Bioorganic & Medicinal Chemistry Letters 23 (2013) 967–974

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Bioorganic & Medicinal Chemistry Letters

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Synthesis and biological evaluation of substituted 2-benzoylpyridinethiosemicarbazones: Novel structure–activity relationships under-pinning their anti-proliferative and chelation efficacy

0960-894X/$ - see front matter � 2012 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.bmcl.2012.12.044

Abbreviations: 3-AP, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone;BpT, 2-benzoylpyridine thiosemicarbazone; DFO, desferrioxamine; 3,4-DiOMe-6-MeBpT, 2-(30 ,40-dimethoxybenzoyl)-6-methylpyridine thiosemicarbazone; 3,4-DiOMe-6-Bp44mT, 2-(30 ,40-dimethoxybenzoyl)-6-methylpyridine 4,4-dimethyl-3-thiosemicarbazone; EDTA, ethylenediaminetetraacetic acid; EtOH, ethanol;IBE, iron-binding equivalent; 6-MeBpT, 2-(6-methylbenzoyl)pyridine thiosem-icarbazone; 6-MeBp4aT, 2-(6-methylbenzoyl)pyridine 4-allyl-3-thiosemicarba-zone; 6-MeBp4eT, 2-(6-methylbenzoyl)pyridine 4-ethyl-3-thiosemicarbazone;6-MeBp44mT, 2-(6-methylbenzoyl)pyridine 4,4-dimethyl-3-thiosemicarbazone;6-MeBp4mT, 2-(6-methylbenzoyl)pyridine 4-methyl-3-thiosemicarbazone; 6-MeBp4pT, 2-(6-methylbenzoyl)pyridine 4-phenyl-3-thiosemicarbazone; 4-OMe-6-MeBpT, 2-(40-methoxybenzoyl)-6-methylpyridine thiosemicarbazone;4-OMe-6-MeBp4aT, 2-(40-methoxybenzoyl)-6-methylpyridine 4-allyl-3-thio-semicarbazone; 4-OMe-6-MeBp4eT, 2-(40-methoxybenzoyl)-6-methylpyridine 4-ethyl-3-thiosemicarbazone; 4-OMe-6-MeBp44mT, 2-(40-methoxybenzoyl)-6-meth-ylpyridine 4,4-dimethyl-3-thiosemicarbazone; 4-OMe-6-MeBp4mT, 2-(40-methoxy-benzoyl)-6-methylpyridine 4-methyl-3-thiosemicarbazone; 4-OMe-6-MeBp4pT,2-(40-methoxybenzoyl)-6-methylpyridine 4-phenyl-3-thiosemicarbazone; ROS,reactive oxygen species; Tf, transferrin.⇑ Corresponding authors. Tel.: +61 2 9385 4698; fax: +61 2 9385 6141 (N.K.); tel.:

+61 2 9036 6548; fax: +61 2 9036 6549 (D.R.R.).E-mail addresses: n.kumar@unsw.edu.au (N. Kumar), d.richardson@med.usyd.

edu.au (D.R. Richardson).� These two authors contributed equally as first authors.

Adeline Y. Lukmantara a,�, Danuta S. Kalinowski b,�, Naresh Kumar a,⇑, Des R. Richardson b,⇑a School of Chemistry, University of New South Wales, Sydney, NSW 2052, Australiab Iron Metabolism and Chelation Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, NSW 2006, Australia

a r t i c l e i n f o a b s t r a c t

Article history:Received 5 November 2012Accepted 13 December 2012Available online 21 December 2012

Keywords:Iron chelators2-BenzoylpyridineThiosemicarbazonesAnti-proliferative activity

The 2-benzoylpyridine thiosemicarbazone (BpT) chelators demonstrate potent anti-proliferative effectsagainst tumor cells. To understand their structure–activity relationships, BpT analogues incorporatingelectron-donating substituents on the pyridine and phenyl rings of the BpT scaffold were designed andrepresent the first attempts to modify the pyridine ring of these thiosemicarbazones. Eight analoguesshowed significantly (p <0.001) greater anti-proliferative activity than the ‘gold-standard’ chelator, des-ferrioxamine. Structure–activity analysis revealed that mono- or di-methoxy substitution at the phenylring resulted in lower anti-proliferative activity, while methoxy substitutions at the phenyl ringenhanced iron chelation efficacy. These important findings facilitate the design of thiosemicarbazoneswith greater anti-tumor activity.

� 2012 Elsevier Ltd. All rights reserved.

