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Research ArticleThiosemicarbazone p-Substituted AcetophenoneDerivatives Promote the Loss of Mitochondrial Δ120595 GSHDepletion and Death in K562 Cells

Felipe S Pessoto1 Cesar H Yokomizo12 Tatiana Prieto1 Cleverton S Fernandes3

Alan P Silva3 Carlos R Kaiser4 Ernani A Basso3 and Iseli L Nantes1

1NanoBioMAv Centro de Ciencias Naturais e Humanas Universidade Federal do ABC Avenida dos Estados 5001Bairro Bangu 09210-580 Santo Andre SP Brazil2Departamento de Biologia Molecular Universidade Federal de Sao Paulo Rua 3 de Maio 100 Vila Clementino04044-020 Sao Paulo SP Brazil3Departamento de Quımica Universidade Estadual de Maringa Avenida Colombo 5790 87020-900 Maringa PR Brazil4Instituto de Quımica-Universidade Federal do Rio de Janeiro (IQ-UFRJ) Edifıcio do Centro de Tecnologia Bloco ACidade Universitaria 21941-909 Rio de Janeiro RJ Brazil

Correspondence should be addressed to Ernani A Basso eabassouemgmailcom and Iseli L Nantes ilnantesgmailcom

Received 25 November 2014 Accepted 3 April 2015

Academic Editor Ange Mouithys-Mickalad

Copyright copy 2015 Felipe S Pessoto et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A series of thiosemicarbazone (TSC) p-substituted acetophenone derivatives were synthesized and chemically characterized Thep-substituents appended to the phenyl group of the TSC structures were hydrogen fluor chlorine methyl and nitro producingcompounds named TSC-H TSC-F TSC-Cl TSC-Me and TSC-NO

2 respectively The TSC compounds were evaluated for their

capacity to induce mitochondrial permeability to deplete mitochondrial thiol content and to promote cell death in the K562 celllineage using flow cytometry and fluorescence microscopy TSC-H TSC-F and TSC-Cl exhibited a bell-shaped dose-responsecurve for the induction of apoptosis in K562 cells due to the change from apoptosis to necrosis as the principal mechanism of celldeath at the highest tested doses TSC-Me and TSC-NO

2exhibited a typical dose-response profile with a half maximal effective

concentration of approximately 10 120583M for cell death Cell death was also evaluated using the 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) assay which revealed lower toxicity of these compounds for peripheral blood mononuclearcells than for K562 cells The possible mechanisms leading to cell death are discussed based on the observed effects of the new TSCcompounds on the cellular thiol content and on mitochondrial bioenergetics

1 Introduction

Thiosemicarbazones (TSCs) are compounds that share theC=N-NH-CS-NH moiety as shown in Figure 1

Thepossibility of a puzzle combination of different groupsat the variable positions 1 2 and 4 makes this class of organiccompounds extremely versatile for use as therapeutic agentsfor a variety of pathological conditions Therefore TSCsand their metal complexes [1 2] possess potent antitumoral[3ndash9] antiviral [2 5 9ndash11] antibacterial [3 5 10 12] andantiparasitic [2 5 11ndash14] properties Anticonvulsant andneurotropic effects are also described in the literature [13

15] In some cases these compounds are used clinicallysuch as the important chemotherapeutic anticancer agent3-aminopyridine-2-carboxaldehyde thiosemicarbazone (ieTriapine) and its derivatives [2 14ndash17]Thus for each specifictherapeutic application a unique structure is appropriated Inthe case of the antifungal and antitumor activities of TSCsthe capacity to complex metal increases drug efficacy Thetransition metal complex of TSCs can redox cycle leadingto the generation of free radical species this process isan important contributing factor for the antitumor activ-ity of these compounds [18] Examples of TSC chelatorswith antitumor activity are the dipyridyl thiosemicarbazone

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2015 Article ID 394367 15 pageshttpdxdoiorg1011552015394367

2 Oxidative Medicine and Cellular Longevity

N N

S

H N

H

123

4R1

R2R3

Figure 1 General structure of thiosemicarbazones with C=N-NH-CS-NH moiety R1 R2 and R3 = alkyl or aryl groups

(ie di-2-pyridyl ketone thiosemicarbazone (DpT)) and 2-benzoylpyridine thiosemicarbazone (BpT) classes In addi-tion to this nonspecific chelator effect a specific activity hasbeen associated with the antitumor activity of TSCs theinhibition of ribonucleotide reductase which is an enzymethat is required for the de novo synthesis of deoxyribonu-cleotides and DNA replication and repair [19] 3-AP is awell-known example of this class of TSC compounds 3-AP has also been suggested to act as a neuroprotectantduring the treatment of neurodegenerative diseases [20ndash22]Regardless of the mechanisms responsible for the diversetherapeutic effects of TSCs these compounds are dependenton drug transfer through the plasma membrane Thereforethe hydrophobic R groups and bulky substituent at the p-position of the rings are important for cytotoxicity In thepresent study we present a series of TSCs with benzyl asthe R3 group p-substituted phenyl as the R2 group andmethyl as the R1 group These groups endow the moleculeswith a high hydrophobicity and favor transport throughmembranes Furthermore the R2 group was appended withdifferent substituents to confer different properties to themolecules (Scheme 1) These TSC compounds were tested onK562 and peripheral blood mononuclear (PBMN) cells todetermine their capacity to induce cell death and identifytheir effects on mitochondrial bioenergetics and thiol redoxbalance

2 Materials and Methods

21 General Chromatography was performed on silica gelMerck 230ndash400 mesh ASTM All melting points were deter-mined using a Microquimica model MQAPF-301 appa-ratus The high-resolution electron spray ionization massspectrometry (HRESIMS) analyses were performed on aQTOF Micro (Waters Manchester UK) mass spectrometerequipped with an ESI source Proton nuclear magneticresonance (1HNMR) spectra were recorded using CDCl

3as a

solvent at ambient temperature using a Varian Mercury Plus(300MHz) with TMS as an internal standard The chemicalshifts (120575) are given in parts per million relative to TMSCarbon-13 nuclear magnetic resonance (13C NMR) spectrawere recorded at 755MHz with the same internal standard

22 Synthesis

221 N(4)-Benzyl-thiosemicarbazide (2) To a solution of 1(500 g 335mmol) in ethanol (EtOH) (15mL) was addedhydrazine (107 g 335mmol) The reaction mixture wasstirred at 90∘C for 12 h The resulting solid was collected by

filtration rinsed with cold EtOH and recrystallized fromethyl acetatemethanol (EtOAc)MeOH (2 1) to afford 2(580 g 95) as white crystals 1HNMR (300MHz dimethylsulfoxide-D6) 120575 876 (br s 1H NH) 830 (br t J = 63Hz1H NHCH

2) 733ndash719 (m 5H Ar H) 471 (d J = 63Hz 2H

NHCH2) 452 (br s 2H NH

2) 13CNMR (75MHz dimethyl

sulfoxide-D6)120575 1826 1391 1280 1273 1266 469NMRdataare in agreement with literature [5 7]

222 General Procedure for the Synthesis and Characteri-zation of Compound 3andash3e (Scheme 1) To a solution of 2(030 g 165mmol) in ethanol was added with stirring asolution of ketone (165mmol) and drops of an aqueoussolution of sulfuric acid (50) The mixture was stirred atroom temperature for 12 h and then was concentrated invacuum The residue was partitioned between CHCl

3and

10 NaHCO3aqueous solution and purified by silica gel

column chromatography to afford 3andash3e [5 7]

223 N(1)-Methylphenyl Ketone-N(4)-benzyl-thiosemicarba-zone-3a (TSC-H) Starting from 2 and methylphenyl ketonethe procedures summarized above provided the title com-pound in 61 yield (028 g) as white crystals after purificationby column chromatography (silica gel CHCl

3EtOAc 100 0

to 2 1) 1H NMR (300MHz CDCl3) 120575 876 (br s 1H NH)

792 (br t J = 6Hz 1H NHCH2) 766ndash763 (m 2H Ar H)

740ndash726 (m 8H Ar H) 498 (d J = 6Hz 2H NHCH2) 227

(s 3H CH3) 13C NMR (75MHz CDCl

3) 120575 1786 (C3) 1472

(C11015840) 1377 (C31015840) 1375 (C6) 1299 (C51015840 and C71015840) 1289 (C8and C10) 1287 (C9) 1280 (C7 and C11) 1265 (C41015840 and C81015840)486 (C5) 138 (C21015840) mp 1574minus1597∘C HRMS (M + H+)2841218 C

16H18N3S+ requires 2841216

224 N(1)-Methyl-para-fluorophenylketone-N(4)-benzyl-thi-osemicarbazone-3b (TSC-F) Starting from 2 and methyl-para-fluorophenylketone the procedures summarized aboveprovided the title compound in 54 yield (027 g) as a whitesolid after purification by column chromatography (silica gelCHCl

3EtOAc 100 0 to 2 1) 1HNMR (300MHz CDCl

3) 120575

882 (br s 1H NH) 788 (br t J = 57Hz 1H NHCH2) 764

(dd J = 90 and JH-F = 51Hz 2H Ar H) 740ndash726 (m 5HAr H) 704 (dd J = 90 and JH-F = 90Hz 2H Ar H) 498(d J = 57Hz 2H NHCH

2) 226 (s 3H CH

3) 13C NMR

(75MHzCDCl3)120575 1785 (C3) 1638 (d JC-F= 2492HzC61015840)

1463 (C11015840) 1377 (C6) 1336 (d JC-F = 38Hz C31015840) 1289(C8 C9 and C10) 1284 (d JC-F = 83Hz C41015840 and C81015840) 1278(C7 and C11) 1157 (d JC-F = 217Hz C51015840 and C71015840) 485(C5) 139 (C21015840) mp 1119minus1145∘CHRMS (M+H+) 3021120C16H17FN3S+ requires 3021122

225 N(1)-Methyl-para-chlorophenylketone-N(4)-benzyl-thi-osemicarbazone-3c (TSC-Cl) Starting from 2 and methyl-para-chlorophenylketone the procedures summarized aboveprovided the title compound in 47 yield (025 g) as a whitesolid after purification by column chromatography (silica gelCHCl


3) 120575

873 (br s 1H NH) 786 (br t J = 6Hz 1H NHCH2) 758 (d

J = 87Hz 2H Ar H) 740ndash728 (m 7H Ar H) 498 (d J =

Oxidative Medicine and Cellular Longevity 3

NCS N

S

H 6

11

7

10

8

9

5 43 N

2S

H

H N 1

R

N

H

Na b

3a (R=H) TSC-H3b (R=F) TSC-F3c (R=Cl) TSC-Cl3d (R=Me) TSC-Me

1 23

1998400

2998400

3998400

4998400

5998400

69984007998400

8998400

9998400

(b) MeCOPh-p-R acidified with a solution of 50 H2SO4 rt 12h

3e (R=NO2) TSC-NO2

CH3NH2

aReagents and conditions (a) NH2NH2 EtOH reflux 12h

Scheme 1 Synthesis of thiosemicarbazones

6Hz 2H NHCH2) 226 (s 3H CH

3) 13C NMR (75MHz

CDCl3) 120575 1786 (C3) 1460 (C11015840) 1376 (C6) 1360 (C31015840) 1359

(C61015840) 1290 (C51015840 and C71015840) 1280 (C8 C9 and C10) 1279 (C7and C11) 1278 (C41015840 and C81015840) 487 (C5) 137 (C21015840) mp 1685ndash1705∘C HRMS (M + H+) 3180819 C

