Identification and Characterization of Thiosemicarbazones with

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Identification and Characterization of Thiosemicarbazones with Antifungal and Antitumor Effects: Cellular Iron Chelation Mediating Cytotoxic Activity Veronika Opletalova´,† Danuta S. Kalinowski,‡ Marcela Vejsova´,§ Jirˇí Kunesˇ,† Milan Pour,† Josef Jampı´lek,| Vladimı´r Buchta,§ and Des R. Richardson*,‡ Faculty of Pharmacy in Hradec Kra´loVe´, Charles UniVersity in Prague, HeyroVske´ho 1203, CZ-500 05 Hradec Kra´loVe´, Czech Republic, Department of Pathology and Bosch Institute, Faculty of Medicine, UniVersity of Sydney, Sydney, New South Wales 2006, Australia, Department of Clinical Microbiology, Charles UniVersity Medical School and Teaching Hospital, Sokolska´ 581, CZ-500 05 Hradec Kra´loVe´, Czech Republic, and ZentiVa, a. s., U KabeloVny 130, CZ-102 37, Prague, Czech Republic ReceiVed May 18, 2008

Thiosemicarbazones derived from acetylpyrazines were prepared by condensing an acetylpyrazine or a ring-substituted acetylpyrazine with thiosemicarbazide. Using the same procedure, N,N-dimethylthiosemicarbazones were synthesized from acetylpyrazines and N,N-dimethylthiosemicarbazide. A total of 20 compounds (16 novel) were chemically characterized and then tested for antifungal effects on eight strains of fungi and also for antitumor activity against SK-N-MC neuroepithelioma cells. The most effective compound identified in terms of both antifungal and antitumor activity was N,N-dimethyl-2-(1-pyrazin2-ylethylidene)hydrazinecarbothioamide (5a). The mechanism of action of this and its related thiosemicarbazones was due, at least in part, to its ability to act as a tridentate ligand that binds metal ions. This was deduced from preparation of the related thiosemicarbazones [acetophenone thiosemicarbazone (6) and acetophenone N,N-dimethylthiosemicarbazone (7)] that do not possess a coordinating ring-N, which plays a vital role in metal ion chelation. Furthermore, 5a and several other thiosemicarbazones that showed high antiproliferative activity were demonstrated to have marked iron (Fe) chelation efficacy. In fact, these agents were highly effective at mobilizing 59Fe from prelabeled SK-N-MC cells and preventing 59 Fe uptake from the serum Fe transport protein, transferrin. In contrast, compounds 6 and 7 that do not possess a tridentate metal-binding site showed little activity. Further studies examining ascorbate oxidation demonstrated that the Fe complexes of the most effective compounds were redox-inactive. Thus, in contrast to other thiosemicarbazones with potent antiproliferative activity, Fe chelation and mobilization rather than free radical generation played a significant role in the cytotoxic effects of the current ligands. Introduction Thiosemicarbazones are well-known chelators of metal ions and many of their biological activities often have been attributed to their ability to form biologically active complexes (1). In fact, many of these agents are typical tridentate ligands, including the compound 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP,1 triapine, Figure 1A), which is undergoing clinical trials (1, 2). Both thiosemicarbazones and their metal complexes have been studied as potential antiviral, antibacterial, antimycobacterial, antiprotozoal, antifungal, and antineoplastic agents (1–6). Furthermore, their anticonvulsant and neurotropic effects also have been reported (3). The * To whom correspondence should be addressed. Tel: +61-2-9036-6548. Fax: +61-2-9036-6549. E-mail: [email protected]. † Charles University in Prague. ‡ University of Sydney. § Charles University Medical School and Teaching Hospital. | Zentiva. 1 Abbreviations: 311, 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone; 3-AP, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone; BpT, 2-benzoylpyridine thiosemicarbazone; DFO, desferrioxamine; DpT, di-2pyridylketone thiosemicarbazone; Dp44mT, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone; IBE, iron-binding equivalent; MIC, minimum inhibitory concentration; MPIH, methyl pyrazinylketone isonicotinoyl hydrazone; PBS, phosphate-buffered saline; RP-HPLC, reverse phase highperformance liquid chromatography.

antifungal properties of thiosemicarbazones can be increased upon complexation with metal ions (3, 6). The R-(N)-heterocyclic carboxaldehyde thiosemicarbazones are potent inhibitors of ribonucleotide reductase, an enzyme required for de novo synthesis of deoxyribonucleotides and DNA replication and repair (1). The best known representative of this class of compounds is 3-AP (Figure 1A), which is currently undergoing phase I and phase II clinical trials as a novel antineoplastic drug (7–9). 3-AP also has been suggested to have some potential as a neuroprotectant for the treatment of neurodegenerative diseases (10–12). However, the cytotoxic properties of 3-AP, even at relatively low concentrations, bring into question the therapeutic potential of any neuroprotectant activity. More recently, it has been shown that thiosemicarbazones including 3-AP bind Fe and then redox cycle, leading to the generation of cytotoxic free radical species that could be important for their antitumor activity (1, 13). Furthermore, development of the thiosemicarbazones of the dipyridyl thiosemicarbazone (di-2-pyridylketone thiosemicarbazone, DpT) and 2-benzoylpyridine thiosemicarbazone (BpT) classes demonstrates that some of these agents exhibit marked and selective antitumor activity in vitro and in vivo and include chelators such as di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT; Figure 1A) (14–17).

10.1021/tx800182k CCC: $40.75  2008 American Chemical Society Published on Web 08/13/2008

Cytotoxicity of Acetylpyrazine Thiosemicarbazones

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Figure 1. Chemical structures of compounds used within the current study. (A) 3-AP, 311, and Dp44mT; (B) thiosemicarbazone derivatives 4a-j; (C) N,N-dimethylthiosemicarbazone derivatives 5a-h; and (D) acetophenone derivatives 6 and 7.

In the current study, we focused on 20 biologically active derivatives of pyrazine thiosemicarbazones (compounds 4a-j; Figure 1B) and N,N-dimethylthiosemicarbazones (compounds 5a-h; Figure 1C) that were prepared in our laboratory. Pyrazine thiosemicarbazones were studied, as previous investigations have

demonstrated that various pyrazine derivatives of similar structure (e.g., pyrazine diazohydroxide) possess a variety of biological effects including antifungal and antitumor activity (18, 19). The syntheses of the compounds examined in this study are illustrated in Scheme 1.

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OpletaloVa´ et al.

Scheme 1. Synthesis of the Studied Compoundsa

R: a ) H, b ) propyl, c ) isopropyl, d ) butyl, e ) isobutyl, f ) tert-butyl, g ) pentyl, and h ) hexyl. Conditions: (i) CH3MgI, diethylether. (ii) Pyruvic acid, AgNo3, (NH4)2S2O8, diluted H2SO4. (iii) Thiosemicarbazide, MeOH, AcOH. (iv) N,N-Dimethylthiosemicarbazide, MeOH, AcOH. a

Considering the importance of Fe chelation and redox cycling in the biological activity of many thiosemicarbazones (2, 13, 15, 16, 20, 21), we prepared acetophenone thiosemicarbazone 6 and acetophenone N,N-dimethylthiosemicarbazone 7 (Figure 1D) for comparison, as these agents do not possess a coordinating ring-N that plays an important role in metal chelation (15, 16). Hence, compounds 6 or 7 could not act as typical tridentate Fe chelators and act as appropriate negative controls. These compounds were tested for various biological activities including their ability to inhibit a variety of fungal species and the neuroepithelioma cell line, SK-N-MC.