Iron is an essential nutrient that is required for many criticalcellular processes including energy generation, cell cycle progres-sion and DNA synthesis.1–4 In mammals, iron can be incorporatedinto heme or iron sulfur clusters, or is stored within the protein,ferritin.1 The physiological levels of iron are carefully regulated un-der normal conditions and the dys-regulation of iron homeostasis

can lead to iron overload disease.3,5 Such conditions have tradition-ally been treated with high-affinity iron chelators, such as desfer-rioxamine (DFO).3

Due to the essential function of iron in cellular proliferation, therole of chelators has expanded beyond the treatment of iron over-load conditions.3,6,7 The increased requirement for iron in rapidlydividing cells compared to normal cells offers a therapeutic avenuefor selective inhibition of cancer growth by iron deprivation. Thus,iron chelators displaying anti-tumor activity in vitro and in vivohave been explored as a potential class of anti-cancertherapeutics.3,6,7

Cancer cells express increased levels of transferrin receptor 1(TfR1), a protein responsible for iron uptake from the iron trans-port protein, transferrin (Tf).3 The iron-containing enzyme, ribonu-cleotide reductase, catalyzes the rate-limiting step of DNAreplication and is a key molecular target for iron chelatingagents.3,7–11 The higher expression of ribonucleotide reductase intumor cells relative to their normal counterparts increases the sen-sitivity of tumor cells to iron chelation.12 The induction of cell cyclearrest at the G1/S phase13–15 by chelators is due, in part, to the inhi-bition of ribonucleotide reductase activity and also to their effectson the expression of molecules involved in cell cycle control (e.g.,cyclin D1).16

A number of iron chelators have demonstrated anti-canceractivity in vitro and in vivo, including clinical trials.12 For example,DFO has shown moderate anti-proliferative effects both in vitroand in vivo (Fig. 1).17–19 Its efficacy is limited by its highly

Figure 1. Line drawings of the structures of the iron chelators, desferrioxamine (DFO), deferiprone, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP), and the 2-acetylpyridine thiosemicarbazone (ApT), di-2-pyridylketone thiosemicarbazone (DpT), 2-benzoylpyridine thiosemicarbazone (BpT) and 2-(3-nitrobenzoyl)pyridinethiosemicarbazone (NBpT) series.

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hydrophilic nature, which results in poor membrane permeabilityand relatively low iron chelation efficacy.3 Other chelators, includ-ing deferiprone (Fig. 1) and a number of its analogues, also demon-strate some anti-proliferative activity against tumor cells inculture.3

Thiosemicarbazone iron chelators have demonstrated greatpromise as anti-cancer agents, with 3-aminopyridine-2-carboxal-dehyde thiosemicarbazone (3-AP or Triapine�; Fig. 1) enteringPhase I and II clinical trials.12,20,21 However, a general lack ofanti-tumor efficacy in human patients and dose-limiting side ef-fects, such as methemoglobinemia and hypoxia, have been re-ported for 3-AP, severely limiting its potential use.12,22,23

Chelators based on hybrid aroylhydrazones and thiosemicarba-zones24,25 have led to the development of potent anti-tumoragents, including the 20-acetylpyridine thiosemicarbazone (ApT),di-20-pyridylketone thiosemicarbazone (DpT), 20-benzoylpyridinethiosemicarbazone (BpT) and 20-(3-nitrobenzoyl)pyridine thiosem-icarbazone (NBpT) series (Fig. 1). These analogues have demon-strated potent and selective anti-tumor activity.24,26–30 Inparticular, when given by the intravenous route, di-20-pyridylke-tone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) or di-2-pyri-dylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC)were able to markedly inhibit the growth of a wide variety ofhuman and murine tumor xenografts in mouse models.6,24,30,31

Additionally, ligands such as DpC and 20-benzoylpyridine 4,4-dimethyl-3-thiosemicarbazone (Bp44mT) have been deliveredorally to nude mice bearing human lung cancer xenografts withoutcausing cardiac toxicity or appreciable weight loss, even whenadministered at much higher doses than those used withDp44mT.24,30,32 Recently, a series of halogenated 20-benzoylpyri-dine thiosemicarbazones (XBpT) were synthesized with some

analogues showing appreciable therapeutic indices for neoplasticover normal cells and potent anti-proliferative effects comparedto the parent BpT group.33

Thiosemicarbazone iron complexes exhibit facile inter-conver-sion between the FeII and FeIII states making them ideal catalystsfor Fenton chemistry.27,29,31,33 Indeed, the ability of these com-pounds to redox cycle and generate reactive oxygen species(ROS) is well correlated to their anti-tumor activity.27,29,31