16H17ClN3S+ requires

3180826

226 N(1)-Methyl-para-methylphenylketone-N(4)-benzyl-thi-osemicarbazone-3d (TSC-Me) Starting from 2 and methyl-para-methylphenylketone the procedures summarizedabove provided the title compound in 45 yield (022 g) aswhite crystals after purification by column chromatography(silica gel CHCl

3EtOAc 100 0 to 2 1) 1HNMR (300MHz

CDCl3) 120575 882 (br s 1H NH) 793 (br t J = 57Hz 1H

NHCH2) 754 (d J = 81Hz 2H Ar H) 740ndash725 (m 5H

Ar H) 716 (d J = 81Hz 2H Ar H) 497 (d J = 57Hz 2HNHCH

2) 234 (s 3H Ar-CH

3) 223 (s 3H CH

3) 13C NMR

(75MHz CDCl3) 120575 1783 (C3) 1474 (C11015840) 1400 (C61015840) 1377

(C6) 1346 (C31015840) 1293 (C51015840 and C71015840) 1288 (C8 and C10)1277 (C7 and C11) 1276 (C9) 1263 (C41015840 and C81015840) 484 (C5)214 (C91015840) 137 (C21015840) mp 1603minus1622∘C HRMS (M + H+)2981369 C


227 N(1)-Methyl-para-nitrophenylketone-N(4)-benzyl-thi-osemicarbazone-3e (TSC-NO

2) Starting from 2 and methyl-

para-nitrophenylketone the procedures summarized aboveprovided the title compound in 26 yield (014 g) as yellowcrystals after purification by column chromatography (silicagel CHCl

3EtOAc 100 0 to 2 1) 1H NMR (300MHz

CDCl3) 120575 880 (br s 1H NH) 822 (d J = 93Hz 2H Ar H)

785 (br t J = 57Hz 1H NHCH2) 780 (d J = 93Hz 2H Ar

H) 740ndash729 (m 5H ArH) 500 (d J = 57Hz 2H NHCH2)

233 (s 3H CH3) 13C NMR (75MHz CDCl

3) 120575 1787 (C3)

1482 (C61015840) 1443 (C11015840) 1434 (C31015840) 1374 (C6) 1291 (C8 andC10) 1281 (C9) 1280 (C7 and C11) 1272 (C51015840 and C71015840) 1240(C41015840 andC81015840) 488 (C5) 137 (C2rsquo) mp 1568ndash1580∘CHRMS(M + H+) 3291071 C

16H17N4O2S+ requires 3291067

23HRESIMSAnalyses Thehigh-resolutionmass spectrom-etry analyses were performed on a QTOF Micro (Waters

Manchester UK) mass spectrometer equipped with an ESIsourceThe analyses were recorded betweenmz 90 and 1000in positive ion mode and the mass spectrometer parameterswere as follows the nebulization gas was set to 500 Lh at140∘C the cone gas set to 50 Lh and the source temperatureset to 100∘C The capillary voltage was set to 45 kV and conevoltage set to 30VThe QTOF acquisition rate was set to 10 swith a 04 s interscan delay and the data processed on theMassLynx 40 software (Waters Manchester UK) Analyteswere acquired using the LockSpray and phosphoric acid (01in acetonitrilewater 1 1) as external and internal standardto ensure accuracy mass The sample solutions (05mgmL)were prepared in acetonitrile with addition of 20120583L of formicacid The analyses were carried out by direct infusion usingan Alliance HT 2795 HPLC system (Waters ManchesterUK) at flow ratio of 150120583Lmin of acetonitrilewater 1 1 andinjection volume of 10 120583L The obtained results are showedin Table 1 HRESIMS mz 2841218 [M + H]+ (Calcd forC16H18N3S+ 2841216) HRESIMS mz 3021120 [M + H]+

(Calcd for C16H17FN3S+ 3021122) HRESIMS mz 3180819

[M + H]+ (Calcd for C16H17ClN3S+ 3180826) HRESIMS

mz 2981369 [M + H]+ (Calcd for C17H20N3S+ 2981372)

HRESIMSmz 3291071 [M +H]+ (Calcd for C16H17N4O2S+

3291067) The mass values are presented in Table 1

24 Determination of Total Thiol Content After 15minincubation under swelling conditions mitochondria weretreated with trichloroacetic acid (5 final concentration) andcentrifuged at 4500 g for 10min The pellet was suspendedwith 1mL of 05M potassium phosphate buffer pH 76 andafter addition of 01mM DTNB absorbance was determinedat 412 nm The amount of accessible sulfhydryl groups wascalculated by measuring the TNB released using a molarextinction coefficient of 13600Mminus1 cmminus1 [23]

25 Determination of K562 Reduced Glutathione Con-tent After 15min incubation under swelling conditionsmitochondria suspension was treated with 05mL of 13trichloroacetic acid and centrifuged at 900 g for 3minAliquots (100 120583L) of the supernatant were mixed with 2mL


Table 1 Calculated and experimental mass compounds obtained by high-resolution mass spectrometry

Code Molecular formula Calculated Experimental Error (ppm)a

3a(TSC-H) C16H18N3S+ 2841216 2841218 070

3b(TSC-F) C16H17FN3S+ 3021122 3021120 minus066

3c(TSC-Cl) C16H17ClN3S+ 3180826 3180819 minus220

3d(TSC-Me) C17H20N3S+ 2981372 2981369 minus101

3e(TSC-NO2) C16H17N4O2S+ 3291067 3291071 122

aAcceptable mass errors le plusmn500 ppm

of 100mMNaH2PO4buffer pH 80 containing 5mMEGTA

One hundred microliters of o-phthalaldehyde (1mgmL) wasadded and the fluorescence was measured 15min later usingthe 350420 nm excitationemission wavelength pair in a F-2500 fluorescence spectrophotometer (Hitachi Ltd TokyoJapan) [24]

26 Isolation of Peripheral Blood Mononuclear (PBMN) CellsThe mononuclear lymphocytes were isolated by densitygradient centrifugation The blood volume was firstly 4-fold diluted with Roswell Park Memorial Institute medium(RPMI) 2mM EDTA 7mL from diluted blood was carefullylayered over 3mL of Histopaque-1077 (Sigma) centrifugedat 400timesg for 40min the mononuclear cloud layer was gentlyaspirated and diluted in 50mLof RPMI centrifuged at 300timesgfor 15min the pellet was suspended in 40mL of RPMI andcentrifuged at 200timesg for 10min (twice) finally the cells weresuspended in an appropriated volume for MTT assay

27 Cell Culture and Cell Viability Assay K562 and mononu-clear cells were cultured in RPMI 1640 medium supple-mented with 10 fetal bovine serum (FBS) 100UmL peni-cillin and 100mgmL streptomycin (Invitrogen CarlsbadCA USA) at 37∘C5 CO

2(Sanyo MCO-20AIC Japan)

For viability experiments cells were maintained in grownmedia at 2 times 105 cellswell for both cells lines and theywere added to 96-well microplates (02mL final volume)in the presence of different concentrations (1120583Mndash2mM) ofTSC-Me and TSC-NO

2and incubated for 3 and 6 h After

incubation time 025mgmL MTT (3-(45-dimethylthiazol-2-yl)-25-diphenyl-2H-tetrazolium bromide) was added andafter 4 h the cells andMTT crystals were solubilized in 01mLof 10 SDS001MHCl overnightThe final absorbance assaywas read at 630 nm (Biochrom Asys Expert Plus CambridgeUK) Cell viability was determined relative to the controlperformed in the absence of TSC compounds and consideredas 100 reference Results are presented as themean plusmn SD ofthree independent experiments

28 Annexin Fluorescein Isothiocyanate7-Amino-actinom-ycin D (V-FITC7-ADD) Double-Staining and Flow Cytom-etry Analysis After treatment with TSC for 6 and 24 h asdescribed above K562 cells were harvested washed with coldPBS and suspended in binding buffer (001M HEPES pH74 014M NaCl and 25mM CaCl

2) at a concentration of

1 times 106 cellsmL The suspensions were transferred to 5-mLtubes and 5 120583L annexin V-FITC and 5 120583gmL 7-ADD wereadded The cells were incubated at 25∘C for 20 minutes andafter the addition of 03mL of binding buffer the analysis wasperformed in a flux cytometer Beckman Coulter model LabCell Quanta SCMPL Control cells were treated only with themedium Data were present as media plusmn SD of the triplicates

29 Cytomorphology Microscopy images of the cells wereobtained using a Widefield Leica DMI 6000B microscope(Leica Microsystems Germany) with an objective HCXPL APO 40x085 coupled to an ultra-fast digital cameraLeica DFC365 FX (Leica Microsystems Germany) Thenuclei in the same visual field were stained with 410158406-diamidino-2-phenylindole (DAPI) (10 120583gmL) and energizedmitochondria were stained by the retention of the dye331015840-dihexyloxacarbocyanine iodide (DiOC

6(3)) incubated at

37∘C for 30min

3 Results and Discussion

31 Synthesis and Chemical Characterization of the TSCsThe five novel compounds TSC-Cl TSC-Me TSC-F TSC-Hand TSC-NO

2were prepared using the procedure described

in Scheme 1 The synthesis of TSCs 3andash3e was based onbenzyl-thiosemicarbazide (2) which was obtained by react-ing hydrazine with benzyl-isothiocyanate (1) in ethanol at90∘C After isolation the ethanol solution of compound2 and the acetophenone derivatives were stirred at roomtemperature in the presence of drops of 50 sulfuric aciduntil the complete consumption of compound 2 occurredTSCs 3andash3e (Scheme 1) were obtained with a good yieldafter purification of the resulting reaction mixture by elutionthrough a silica gel chromatography column Compounds3andash3ewere characterized via the combined use of HRESIMS1D nuclear magnetic resonance (NMR) and 2D NMR andsubsequent comparison with data available in the literature[25ndash27]The 1DNMR spectra of general structure 3 exhibitedsignals at 120575H120575C 223ndash233 (s 3H)137ndash139 from the methylgroup 497ndash500 (d 2H)484ndash488 from the methylenegroup 785ndash7041654ndash1157 from the aromatic rings and 120575H873ndash882 and 786ndash822 from the NH groups in additionthe signals 120575C 1433ndash1474 and 1783ndash1787 originated from theC=N and C=S groups respectively


The log P magnitude was calculated using AdvancedChemistry Development Inc (ACDLabs) ChemSketch soft-ware Toronto ON Canada and the following values wereobtained 419 plusmn 089 389 plusmn 059 365 plusmn 062 343 plusmn 059and 339 plusmn 060 The high values of logP indicated thatthese compounds are highly hydrophobic and have a highaffinity for the lipid bilayers of biological membranes Thesecompounds were then tested for their capacity to promotecell death in associationwithmitochondrial permeability anddepletion of thiol content

32 Effect of TSCs onMitochondrial Permeability and the TotalContent of Protein Thiol Groups The TSC compounds weretested in cultured K562 cells at the concentrations of 10 100and 1000 120583MThe cells were incubatedwith TSC compoundsand the progression of apoptosis was analyzed using a flowcytometer with the annexin V-FITC7-AAD kit Cells losemembrane phospholipid asymmetry during the early step ofapoptosis and phosphatidylserine (PS) which is a negativelycharged phospholipid that is located in the inner surface ofthe plasma membrane appears on the external cell surfaceDuring this step cell membrane integrity is preservedFluorescein-labeled annexin V binds preferentially to PS andstains dying cells before they hydrolyze their DNA and losetheir morphology thus annexin V staining characterizescells during early apoptosis As apoptosis progresses the cellsare stained by the DNA-specific viability dye 7-AAD [28ndash32] Thus in the presence of both annexin V and 7-AADit is possible to distinguish different cell populations Cellsundergoing early apoptosis are stained only with annexin Vlate apoptotic cells are stained with both annexin V and 7-AAD necrotic cells are stained only with 7-AAD and livecells are not stained by any dye Figures 2(a) 2(b) 2(c)2(d) and 2(e) present the results obtained using TSC-ClTSC-Me TSC-F TSC-H and TSC-NO