Experimental Procedures Chemicals and Reagents. Desferrioxamine was purchased from Novartis. 3-AP was a gift from Vion Pharmaceuticals Inc. (New Haven, CT). The chelators, Dp44mT and 2-hydroxy-1naphthaldehyde isonicotinoyl hydrazone (311; Figure 1A), were prepared by Schiff base condensation and characterized as described previously (16, 22). Commercially available analytical grade thiosemicarbazide (Lachema, Brno, Czech Republic), N,Ndimethylthiosemicarbazide (Acros-Organics, Geel, Belgium), and acetophenone (Reachim, Belarus, Russia) were used. Synthesis of the starting acetylpyrazines was performed using methods reported previously (Scheme 1). Pyrazine-2-carbonitriles 1a-h were converted to the corresponding ketones 2a-h by the Grignard reaction (23). 5-Acetylpyrazine-2-carbonitrile (2i) and 3-acetyl-5-tert-butylpyrazine-2-carbonitrile (2j) were prepared by homolytic acetylation of corresponding nitriles (24) (Scheme 1). Instrumentation. Analytical samples were dried over anhydrous phosphorus pentoxide under reduced pressure at room temperature. Melting points were determined with a Boe¨tius apparatus and are uncorrected. UV spectra (λ, nm) were determined on a Waters photodiode array detector 2996 (Waters Corp., Milford, MA) in ca. 9 × 10-4 M methanolic solution.

The logarithm of the molar absorption coefficient () was calculated for the λmax of individual compounds. IR spectra were recorded in KBr pellets on a Nicolet Impact 400 spectrophotometer. Characteristic wavenumbers are given in cm-1. 1H and 13 C NMR spectra were recorded at ambient temperature on a Varian Mercury-Vx BB 300 spectrometer operating at 300 MHz for 1H and 75 MHz for 13C. Chemical shifts were recorded as δ values in ppm and were indirectly referenced to tetramethylsilane (TMS) via the solvent signal (2.49 for 1H and 39.7 for 13 C in DMSO-d6 and 7.26 for 1H and 77.0 for 13C in CDCl3). Coupling constants (J) are given in Hz. Synthesis. General Procedure for Preparation of Thiosemicarbazones and N,N-Dimethylthiosemicarbazones. Arylmethylketone (0.01 mol) and thiosemicarbazide or N,Ndimethylthiosemicarbazide (0.01 mol) were dissolved in methanol (10-15 mL). Three drops of concentrated acetic acid were added, and the mixture was heated at reflux for 5 h. After it was cooled, the product was removed by filtration and crystallized from ethanol. Analyses. (E)-2-(1-Pyrazin-2-ylethylidene)hydrazinecarbothioamide (4a). Off-white, crystalline solid. Yield, 73%. Mw, 195.24. mp, 225-228 °C; dec [ref 25, 226-227 °C dec; ref 26, 225-226 °C dec]. Log K, 0.14. UV (λmax/log ): 314.1/ 3.33. IR (KBr): 3373, 3254, 3174 (NH), 1619 (CdN). 1H NMR (300 MHz, DMSO-d6): δ 10.48 (bs, 1H, NH), 9.64 (d, 1H, J ) 1.4, H-3), 8.61-8.56 (m, 2H, H-5, H-6), 8.47 (bs, 1H, NH2), 8.31 (bs, 1H, NH2), 2.35 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6) δ 179.4, 150.3, 146.4, 144.1, 143.5, 143.3, 12.1. (E)-2-[1-(5-Propylpyrazin-2-yl)ethylidene]hydrazinecarbothioamide (4b). White, crystalline solid. Yield, 72%. Mw, 237.32. mp, 202-207 °C; dec. Log K, 0.72. UV (λmax/log ): 320.0/3.28. IR (KBr): 3392, 3258, 3181 (NH); 1613 (C)N). 1 H NMR (300 MHz, DMSO-d6): δ 10.42 (bs, 1H, NH), 9.51 (d, 1H, J ) 1.4, H-3), 8.47 (d, 1H, J ) 1.4, H-6), 8.43 (bs, 1H, NH2), 8.24 (bs, 1H, NH2), 2.74 (t, 2H, J ) 7.4, CH2) 2.34 (s,


3H, CH3), 1.76-1.61 (m, 2H, CH2), 0.89 (t, 3H, J ) 7.4, CH3). 13 C NMR (75 MHz, DMSO-d6): δ 179.3, 156.4, 147.8, 146.7, 142.4, 142.3, 36.5, 22.2, 13.8, 12.2. (E)-2-[1-(5-Isopropylpyrazin-2-yl)ethylidene]hydrazinecarbothioamide (4c). Off-white, crystalline solid. Yield, 74%. Mw, 237.32. mp, 213-215 °C; dec. Log K, 0.70. UV spectrum (λmax/log ): 316.5/3.31. IR (KBr): 3403, 3266, 3170 (NH), 1606 (CdN). 1H NMR (300 MHz, DMSO-d6): δ 10.44 (bs, 1H, NH), 9.50 (d, 1H, J ) 1.37, H-3), 8.51 (d, 1H, J ) 1.37, H-6), 8.43 (bs, 1H, NH2), 8.23 (bs, 1H, NH2), 3.18-3.03 (m, 1H, CH), 2.34 (s, 3H, CH3), 1.24 (d, 6H, J ) 6.87, CH3). 13C NMR (75 MHz, DMSO-d6): δ 179.3, 161.0, 148.1, 146.7, 142.3, 141.0, 33.1, 22.2, 12.2. (E)-2-[1-(5-Butylpyrazin-2-yl)ethylidene]hydrazinecarbothioamide (4d). Off-white, crystalline solid. Yield, 69%. Mw, 251.35. mp, 201-204 °C; dec. Log K, 1.05. UV (λmax/log ): 323.6/3.24. IR (KBr): 3389, 3258, 3188 (NH), 1612 (CdN). 1 H NMR (300 MHz, DMSO-d6): δ 10.42 (bs, 1H, NH), 9.50 (d, 1H, J ) 1.4, H-3), 8.47 (d, 1H, J ) 1.4, H-6), 8.43 (bs, 1H, NH2), 8.24 (bs, 1H, NH2), 2.76 (t, 2H, J ) 7.7, CH2) 2.34 (s, 3H, CH3), 1.71-1.58 (m, 2H, CH2), 1.37-1.22 (m, 2H, CH2), 0.88 (t, 3H, J ) 7.7, CH3). 13C NMR (75 MHz, DMSO-d6): δ 179.3, 156.6, 147.8, 146.7, 142.4, 142.3, 34.1, 31.1, 21.9, 13.9, 12.1. (E)-2-[1-(5-Isobutylpyrazin-2-yl)ethylidene]hydrazinecarbothioamide (4e). White, crystalline solid. Yield, 64%. Mw, 251.35. mp, 225-230 °C; dec. Log K, 0.98. UV (λmax/log : 322.4/3.18. IR (KBr): 3392, 3258, 3143 (NH), 1613 (CdN). 1 H NMR (300 MHz, DMSO-d6): δ 10.42 (bs, 1H, NH), 9.52 (d, 1H, J ) 1.4, H-3), 8.44 (d, 1H, J ) 1.4, H-6), 8.43 (bs, 1H, NH2), 8.25 (bs, 1H, NH2), 2.64 (d, 2H, J ) 6.9, CH2) 2.34 (s, 3H, CH3), 2.14-1.95 (m, 1H, CH), 0.87 (d, 6H, J ) 6.9, CH3). 13 C NMR (75 MHz, DMSO-d6): δ 179.3, 155.8, 147.8, 146.7, 142.8, 142.5, 43.5, 28.6, 22.3, 12.1. (E)-2-[1-(5-tert-Butylpyrazin-2-yl)ethylidene]hydrazinecarbothioamide (4f). White, crystalline solid. Yield, 68%. Mw, 251.35. mp, 225-228 °C; dec. Log K, 1.02. UV (λmax/log ): 321.2/3.09. IR (KBr): 3407, 3254, 3174 (NH), 1609 (CdN). 1 H NMR (300 MHz, DMSO-d6): δ 10.45 (bs, 1H, NH), 9.52 (d, 1H, J ) 1.4, H-3), 8.66 (d, 1H, J ) 1.4, H-6), 8.42 (bs, 1H, NH2), 8.22 (bs, 1H, NH2), 2.35 (s, 3H, CH3), 1.34 (s, 9H, CH3). 13 C NMR (75 MHz, DMSO-d6): δ 179.3, 162.9, 147.6, 146.5, 141.7, 139.3, 36.4, 29.7, 12.2. (E)-2-[1-(5-Pentylpyrazin-2-yl)ethylidene]hydrazinecarbothioamide (4g). White, crystalline solid. Yield, 64%. Mw, 265.38. mp, 185-190 °C; dec. Log K, 1.37. UV (λmax/log ): 322.4/3.19. IR (KBr): 3391, 3262, 3177 (NH), 1611 (CdN). 1 H NMR (300 MHz, DMSO-d6): δ 10.42 (bs, 1H, NH), 9.50 (d, 1H, J ) 1.37, H-3), 8.47 (d, 1H, J ) 1.37, H-6), 8.43 (bs, 1H, NH2), 8.24 (bs, 1H, NH2), 2.76 (t, 2H, J ) 7.42, CH2) 2.34 (s, 3H, CH3), 1.73-1.60 (m, 2H, CH2), 1.37-1.19 (m, 4H, CH2), 0.84 (t, 3H, J ) 7.14, CH3). 13C NMR (75 MHz, DMSO-d6): δ 179.3, 156.7, 147.8, 146.7, 142.4, 142.3, 34.4, 31.0, 28.6, 22.1, 14.1, 12.1. (E)-2-[1-(5-Hexylpyrazin-2-yl)ethylidene]hydrazinecarbothioamide (4h). White, crystalline solid. Yield, 75%. Mw, 279.40. mp, 158-160 °C; dec. Log K, 1.72. UV (λmax/log ): 322.4/3.16. IR (KBr): 3390, 3258, 3181 (NH), 1612 (CdN). 1 H NMR (300 MHz, DMSO-d6): δ 10.40 (bs, 1H, NH), 9.50 (d, 1H, J ) 1.4, H-3), 8.47 (d, 1H, J ) 1.4, H-6), 8.41 (bs, 1H, NH2), 8.23 (bs, 1H, NH2), 2.76 (t, 2H, J ) 7.6, CH2) 2.34 (s, 3H, CH3), 1.71-1.56 (m, 2H, CH2), 1.38-1.17 (m, 6H, CH2), 0.83 (t, 3H, J ) 6.9, CH3). 13C NMR (75 MHz, DMSO-d6): δ