Moreover, the FeII/III redox potentials of the thiosemicarbazonecomplexes can be fine-tuned by the incorporation of electron-donating or electron-withdrawing substituents on the thiosemi-carbazone scaffold.26 Interestingly, for the halogenated XBpTligands, an important structure–activity relationship was deduced,where the addition of halogens led to a positive correlationbetween intracellular ROS generation and anti-proliferative activ-ity. In fact, the halogenated ligands were more effective than theirrelatively hydrophilic BpT parent compounds.33

Considering the promising results exhibited by the thiosemicar-bazone chelators, it was desirable to investigate the effect ofincorporating additional substituents on the pyridine and/orphenyl rings to gain further insight into the structure–activityrelationship of these compounds. In this current study, novel ironchelators based on 2-benzoyl-6-methylpyridine thiosemicarba-zone (6-MeBpT), 2-(30,40-dimethoxybenzoyl)-6-methylpyridinethiosemicarbazone (3,4-DiOMe-6-MeBpT) and 2-(40-methoxyben-zoyl)-6-methylpyridine thiosemicarbazone (4-OMe-6-MeBpT)were synthesized (Scheme 1) and examined for their anti-prolifer-ative activity. In addition, their ability to mobilize intracellular ironand inhibit iron uptake in cancer cells was evaluated. Structure–activity analysis revealed that the incorporation of a single methylsubstituent at the pyridine ring of the benzoylpyridine scaffold

Scheme 1. Synthesis of substituted 2-benzoylpyridine thiosemicarbazones. Reagents and conditions: HCl/EtOH, reflux.

A. Y. Lukmantara et al. / Bioorg. Med. Chem. Lett. 23 (2013) 967–974 969

resulted in the highest anti-proliferative activity of all three novelseries. The presence of two methoxy substituents at the non-coor-dinating phenyl ring yielded enhanced ability to increase 59Femobilization from cells and inhibit 59Fe uptake from the physiolog-ical iron transport protein, Tf. Importantly, these analogues repre-sent the first attempt to understand the effect of modification ofthe pyridine ring of the BpT scaffold on their activity.

The target substituted 2-benzoylpyridine thiosemicarbazonederivatives were synthesized using literature methods.29,34,35

The Schiff base condensation reactions between 2-benzoyl-6-methylpyridine, 2-(30,40-dimethoxybenzoyl)-6-methylpyridine or2-(40-methoxybenzoyl)-6-methylpyridine with the appropriatethiosemicarbazide yielded the corresponding thiosemicarbazones6-MeBpT (1a–f), 3,4-DiOMe-6-MeBpT (2a–f) and 4-OMe-6-MeBpT(3a–f), all as crystalline compounds (Scheme 1). The ligands weresparingly soluble in water, but displayed greater solubility inDMSO, DCM or CHCl3. X-ray crystal structures of 6-MeBp44mT(1e), 3,4-DiOMe-6-MeBp4mT (2b) and 4-OMe-6-MeBp4mT (3b)were obtained as representative members of each class of thesubstituted 6-MeBpT analogues (Fig. 2A–C) and their crystal datais presented in Table 1.

ORTEP views of the three compounds 1e, 2b and 3b (only one ofthe two molecules in the asymmetric unit, molecule A) are shownin Figure 2. A single intra-molecular N–H. . .N hydrogen bond ispresent in 1e, while 2 intra-molecular N–H. . .N hydrogen bondsare observed in 3b (as previously determined in the related ligand,NBpT).27 The intra-molecular hydrogen bonds observed in both 1eand 3b between the pyridine nitrogen and the thioamide protonare able to form due to the Z isomeric configuration of the ligands(about the imine bond). Although both 1e and 3b are Z isomers, theconfiguration of their thioamide moieties differ, having an anti,synand anti,anti orientation regarding their donor atoms (pyridylN/imine N and imine N/S), respectively (Fig. 2).