2 respectively The

insets show the 3D structures of the respective TSCs Thecompounds are arranged from (a) to (e) in decreasing orderof the logPmagnitude which was calculated using ACDlabChemSketch software All probed TSC compounds wereable to trigger cell death but these compounds had uniqueprofiles

Figure 2 reveals three different patterns of dose-dependent cell death thatwere promoted byTSC compoundsThe compounds TSC-Cl TSC-Me TSC-F and TSC-Hexhibited bell-shaped profiles of their dose-response effectsTSC-Cl and TSC-Me had the highest calculated logP valuesand exhibited very similar dose-response curves After a6 h incubation with 100 120583M TSC-Cl sim80 of the K562cells were in the death process with equal contributionsfrom cells in early apoptosis and cells in late apoptosisK562 cells that were incubated with 1mM TSC-Cl remainedsim50 viable and necrosis was the predominant mechanismof cell death This peculiar dose-response effect with aswitch from apoptosis to necrosis could result from theaggregation of the TSC molecules at high concentrationsThis aggregation could result in the formation of someparticles with a lower availability for endocytosis Howeverthe eventual endocytosis of aggregated particles by some

cells could result in a high individual dosage leading tonecrosis TSC-Me exhibited a dose-response profile similarto that of TSC-Cl but the percentage of viable cells decreasedwith increasing TSC-Me concentrations with saturation at1mM For TSC-Me the switch from apoptosis to necrosiswas not observed although a significant increase in the cellpopulation undergoing necrosis was observed at a dose of100 120583M

The dose-response data obtained for TSC-H and TSC-F which had intermediate calculated logP values werenearly identical For those compounds early apoptosis wasthe predominant event observed in K562 cells after a 6 hincubation in the presence of 10 or 100120583M TSC At the latterconcentration more than 80 of the cells were undergoingearly apoptosis after a 6 h incubation However after a 6 hincubationwith 1mMTSC-Hor TSC-F 40of theK562 cellsremained viable and the remaining 60 of the cells whichwere detected in a death process were in early apoptosisapoptosis or necrosis which was the most abundant popula-tionwhen the cells were treated with TSC-HThe cells treatedwith 1mM TSC-F were undergoing apoptosis and necrosiswith similar percentages

K562 cells that were incubated with different doses ofTSC-NO

2 which had the lowest calculated logP value

exhibited early apoptosis as the predominant mechanism ofcell death at all of the tested doses the percentage of viablecells decreased in a dose-dependent manner For TSC-NO

2

the EC50 for apoptosis was sim10 120583M which is lower than or inthe range of other TSC compounds reported in the literature[33ndash36]

To investigate the possible involvement of mitochondrialbioenergetics in the induction of apoptosis by the investigatedTSC compounds the mitochondrial membrane potential(Δ120595119898) was assessed using fluorescence microscopy of cells

labeled with the dye DiOC6(3) DiOC

6(3) is a cell-permeant

green-fluorescent lipophilic dye which selectively stains theenergized mitochondria of live cells at low concentrations[37ndash42]

Figure 3 shows the effects of TSC compounds on theΔ120595119898

and nuclear morphology of K562 cells Panel (a) shows arepresentative image of a control sample of K562 cells aftera 6 h incubation in the absence of TSC compounds In theabsence of TSC compounds K562 cells appeared normalwith round and homogenous nuclei Green fluorescence inthe majority of cells indicates that the mitochondria arecoupled Panels (b) and (c) show that significant loss of Δ120595

119898

was observed after a 3 h incubation in the presence of 001and 01mM of the studied TSC compounds that is TSC-Cl TSC-Me TSC-F TSC-H and TSC-NO

2 However at the

concentration of 1mM 1 h of incubation was enough for acomplete disruption of Δ120595

119898of K562 cells that were treated

with the TSC compounds Panel (d) shows images of K562cells after a 6 h incubation in the presence of 1mMof the TSCcompounds The images are consistent with the flow cytom-etry results K562 cells treated with 1mM concentrations ofTSC-Cl TSC-Me TSC-F and TSC-H exhibited character-istics of necrosis with nuclear fragmentation Consistentlycharacteristics of necrosis were not detected for cells treatedwith TSC-NO

2 The green fluorescence present in these


419

Cell

s (

)80

60

40

20

0

Con

trol

TSC-

Cl001

mM

TSC-

Cl01

mM

TSC-

Cl1

mM

(a)

389

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

Me

001

mM

TSC-

Me

01

mM

TSC-

Me

1m

M

(b)

365

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

F001

mM

TSC-

F01

mM

TSC-

F1

mM

(c)

343

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

H001

mM

TSC-

H01

mM

TSC-

H1

mM

(d)

339

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

NO

2

001

mM

TSC-

NO

201

mM

TSC-

NO

21

mM

(e)

Figure 2 Cell death apoptosis and necrosis were triggered in K562 cells by TSC compounds in a dose-dependent manner K562 cells wereincubated with 001 01 and 10mmolL of (a) TSC-Cl (b) TSC-Me (c) TSC-F (d) TSC-H and (e) TSC-NO

2for 6 h Apoptosis and necrosis

were evaluated via flow cytometric analysis of phosphatidylserine externalization (ie annexin V binding) and cell membrane integrity (ie7-AAD staining) White columns correspond to the percentage of the viable cell population light gray columns correspond to early apoptoticcells gray columns correspond to late apoptotic cells and black columns correspond to necrotic cells The insets show the structures of thecompounds with the corresponding calculated logP values


100120583m

(a)

TSC-Cl

TSC-Me

TSC-F

100120583m

(b)

100120583m

TSC-H

TSC-NO2

(c)

TSC-Cl TSC-Me TSC-F

TSC-H TSC-NO2

100 120583m

100120583m 100120583m

(d)

Figure 3 Effect of TSC compounds on mitochondrial membrane potential and nuclear morphology after exposure to TSC compoundsSimultaneous measurements of Δ120595

119898(DiOC

6(3) green fluorescence) and nuclear morphology (DAPI blue fluorescence) were made in K562

cells after different incubation times in the absence (control) and in the presence of TSC compounds Bleaching of the green fluorescence ofDiOC

6(3) indicates the disruption of Δ120595

119898 Panel (a) shows an image from a control sample of K562 cells after 6 h incubation in the absence

of TSC compounds From left to center panel (b) shows images of K562 cells after 3 h incubation in the presence of 001 or 01mM of theindicated TSC compounds (ie TSC-Cl TSC-Me and TSC-F) the right side shows images of K562 cells after 1 h incubation in the presenceof 1mM of the same compounds From left to center panel (c) shows images of K562 cells after 3 h incubation in the presence of 001 or01mM of the indicated TSC compounds (ie TSC-H and TSC-NO

2) the right side shows images of K562 cells after 1 h incubation in the

presence of 1mM of the same compounds Panel (d) shows images of K562 cells after 6 h incubation in the presence of 1mM of the indicatedTSC compounds that is TSC-Cl TSC-Me TSC-F TSC-H and TSC-NO

2

conditions can be explained by the behavior of DiOC6(3)

Once DiOC6(3) is detached by depolarized mitochondria

it binds to other biological membranes such as membranesfrom fragmented organelles It is not uncommon that theloss of Δ120595

119898is associated with the depletion of cellular thiol

content [43] Therefore the membrane protein thiol andglutathione (GSH) content of K562 cells was analyzed afterincubation with the TSC compounds (Figure 4)

TheTSC compounds did not promote significant changesin the membrane protein thiol content (data not shown)However a dose-dependent decrease in GSH content wasobserved for K562 cells treated with the TSC compoundsA direct correlation between the extension of apoptosisand the disruption of Δ120595

119898and the depletion of GSH

was not observed for cells treated with TSC compoundsInterestingly TSC-NO

2had peculiar and consistent effects

on the GSH content and Δ120595119898

of K562 cells TSC-NO2

was the only TSC compound that was able to promotecell death exclusively by apoptosis with significant GSHdepletion at low concentrations (10 120583M) The depletionof GSH by the TSC compounds studied here could berelated to the activation of the ABCB1 transporter andquinone reductase 2 (QR2) activity TSCs with a para sub-stituted phenyl moiety which are exemplified by the struc-ture of 1-isatin-4-(40-methoxyphenyl)-3-thiosemicarbazone(NSC73306 Figure 5) have been reported to be a target fortheABCB1 transporter whosemechanism involvesGSHcon-sumption Differently the activity of QR2 involves generationof hydroxyl radical [44]

A more plausible mechanism for the depletion of GSHthat is promoted by TSC compounds particularly TSC-NO2 is a nonenzymatic reaction with the peptide thiol


TSC (120583M)

TSC-Cl

Relat

ive (

)

10

05

000 10 100 1000

(a)Re

lativ

e (

)

TSC-Me10

05

00

TSC (120583M)

0 10 100 1000

(b)

TSC-F

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(c)

TSC-H

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(d)

TSC-NO2

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(e)

Figure 4 Depletion of GSH in K562 cells by the indicated TSC compounds GSH levels in K562 cells were determined based onfluorescence after 15min incubation with o-phthalaldehyde (1mgmL) The fluorescence measurements were determined using the 350420-nm excitationemission wavelength pair in an F-2500 fluorescence spectrophotometer as described in Section 2


wwwacdlabscom

Figure 5 3D structure of the compound NSC73306

groups This putative mechanism is inspired by the mech-anism proposed by Tiwari et al [45] for the formationof nitroso-intermediates of nitroaromatic compounds withantituberculosis (TB) activity Nitroaromatic compoundssuch as 13-benzothiazin-4-ones (BTZs) and similar com-pounds generate nitroso-intermediates in vivo that are ableto cause suicide inhibition of decaprenylphosphoryl-120573-D-ribose 21015840 oxidase (DprE1) This enzyme inhibition causesthe death of Mycobacterium tuberculosis because DprE1isresponsible for the biosynthesis of arabinogalactan in thecell wall According to computational studies the authorsdemonstrated that the nonsubstituted aromatic carbons ofnitroaromatic compounds are the most electron-deficientand the most susceptible to nucleophilic attack Thereforenucleophiles such as thiolates of glutathione might inducethe nonenzymatic reduction of the nitro groups in thestructure of these compounds (Scheme 2) This mechanisminvolves the addition of the nucleophiles at the nonsubsti-tuted electron-deficient aromatic carbon that is present inthese compounds

The fate of the nitrocompound after the reaction withGSH might be the generation of NO∙ which is consistentwith the induction of cell death in cultured cells Anotherpossibility is the formation of a nitroso derivative that alsoexerts toxic effects on cells

To investigate the capacity of the TSC compounds toreact nonenzymatically with the thiolate groups of proteinsand peptides two representative TSC compounds (ie TSC-Me and TSC-NO

2) were incubated with glutathione and

electronic absorbance spectral analyses of air- and nitrogen-equilibrated solutions were performed (Figures 6(a) and6(b)) These TSC compounds were chosen based on thepresence of an electron donor and an electron-withdrawingp-substituent in TSC-Me and TSC-NO