179.3, 156.6, 147.8, 146.7, 142.4, 142.3, 34.4, 31.2, 28.8, 28.4, 22.1, 14.1, 12.1. (E)-2-[1-(5-Cyanopyrazin-2-yl)ethylidene]hydrazinecarbothioamide (4i). Yellow, crystalline solid. Yield, 76%. Mw, 220.25. mp, 238-242 °C; dec. Log K, 0.31. UV (λmax/log ): 340.3/3.40. IR (KBr): 3365, 3277, 3178 (NH), 2236 (CN), 1598 (CdN). 1H NMR (300 MHz, DMSO-d6): δ 10.71 (bs, 1H, NH), 9.84 (d, 1H, J ) 1.4, H-3), 9.14 (d, 1H, J ) 1.4, H-6), 8.63 (bs, 1H, NH2), 8.47 (bs, 1H, NH2), 2.35 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6): δ 179.6, 152.7, 147.4, 144.9, 144.4, 127.9, 116.8, 11.9. (E)-2-[1-(6-tert-Butyl-3-cyanopyrazin-2-yl)ethylidene]hydrazinecarbothioamide (4j). Off-white, crystalline solid. Yield, 68%. Mw, 276.36. mp, 170-173 °C; dec. Log K, 1.30. UV (λmax/log ): 322.4/2.72. IR (KBr): 3389, 3261, 3168 (NH), 2229 (CN), 1603 (CdN). 1H NMR (300 MHz, CDCl3): δ 9.13 (bs, 1H, NH), 8.70 (s, 1H, H-5), 6.68 (bs, 1H, NH2), 2.45 (s, 3H, CH3), 1.43 (s, 9H, CH3). 13C NMR (75 MHz, CDCl3): δ 179.9, 166.2, 149.9, 143.8, 140.7, 122.8, 118.7, 37.4, 29.4, 11.0. (E)-N,N-Dimethyl-2-(1-pyrazin-2-ylethylidene)hydrazinecarbothioamide (5a). Orange, crystalline solid. Yield, 59%. Mw, 223.30. mp, 162-165 °C; dec [ref 26, 158-161 °C; dec, ref 27, 164-167 °C]. Log K, 0.23. UV (λmax/log ): 314.1/ 3.32. IR (KBr): 3135 (NH); 1609 (CdN). 1H NMR (300 MHz, DMSO-d6): δ 9.80 (bs, 1H, NH), 9.21 (d, 1H, J ) 1.4, H-3), 8.63-8.60 (m, 1H, H-5), 8.59 (d, 1H, J ) 2.5, H-6), 3.31 (s, 6H, N(CH3)2), 2.35 (s, 3H, CH3). (E)-N,N-Dimethyl-2-[1-(5-propylpyrazin-2-yl)ethylidene]hydrazinecarbothioamide (5b). Yellow, crystalline solid. Yield, 30%. Mw, 265.38. mp, 250-254 °C; dec. Log K, 1.51. UV (λmax/log ): 352.3/3.23. IR (KBr): 3170, 3127 (NH), 1609 shoulder, 1592 (CdN). 1H NMR (300 MHz, DMSO-d6): δ 11.48 (bs, 1H, NH), 9.03 (d, 1H, J ) 1.4, H-3), 8.46 (d, 1H, J ) 1.4, H-6), 2.97 (s, 6H, NCH3), 2.74 (t, 2H, J ) 7.3, CH2), 2.29 (s, 3H, CH3), 1.77-1.61 (m, 2H, CH2), 0.90 (t, 3H, J ) 7.3, CH3). (E)-2-[1-(5-Isopropylpyrazin-2-yl)ethylidene]-N,N-dimethylhydrazinecarbothioamide (5c). Yellow, crystalline solid. Yield, 49%. Mw, 265.38. mp, 257-261 °C; dec. Log K, 1.52. UV (λmax/log ): 352.3/3.43. IR (KBr): 3173, 3127 (NH), 1608 shoulder, 1590 (CdN). 1H NMR (300 MHz, DMSO-d6): δ 11.50 (bs, 1H, NH), 9.06-9.03 (m, 1H, H-3), 8.51-8.49 (m, 1H, H-6), 3.20-3.04 (m, 1H, CH), 2.96 (s, 6H, NCH3), 2.29 (s, 3H, CH3), 1.25 (d, 6H, J ) 6.9, CH3). (E)-2-[1-(5-Butylpyrazin-2-yl)ethylidene]-N,N-dimethylhydrazinecarbothioamide (5d). Yellow, crystalline solid. Yield, 32%. Mw, 279.40. mp, 220-224 °C; dec. Log K, 1.87. UV (λmax/log ): 353.5/3.26. IR (KBr): 3172, 3127 (NH), 1608 shoulder, 1591 (CdN). 1H NMR (300 MHz, DMSO-d6): δ 11.48 (bs, 1H, NH), 9.05-9.02 (m, 1H, H-3), 8.49-8.45 (m, 1H, H-6), 2.96 (s, 6H, NCH3), 2.76 (t, 2H, J ) 7.4, CH2), 2.29 (s, 3H, CH3), 1.73-1.58 (m, 2H, CH2), 1.39-1.21 (m, 2H, CH3), 0.89 (t, 3H, J ) 7.4, CH3). (E)-2-[1-(5-Isobutylpyrazin-2-yl)ethylidene]-N,N-dimethylhydrazinecarbothioamide (5e). Yellow, crystalline solid. Yield, 38%. Mw, 279.40. mp, 230-232 °C; dec. Log K, 1.79. UV (λmax/log ): 353.51/3.18. IR (KBr): 3127 (NH), 1609 shoulder, 1590 (CdN). 1H NMR (300 MHz, DMSO-d6): δ 11.51 (bs, 1H, NH), 9.04 (d, 1H, J ) 1.2, H-3), 8.44 (d, 1H, J ) 1.2, H-6), 2.96 (s, 6H, NCH3), 2.64 (d, 2H, J ) 6.9, CH2), 2.29 (s, 3H, CH3), 2.14-1.95 (m, 1H, CH), 0.88 (d, 6H, J ) 6.9, CH3).