In contrast, the presence of Cl� in the hydrochloride salt of 2bresults in two N–H. . .Cl and a C–H. . .Cl interaction, with the ligandhaving the E isomer (syn,anti) configuration. Hydrogen bondingH. . .N distances are in the range 1.95–2.22 Å and the H (N–H. . .N) angle varies from 106� to 132� and are in agreement withthe values previously observed for the chelator, NBpT.27

The FeIII complexes of each series were synthesized and charac-terized. The FeII complexes are known to appear as an invariablygreen color due to the charge transfer transition that occurs at�640 nm.27 The FeIII complexes appeared dark brown due to theabsence of this transition.27 As the ligands are tridentate, theyare expected to form 2:1 ligand:metal complexes.27,36

It has been shown that the anti-proliferative activity of the BpTand DpT series and related ligands are linked with their capabilityto facilitate Fenton chemistry upon complexation with intracellu-lar iron.27,29,37 Considering this, cyclic voltammetry of a selectrange of the iron complexes of the substituted 6-MeBpT analoguesin MeCN:H2O (7:3) was carried out to determine the FeII/III redoxpotentials of the complexes (Table 2). The MeCN:H2O solvent mix-ture was used to enable sufficient solubility and to ensure consis-tency with other studies examining the FeII/III redox potentials ofsimilar thiosemicarbazones.27,33

The complexes exhibited totally reversible FeII/III redox poten-tials with significant cathodic shifts in comparison to previouslyreported thiosemicarbazone chelators.27 Generally, the redoxpotentials of the substituted thiosemicarbazone complexes werecathodically shifted by 56–121 mV relative to their correspondingunsubstituted BpT counterparts (Table 2). The iron complexes ofthe Bp4pT analogues (2f and 1f), containing a phenyl substituentat the terminal nitrogen atom, and the substituted BpT analogue,1a, exhibited the highest redox potentials at +59, +72 and+64 mV versus the normal hydrogen electrode (NHE), respectively.All other novel complexes tested showed redox values of under

Figure 2. Crystal structures of (A) 1e, (B) 2b, (C) 3b.

Table 1Crystal data

Crystal data A (1e) B (2b) C (3b)

Chemical formula C16H18N4S C17H21ClN4O2S C16H18N4OSMr 298.40 380.89 314.40Crystal system, space group Monoclinic, P21/c Triclinic, P�1 Monoclinic, P21/nTemperature (K) 152 150 152a, b, c (Å) 8.0822 (4), 19.8431 (10), 10.1669 (5) 8.7706 (3), 9.7823 (3), 12.3795 (6) 15.8408 (5), 10.7549 (3), 18.5257 (6)a, b, c (�) 90, 110.804 (2), 90 97.491 (2), 102.165 (2), 112.058 (1) 90, 92.249 (1), 90V (Å3) 1524.22 (13) 936.19 (6) 3153.72 (17)Z 4 2 8Radiation type Mo Ka Mo Ka Mo Kal (mm�1) 0.21 0.33 0.21Crystal size (mm) 0.54 � 0.18 � 0.06 0.24 � 0.22 � 0.07 0.38 � 0.28 � 0.14CCDC No. 895055 895053 895054

Data collectionDiffractometer Bruker kappa APEXII CCD

diffractometerBruker kappa APEXII CCD diffractometer Bruker Kappa APEXII CCD

diffractometerAbsorption correction Multi-scan SADABS (Bruker, 2001) Multi-scan SADABS (Bruker, 2001) Multi-scan SADABS (Bruker, 2001)Tmin, Tmax 0.894, 0.988 0.924, 0.976 0.923, 0.972No. of measured, independent

and observed[I >2r(I)] reflections

10,337, 2677, 2310 12,503, 3257, 2908 20,488, 5517, 4637

Rint 0.028 0.046 0.035(sin h/k)max (�1) 0.595 0.595 0.595

RefinementR[F2 >2r(F2)], wR(F2), S 0.037, 0.145, 1.20 0.036, 0.120, 1.97 0.034, 0.100, 1.05No. of reflections 2677 3257 5517No. of parameters 193 234 403No. of restraints 0 0 0H-atom treatment H-atom parameters constrained H-atoms treated by a mixture of independent

and constrained refinementH-atom parameters constrained

Dmax, Dmin (e �3) 0.37, �0.21 0.39, �0.24 0.26, �0.28

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+30 mV versus NHE. The cathodic shifts in potential observed forthe substituted 6-MeBpT chelators could be rationalized on the ba-sis of the electron-donating properties of the methyl and methoxysubstituents in the pyridine and phenyl rings. In summary, thereversible electrochemical behavior of the iron complexes suggeststhat substituted 6-MeBpT chelators are able to undergo reversibleredox cycling. Hence, these compounds may exert their anti-prolif-erative effects, at least in part, via the generation of intracellularROS.