2 respectively

Figure 6(a) shows that TSC-NO2and TSC-Me exhibited

time-dependent bleaching after incubation with GSH in anN2-equilibrated solution Figure 6(b) shows that the bleach-

ing of TSC-NO2is mitigated by the presence of molecular

oxygen Similar results were obtained for TSC-Me in anair-equilibrated solution (data not shown) The occurrenceof TSC bleaching is consistent with the loss of aromaticconjugation that is expected to occur if the reaction presented

in Scheme 2 occurred However in the case of TSC-Methe mechanism described for TSC-NO

2is not expected

and the bleaching could be caused by the reduction of theTSC moiety as described by Andrade and Temperini [46]The exclusive capacity of TSC-NO

2to deplete GSH at low

concentrations in association with the occurrence of celldeath exclusively by apoptosis suggests that for the nitroTSCthe mechanism described in Scheme 2 might play a role Thereduction of TSC produces thiocarbamide (thiourea) [47]whose toxic effects are well-known [48ndash50]

TSCs that possess a coordinating ring-N were previouslyreported to be efficient metal chelators due to their capacityto form a tridentate complex [51] These TSC compoundsexhibited high antiproliferative activity associated with asignificant iron (Fe) chelation ability This antiproliferativeeffect in association with iron chelation was corroboratedby probing the effect of related TSCs that do not possess acoordinating ring-N (ie acetophenone thiosemicarbazoneand acetophenone NN-dimethylthiosemicarbazone) Thesecompounds exhibited little activity The antiproliferativeeffect of metal chelator TSCs results from Fe chelation andmobilization The literature has also attributed this antipro-liferative effect to the generation of free radicals via the redoxcycling of Fe complexesThe TSC compounds investigated inthe present study do not have a coordinating ring-N in theirstructures thus these compounds can form only a bidentatecomplex as shown as an inset of Figure 6

In addition these compounds exhibit high hydrophobic-ity (log P gt 30) thatmight contribute to the permeabilizationof biological membranes Therefore synergistic effects ofmetal chelation changes in membrane fluidity and GSHdepletion might contribute to cell death

The capacity for metal chelation was tested for TSC-NO2

and TSC-Me The incubation of TSC-NO2with ferrous sul-

fate led to spectral changes for the compound (Figure 7(a))Figure 7(a) demonstrates that immediately after the additionof a molar excess of ferrous sulfate (50 120583M) 15 120583M TSC-NO2exhibited a narrowing of the band peaking at 340 nm in

association with an increase in intensity The narrowing andincreased intensity of the 340 nmband are consistent with theconversion of aggregate forms of TSC-NO

2to themonomeric

form This possibility was reinforced by the titration ofcrescent concentrations of TSC-NO

2in solution showing

that increases in TSC-NO2concentration led to a red shift

and a broadening of the 340 nm band (data not shown) Thedisaggregation of TSC-NO

2molecules after ferrous sulfate

addition is suggestive of the formation of metal complexesAftersim40min of incubation of TSC-NO

2with ferrous sulfate

the compound exhibited a progressive reduction of the bandthat peaked at 340 nm in association with an increase inthe A

240nmA

340nm ratio as shown in panel (b) These

results are consistent with the reduction of TSC compoundsby ferrous ions [46] Consistent with the formation of abidentate metal complex the TSC compounds were unableto compete with bathophenanthroline The spectrum ofbathophenanthroline obtained immediately after its additionto a fresh solution of ferrous sulfate in the absence of TSC-NO2(Figure 7(c) red line) presented the same intensity of

that obtained in the presence of the TSC compound (not


N N

S

N

O

H

H

H

H

H

HH

HH

HH

H

H

H

H

H

O

O

O

N

ON

O

H

ONH

HH

H H

HHH

H

HH

HH

O

O

O

N

ON

SO

H

ON

H

HH HH

HHH

H

HH

HH

N N

S

N

O

H

H

H

H

H

HH

HH

HHH

H

H

H

H

H

Ominus

Ominus

Ominus Ominus

Ominus

Ominus

SminusN+

N+

Scheme 2 Mechanism of the reaction of the thiol group cysteine lateral chain with a nitrophenyl moiety (based on Tiwari et al [45])

Abso

rban

ce

Wavelength (nm)

A (n

orm

aliz

ed)

Time (min)TSC-Me (304nm)TSC-NO2 (340nm)

012

008

004

000240 300 360 420 480 540

10

08

06

040000

0 2 4 6 8 10

(a)

Abso

rban

ce

Wavelength (nm)

012

008

004

000240 300 360 420 480 540

(b)

Figure 6 Reaction of TSC compounds with GSH (a) Time-dependent bleaching of 10120583M TSC-NO2incubated with 20120583M GSH in a

nitrogen-equilibrated solution (b) Time-dependent bleaching of 10 120583MTSC-NO2incubated with 20120583MGSH in an air-equilibrated solution

The inset of (a) shows the kinetics of TSC-Me (gray ball) and TSC-NO2(black ball) bleaching at 304 and 340 nm respectively in nitrogen-

equilibrated solutions

shown) However the addition of bathophenanthroline to aferrous sulfate solution incubated for 6 h with 15120583M TSC-NO2presented an absorbance intensity (Figure 7(c) black

line) consistent with a reduction of the ferrous ion content(sim15) compared to the control which was incubated for 6 hin the absence of TSC-NO

2(Figure 7(c) gray line) A 15

decrease in ferrous ion content corresponds to the oxidationof sim75 120583M of ferrous sulfate and the reduction of an equalconcentration of TSC-NO

2 This calculation is consistent

with the observed sim50 decrease in the TSC-NO2(15 120583M)

band that occurred after a 6 h incubation with ferrous sulfateThe bleaching of TSC-NO

2and TSC-Mewas also observed in

an air-equilibrated solution (data not shown) and after a 24 hincubation both of the TSC compounds exhibited intensebleachingThese results suggest that complex redox processescan occur with TSC compounds in cells The elucidation of

these processes and the identification of the products arenot the focus of the present study and these topics will beinvestigated in the future

Considering the capacity of TSC compounds to promotethe death of K562 cells it was important to investigate thecomparative toxic effects of these compounds for peripheralblood mononuclear cells (PBMNs) A comparison of thesetoxic effects was performed for cells treatedwith TSC-Me andTSC-NO

2using MTT assays Figure 8 demonstrated that in

contrast to the observations made for K562 cells (ie sim25viability) more than 50 of the PBMNs remained viableeven after a 6 h incubation with 2mM TSC-NO

2and TSC-

MeTaken together the present study demonstrated the effi-

ciency of new TSC compounds to kill K562 cells withpeculiarities for the nitrocompound TSC-NO

2 Aromatic


Abso

rban

ce

Wavelength (nm)

025

020

015

010

005

000250 300 350 400 450 500 550 wwwacdlabscom

(a)

Time (min)

A340

(nm

)

A240

nmA

340

nmra

tio

020

018

016

014

012

010

0000 50 100 150 200 250 300 350

18

16

14

12

000

(b)

Abso

rban

ce

Wavelength (nm)350 400 450 500 550 600 650

075

050

025

000

(c)

Figure 7 Spectral changes of TSC compounds associated with metal complexation (a) Spectral changes of TSC-NO2after the addition

of ferrous sulfate The red line corresponds to the initial spectrum before metal addition The structure of TSC-NO2complexed with Fe2+

is shown beside the spectrum (b) A progressive decrease of the band that peaks at 340 nm and increase of the A240

nmA340

nm ratio areassociated with the disaggregation of TSC-NO

2and the formation of metal complexes Panel (c) black line shows the spectrum obtained

when bathophenanthroline is added to themedium containing ferrous sulfate after 6 h incubation of with TSC-NO2The red line corresponds

to the spectrum of bathophenanthroline added to a fresh solution of ferrous sulfate and the gray line corresponds to the control that isbathophenanthroline added to a solution of ferrous sulphate incubated for 6 h in the absence of TSC-NO

2

nitrocompounds contain a NO2group and are used in a wide

range of therapeutic applications including antineoplasticactivity [52ndash54] The biological activity of nitrocompoundsappears to involve three important steps passage throughbiological membranes cellular metabolism and reactivitywith biomolecules Regarding the first step hydrophobicityis a characteristic shared by the four TSC compounds studiedhere Inside cells the mechanism of action was influencedby the different bulky substituents present at the R2 groupIn the case of TSC-NO

2 the predominance of apoptosis

associated with GSH depletion even at lower concentrationsof the compound is consistent with enzymatic bioreductionof the NO

2group that generate R-NO

2

∙ [55ndash57] The fate ofR-NO

2

∙ is dependent on the oxygenation state of the cellsIn aerobic conditions the reversion of R-NO

2

∙ to R-NO2

by molecular oxygen characterizes a futile cycle with thegeneration of the superoxide ion In anaerobic conditionsthe complete reduction to the amine derivative involves theformation of different intermediates including free radicalsand compounds that interact with DNA and are acceptorsfor thiol groups Therefore nitrocompounds are frequentlydescribed as efficient against solid tumors in which hypoxiais commonly present However in the present study TSC-NO2was efficient in aerobic conditions which is consistent

with the formation of superoxide ions in the futile cycleleading to GSH depletion The regeneration of TSC-NO

2in

the futile cycle is consistent with the depletion of GSH by lowconcentrations of TSC-NO

2 Another probable mechanism

that operates in aerobic conditions is the alkylation ofGSH as described for nitrocompounds [45] In this regard


Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)

TSC-Me 3h TSC-NO2 3h


100

80

60

40

2000

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(a)

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(b)

Figure 8 Viability of K562 and PBMN cells after 3 and 6 h of incubation with TSC-Me and TSC-NO2 (a) K562 and (b) PBMN cell viability

was evaluated by the MTT reduction test Cells (2 times 105 per well) were incubated with different concentrations of drug for 3 and 6 h Afterthe incubation MTT was added and the absorbance at 630 nm was measured The percentage of viable cells after treatment with TSC-Meand TSC-NO

2is expressed relative to the control (absence of TSC compounds) which was considered as 100 Results presented are means

plusmn SD of three independent experiments


this mechanism could operate for the other studied TSCcompounds that is consistent with significant GSH depletiononly at concentrations ge 50120583M The present study providedconsiderable insight into the mechanisms by which theseTSC-derivatives exert their effects and these investigationsshould be continued in future specific and detailed studies

4 Conclusion

The new TSC compounds presented here exhibit an inno-vative structure with benzyl as the R3 group phenyl asthe R2 group and methyl as the R1 group This structureendows high hydrophobicity to the molecules and favorstransport through membranes The activity of these com-pounds against K562 cells was modulated by the differentbulky substituents appended to the R2 group The presenceof the group appended to the R2 position was crucial forthe induction of cell death by the compounds All of theTSC compounds studied here promoted the disruption ofthe mitochondrial potential prior to the death of K562cells Apoptosis was favored for compounds with a lowerlog P value and compounds that were unable to chelateiron Exclusively for TSC-NO

2 cell death was associated

with metabolic effects related to GSH depletion even at thelowest concentration Therefore the effects of those TSCcompounds were modulated by the R2 group leading todifferent metabolic routes in the cells The significantly lowertoxic effects for PBMNs indicate that these compounds havepotential for use as therapeutic agents

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank FAPESP (201207456-7) CNPq (ConselhoNacional de Desenvolvimento Cientıfico e Tecnologico)CAPES (Coordenacao de Aperfeicoamento de Pessoal deNıvel Superior) and Nucleo de Bioquimica e Biotecnolo-giaUniversidade Federal do ABC

