc Dalton Trans 1975;295-300.

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47. Domiano P, Pelizzi C, Predieri G, Vignali, C, Palla G. Aroylhydrazones as chelating agents: dl°-metal complexes of pyridine-2-carbaldehyde salicyloylhydrazone and x-ray structure of the zinc derivative. Polyhedron 1984;3:281-6. 48. Bacchi A, Carcelli M, Costa M, Pelgatti P, Pelizzi C, Pelizzi G. Versatile ligand behaviour of phenyl 2-pyridyl ketone benzoylhydrazone in palladium(II) complexes. J Chem Soc Dalton Trans 1996;4239-44. 49. Porter JB, Gyparaki M, Burke LC, Huehns ER, Sarpong P, Saez V, et al. Iron mobilization from hepatocyte monolayer cultures by chelators: the importance of membrane permeability and the iron-binding constant. Blood 1988;72:1497503. 50. Ponka P, Richardson DR, Edward JT, Chubb FL. Iron chelators of the pyridoxal isonicotinoyl hydrazone class. Relationship of the lipophilicity of the apochelator to its ability to remove iron from reticulocytes in vitro. Can J Physiol Pharmacol 1994;72:659-66. 51. Thelander L, Reichard R The reduction of ribonucleotides. Annu Rev Biochem 1979;48:133-58. 52. Richardson DR, Bernhardt PV. Crystal and molecular structure of 2-hydroxy- 1-naphthaldehyde isonicotinoyl hydrazone (NIH) and its iron(III) complex: an iron chelator with antiturnout activity. J Biol Inorg Chem 1999;4:266-73.