The ability of the thiosemicarbazone analogues to inhibit cellu-lar proliferation was assessed using SK-N-MC neuroepitheliomacells as the effect of iron chelators on this cell line has been wellcharacterized.6,28,31 The anti-cancer activity of the novel chelators

were compared to the positive controls, DFO and Dp44mT, whichhad IC50 values of 21.6 ± 0.3 lM and 0.004 ± 0.001 lM (Table 3),respectively.6,28,31 Significantly, eight of the 18 tested thiosemicar-bazone derivatives exhibited significantly (p <0.001) greater anti-proliferative activity than DFO. The other ligands did not displayappreciable activity at the highest thiosemicarbazone concentra-tion tested (IC50 >6.25 lM; Table 3). The 6-MeBpT derivatives(1a–f), with a single methyl substituent on the pyridine ring, pos-sessed the highest anti-proliferative activity of the three series,with four of the tested analogues exhibiting IC50 values under5.32 lM. The ligand, 2-benzoyl-6-methylpyridine 4,4-dimethyl-3-thiosemicarbazone (6-MeBp44mT; 1e), was the most potent ana-logue developed in the present study. Indeed, this chelator had

Table 2FeII/III redox potentials (MeCN:H2O, 7:3) of the iron complexes of the substituted 6-MeBpT chelators

Ligand FeII/III redox couple(mV vs NHE)

[Fe(BpT)2] +120a

[Fe(Bp4mT)2] +108a

[Fe(Bp44mT)2] +119a

[Fe(Bp4eT)2] +99a

[Fe(Bp4aT)2] +117a

[Fe(Bp4pT)2] +180a

[Fe(6-MeBpT)2] (1a) +64[Fe(6-MeBp4mT)2] (1b) +1[Fe(6-MeBp4eT)2] (1c) +3[Fe(6-MeBp4aT)2] (1d) +12[Fe(6-MeBp44mT)2] (1e) +18[Fe(6-MeBp4pT)2] (1f) +72

[Fe(3,4-DiOMe-6-MeBp4mT)2] (2b) +12[Fe(3,4-DiOMe-6-MeBp4aT)2] (2d) +8[Fe(3,4-DiOMe-6-MeBp4pT)2] (2f) +59

[Fe(4-OMe-6-MeBp4mT)2] (3b) +23[Fe(4-OMe-6-MeBp4eT)2] (3c) 0[Fe(4-OMe-6-MeBp4aT)2] (3d) +30

a FeII/III redox potentials previously reported in Ref. 27 and used herein forcomparison to the newly synthesized 6-MeBpT chelators.

A. Y. Lukmantara et al. / Bioorg. Med. Chem. Lett. 23 (2013) 967–974 971

an IC50 value of 0.009 ± 0.001 lM, which was similar to that iden-tified for Dp44mT (Table 3).

By comparison, the thiosemicarbazone analogues bearing 3,4-dimethoxy (2a–f) or 4-methoxy (3a–f) substitution on the phenylring displayed more modest anti-proliferative activity. Of thesetwo series, the 3,4-DiOMe-6-MeBp44mT, 4-OMe-6-MeBp44mT(2e, 3e), 3,4-DiOMe-6-MeBp4pT and 4-OMe-6-MeBp4pT ( 2f, 3f)derivatives showed the most potent anti-proliferative activity(IC50: 0.62–2.48 lM). Interestingly, the activity of the most potentanalogue, 1e (IC50: 0.009 lM), was 244–276 times greater thanthat of 2e (IC50: 2.48 lM) or 3e (IC50: 2.20 lM). This observationsuggested an important structure–activity relationship. That is,the incorporation of a mono-methoxy or di-methoxy substitution

Table 3Anti-proliferative activity of the substituted 6-MeBpT chelators or iron complexes agaidetermined by the MTT assay

Ligand

SK-N-MC Feag

DFO 21.63 ± 0.32Dp44mT 0.004 ± 0.001

6-MeBpT (1a) >6.25 >66-MeBp4mT (1b) 5.32 ± 0.40 >66-MeBp4eT (1c) 0.88 ± 0.33 4.6-MeBp4aT (1d) >6.25 >66-MeBp44mT (1e) 0.009 ± 0.001 0.6-MeBp4pT (1f) 2.80 ± 0.36 5.

3,4-DiOMe-6-MeBpT (2a) >6.25 ND3,4-DiOMe-6-MeBp4mT (2b) >6.25 >63,4-DiOMe-6-MeBp4eT (2c) >6.25 ND3,4-DiOMe-6-MeBp4aT (2d) >6.25 >63,4-DiOMe-6-MeBp44mT (2e) 2.48 ± 0.08 ND3,4-DiOMe-6-MeBp4pT (2f) 0.62 ± 0.27 0.