References

[1] J S Casas M S Garcıa-Tasende and J Sordo ldquoMain groupmetal complexes of semicarbazones and thiosemicarbazones Astructural reviewrdquoCoordinationChemistry Reviews vol 209 no1 pp 197ndash261 2000

[2] H Beraldo ldquoSemicarbazones and thiosemicarbazones theirwide pharmacological profile and clinical applicationsrdquoQuımica Nova vol 27 no 3 pp 461ndash471 2004

[3] H Beraldo and D Gambino ldquoThe wide pharmacologicalversatility of semicarbazones thiosemicarbozones and theirmetal complexesrdquoMini-Reviews in Medicinal Chemistry vol 4no 1 pp 31ndash39 2004

[4] S Cunha and T L D Silva ldquoOne-pot and catalyst-free synthesisof thiosemicarbazones viamulticomponent coupling reactionsrdquoTetrahedron Letters vol 50 no 18 pp 2090ndash2093 2009

[5] R P Tenorio A J S Goes J G De Lima A R De Faria A JAlves and T M De Aquino ldquoThiosemicarbazones preparationmethods synthetic applications and biological importancerdquoQuimica Nova vol 28 no 6 pp 1030ndash1037 2005

[6] T R Todorovic A Bacchi D M Sladic et al ldquoSynthesischaracterization and biological activity evaluation of Pt(II)Pd(II) Co(III) and Ni(II) complexes with N-heteroaromaticselenosemicarbazonesrdquo Inorganica Chimica Acta vol 362 no10 pp 3813ndash3820 2009

[7] A P da Silva M V Martini C M A de Oliveira et al ldquoAnti-tumor activity of (-)-120572-bisabolol-based thiosemicarbazonesagainst human tumor cell linesrdquo European Journal of MedicinalChemistry vol 45 no 7 pp 2987ndash2993 2010

[8] M A Soares J A Lessa I C Mendes et al ldquoN 4-Phenyl-substituted 2-acetylpyridine thiosemicarbazones cytotoxicityagainst human tumor cells structure-activity relationship stud-ies and investigation on themechanism of actionrdquo Bioorganic ampMedicinal Chemistry vol 20 no 11 pp 3396ndash3409 2012

[9] C R Kowol R Eichinger M A Jakupec M Galanski V BArion and B K Keppler ldquoEffect of metal ion complexationand chalcogen donor identity on the antiproliferative activityof 2-acetylpyridine NN-dimethyl(chalcogen)semicarbazonesrdquoJournal of Inorganic Biochemistry vol 101 no 11-12 pp 1946ndash1957 2007

[10] Y Teitz D Ronen A Vansover T Stematsky and J L RiggsldquoInhibition of human immunodeficiency virus by n-methy-lisatin-beta4101584041015840-diethylthiosemicarbazone and N-allylisatin-beta-4101584041015840-diallythiosemicarbazonerdquoAntiviral Research vol 24no 4 pp 305ndash314 1994

[11] D H Rich J Green M V Toth G R Marshall and S B HKent ldquoHydroxyethylamine analogues of the p17p24 substratecleavage site are tight-binding inhibitors of HIV proteaserdquoJournal of Medicinal Chemistry vol 33 no 5 pp 1285ndash12881990

[12] N C Kasuga K Sekino M Ishikawa et al ldquoSynthesisstructural characterization and antimicrobial activities of 12zinc(II) complexes with four thiosemicarbazone and two semi-carbazone ligandsrdquo Journal of Inorganic Biochemistry vol 96no 2-3 pp 298ndash310 2003

[13] N Bharti K Husain M T Gonzalez Garza et al ldquoSynthe-sis and in vitro antiprotozoal activity of 5-nitrothiophene-2-carboxaldehyde thiosemicarbazone derivativesrdquo Bioorganic ampMedicinal Chemistry Letters vol 12 no 23 pp 3475ndash3478 2002

[14] M C Pirrung S V Pansare K D Sarma K A Keithand E R Kern ldquoCombinatorial optimization of isatin-120573-thiosemicarbazones as anti-poxvirus agentsrdquo Journal of Medic-inal Chemistry vol 48 no 8 pp 3045ndash3050 2005

[15] K Kadaba Pankaja and Z Lin ldquoHydrazones hydrazinessemicarbazones and thiosemicarbazones derived from pyridylketones as anticonvulsant drugs and excitatory amino acidantagonistsrdquo 1997

[16] R A Finch M-C Liu S P Grill et al ldquoTriapine (3-aminop-yridine-2-carboxaldehyde-thiosemicarbazone) a potent inhi-bitor of ribonucleotide reductase activity with broad spectrumantitumor activityrdquoBiochemical Pharmacology vol 59 no 8 pp983ndash991 2000

[17] VOpletalova D S KalinowskiMVejsova et al ldquoIdentificationand characterization of thiosemicarbazoneswith antifungal andantitumor effects cellular iron chelation mediating cytotoxicactivityrdquo Chemical Research in Toxicology vol 21 no 9 pp1878ndash1889 2008


[18] J M Myers Q Cheng W E Antholine et al ldquoRedox acti-vation of Fe(III)-thiosemicarbazones and Fe(III)-bleomycin bythioredoxin reductase specificity of enzymatic redox centersand analysis of reactive species formation by ESR spin trappingrdquoFree Radical Biology amp Medicine vol 60 pp 183ndash194 2013

[19] Y Yu D S Kalinowski Z Kovacevic et al ldquoThiosemicar-bazones from the old to new iron chelators that are more thanjust ribonucleotide reductase inhibitorsrdquo Journal of MedicinalChemistry vol 52 no 17 pp 5271ndash5294 2009

[20] Z-G Jiang M S Lebowitz and H A Ghanbari ldquoNeuropro-tective activity of 3-aminopyridine-2-carboxaldehyde thiosemi-carbazone (PAN-811) a cancer therapeutic agentrdquo CNS DrugReviews vol 12 no 1 pp 77ndash90 2006

[21] R-W Chen C Yao X C M Lu et al ldquoPAN-811 (3-Aminopyridine-2-carboxaldehyde thiosemicarbazone) a novelneuroprotectant elicits its function in primary neuronal cul-tures by up-regulating Bcl-2 expressionrdquo Neuroscience vol 135no 1 pp 191ndash201 2005

[22] R-WChen X-CM Lu C Yao et al ldquoPAN-811 provides neuro-protection against glutamate toxicity by suppressing activationof JNK and p38MAPKrdquoNeuroscience Letters vol 422 no 1 pp64ndash67 2007

[23] D Cavallini M T Graziani and S Dupre ldquoDetermination ofdisulphide groups in proteinsrdquo Nature vol 212 no 5059 pp294ndash295 1966

[24] P J Hissin and R Hilf ldquoA fluorometric method for determina-tion of oxidized and reduced glutathione in tissuesrdquo AnalyticalBiochemistry vol 74 no 1 pp 214ndash226 1976

[25] K R Watts J Ratnam K-H Ang et al ldquoAssessing the try-panocidal potential of natural and semi-synthetic diketopiper-azines from two deep water marine-derived fungirdquo Bioorganicamp Medicinal Chemistry vol 18 no 7 pp 2566ndash2574 2010

[26] M S Sarma S Mazumder D Ghosh A Roy A Duthieand E R T Tiekink ldquoSynthesis spectroscopic characteriza-tion and biocidal activity of some diorganotin(IV) comp-lexes of salicylaldehydethiosemicarbazones and relatedligands Molecular and supramolecular structures of[R2Sn(OArCH=NminusN-CSNH

2)] where R = Me Ph and Ar =

minusC6H4minusC6H3(5-Br) andC

6H3(5-Cl) and of [Me

2SnOC

6H3(5-

Br)CH=NminusN=CSNH2]sdotOH2rdquo Applied Organometallic Chem-

istry vol 21 no 10 pp 890ndash905 2007[27] J Stariat V Sestak K Vavrova et al ldquoLC-MSMS identification

of the principal in vitro and in vivo phase i metabolites of thenovel thiosemicarbazone anti-cancer drug Bp4eTrdquo Analyticaland Bioanalytical Chemistry vol 403 no 1 pp 309ndash321 2012

[28] D Wlodkowic W Telford J Skommer and Z DarzynkiewiczldquoApoptosis and beyond cytometry in studies of programmedcell deathrdquoMethods in Cell Biology vol 103 pp 55ndash98 2011

[29] Z Darzynkiewicz G Juan X Li W Gorczyca T Murakamiand F Traganos ldquoCytometry in cell necrobiology analysis ofapoptosis and accidental cell death (necrosis)rdquo Cytometry vol27 no 1 pp 1ndash20 1997

[30] C H E Homburg M De Haas A E G K Von dem Borne AJ Verhoeven C P M Reutelingsperger and D Roos ldquoHumanneutrophils lose their surface Fc120574RIII and acquire Annexin Vbinding sites during apoptosis in vitrordquo Blood vol 85 no 2 pp532ndash540 1995

[31] H M Hanauske-Abel D Saxena P E Palumbo et al ldquoDrug-induced reactivation of apoptosis abrogates HIV-1 infectionrdquoPLoS ONE vol 8 no 9 Article ID e74414 2013

[32] T Yin Y-L Wu H-P Sun et al ldquoCombined effects of As4S4

and imatinib on chronic myeloid leukemia cells and BCR-ABLoncoproteinrdquo Blood vol 104 no 13 pp 4219ndash4225 2004

[33] J A Lessa M A Soares R G dos Santos et al ldquoGallium(III)complexes with 2-acetylpyridine-derived thiosemicarbazonesantimicrobial and cytotoxic effects and investigation on theinteractions with tubulinrdquo Biometals vol 26 pp 151ndash165 2013

[34] S Tardito O Bussolati M Maffini et al ldquoThioamido coordi-nation in a thioxo-124-triazole copper(II) complex enhancesnonapoptotic programmed cell death associated with copperaccumulation and oxidative stress in human cancer cellsrdquoJournal of Medicinal Chemistry vol 50 no 8 pp 1916ndash19242007

[35] E Galeano E Nieto A I Garcıa-Perez M D Delgado MPinilla and P Sancho ldquoEffects of the antitumoural dequaliniumon NB4 and K562 human leukemia cell lines mitochondrialimplication in cell deathrdquo Leukemia Research vol 29 no 10 pp1201ndash1211 2005

[36] A P Rebolledo M Vieites D Gambino et al ldquoPalladium(II)complexes of 2-benzoylpyridine-derived thiosemicarbazonesspectral characterization structural studies and cytotoxic activ-ityrdquo Journal of Inorganic Biochemistry vol 99 no 3 pp 698ndash706 2005

[37] R K Emaus R Grunwald and J J Lemasters ldquoRhodamine123 as a probe of transmembrane potential in isolated rat-livermitochondria spectral andmetabolic propertiesrdquo Biochimica etBiophysica Acta vol 850 no 3 pp 436ndash448 1986

[38] L V Johnson M L Walsh B J Bockus and L B ChenldquoMonitoring of relative mitochondrial membrane potential inliving cells by fluorescence microscopyrdquo The Journal of CellBiology vol 88 no 3 pp 526ndash535 1981

[39] S Goldstein and L B Korczack ldquoStatus of mitochondria inliving human fibroblasts during growth and senescence in vitrouse of the laser dye rhodamine 123rdquoThe Journal of Cell Biologyvol 91 no 2 pp 392ndash398 1981

[40] J E OrsquoConnor J L Vargas B F Kimler J Hernandez-Yago andS Grisolia ldquoUse of Rhodamine 123 to investigate alterations inmitochondrial activity in isolated mouse liver mitochondriardquoBiochemical and Biophysical Research Communications vol 151no 1 pp 568ndash573 1988