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(E)-2-[1-(5-tert-Butylpyrazin-2-yl)ethylidene]-N,N-dimethylhydrazinecarbothioamide (5f). Yellow, crystalline solid. Yield, 45%. Mw, 279.40. mp, 259-261 °C; dec. Log K, 1.91. UV (λmax/log ): 352.31/3.03. IR (KBr): 3122 (NH), 1588 (CdN). 1H NMR spectrum (300 MHz, DMSO-d6): δ 11.51 (bs, 1H, NH), 9.05 (d, 1H, J ) 1.5, H-3), 8.65 (d, 1H, J ) 1.5, H-6), 2.96 (s, 6H, NCH3), 2.29 (s, 3H, CH3), 1.34 (s, 9H, CH3). (E)-N,N-Dimethyl-2-[1-(5-pentylpyrazin-2-yl)ethylidene]hydrazinecarbothioamide (5g). Yellow, crystalline solid. Yield, 26%. Mw, 293.43. mp, 217-220 °C; dec. Log K, 2.20. UV (λmax/log ): 353.5/2.97. IR (KBr): 3131 (NH), 1612 shoulder, 1592 (CdN). 1H NMR (300 MHz, DMSO-d6): δ 11.50 (bs, 1H, NH), 9.02 (s, 1H, H-3), 8.44 (s, 1H, H-6), 2.96 (s, 6H, NCH3), 2.74 (t, 2H, J ) 7.6, CH2), 2.28 (s, 3H, CH3), 1.76-1.57 (m, 2H, CH2), 1.39-1.17 (m, 4H, CH2), 0.84 (t, 3H, J ) 6.5, CH3). (E)-2-[1-(5-Hexylpyrazin-2-yl)ethylidene]-N,N-dimethylhydrazinecarbothioamide (5h). Yellow, crystalline solid. Yield, 35%. Mw, 307.46. mp, 206-209 °C; dec. Log K, 2.62. UV (λmax/log ): 352.3/2.94. IR (KBr): 3127 (NH), 1612 shoulder, 1592 (CdN). 1H NMR (300 MHz, DMSO-d6): δ 11.50 (bs, 1H, NH), 9.02 (d, 1H, J ) 1.5, H-3), 8.44 (d, 1H, J ) 1.5, H-6), 2.96 (s, 6H, NCH3), 2.73 (t, 2H, J ) 7.6, CH2), 2.28 (s, 3H, CH3), 1.75-1.55 (m, 2H, CH2), 1.36-1.16 (m, 6H, CH2), 0.83 (t, 3H, J ) 6.9, CH3). (E)-2-(1-Phenylethylidene)hydrazinecarbothioamide (6). Off-white, crystalline solid. Yield, 45%. Mw, 193.27. mp, 115-117 °C; dec [ref 28, 116-117 °C]. Log K, 0.57. UV (λmax/ log ): 285.5/3.37. IR (KBr): 3412, 3265, 3153 (NH), 1589 (CdN). 1H NMR (300 MHz, DMSO-d6): δ 10.22 (bs, 1H, NH), 8.28 (bs, 1H, NH2), 7.96-7.87 (m, 3H, H-2, H-6, NH2), 7.40-7.34 (m, 3H, H-3, H-4, H-5), 2.29 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6): δ 179.1, 148.1, 137.9, 129.4, 128.5, 126.8, 14.2. (E)-N,N-Dimethyl-2-(1-phenylethylidene)hydrazinecarbothioamide (7). Yellow, crystalline solid. Yield, 40%. Mw, 221.32. mp, 243-247 °C; dec. Log K, 1.54. UV (λmax/log ): 324.8/3.38. IR (KBr): 3135 (NH), 1609 shoulder, 1591 (CdN). 1 H NMR (300 MHz, DMSO-d6): δ 11.07 (bs, 1H, NH), 7.75-7.67 (m, 2H, H-2, H-6), 7.44-7.28 (3H, H-3, H-4, H-5), 2.94 (s, 6H, NCH3), 2.25 (s, 3H, CH3). Log K Determination Using Reverse Phase High-Performance Liquid Chromatography (RP-HPLC). Log K determinations of the compounds was performed using RP-HPLC. The RP-HPLC equipment consisted of a Waters Alliance 2695 XE HPLC separation module, Waters photodiode array detector 2996, and a Symmetry C18 chromatographic column (5 µm, 4.6 mm × 250 mm, Part #WAT054275; Waters Corp., Milford, MA). The HPLC separation process was monitored using Millennium32 Chromatography Manager Software, Waters, 2004 (Waters Corp.). A mixture of MeOH p.a. (50%) and Milli-Q grade water (50%) was used as a mobile phase. The total flow of the column was 0.9 mL/min, the injection was 30 µL, the column temperature was 25 °C, the sample temperature was 10 °C, and the detection wavelength was 210 nm. Log K values were calculated from RP-HPLC data. The capacity factors K were calculated using the Millennium32 Chromatography Manager Software according to the formula K ) (tR - tD)/tD, where tR is the retention time of the solute, whereas tD denotes the dead time obtained via an unretained analyte. Retention times (tR) were measured in minutes. A KI methanolic solution was used for the dead time (tD) determination.

OpletaloVa´ et al.

Biological Methods. Evaluation of In Vitro Antifungal Activity. The antifungal activity of all compounds was evaluated by the microdilution broth method (29). The organisms examined included Candida albicans ATCC 44859 (American type Culture Collection, Manassas, VA), Candida tropicalis 156, Candida krusei E 28, Candida glabrata 20/I, Trichosporon asahii 1188, Aspergillus fumigatus 231, Absidia corymbifera 272, and Trichophyton mentagrophytes 445 (these clinical isolates were obtained from the Department of Clinical Microbiology, University Hospital and Faculty of Medicine, Charles University, Prague, Czech Republic). All strains were subcultured on Sabouraud dextrose agar (SDA; Difco/Becton Dickinson, Detroit, MI) and maintained on the same medium at 4 °C. Prior to testing, each strain was passaged onto SDA, and fungal innocula were prepared by suspending yeasts, conidia, or sporangiospores in sterile 0.85% saline. The cell density was adjusted using a Bu¨rker’s chamber to yield a stock suspension of (1.0 ( 0.2) × 105 CFU/mL. The final innoculum was made by 1:20 dilution of the stock suspension with the test medium. The compounds were dissolved in DMSO, and the antifungal activity was determined in RPMI 1640 media (Sevapharma, Prague, Czech Republic) buffered to pH 7.0 with 0.165 M 3-morpholinopropane-1-sulfonic acid (Sigma-Aldrich, St. Louis, MO). Controls consisted of medium and DMSO alone. The final concentration of DMSO in the test medium did not exceed 1% (v/v) of the total solution composition. The minimum inhibitory concentration (MIC), defined as 80% inhibition of fungal growth as compared to control, was determined after 24 and 48 h of static incubation at 35 °C. In the case of T. mentagrophytes, the MICs were recorded after 72 and 120 h due to its slow growth rate. Fluconazole (Pfizer, New York, NY) and amphotericin B (Sigma-Aldrich) were used as reference antifungal drugs. All determinations were performed in triplicate. Evaluation of Antiproliferative Activity against Neoplastic Cells. The effect of the compounds on cellular proliferation was determined by the MTT [1-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium] assay using standard techniques (15, 30). The human neuroepithelioma cell line, SK-N-MC (American Type Culture Collection, Manassas, VA), was seeded in 96-well microtiter plates at 1.5 × 104 cells/well in medium containing human diferric transferrin (Tf) at 1.25 µM and compounds at a range of concentrations (0-6.25 µM). Control samples contained medium with Tf (1.25 µM) without the compounds. The compounds 3-AP, 311 (Figure 1A), desferrioxamine (DFO; Figure 2), and Dp44mT were also included as internal controls, as their effects are wellcharacterized in this cell line (15, 16). The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air for 72 h. After incubation, 10 µL of MTT (5 mg/mL) was added to each well and further incubated at 37 °C for 2 h. After solubilization of the cells with 100 µL of 10% SDS-50% isobutanol in 0.01 M HCl, the plates were read at 570 nm using a scanning multiwell spectrophotometer. The results, which were the means of three replicates, were expressed as a percentage of the control. The inhibitory concentration (IC50) was defined as the compound concentration necessary to reduce the absorbance to 50% of the untreated control. As shown previously, MTT absorbance was proportional to SK-N-MC cell number (30). Preparation of 59Fe-Tf. Tf (Sigma) was labeled with 59Fe (Dupont NEN, MA) to produce fully saturated diferric Tf (59FeTf), as previously described (31, 32).