4-OMe-6-MeBpT (3a) >6.25 ND4-OMe-6-MeBp4mT (3b) >6.25 1.4-OMe-6-MeBp4eT (3c) >6.25 >64-OMe-6-MeBp4aT (3d) >6.25 0.4-OMe-6-MeBp44mT (3e) 2.20 ± 0.55 ND4-OMe-6-MeBp4pT (3f) 1.75 ± 0.48 ND

Cells were seeded and allowed to attach to wells for 24 h and then incubated for 72experiments). Calculated partition coefficients are shown for comparison. ND = Not dete

at the non-coordinating phenyl ring resulted in lower anti-prolifer-ative activity. Furthermore, 4,4-dimethyl (1e, 2e, 3e) or phenyl (1f,2f, 3f) substitution at the terminal N4 atom of the thiosemicarba-zone scaffold consistently yielded higher anti-proliferative activityacross the three series of chelators. This could be due to the influ-ence of the substituents on the lipophilicity of the ligands, result-ing in increased membrane permeability. A plot of IC50 valuesagainst calculated octanol:water partition coefficients (logPcalc)suggests that the most active ligands have an optimal lipophilicrange between 3.8 and 5 (Fig. 3). This indicated that lipophilicityplays an important role in determining the anti-proliferative activ-ity of thiosemicarbazone chelators.3,38

Additionally, the effect of iron complexation on the anti-prolif-erative behavior of the substituted 6-MeBpT were determinedagainst SK-N-MC neuroepithelioma cells. Upon examining the ironcomplexes of the 6-MeBpT derivatives with a single methyl substi-tuent on the pyridine ring (1a–f), a decrease in the anti-prolifera-tive activity of the complexes relative to the ligands wasobserved (Table 3). The lowered potency of the complexes couldbe caused by the inability of the pre-formed complex to chelateintracellular iron and inhibit the action of key iron-dependent en-zymes such as ribonucleotide reductase.29

Interestingly, three iron complexes of ligands of the 3,4-DiOMe-6-MeBpT and 4-OMe-6-MeBpT series, 2f, 3b and 3d demonstrateda decrease in their IC50 values in comparison to the ligand alone.This represented a 4- to 20-fold increase in anti-tumor efficacywhen compared to the free ligands (Table 3). The reason for this in-creased activity relative to their free ligand described above is un-clear. However, a possible explanation may relate to the higherlipophilicity of the iron complex due to the shielding of the liganddonor atoms from the solvent environment. This could result inimproved membrane permeability of the compounds.27,39 Thus,the pre-complexation of the iron may increase the intracellularlevels of redox-active complex within the cell, resulting in greateranti-proliferative activity.27,37,39,40 On the other hand, the otherexamined iron complexes of 2b, 2d and 3c showed no significantdifference in the anti-proliferative activity between the ligandand the complex in SK-N-MC cells (Table 3). The reason for the

nst SK-N-MC neuroepithelioma cells and the mortal MRC-5 fibroblast cell line as

Average IC50 (lM)

III (L)2ClO4 complexainst SK-N-MC

MRC-5 logPcalc

.25 >6.25 2.95

.25 >6.25 3.4731 ± 0.09 >6.25 3.81.25 >6.25 4.31

58 ± 0.37 0.021 ± 0.001 3.8578 ± 0.44 >6.25 5.14

>6.25 2.7.25 >6.25 3.22

>6.25 3.56.25 >6.25 3.35

>6.25 3.635 ± 0.03 2.11 ± 1.35 4.88

>6.25 2.8341 ± 0.45 >6.25 3.35.25 >6.25 3.68

31 ± 0.03 >6.25 2.19>6.25 3.723.81 ± 0.90 5.01

h at 37 �C with control medium or the chelators. Results are mean ± SD (threermined.

Figure 3. Relationship between the anti-proliferative activity (IC50) and lipophil-icity (logPcalc) of the substituted 6-MeBpT chelators.

Figure 4. The effect of the chelators on: (A) 59Fe mobilization from prelabeled SK-N-MC neuroepithelioma cells and (B) 59Fe uptake from 59Fe-transferrin (59Fe–Tf) bySK-N-MC neuroepithelioma cells. In iron mobilization studies, cells were prelabeledwith 59Fe–Tf (0.75 lM) for 3 h/37 �C, washed four times with ice cold PBS andsubsequently incubated with control medium or chelators (25 lM) for 3 h/37 �C. Iniron uptake assays, cells were labeled with 59Fe–Tf (0.75 lM) for 3 h/37 �C in theabsence or presence of chelators (25 lM), washed four times on ice and theinternalization of 59Fe assessed (see Supplementary data). Results are mean ± SD ofthree experiments with three determinations in each experiment.