[41] P X Petit J E OrsquoConnor D Grunwald and S C BrownldquoAnalysis of the membrane potential of rat- and mouse-livermitochondria by flow cytometry and possible applicationsrdquoEuropean Journal of Biochemistry vol 194 no 2 pp 389ndash3971990

[42] G Juan M Cavazzoni G T Saez and J E OrsquoConnor ldquoAfast kinetic method for assessing mitochondrial membranepotential in isolated hepatocytes with rhodamine 123 and flowcytometrylrdquo Cytometry vol 15 no 4 pp 335ndash342 1994

[43] I L Nantes T Rodrigues A C F Caires et al ldquoSpecific effectsof reactive thiol drugs on mitochondrial bioenergeticsrdquo Journalof Bioenergetics and Biomembranes vol 43 no 1 pp 11ndash18 2011

[44] K Reybier P Perio G Ferry et al ldquoInsights into the redox cycleof human quinone reductase 2rdquo Free Radical Research vol 45no 10 pp 1184ndash1195 2011

[45] R Tiwari G C Moraski V Krchnak et al ldquoThiolates chem-ically induce redox activation of BTZ043 and related potentnitroaromatic anti-tuberculosis agentsrdquo Journal of the AmericanChemical Society vol 135 no 9 pp 3539ndash3549 2013

[46] G F S Andrade and M L A Temperini ldquoThe adsorptionand faradaic processes of formylferrocene thiosemicarbazone


monitored by in situ SERS and UV-VIS spectroscopiesrdquo Journalof Solid State Electrochemistry vol 11 no 11 pp 1497ndash1503 2007

[47] D M Ziegler ldquoIntermediate metabolites of thiocarbamidesthioureylenes and thioamides mechanism of formation andreactivityrdquoBiochemical Society Transactions vol 6 no 1 pp 94ndash96 1978

[48] K Takegawa K Mitsumori H Onodera et al ldquoUDP-GTinvolvement in the enhancement of cell proliferation in thyroidfollicular cell proliferative lesions in rats treated with thioureaand vitamin Ardquo Archives of Toxicology vol 71 no 11 pp 661ndash667 1997

[49] K Mitsumori H Onodera M Takahashi et al ldquoPromotingeffect of large amounts of vitamin A on cell proliferation ofthyroid proliferative lesions induced by simultaneous treatmentwith thioureardquo Cancer Letters vol 103 no 1 pp 19ndash31 1996

[50] DFG ldquoCommission for the investigation of health hazards ofchemical compounds in the work area Deustche Forschungs-gemeinschaft list of MAK and BAT values maximum concen-trations and biological tolerance values at the workplacerdquo TechRep 4 Wiley-VCH Bonn Germany 2012

[51] D S Kalinowski Y Yu P C Sharpe et al ldquoDesign syn-thesis and characterization of novel iron chelators structure-activity relationships of the 2-benzoylpyridine thiosemicar-bazone series and their 3-nitrobenzoyl analogues as potentantitumor agentsrdquo Journal of Medicinal Chemistry vol 50 no15 pp 3716ndash3729 2007

[52] W Raether and H Hanel ldquoNitroheterocyclic drugs with broadspectrum activityrdquo Parasitology Research vol 90 no 1 pp S19ndashS39 2003

[53] AKorolkovas and JH BurckhalterQuımica Farmaceutica vol1 Guanabara Koogan Rio de Janeiro Brasil 1988

[54] P Herrlich andM Schweiger ldquoNitrofurans a group of syntheticantibiotics with a new mode of action discrimination ofspecific messenger RNA classesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 73 no10 pp 3386ndash3390 1976

[55] J H Tocher ldquoReductive activation of nitroheterocyclic com-poundsrdquoGeneral Pharmacology vol 28 no 4 pp 485ndash487 1997

[56] C E Viode N Bettache N Cenas et al ldquoEnzymatic reductionstudies of nitroheterocyclesrdquo Biochemical Pharmacology vol 57no 5 pp 549ndash557 1999

[57] J D Maya B K Cassels P Iturriaga-Vasquez et al ldquoMode ofaction of natural and synthetic drugs againstTrypanosoma cruziand their interaction with the mammalian hostrdquo ComparativeBiochemistry and Physiology Part A Molecular amp IntegrativePhysiology vol 146 no 4 pp 601ndash620 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


N N

S

H N

H

123

4R1

R2R3

Figure 1 General structure of thiosemicarbazones with C=N-NH-CS-NH moiety R1 R2 and R3 = alkyl or aryl groups

(ie di-2-pyridyl ketone thiosemicarbazone (DpT)) and 2-benzoylpyridine thiosemicarbazone (BpT) classes In addi-tion to this nonspecific chelator effect a specific activity hasbeen associated with the antitumor activity of TSCs theinhibition of ribonucleotide reductase which is an enzymethat is required for the de novo synthesis of deoxyribonu-cleotides and DNA replication and repair [19] 3-AP is awell-known example of this class of TSC compounds 3-AP has also been suggested to act as a neuroprotectantduring the treatment of neurodegenerative diseases [20ndash22]Regardless of the mechanisms responsible for the diversetherapeutic effects of TSCs these compounds are dependenton drug transfer through the plasma membrane Thereforethe hydrophobic R groups and bulky substituent at the p-position of the rings are important for cytotoxicity In thepresent study we present a series of TSCs with benzyl asthe R3 group p-substituted phenyl as the R2 group andmethyl as the R1 group These groups endow the moleculeswith a high hydrophobicity and favor transport throughmembranes Furthermore the R2 group was appended withdifferent substituents to confer different properties to themolecules (Scheme 1) These TSC compounds were tested onK562 and peripheral blood mononuclear (PBMN) cells todetermine their capacity to induce cell death and identifytheir effects on mitochondrial bioenergetics and thiol redoxbalance

2 Materials and Methods

21 General Chromatography was performed on silica gelMerck 230ndash400 mesh ASTM All melting points were deter-mined using a Microquimica model MQAPF-301 appa-ratus The high-resolution electron spray ionization massspectrometry (HRESIMS) analyses were performed on aQTOF Micro (Waters Manchester UK) mass spectrometerequipped with an ESI source Proton nuclear magneticresonance (1HNMR) spectra were recorded using CDCl

3as a

solvent at ambient temperature using a Varian Mercury Plus(300MHz) with TMS as an internal standard The chemicalshifts (120575) are given in parts per million relative to TMSCarbon-13 nuclear magnetic resonance (13C NMR) spectrawere recorded at 755MHz with the same internal standard

22 Synthesis

221 N(4)-Benzyl-thiosemicarbazide (2) To a solution of 1(500 g 335mmol) in ethanol (EtOH) (15mL) was addedhydrazine (107 g 335mmol) The reaction mixture wasstirred at 90∘C for 12 h The resulting solid was collected by

filtration rinsed with cold EtOH and recrystallized fromethyl acetatemethanol (EtOAc)MeOH (2 1) to afford 2(580 g 95) as white crystals 1HNMR (300MHz dimethylsulfoxide-D6) 120575 876 (br s 1H NH) 830 (br t J = 63Hz1H NHCH

2) 733ndash719 (m 5H Ar H) 471 (d J = 63Hz 2H

NHCH2) 452 (br s 2H NH

2) 13CNMR (75MHz dimethyl

sulfoxide-D6)120575 1826 1391 1280 1273 1266 469NMRdataare in agreement with literature [5 7]

222 General Procedure for the Synthesis and Characteri-zation of Compound 3andash3e (Scheme 1) To a solution of 2(030 g 165mmol) in ethanol was added with stirring asolution of ketone (165mmol) and drops of an aqueoussolution of sulfuric acid (50) The mixture was stirred atroom temperature for 12 h and then was concentrated invacuum The residue was partitioned between CHCl

3and

10 NaHCO3aqueous solution and purified by silica gel

column chromatography to afford 3andash3e [5 7]

223 N(1)-Methylphenyl Ketone-N(4)-benzyl-thiosemicarba-zone-3a (TSC-H) Starting from 2 and methylphenyl ketonethe procedures summarized above provided the title com-pound in 61 yield (028 g) as white crystals after purificationby column chromatography (silica gel CHCl

3EtOAc 100 0

to 2 1) 1H NMR (300MHz CDCl3) 120575 876 (br s 1H NH)

792 (br t J = 6Hz 1H NHCH2) 766ndash763 (m 2H Ar H)

740ndash726 (m 8H Ar H) 498 (d J = 6Hz 2H NHCH2) 227

(s 3H CH3) 13C NMR (75MHz CDCl

3) 120575 1786 (C3) 1472

(C11015840) 1377 (C31015840) 1375 (C6) 1299 (C51015840 and C71015840) 1289 (C8and C10) 1287 (C9) 1280 (C7 and C11) 1265 (C41015840 and C81015840)486 (C5) 138 (C21015840) mp 1574minus1597∘C HRMS (M + H+)2841218 C


224 N(1)-Methyl-para-fluorophenylketone-N(4)-benzyl-thi-osemicarbazone-3b (TSC-F) Starting from 2 and methyl-para-fluorophenylketone the procedures summarized aboveprovided the title compound in 54 yield (027 g) as a whitesolid after purification by column chromatography (silica gelCHCl


3) 120575

882 (br s 1H NH) 788 (br t J = 57Hz 1H NHCH2) 764

(dd J = 90 and JH-F = 51Hz 2H Ar H) 740ndash726 (m 5HAr H) 704 (dd J = 90 and JH-F = 90Hz 2H Ar H) 498(d J = 57Hz 2H NHCH

2) 226 (s 3H CH

3) 13C NMR

(75MHzCDCl3)120575 1785 (C3) 1638 (d JC-F= 2492HzC61015840)

1463 (C11015840) 1377 (C6) 1336 (d JC-F = 38Hz C31015840) 1289(C8 C9 and C10) 1284 (d JC-F = 83Hz C41015840 and C81015840) 1278(C7 and C11) 1157 (d JC-F = 217Hz C51015840 and C71015840) 485(C5) 139 (C21015840) mp 1119minus1145∘CHRMS (M+H+) 3021120C16H17FN3S+ requires 3021122

225 N(1)-Methyl-para-chlorophenylketone-N(4)-benzyl-thi-osemicarbazone-3c (TSC-Cl) Starting from 2 and methyl-para-chlorophenylketone the procedures summarized aboveprovided the title compound in 47 yield (025 g) as a whitesolid after purification by column chromatography (silica gelCHCl


3) 120575

873 (br s 1H NH) 786 (br t J = 6Hz 1H NHCH2) 758 (d

J = 87Hz 2H Ar H) 740ndash728 (m 7H Ar H) 498 (d J =


NCS N

S

H 6

11

7

10

8

9

5 43 N

2S

H

H N 1

R

N

H

Na b


1 23

1998400

2998400

3998400

4998400

5998400

69984007998400

8998400

9998400


3e (R=NO2) TSC-NO2

CH3NH2




3) 13C NMR (75MHz

CDCl3) 120575 1786 (C3) 1460 (C11015840) 1376 (C6) 1360 (C31015840) 1359



3180826






2) 234 (s 3H Ar-CH

3) 223 (s 3H CH

3) 13C NMR

(75MHz CDCl3) 120575 1783 (C3) 1474 (C11015840) 1400 (C61015840) 1377











3) 120575 1787 (C3)