Figure 2. Structures of the iron chelators DFO, deferiprone, CP94, and deferasirox.

Effect of Thiosemicarbazones on 59Fe Efflux from SK-NMC Cells. Iron efflux experiments examining the ability of chelators to mobilize 59Fe from SK-N-MC cells were performed using established techniques (30, 33). The cells were initially prelabeled with 59Fe-Tf (0.75 µM) for 3 h at 37 °C and then washed four times with ice-cold phosphate-buffered saline (PBS). The cells were then incubated with either control medium or medium containing the chelator (50 µM) for 3 h at 37 °C. Subsequently, the overlying supernatant containing released 59Fe was then separated from the cells using a pasteur pipet and placed in γ-counting tubes. The cells were removed from the plate in 1 mL of PBS using a plastic spatula and added to separate γ-counting tubes. Radioactivity was measured in both the cell pellet and the supernatant using a γ-scintillation counter (Wallac Wizard 3, Turku, Finland). In these studies, the novel ligands were compared to the well-characterized chelators, DFO and Dp44mT (positive controls) (13, 17). Effect of Thiosemicarbazones at Preventing 59Fe Uptake from 59Fe-Tf. The ability of the chelator to prevent cellular 59 Fe uptake from the serum Fe transport protein 59Fe-Tf was examined using established techniques (13, 30). Cells were incubated with 59Fe-Tf (0.75 µM) for 3 h at 37 °C in the presence of each of the chelators (50 µM). The cells were then washed four times with ice-cold PBS, and internalized 59Fe was determined by standard techniques by incubating the cell monolayer for 30 min at 4 °C with the general protease, Pronase (1 mg/mL; Sigma) (31, 32). The cells were removed from the monolayer using a plastic spatula and centrifuged for 1 min at 14000 rpm. The supernatant represented membrane-bound Pronase-sensitive 59Fe that was released by the protease, while the internalized 59Fe was the Pronase-insensitive fraction (31, 32). The novel ligands were compared to the previously characterized chelators, DFO and Dp44mT (13, 17).

Ability of the Chelators to Directly Remove 59Fe from Tf. To examine the efficacy of the chelators at directly removing 59 Fe from the Fe-binding sites of 59Fe-Tf, dialysis experiments were performed using an established procedure (33). Labeled 59 Fe-Tf (0.75 µM) was incubated with each chelator (50 µM) for 3 h at 37 °C to mimic the incubation in the uptake experiments previously described. The solution was then placed in a dialysis sac, and the contents dialysed with constant mixing for 24 h at 4 °C. The dialysate and sac were separated, and the radioactivity was assessed in each using the γ-counter described above. After dialysis, the 59Fe in the dialysate represented 59Fe that has been removed from 59Fe-Tf by the chelator, while 59Fe in the sack was Tf-bound 59Fe. Ascorbate Oxidation Assay. An established protocol was used to measure ascorbate oxidation (13). Ascorbic acid (0.1 mM) was prepared immediately prior to an experiment and incubated in the presence of Fe(III) (10 µM; added as FeCl3), a 50-fold molar excess of citrate (500 µM), and the chelator (1-60 µM). Absorbance at 265 nm was measured after 10 and 40 min at room temperature, and the decrease in intensity between these time points was calculated. The results were expressed as iron-binding equivalents (IBE). This was done due to the different coordination modes of the ligands to Fe, that is, EDTA and DFO were hexadentate and formed 1:1 ligand:Fe complexes, while Dp44mT, 4f,h, and 5a were tridentates resulting in 2:1 complexes. A range of ligand:Fe IBE ratios were used, namely, 0.1, 1, or 3. An IBE of 0.1 represented an excess of Fe to chelator, that is, one hexadentate chelator or two tridentate chelators in the presence of 10 Fe atoms. An IBE of one was equivalent to the complete filling of the coordination sphere, that is, Fe:EDTA 1:1 or Fe:Dp44mT 1:2. An IBE of three represented an excess of chelator to Fe and was equal to

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either three hexadentate or six tridentate ligands in the presence of one Fe atom (15, 16). Statistical Analysis. Results were presented as means ( SD. Data were compared using the Student’s t test. Results were considered statistically significant when p > 0.05.

Results and Discussion Synthesis and Chemical Characterization of the Thiosemicarbazones. The 20 title compounds were prepared using the procedure reported by Easmon and co-workers (26). Compounds 4a, 5a, 6, and 7 have been prepared previously, while the remaining 16 thiosemicarbazones are novel. Analytically pure products were obtained by recrystallization from absolute ethanol. On the basis of 1H and 13C NMR spectra, the compounds were greater than 98% pure, which was also confirmed by HPLC. Analysis of IR spectra showed that the compounds reported in the present paper exist in the thioxo form in the solid state (KBr pellets) because the S-H absorption bands at 2600-2550 cm-1 are missing in their IR spectra (34). Discrimination between E- and Z-isomers can be readily achieved on the basis of the chemical shifts of the hydrazono N-H proton. In thiosemicarbazones derived from R-(N)-heterocyclic carbaldehydes and ketones, the dN-NH- proton of the Z-isomer is involved in a stable, intramolecular hydrogen bond with the heterocyclic nitrogen, which results in a marked downfield shift to δ 14-15 ppm, while the E-isomer gives the corresponding signal at about δ 10 ppm (35, 36). In 1H NMR spectra of 4a-j and 6, as well as that of 5a-h and 7a, only one set of signals was observed with the chemical shifts of the hydrazono N-H proton in the range of 9.13-10.71. Hence, it can be concluded that the compounds were obtained as single E-isomers. Econfiguration of the thiosemicarbazones was further supported by 13C NMR spectra. These studies demonstrated that the chemical shift for the methyl group was δ 11.0-12.1 ppm for thiosemicarbazones derived from acetylpyrazines (4a-j) and δ 14.2 ppm for acetophenone-thiosemicarbazone 6. In contrast, the reported data for Z-izomers are ∼21 ppm (36). 13C NMR spectra of N,N-dimethylthiosemicarbazones 5a-h and 7 could not be interpreted because the solubility of these compound in DMSO was rather low. In fact, they separated from solution during the relatively long time required for acquiring 13C NMR spectra. These compounds were then tested for their antifungal and antiproliferative activities. Antifungal Activity In Vitro. Some thiosemicarbazones and their metal complexes show antifungal activity (37, 38). However, a systematic study of the antifungal effects of acetylpyrazine-thiosemicarbazones has not been reported. Therefore, we tested all compounds against C. albicans ATCC 44859, C. tropicalis 156, C. krusei E 28, C. glabrata 20/I, T. asahii 1188, A. fumigatus 231, A. corymbifera 272, and T. mentagrophytes 445 by the microdilution broth method (Table 1). Most thiosemicarbazones 4a-h derived from acetylpyrazine or its 5-alkylated congeners exhibited weak (MIC > 50 µM) to moderate (MIC > 10-50 µM) potency against Candida spp., T. asahii, and T. mentagrophytes (Table 1). Overall, the most potent compounds were 4g,h and particularly 5a. The efficacy of 5a was similar to the clinically used agent fluconazole when assessed against C. albicans ATCC 44859, C. tropicalis 156, and T. asahii 1188. Importantly, 5a showed much greater activity than fluconazole against C. krusei E 28, C. glabrata 20/I, A. fumigatus, A. corymbifera 272, and T. mentagrophytes 445.