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difference in response between these groups of ligands may relateto properties such as the relative change in lipophilicity betweenthese chelators upon complexation with iron, as this property isknown to markedly affect anti-proliferative activity.27,41

For the chelators to be considered as potential anti-tumoragents, they must exhibit potent anti-proliferative activity towardsneoplastic cells, while having markedly less activity against normalcells. Thus, the effects of the substituted 6-MeBpT ligands on theproliferation of mortal human MRC-5 fibroblasts was examinedin order to determine whether these compounds exhibited selec-tivity for tumor cells (Table 3). Comparison of the anti-proliferativeactivity of the chelators against SK-N-MC cells and MRC-5 fibro-blasts indicated that the neoplastic cells were more sensitive tothe chelators compared to the normal cells. Of the analogues thatdisplayed anti-cancer activity in SK-N-MC cells, their anti-prolifer-ative effects were reduced in normal MRC-5 cells by 1.2- to 7.1-fold(Table 3). These observations demonstrate that the mortal cellswere less susceptible to these agents than the tumor cells.

Previous studies have indicated that iron chelation efficacy is animportant factor determining the anti-proliferative activity of li-gands.3,38 Hence, the ability of the chelators to mobilize intracellu-lar 59Fe from pre-labeled SK-N-MC neuroepithelioma cells wasassessed and compared to the positive control chelators, DFO andDp44mT, that have been well characterized.31,38 The majority ofthe substituted 6-MeBpT chelators demonstrated higher 59Femobilization compared to DFO, which released only 15 ± 1% ofintracellular 59Fe (Fig. 4A). In contrast, Dp44mT was the most effec-tive ligand examined in terms of mobilizing cellular 59Fe. Of all thenewly synthesized ligands, 2e and 3b were the most effective,resulting in the release of 41 ± 1% and 42 ± 1% of cellular 59Fe,respectively (Fig. 4A). Generally, the 6-MeBpT derivatives with asingle methyl substituent on the pyridine ring (1a–f) were the leastactive of the newly synthesized analogues, leading to the release of9–26% of cellular 59Fe. In comparison, chelators belonging to the3,4-DiOMe-6-MeBpT (2a–f) and 4-OMe-6-MeBpT (3a–f) seriesgenerally showed higher mobilization efficacy, resulting in the re-lease of 21–42% and 15–45% of cellular 59Fe, respectively.

Hence, these results suggested that the presence of methoxysubstitution on the phenyl ring afforded chelators with higher59Fe mobilization activity. Another interesting structure–activityrelationship was apparent when a phenyl group was added tothe R2 position, with this leading to a decrease in cellular 59Fe re-lease by the three series of substituted BpT analogues (Fig. 4A). Asimilar reduction in chelation efficacy has also been observed in re-lated thiosemicarbazone chelators upon the addition of a phenylgroup to this position.25

The efficiency of the anti-proliferative activity of chelators isknown to involve both the simultaneous release of iron from cells

and their ability to inhibit iron uptake from Tf.26–29,38,42 Thus, theefficacy of the substituted BpT series to inhibit 59Fe uptake fromthe serum iron-binding protein, Tf, was assessed in SK-N-MC neu-roepithelioma cells. As shown in previous studies,3,38 the controlchelator, DFO, had limited effect on 59Fe uptake from Tf, reducingit to only 80 ± 3% of the control (Fig. 4B). In contrast, the other po-sitive control, Dp44mT, was highly efficient, markedly reducing59Fe uptake to 5 ± 1% of the control.

Fifteen out of the 18 newly synthesize BpT chelators reduced59Fe uptake to between 14% and 42% of the control (Fig. 4B). Themost potent ligands, namely 2e and 3b, inhibited 59Fe uptake to14 ± 1% and 22 ± 1% of the control, respectively. Notably, 2e and3b were also the most effective chelators in the 59Fe efflux assay(Fig. 4A). As observed in 59Fe efflux studies, the chelators, 1f, 2fand 3f, that bear the phenyl group at R2, were the least effectiveat reducing iron uptake from 59Fe–Tf, reducing it to only 58–77%of the control (Fig. 4B).