3a(TSC-H) C16H18N3S+ 2841216 2841218 070




3e(TSC-NO2) C16H17N4O2S+ 3291067 3291071 122














6(3)) incubated at

37∘C for 30min








2 respectively The








2








119898


2 However at the






419

Cell

s (

)80

60

40

20

0

Con

trol

TSC-

Cl001

mM

TSC-

Cl01

mM

TSC-

Cl1

mM

(a)

389

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

Me

001

mM

TSC-

Me

01

mM

TSC-

Me

1m

M

(b)

365

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

F001

mM

TSC-

F01

mM

TSC-

F1

mM

(c)

343

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

H001

mM

TSC-

H01

mM

TSC-

H1

mM

(d)

339

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

NO

2

001

mM

TSC-

NO

201

mM

TSC-

NO

21

mM

(e)





100120583m

(a)

TSC-Cl

TSC-Me

TSC-F

100120583m

(b)

100120583m

TSC-H

TSC-NO2

(c)

TSC-Cl TSC-Me TSC-F

TSC-H TSC-NO2

100 120583m

100120583m 100120583m

(d)


119898(DiOC








2















TSC (120583M)

TSC-Cl

Relat

ive (

)

10

05

000 10 100 1000

(a)Re

lativ

e (

)

TSC-Me10

05

00

TSC (120583M)

0 10 100 1000

(b)

TSC-F

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(c)

TSC-H

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(d)

TSC-NO2

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(e)



wwwacdlabscom







2 respectively






2is not expected










2to themonomeric









240nmA





N N

S

N

O

H

H

H

H

H

HH

HH

HH

H

H

H

H

H

O

O

O

N

ON

O

H

ONH

HH

H H

HHH

H

HH

HH

O

O

O

N

ON

SO

H

ON

H

HH HH

HHH

H

HH

HH

N N

S

N

O

H

H

H

H

H

HH

HH

HHH

H

H

H

H

H

Ominus

Ominus

Ominus Ominus

Ominus

Ominus

SminusN+

N+


Abso

rban

ce

Wavelength (nm)

A (n

orm

aliz

ed)


012

008

004

000240 300 360 420 480 540

10

08

06

040000

0 2 4 6 8 10

(a)

Abso

rban

ce

Wavelength (nm)

012

008

004

000240 300 360 420 480 540

(b)


















2and TSC-



2 Aromatic


Abso

rban

ce

Wavelength (nm)

025

020

015

010

005

000250 300 350 400 450 500 550 wwwacdlabscom

(a)

Time (min)

A340

(nm

)

A240

nmA

340

nmra

tio

020

018

016

014

012

010

0000 50 100 150 200 250 300 350

18

16

14

12

000

(b)

Abso

rban

ce

Wavelength (nm)350 400 450 500 550 600 650

075

050

025

000

(c)




nmA340





2






2


2


2

∙ to R-NO2



2in





Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

2000

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(a)

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(b)







4 Conclusion






Acknowledgments


References































2SnOC

6H3(5-










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NCS N

S

H 6

11

7

10

8

9

5 43 N

2S

H

H N 1

R

N

H

Na b


1 23

1998400

2998400

3998400

4998400

5998400

69984007998400

8998400

9998400


3e (R=NO2) TSC-NO2

CH3NH2




3) 13C NMR (75MHz

CDCl3) 120575 1786 (C3) 1460 (C11015840) 1376 (C6) 1360 (C31015840) 1359



3180826






2) 234 (s 3H Ar-CH

3) 223 (s 3H CH

3) 13C NMR

(75MHz CDCl3) 120575 1783 (C3) 1474 (C11015840) 1400 (C61015840) 1377











3) 120575 1787 (C3)















3a(TSC-H) C16H18N3S+ 2841216 2841218 070




3e(TSC-NO2) C16H17N4O2S+ 3291067 3291071 122














6(3)) incubated at

37∘C for 30min








2 respectively The








2








119898


2 However at the






419

Cell

s (

)80

60

40

20

0

Con

trol

TSC-

Cl001

mM

TSC-

Cl01

mM

TSC-

Cl1

mM

(a)

389

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

Me

001

mM

TSC-

Me

01

mM

TSC-

Me

1m

M

(b)

365

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

F001

mM

TSC-

F01

mM

TSC-

F1

mM

(c)

343

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

H001

mM

TSC-

H01

mM

TSC-

H1

mM

(d)

339

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

NO

2

001

mM

TSC-

NO

201

mM

TSC-

NO

21

mM

(e)





100120583m

(a)

TSC-Cl

TSC-Me

TSC-F

100120583m

(b)

100120583m

TSC-H

TSC-NO2

(c)

TSC-Cl TSC-Me TSC-F

TSC-H TSC-NO2

100 120583m

100120583m 100120583m

(d)


119898(DiOC








2















TSC (120583M)

TSC-Cl

Relat

ive (

)

10

05

000 10 100 1000

(a)Re

lativ

e (

)

TSC-Me10

05

00

TSC (120583M)

0 10 100 1000

(b)

TSC-F

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(c)

TSC-H

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(d)

TSC-NO2

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(e)



wwwacdlabscom







2 respectively






2is not expected










2to themonomeric









240nmA





N N

S

N

O

H

H

H

H

H

HH

HH

HH

H

H

H

H

H

O

O

O

N

ON

O

H

ONH

HH

H H

HHH

H

HH

HH

O

O

O

N

ON

SO

H

ON

H

HH HH

HHH

H

HH

HH

N N

S

N

O

H

H

H

H

H

HH

HH

HHH

H

H

H

H

H

Ominus

Ominus

Ominus Ominus

Ominus

Ominus

SminusN+

N+


Abso

rban

ce

Wavelength (nm)

A (n

orm

aliz

ed)


012

008

004

000240 300 360 420 480 540

10

08

06

040000

0 2 4 6 8 10

(a)

Abso

rban

ce

Wavelength (nm)

012

008

004

000240 300 360 420 480 540

(b)


















2and TSC-



2 Aromatic


Abso

rban

ce

Wavelength (nm)

025

020

015

010

005

000250 300 350 400 450 500 550 wwwacdlabscom

(a)

Time (min)

A340

(nm

)

A240

nmA

340

nmra

tio

020

018

016

014

012

010

0000 50 100 150 200 250 300 350

18

16

14

12

000

(b)

Abso

rban

ce

Wavelength (nm)350 400 450 500 550 600 650

075

050

025

000

(c)




nmA340





2






2


2


2

∙ to R-NO2



2in





Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

2000

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(a)

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(b)







4 Conclusion






Acknowledgments


References































2SnOC

6H3(5-










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















3a(TSC-H) C16H18N3S+ 2841216 2841218 070




3e(TSC-NO2) C16H17N4O2S+ 3291067 3291071 122














6(3)) incubated at

37∘C for 30min








2 respectively The








2








119898


2 However at the






419

Cell

s (

)80

60

40

20

0

Con

trol

TSC-

Cl001

mM

TSC-

Cl01

mM

TSC-

Cl1

mM

(a)

389

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

Me

001

mM

TSC-

Me

01

mM

TSC-

Me

1m

M

(b)

365

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

F001

mM

TSC-

F01

mM

TSC-

F1

mM

(c)

343

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

H001

mM

TSC-

H01

mM

TSC-

H1

mM

(d)

339

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

NO

2

001

mM

TSC-

NO

201

mM

TSC-

NO

21

mM

(e)





100120583m

(a)

TSC-Cl

TSC-Me

TSC-F

100120583m

(b)

100120583m

TSC-H

TSC-NO2

(c)

TSC-Cl TSC-Me TSC-F

TSC-H TSC-NO2

100 120583m

100120583m 100120583m

(d)


119898(DiOC








2















TSC (120583M)

TSC-Cl

Relat

ive (

)

10

05

000 10 100 1000

(a)Re

lativ

e (

)

TSC-Me10

05

00

TSC (120583M)

0 10 100 1000

(b)

TSC-F

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(c)

TSC-H

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(d)

TSC-NO2

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(e)



wwwacdlabscom







2 respectively






2is not expected










2to themonomeric









240nmA





N N

S

N

O

H

H

H

H

H

HH

HH

HH

H

H

H

H

H

O

O

O

N

ON

O

H

ONH

HH

H H

HHH

H

HH

HH

O

O

O

N

ON

SO

H

ON

H

HH HH

HHH

H

HH

HH

N N

S

N

O

H

H

H

H

H

HH

HH

HHH

H

H

H

H

H

Ominus

Ominus

Ominus Ominus

Ominus

Ominus

SminusN+

N+


Abso

rban

ce

Wavelength (nm)

A (n

orm

aliz

ed)


012

008

004

000240 300 360 420 480 540

10

08

06

040000

0 2 4 6 8 10

(a)

Abso

rban

ce

Wavelength (nm)

012

008

004

000240 300 360 420 480 540

(b)


















2and TSC-



2 Aromatic


Abso

rban

ce

Wavelength (nm)

025

020

015

010

005

000250 300 350 400 450 500 550 wwwacdlabscom

(a)

Time (min)

A340

(nm

)

A240

nmA

340

nmra

tio

020

018

016

014

012

010

0000 50 100 150 200 250 300 350

18

16

14

12

000

(b)

Abso

rban

ce

Wavelength (nm)350 400 450 500 550 600 650

075

050

025

000

(c)




nmA340





2






2


2


2

∙ to R-NO2



2in





Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

2000

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(a)

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(b)







4 Conclusion






Acknowledgments


References































2SnOC

6H3(5-










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















2 respectively The








2








119898


2 However at the






419

Cell

s (

)80

60

40

20

0

Con

trol

TSC-

Cl001

mM

TSC-

Cl01

mM

TSC-

Cl1

mM

(a)

389

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

Me

001

mM

TSC-

Me

01

mM

TSC-

Me

1m

M

(b)

365

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

F001

mM

TSC-

F01

mM

TSC-

F1

mM

(c)

343

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

H001

mM

TSC-

H01

mM

TSC-

H1

mM

(d)

339

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

NO

2

001

mM

TSC-

NO

201

mM

TSC-

NO

21

mM

(e)





100120583m

(a)

TSC-Cl

TSC-Me

TSC-F

100120583m

(b)

100120583m

TSC-H

TSC-NO2

(c)

TSC-Cl TSC-Me TSC-F

TSC-H TSC-NO2

100 120583m

100120583m 100120583m

(d)


119898(DiOC








2















TSC (120583M)

TSC-Cl

Relat

ive (

)

10

05

000 10 100 1000

(a)Re

lativ

e (

)

TSC-Me10

05

00

TSC (120583M)

0 10 100 1000

(b)

TSC-F

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(c)

TSC-H

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(d)

TSC-NO2

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(e)



wwwacdlabscom







2 respectively






2is not expected










2to themonomeric









240nmA





N N

S

N

O

H

H

H

H

H

HH

HH

HH

H

H

H

H

H

O

O

O

N

ON

O

H

ONH

HH

H H

HHH

H

HH

HH

O

O

O

N

ON

SO

H

ON

H

HH HH

HHH

H

HH

HH

N N

S

N

O

H

H

H

H

H

HH

HH

HHH

H

H

H

H

H

Ominus

Ominus

Ominus Ominus

Ominus

Ominus

SminusN+

N+


Abso

rban

ce

Wavelength (nm)

A (n

orm

aliz

ed)


012

008

004

000240 300 360 420 480 540

10

08

06

040000

0 2 4 6 8 10

(a)

Abso

rban

ce

Wavelength (nm)

012

008

004

000240 300 360 420 480 540

(b)


















2and TSC-



2 Aromatic


Abso

rban

ce

Wavelength (nm)