OpletaloVa´ et al.

Considering the structure-activity relationships of these agents, it is apparent that slight changes in structure resulted in marked alterations in biological activity (Table 1). Generally, for the thiosemicarbazone series 4a-j, it appeared that high lipophilicity and intracellular access were criterion for activity. In this group of compounds (Figure 1B), the two most lipophilic molecules, namely, 4g (log K ) 1.37) and 4h (log K ) 1.72), possessed the greatest antifungal activity. Both 4g and 4h have a long alkyl chain in position 5 of the pyrazine ring (Figure 1B). Hence, the lipophilicity of these agents may be suitable for penetrating the fungal cell wall. Hydrophobicity is an important criterion for intracellular access and cytotoxic activity of thiosemicarbazones and other Fe chelators (30, 39). It is also possible that due to their lipophilicity, these agents may exhibit surface-active properties, causing unfavorable changes in fungal membranes that induce cytotoxicity (40, 41). Clearly, hydrophobicity was not the only important factor that was vital for activity. In fact, the ability of thiosemicarbazones to act as metal chelators is well-known and plays a critical role in their biological activity (1). Considering this, it is important to note that the acetophenone derivatives 6 and 7 were practically ineffective (Table 1). This is probably because these compounds lack the coordinating pyrazinyl nitrogen that is vital for tridentate binding of metal ions (16). Furthermore, the ability of thiosemicarbazones to bind metals leads to complexes, some of which have been shown to redox cycle to generate cytotoxic free radicals that mediate, in part, the antiproliferative activity of these compounds (13, 16, 17). The hypothesis that other factors apart from lipophilicity play an important part of the activity of these thiosemicarbazones is also demonstrated by examining the properties of 4j. The hydrophobicity of this latter molecule (log K ) 1.30) approaches that of the highly active compound 4g; yet, its antifungal efficacy is very low (Table 1). The potency of 4j, which showed appreciable lipophilicity, appeared to be negatively influenced by the substitution with a tert-butyl group. Our previous results with chalcones (42, 43) indicated that derivatives with nonbranched alkyl substituents on the pyrazine ring usually exhibit better antifungal properties than their analogues substituted with branched alkyls. Furthermore, the electron-withdrawing cyano group on the pyrazine ring may also increase the electrochemical potential of the Fe complex, reducing its ability to redox cycle and generate cytotoxic complexes. A similar effect of an electron-withdrawing nitro group in thiosemicarbazones has been demonstrated in previous studies (15). The low antifungal activity of 4i is probably due to its low lipophilicity (log K ) 0.31), which is imparted by the cyano group, probably leading to its inability to penetrate into fungal cells. The structure-activity relationships of the thiosemicarbazone derivatives (Figure 1B) appeared to be quite different to the N,N-dimethylthiosemicarbazone analogues (Figure 1C) that generally (apart from compound 5a) were more lipophilic than their thiosemicarbazone homologues. As a consequence of their greater lipophilicity, for many N,N-dimethylthiosemicarbazones, the precise MIC’s could not been determined due to their poor solubility and precipitation in cultivation medium. Of interest, compound 5a had high activity at inhibiting the growth of all fungal strains despite its relatively low lipophilicity (log K ) 0.23). In fact, 5a was the most hydrophilic member of the N,Ndimethylthiosemicarbazones series, the remainder of which had hydrophobicity that ranged between log K 1.51 and 2.62. Notably, the lipophilicity of these latter analogues was greater than the thiosemicarbazones 4g,h that showed pronounced efficacy. This suggested that the higher lipophilicity of most of



Table 1. Antifungal Activity of the Thiosemicarbazones in Comparison to Fluconazole and Amphotericin B after an Incubation of 24 and 48 ha MIC/IC80 (µmol L-1)

compound 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 5a 5b 5c 5d 5e 5f 5g 5h 6 7 fluconazole amphotericin B

C. albicansATCC 44859 24 h 48 h

C. tropicalis 156 24 h 48 h

C. krusei E 28 24 h 48 h

C. glabrata 20/I 24 h 48 h

T. asahii 1188 24 h 48 h

A. fumigatus 231 24 h 48 h

A. corymbifera 272 24 h 48 h

T. mentagrophytes 445 72 h 120 h

36 ( 19 36 ( 19 21 ( 7 21 ( 7 13 ( 4 16 ( 1 10 ( 4 10 ( 4 13 ( 4 18 ( 10 26 ( 26 26 ( 26 4(1 7(2 2(1 3(1 >50 >50 >50 >50 0.98 ( 0.01 1.30 ( 0.46 39 ( 23 >50 >50 >50 >50 >50 13 ( 4 >50 8(1 16 ( 1 >50 >50 >50 >50 >50 >50 >50 >50 1(1 2(1 0.03 ( 0.01 0.06 ( 0.02

>50 >50 >50 >50 23 ( 11 26 ( 7 36 ( 19 42 ( 15 42 ( 5 42 ( 5 42 ( 15 42 ( 15 12 ( 6 12 ( 6 8(6 12 ( 6 >50 >50 >50 >50 2.93 ( 0.98 2.93 ( 1.38 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 3(1 5(2 0.09 ( 0.03 0.11 ( 0.03

>50 >50 >50 >50 31 ( 1 42 ( 15 26 ( 7 31 ( 1 31 ( 1 31 ( 1 26 ( 7 31 ( 1 13 ( 4 16 ( 1 5(2 8(1 >50 >50 >50 >50 0.98 ( 0.01 1.63 ( 0.46 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 0.14 ( 0.01 0.18 ( 0.06

26 ( 7 >50 16 ( 1 21 ( 7 8(1 16 ( 1 16 ( 11 16 ( 11 13 ( 4 12 ( 4 10 ( 4 29 ( 24 5(2 7(2 2(1 4(1 >50 >50 >50 >50 0.98 ( 0.01 1.95 ( 0.01 >50 >50 44 ( 26 >50 >50 >50 >50 >50 12 ( 4 >50 >50 >50 >50 >50 >50 >50 >50 >50 22 ( 6 >50 0.03 ( 0.01 0.09 ( 0.03

>50 >50 31 ( 1 31 ( 1 31 ( 1 31 ( 1 21 ( 7 21 ( 7 21 ( 7 31 ( 1 21 ( 7 31 ( 1 8(1 13 ( 4 4(1 6(2 >50 >50 >50 >50 1.95 ( 0.01 1.95 ( 0.01 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 4(2 9(3 1.08 ( 0.01 1.80 ( 0.51

>50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 26 ( 7 >50 22 ( 13 23 ( 11 >50 >50 >50 >50 5.86 ( 1.96 5.86 ( 2.76 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 0.18 ( 0.06 0.23 ( 0.06

>50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 26 ( 7 36 ( 20 12 ( 6 13 ( 4 >50 >50 >50 >50 11.72 ( 3.91 8.46 ( 5.60 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 1.08 ( 0.01 2.16 ( 0.01