The relationship between lipophilicity and the ability of thechelators to bind cellular iron pools were examined by plotting

A. Y. Lukmantara et al. / Bioorg. Med. Chem. Lett. 23 (2013) 967–974 973

59Fe efflux or 59Fe uptake activity against logPcalc values (Fig. 5Aand B).3,43 Chelators with logPcalc values in the range of 2.5–4.0were the most effective at inhibiting 59Fe uptake and mobilizingcellular 59Fe. This probably reflects the importance of optimal lipo-philicity in facilitating the penetration of the chelators and theircomplexes through cellular membranes, enhancing iron mobiliza-tion or inhibition of iron uptake by cells. It is important to note thathighly lipophilic chelators, such as 1f, 2f and 3f, that contain thephenyl group at R2 (logPcalc �5; Table 3), may form hydrophobiciron complexes that become trapped within cellular mem-branes,25,27 reducing their iron chelation efficacy.

The current studies have highlighted several important struc-ture–activity relationships that are critical for the design of potentthiosemicarbazone-based iron chelators for the treatment of can-cer. The novel 6-MeBpT analogues represent the first attempt tounderstand the effect of substitution on the pyridine ring of thisclass of thiosemicarbazones.

Of the 18 novel substituted 6-MeBpT analogues synthesized,eight of these agents exhibited anti-proliferative activity againstSK-N-MC neuroepithelioma cells that was markedly greater thanDFO. The most effective agent prepared was analogue 1e, whichshowed anti-proliferative activity that was comparable to the wellcharacterized ligand, Dp44mT. All of the 6-MeBpT analogues wereable to mobilize intracellular 59Fe and prevent 59Fe uptake from Tf.In fact, all of the ligands showed similar or greater iron chelationefficacy than DFO, with compound 2e being the most effective li-gand in terms of inhibiting iron uptake and increasing iron mobili-zation from cells.

Structure–activity analysis revealed that the 6-MeBpT deriva-tives (1a–f) with a single methyl substituent on the pyridine ringpossessed the highest anti-proliferative activity of the three seriesof substituted BpT ligands. Incorporation of a mono-methoxy ordi-methoxy substitution on the non-coordinating phenyl ringresulted in lower anti-proliferative activity. In addition, the 4,4-dimethyl (1e, 2e, 3e) or phenyl (1f, 2f, 3f) substitution at the termi-nal N4 atom of the thiosemicarbazone scaffold consistently yielded

Figure 5. The relationship between (A) 59Fe mobilization and lipophilicity (logPcalc)and (B) 59Fe uptake and lipophilicity (logPcalc) of the novel substituted BpT ligands.

higher anti-proliferative activity across the three series of chela-tors. This could be due to the influence of the substituent groupson the lipophilicity of the chelators, which has been shown to playan important role in the activity of these agents. A plot of IC50 val-ues against logPcalc suggested that the optimal lipophilic range wasbetween logPcalc values of 3.8–5 (Fig. 3).

Similarly, examining the relationship between lipophilicity andiron chelation efficacy demonstrated that chelators with logPcalc

values in the range of 2.5–4.0 were the most effective at inhibiting59Fe uptake and mobilizing cellular 59Fe. Overall, this dependencelikely reflects the importance of optimal lipophilicity in facilitatingligand penetration through cell membranes, enhancing iron mobi-lization or inhibition of iron uptake by cells. Reduced iron chelationefficacy was observed for the highly lipophilic chelators (i.e., 1f, 2fand 3f) that contain a phenyl group at R2 (logPcalc �5). Their de-creased activity may be explained by the formation of hydrophobiciron complexes that may become trapped within membranes dueto their affinity for the lipid environment.25,27

Overall, these results elucidate the effect of substitution on thenon-coordinating phenyl ring on the activity of the BpT ligands andprovide new information useful for the synthesis of future com-pounds with high efficacy.

Acknowledgments

This work was supported by a Project Grant from the NationalHealth and Medical Research Council (NHMRC) of Australia toD.R.R. [Grant 632778]; a NHMRC Senior Principal Research Fellow-ship to D.R.R. [Grant 571123]; a Cancer Institute New South WalesEarly Career Development Fellowship to D.S.K. [Grant 08/ECF/1-30]; a Cancer Institute Research Innovation Grant to D.R.R. andD.S.K. [Grant 10/RFG/2-50]. The research was additionally sup-ported by an ARC Discovery Grant (DP1095159) and LinkageProject Grant (LP110200635). We kindly thank Mr. Mohan Bhadh-ade (Crystallography Laboratory, Analytical Centre, The Universityof New South Wales, Sydney, Australia) for collecting X-ray crystalstructure data.

Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.12.044.

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