025

020

015

010

005

000250 300 350 400 450 500 550 wwwacdlabscom

(a)

Time (min)

A340

(nm

)

A240

nmA

340

nmra

tio

020

018

016

014

012

010

0000 50 100 150 200 250 300 350

18

16

14

12

000

(b)

Abso

rban

ce

Wavelength (nm)350 400 450 500 550 600 650

075

050

025

000

(c)




nmA340





2






2


2


2

∙ to R-NO2



2in





Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

2000

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(a)

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(b)







4 Conclusion






Acknowledgments


References































2SnOC

6H3(5-










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















419

Cell

s (

)80

60

40

20

0

Con

trol

TSC-

Cl001

mM

TSC-

Cl01

mM

TSC-

Cl1

mM

(a)

389

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

Me

001

mM

TSC-

Me

01

mM

TSC-

Me

1m

M

(b)

365

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

F001

mM

TSC-

F01

mM

TSC-

F1

mM

(c)

343

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

H001

mM

TSC-

H01

mM

TSC-

H1

mM

(d)

339

Cell

s (

)

100

80

60

40

20

0

Con

trol

TSC-

NO

2

001

mM

TSC-

NO

201

mM

TSC-

NO

21

mM

(e)





100120583m

(a)

TSC-Cl

TSC-Me

TSC-F

100120583m

(b)

100120583m

TSC-H

TSC-NO2

(c)

TSC-Cl TSC-Me TSC-F

TSC-H TSC-NO2

100 120583m

100120583m 100120583m

(d)


119898(DiOC








2















TSC (120583M)

TSC-Cl

Relat

ive (

)

10

05

000 10 100 1000

(a)Re

lativ

e (

)

TSC-Me10

05

00

TSC (120583M)

0 10 100 1000

(b)

TSC-F

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(c)

TSC-H

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(d)

TSC-NO2

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(e)



wwwacdlabscom







2 respectively






2is not expected










2to themonomeric









240nmA





N N

S

N

O

H

H

H

H

H

HH

HH

HH

H

H

H

H

H

O

O

O

N

ON

O

H

ONH

HH

H H

HHH

H

HH

HH

O

O

O

N

ON

SO

H

ON

H

HH HH

HHH

H

HH

HH

N N

S

N

O

H

H

H

H

H

HH

HH

HHH

H

H

H

H

H

Ominus

Ominus

Ominus Ominus

Ominus

Ominus

SminusN+

N+


Abso

rban

ce

Wavelength (nm)

A (n

orm

aliz

ed)


012

008

004

000240 300 360 420 480 540

10

08

06

040000

0 2 4 6 8 10

(a)

Abso

rban

ce

Wavelength (nm)

012

008

004

000240 300 360 420 480 540

(b)


















2and TSC-



2 Aromatic


Abso

rban

ce

Wavelength (nm)

025

020

015

010

005

000250 300 350 400 450 500 550 wwwacdlabscom

(a)

Time (min)

A340

(nm

)

A240

nmA

340

nmra

tio

020

018

016

014

012

010

0000 50 100 150 200 250 300 350

18

16

14

12

000

(b)

Abso

rban

ce

Wavelength (nm)350 400 450 500 550 600 650

075

050

025

000

(c)




nmA340





2






2


2


2

∙ to R-NO2



2in





Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

2000

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(a)

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(b)







4 Conclusion






Acknowledgments


References































2SnOC

6H3(5-










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100120583m

(a)

TSC-Cl

TSC-Me

TSC-F

100120583m

(b)

100120583m

TSC-H

TSC-NO2

(c)

TSC-Cl TSC-Me TSC-F

TSC-H TSC-NO2

100 120583m

100120583m 100120583m

(d)


119898(DiOC








2















TSC (120583M)

TSC-Cl

Relat

ive (

)

10

05

000 10 100 1000

(a)Re

lativ

e (

)

TSC-Me10

05

00

TSC (120583M)

0 10 100 1000

(b)

TSC-F

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(c)

TSC-H

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(d)

TSC-NO2

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(e)



wwwacdlabscom







2 respectively






2is not expected










2to themonomeric









240nmA





N N

S

N

O

H

H

H

H

H

HH

HH

HH

H

H

H

H

H

O

O

O

N

ON

O

H

ONH

HH

H H

HHH

H

HH

HH

O

O

O

N

ON

SO

H

ON

H

HH HH

HHH

H

HH

HH

N N

S

N

O

H

H

H

H

H

HH

HH

HHH

H

H

H

H

H

Ominus

Ominus

Ominus Ominus

Ominus

Ominus

SminusN+

N+


Abso

rban

ce

Wavelength (nm)

A (n

orm

aliz

ed)


012

008

004

000240 300 360 420 480 540

10

08

06

040000

0 2 4 6 8 10

(a)

Abso

rban

ce

Wavelength (nm)

012

008

004

000240 300 360 420 480 540

(b)


















2and TSC-



2 Aromatic


Abso

rban

ce

Wavelength (nm)

025

020

015

010

005

000250 300 350 400 450 500 550 wwwacdlabscom

(a)

Time (min)

A340

(nm

)

A240

nmA

340

nmra

tio

020

018

016

014

012

010

0000 50 100 150 200 250 300 350

18

16

14

12

000

(b)

Abso

rban

ce

Wavelength (nm)350 400 450 500 550 600 650

075

050

025

000

(c)




nmA340





2






2


2


2

∙ to R-NO2



2in





Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

2000

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(a)

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(b)







4 Conclusion






Acknowledgments


References































2SnOC

6H3(5-










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















TSC (120583M)

TSC-Cl

Relat

ive (

)

10

05

000 10 100 1000

(a)Re

lativ

e (

)

TSC-Me10

05

00

TSC (120583M)

0 10 100 1000

(b)

TSC-F

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(c)

TSC-H

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(d)

TSC-NO2

Rela

tive (

)

10

05

00

TSC (120583M)

0 10 100 1000

(e)



wwwacdlabscom







2 respectively






2is not expected










2to themonomeric









240nmA





N N

S

N

O

H

H

H

H

H

HH

HH

HH

H

H

H

H

H

O

O

O

N

ON

O

H

ONH

HH

H H

HHH

H

HH

HH

O

O

O

N

ON

SO

H

ON

H

HH HH

HHH

H

HH

HH

N N

S

N

O

H

H

H

H

H

HH

HH

HHH

H

H

H

H

H

Ominus

Ominus

Ominus Ominus

Ominus

Ominus

SminusN+

N+


Abso

rban

ce

Wavelength (nm)

A (n

orm

aliz

ed)


012

008

004

000240 300 360 420 480 540

10

08

06

040000

0 2 4 6 8 10

(a)

Abso

rban

ce

Wavelength (nm)

012

008

004

000240 300 360 420 480 540

(b)


















2and TSC-



2 Aromatic


Abso

rban

ce

Wavelength (nm)

025

020

015

010

005

000250 300 350 400 450 500 550 wwwacdlabscom

(a)

Time (min)

A340

(nm

)

A240

nmA

340

nmra

tio

020

018

016

014

012

010

0000 50 100 150 200 250 300 350

18

16

14

12

000

(b)

Abso

rban

ce

Wavelength (nm)350 400 450 500 550 600 650

075

050

025

000

(c)




nmA340





2






2


2


2

∙ to R-NO2



2in





Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

2000

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(a)

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(b)







4 Conclusion






Acknowledgments


References































2SnOC

6H3(5-










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















wwwacdlabscom







2 respectively






2is not expected










2to themonomeric









240nmA





N N

S

N

O

H

H

H

H

H

HH

HH

HH

H

H

H

H

H

O

O

O

N

ON

O

H

ONH

HH

H H

HHH

H

HH

HH

O

O

O

N

ON

SO

H

ON

H

HH HH

HHH

H

HH

HH

N N

S

N

O

H

H

H

H

H

HH

HH

HHH

H

H

H

H

H

Ominus

Ominus

Ominus Ominus

Ominus

Ominus

SminusN+

N+


Abso

rban

ce

Wavelength (nm)

A (n

orm

aliz

ed)


012

008

004

000240 300 360 420 480 540

10

08

06

040000

0 2 4 6 8 10

(a)

Abso

rban

ce

Wavelength (nm)

012

008

004

000240 300 360 420 480 540

(b)


















2and TSC-



2 Aromatic


Abso

rban

ce

Wavelength (nm)

025

020

015

010

005

000250 300 350 400 450 500 550 wwwacdlabscom

(a)

Time (min)

A340

(nm

)

A240

nmA

340

nmra

tio

020

018

016

014

012

010

0000 50 100 150 200 250 300 350

18

16

14

12

000

(b)

Abso

rban

ce

Wavelength (nm)350 400 450 500 550 600 650

075

050

025

000

(c)




nmA340





2






2


2


2

∙ to R-NO2



2in





Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

2000

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(a)

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(b)







4 Conclusion






Acknowledgments


References































2SnOC

6H3(5-










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















N N

S

N

O

H

H

H

H

H

HH

HH

HH

H

H

H

H

H

O

O

O

N

ON

O

H

ONH

HH

H H

HHH

H

HH

HH

O

O

O

N

ON

SO

H

ON

H

HH HH

HHH

H

HH

HH

N N

S

N

O

H

H

H

H

H

HH

HH

HHH

H

H

H

H

H

Ominus

Ominus

Ominus Ominus

Ominus

Ominus

SminusN+

N+


Abso

rban

ce

Wavelength (nm)

A (n

orm

aliz

ed)


012

008

004

000240 300 360 420 480 540

10

08

06

040000

0 2 4 6 8 10

(a)

Abso

rban

ce

Wavelength (nm)

012

008

004

000240 300 360 420 480 540

(b)


















2and TSC-



2 Aromatic


Abso

rban

ce

Wavelength (nm)

025

020

015

010

005

000250 300 350 400 450 500 550 wwwacdlabscom

(a)

Time (min)

A340

(nm

)

A240

nmA

340

nmra

tio

020

018

016

014

012

010

0000 50 100 150 200 250 300 350

18

16

14

12

000

(b)

Abso

rban

ce

Wavelength (nm)350 400 450 500 550 600 650

075

050

025

000

(c)




nmA340





2






2


2


2

∙ to R-NO2



2in





Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

2000

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(a)

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(b)







4 Conclusion






Acknowledgments


References































2SnOC

6H3(5-










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Abso

rban

ce

Wavelength (nm)

025

020

015

010

005

000250 300 350 400 450 500 550 wwwacdlabscom

(a)

Time (min)

A340

(nm

)

A240

nmA

340

nmra

tio

020

018

016

014

012

010

0000 50 100 150 200 250 300 350

18

16

14

12

000

(b)

Abso

rban

ce

Wavelength (nm)350 400 450 500 550 600 650

075

050

025

000

(c)




nmA340





2






2


2


2

∙ to R-NO2



2in





Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

2000

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(a)

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(b)







4 Conclusion






Acknowledgments


References































2SnOC

6H3(5-










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

lls (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

2000

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(a)

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

Viab

le ce

ll (r

elat

ive t

o th

e con

trol)

()

[TSC] (120583M) [TSC] (120583M)

[TSC] (120583M)[TSC] (120583M)



100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

00

100

80

60

40

20

000 1 5 10 50 100 1000 20000 1 5 10 50 100 1000 2000

0 1 5 10 50 100 1000 2000 0 1 5 10 50 100 1000 2000

(b)







4 Conclusion






Acknowledgments


References































2SnOC

6H3(5-










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















4 Conclusion






Acknowledgments


References































2SnOC

6H3(5-










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























2SnOC

6H3(5-










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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