>50 >50 36 ( 19 36 ( 19 31 ( 1 31 ( 1 26 ( 7 26 ( 7 21 ( 7 36 ( 19 16 ( 1 36 ( 19 13 ( 4 13 ( 4 4(1 6(2 >50 >50 >50 >50 2.28 ( 1.21 2.28 ( 1.21 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 17 ( 6 26 ( 1 1.08 ( 0.01 1.08 ( 0.01

a For T. mentagrophytes 445, incubation periods of 72 and 120 h were necessary due to its slow growth rate. Results are means ( SD (three experiments). Activity >50 µM was considered very low as compared to the clinically used agents, fluconazole and amphotericin B.

the N,N-dimethylthiosemicarbazones did not lead to enhanced antifungal activity. While high lipophilicity can lead to increased intracellular access, it can also result in membrane retention of some compounds and thus lower biological activity (44). Thus, the concept of “optimal lipophilicity” appears relevant and very different for both the thiosemicarbazones (4a-j; Figure 1B) and the N,N-dimethylthiosemicarbazones (5a-h; Figure 1C), where maximal antifungal activity was observed at log K of 1.37-1.72 and 0.23, respectively. Another significant finding was that 4h and 5a showed promising activity against A. corymbifera (Table 1). This filamentous fungus is a relatively rare cause of the opportunistic infection, zygomycosis (also known as mucormycosis) (45). Itraconazole and posaconazole are the marketed triazole drugs with in vitro activity against mucorales molds. However, only posaconazole is an efficient antifungal against mucormycosis (45–47). In this study, the MICs against A. corymbifera after a 24 h incubation were 1.08 ( 0.01 µM for amphotericin B, 12 ( 5 µM for 4h, and 12 ( 4 µM for 5a. Using a similar microdilution broth method as performed herein, the reported MICs (48) of amphotericin B, itraconazole, and posaconazole after 24 h were 1.08, 7.09, and 7.17 µM, respectively. Hence,

it can be concluded that 4h and 5a do not reach the effectiveness of amphotericin B, but they inhibit the growth of A. corymbifera at concentrations only slightly higher than itraconazole and posaconazole. However, the mechanisms underlying the antifungal effects of the thiosemicarbazones are probably different from these commercial antifungal agents and could be related, in part, to their ability to act as effective metal chelators. Considering this, it is notable that derivatives 4i,j, and 5d,g,h have the expected good metal-chelating capability but are as poorly active as compounds 6 and 7 that are not ligands. This indicates that other factors, such as lipophilicity, etc., are also vital for activity. In view of the potential of chelators as antifungal agents, patients treated with the Fe chelator DFO (Figure 2) have a markedly increased incidence of invasive mucormycosis (49). In fact, Rhizopus spp. actually utilize DFO as a siderophore to supply previously unavailable Fe to the fungus, and this ligand worsens survival of animals infected with these microbes (50). In contrast, synthetic chelators such as deferiprone and compound 94 (CP94; Figure 2), which cannot be used as siderophores, do not exacerbate mucormycosis infection (50). Chelators such as CP94 (50) and the orally effective Fe chelator,

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Table 2. Antiproliferative Activity of the Compounds Using the SK-N-MC Neuroepithelioma Cell Line after a 72 h Incubation at 37 °C with the Compoundsa compound 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 5a 5b 5c 5d 5e 5f 5g 5h 6 7 3-AP DFO 311 Dp44mT a

IC50 (µM) >6.25 2.08 ( 1.40 2.90 ( 0.93 0.26 ( 0.11 0.59 ( 0.13 0.18 ( 0.01 0.52 ( 0.22 0.18 ( 0.09 >6.25 >6.25 0.01 ( 0.01 4.51 ( 0.47 4.78 ( 0.14 3.54 ( 0.48 4.23 ( 0.59 2.75 ( 0.21 2.35 ( 0.11 1.13 ( 0.04 >6.25 >6.25 0.36 ( 0.05 9.67 ( 1.50 0.68 ( 0.03 0.008( 0.001

Results are means ( SD (three experiments).

deferasirox (Figure 2) (51), have been investigated as antifungal agents. Therefore, the potential of the more effective thiosemicarbazones synthesized in this study deserves to be carefully assessed in vivo in animal models. Antiproliferative Activity Against Tumor Cells in Culture. The antiproliferative activity of the thiosemicarbazones was also assessed against neoplastic cells using the human SK-N-MC neuroepithelioma cell line. This cell type was chosen as its response to chelators with antiproliferative activity is wellcharacterized (14, 17, 30, 52). The antitumor efficacy of our newly synthesized compounds was compared to well-characterized chelators that act as positive controls, including 3-AP, 311, Dp44mT, and DFO (Figures 1A and 2). Both 3-AP and 311 displayed moderate antitumor efficacy (IC50, 0.36 and 0.68 µM, respectively; Table 2), as shown previously (13, 22). In contrast, the highly hydrophilic chelator, DFO, showed poor antiproliferative activity (IC50, 9.67 µM), as demonstrated before (13, 30). On the other hand, Dp44mT demonstrated potent antiproliferative effects (IC50, 0.008 µM), as found in previous studies (14, 16). Compound 5a showed the most promising anticancer activity (IC50, 0.01 µM) of all of the thiosemicarbazones assessed, with antiproliferative effects comparable to Dp44mT (IC50, 0.008 µM; Table 2). The activity of 5a is surprising considering that its closely related analogue, 4a, showed very little efficacy (IC50 > 6.25 µM; Table 2). It is significant that 5a also showed the greatest antifungal activity, suggesting that it has favorable chemical properties to inhibit proliferation in very different cell types. The potent antitumor activity of 5a warrants that this compound be further investigated in vivo as an antineoplastic agent. The majority of the remaining compounds showed moderate antitumor effects, with IC50 values ranging between 0.18 and 4.51 µM (Table 2). In general, as shown for antifungal activity (Table 1), the 4-unsubstituted thiosemicarbazones (Figure 1B) were more effective than their 4,4-dimethyl thiosemicarbazone counterparts (Figure 1C) at inhibiting SK-N-MC cell proliferation. Deviating from this rule, the poor activity of 4a in

Figure 3. Tridentate thiosemicarbazones 4f,h and 5a are effective Fe chelators increasing: (A) 59Fe efflux and decreasing (B) 59Fe uptake from 59Fe-Tf by SK-N-MC neuroepithelioma cells. In contrast, 6 and 7 showed very little antitumor activity and are not tridentate and do not markedly increase 59Fe efflux or inhibit 59Fe uptake from 59Fe-Tf. (A) SK-N-MC cells were prelabeled with 59Fe-Tf (0.75 µM) for 3 h at 37 °C, washed, and then reincubated for 3 h at 37 °C in the presence of the chelators (50 µM). (B) SK-N-MC cells were incubated with 59FeTf (0.75 µM) for 3 h at 37 °C in the presence of the chelators (50 µM) (see the Experimental Procedures for details). Results are means ( SD of three determinations in a typical experiment of two performed.

comparison to 5a is notable. This was despite their similar structure and indicates the importance, at least in part, of an optimal lipophilic-hydrophilic balance. Furthermore, the hydrophilic compounds or those possessing strong electronwithdrawing groups, namely, 4a,i,j, demonstrated no appreciable activity in the current study (IC50 > 6.25 µM; Table 2). The same compounds also showed little antifungal activity (Table 1). This suggested that lipophilicity and membrane permeability are crucial for the activity of these compounds. As previously discussed for antifungal activity, lipophilicity is not the only factor that is vital to the efficacy of these compounds. Indeed, the acetophenone thiosemicarbazones, 6 and 7, which lack the Fe-binding site, also displayed no appreciable antitumor effects (IC50 > 6.25 µM; Table 2) as well as no appreciable antifungal activity (Table 1). This suggested that the metal-binding activity of the acetylpyrazine analogues plays an integral role in their antiproliferative activity both in fungi and in human tumor cells. This is confirmed by the fact that a number of metal complexes of thiosemicarbazones possess antifungal effects (53, 54), and the ability of the compounds to diffuse into cells to form complexes may be crucial for their efficacy. Mechanism of Antiproliferative Activity in Tumor Cells. Chelation Efficacys59Fe Efflux and Uptake Studies. To understand the mechanism of antiproliferative activity of the


Figure 4. Chelators at IBE ratios of 0.1, 1, and 3 were incubated in the presence of FeIII (10 µM) and ascorbate (100 µM). The UV absorbance at 265 nm was recorded after 10 and 40 min, and the difference between the time points was calculated. The effects of 4f,h and 5a were compared to the positive controls DFO, EDTA, and Dp44mT. Results are the means ( SD (three experiments).

three most effective compounds identified against cancer cells, namely, 4f,h and 5a (Table 2), we examined the Fe chelation efficacy of these agents by assessing their ability to induce 59Fe release from prelabeled SK-N-MC tumor cells (Figure 3A) and prevent 59Fe uptake from the physiological Fe transport protein, 59 Fe-Tf (Figure 3B). These effective compounds were compared to 6 and 7, which act as negative controls as they do not have a Fe-binding site. These studies were done using standard procedures in our laboratory (16, 17, 30) and provide data on the ability of these agents to permeate cell membranes and chelate intracellular Fe pools. In 59Fe efflux studies, we examined the ability of 4f,h and 5a to mobilize 59Fe from prelabeled SK-N-MC cells in comparison to 6 and 7 that do not possess a tridentate Fe-binding site. As relative internal controls, we assessed the efficacy of the well-characterized chelators, DFO and Dp44mT, at mobilizing cellular 59Fe (15, 17, 55). As shown in previous studies, cells incubated with control medium released very little 59Fe (4 ( 1% of total cell 59Fe), while DFO showed limited activity resulting in the mobilization of 14 ( 1% of total cell 59Fe (17) (Figure 3A). In contrast to DFO, Dp44mT was far more effective and markedly increased total cell 59Fe release to 42 ( 2%, as shown previously (15, 17, 55). Compounds 4f,h and 5a increased 59Fe efflux to 40 ( 1, 33 ( 1, and 43 ( 2%, respectively, demonstrating their high efficacy as Fe chelators (Figure 3A). Compounds 6 and 7 that do not possess a complete tridentate binding site were only slightly more effective than control medium, leading to the release of 7 ( 1% of total cell 59 Fe. The ability of the chelator to prevent 59Fe uptake from 59FeTf was then assessed (Figure 3B). As shown in previous studies (15, 30), DFO showed little ability to reduce 59Fe uptake decreasing it to 73 ( 5% of the control, while Dp44mT was


highly effective reducing 59Fe uptake to 6 ( 1% of the control. Compounds 4f,h and 5a showed efficacy similar to Dp44mT reducing 59Fe uptake to 8-9% of the control. In contrast, 6 and 7 were poorly effective, having activity that was similar to DFO and significantly (p < 0.001) less than 4f,h and 5a (Figure 3B). Collectively, these results demonstrate that Fe chelation could be involved in the cytotoxic activity observed with these compounds. To assess if the ability of 4f,h and 5a at reducing the 59Fe uptake from 59Fe-Tf was due to the chelators directly removing 59 Fe from 59Fe-Tf, dialysis studies were performed. Control media incubated with 59Fe-Tf led to the release of 3 ( 2% (three determinations) of Tf-bound 59Fe into the dialysate. This may be due to the presence of low Mr labilizing agents within the medium that aid in Fe release from the protein. Incubation of 4f,h and 5a (50 µM) with 59Fe-Tf in media resulted in the release of 4 ( 4, 1 ( 1, and 2 ( 1% (3 determinations) of 59Fe into the dialysate, which was not significantly (p > 0.05) different from that of control media. Hence, these studies suggested that as found for other chelators of similar structure (33, 56), 4f,h and 5a could not directly remove 59Fe from the Fe-binding sites of Tf. Hence, to inhibit 59Fe uptake from 59FeTf, these chelators must act at a distal site, for example, where 59 Fe is released from 59Fe-Tf within the cell (e.g., in Tfcontaining endosomes) (57). Oxidation of Ascorbate. Considering the ability of 4f,h and 5a to bind cellular Fe pools (Figure 3A,B) and because some thiosemicarbazone Fe complexes have been shown to be redox active (13, 15, 16), further studies were initiated to determine the ability of the compound to oxidize the physiological substrate, ascorbate. Assessment of this reaction has provided a useful indication of the redox activity of a variety of chelators in previous investigations (13, 15, 16). In addition, DFO that does not induce ascorbate oxidation, as well as EDTA and Dp44mT that markedly increase this parameter, were also examined as relevant controls (16). As shown previously (13), the internal control, DFO, prevented ascorbate oxidation, decreasing it to 37 and 23% at an IBE of 1 and 3, respectively (Figure 4). The positive controls, EDTA and Dp44mT markedly increased ascorbate oxidation, as expected (16), as the IBE increased (Figure 4). Compounds 4f,h and 5a all showed protective effects, decreasing ascorbate oxidation to 14-78% of the control at an IBE of 3 (Figure 4). Unlike the thiosemicarbazones of the DpT and BpT series that markedly stimulate ascorbate oxidation (2, 15, 16), the current results suggest that redox activity is not a mechanism by which 4f,h and 5a mediate their cytotoxic effects. Considered with the Fe chelation data, it can be suggested that the cytotoxic effects of the most effective compounds in this study (i.e., 4f,h and 5a) are due, at least in part, to their ability to effectively chelate and mobilize intracellular Fe that is vital for proliferation. It is of interest to note that structurally similar ligands of the methyl pyrazinylketone isonicotinoyl hydrazone (MPIH) series

Figure 5. Structure of the most effective ligand assessed in this study, namely, 5a, in comparison to MPIH.

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(Figure 5) also demonstrated protective effects against ascorbate oxidation (58). This was attributed to the electron-withdrawing effects of the noncoordinating pyrazinyl nitrogen atom, resulting in higher redox potentials and irreversible electrochemistry (58). This effect could explain the lack of redox activity evident in the most active compounds of the current investigation. This is clearly in marked contrast to thiosemicarbazones derived from the pyridyl group, where there are potent redox effects that contribute to their antiproliferative activity (2, 15–17). This suggests that the pyrazinyl moiety of the currently studied thiosemicarbazones could result in redox-inactive Fe complexes that do not induce ascorbate oxidation. As such, the pyrazinyl group is less desirable as a structural component as compared to the pyridyl moiety that results in marked redox activity and antiproliferative efficacy (2, 14, 16, 17, 58).

Conclusions The current investigation has identified several thiosemicarbazones that demonstrate marked antifungal and antitumor activity. From the 20 derivatives assessed, 5a was the most active and showed potent activity at mobilizing 59Fe from prelabeled neoplastic cells and inhibiting 59Fe uptake from 59FeTf. The role of Fe chelation in the mechanism of action of these compounds was demonstrated by the synthesis of 6 and 7 that cannot bind Fe as tridentate ligands and showed very little biological activity. Importantly, the inability of the Fe complexes of 4f,h and 5a to mediate the oxidation of ascorbate suggests that chelation and mobilization of intracellular Fe play a significant role in their cytotoxic effects. The presence of the pyrazinyl ring plays a likely role in preventing redox activity, which limits the antiproliferative activity of these analogues as seen with the MPIH series of chelators (58). Additional studies substituting the pyrazinyl group with the pyridyl moiety could lead to thiosemicarbazones with even greater antitumor and antifungal activities. Acknowledgment. The study was supported by the Czech Ministry of Education, Youth and Sports (Research Project MSM0021620822), project grants, and a Fellowship from the National Health and Medical Research Council of Australia and a Discovery Grant from the Australian Research Council. We thank Louise Eckman for performing dialysis experiments.

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Identification and Characterization of Thiosemicarbazones with

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