Thiazole Derivatives

molecules

Article Cytotoxicity and Antioxidant Potential of Novel 2-(2-((1H-indol-5yl)methylene)-hydrazinyl)-thiazole Derivatives

Adriana Grozav 1, Ioan-Dan Porumb 2, Luiza Ioana G˘ain˘a 2,*, Lorena Filip 3,* and Daniela Hanganu 4

1 Department of Organic Chemistry, Faculty of Pharmacy, “Iuliu Ha¸tieganu”University of Medicine and Pharmacy, 8 Victor Babes, Cluj-Napoca RO-400012, Romania; [email protected] 2 Research Center on Fundamental and Applied Heterochemistry, Faculty of Chemistry and Chemical Engineering, “Babe¸s-Bolyai”,University, M. Kogalniceanu 1, Cluj-Napoca RO-400028, Romania; [email protected] 3 Department of Bromatology, Hygiene, Nutrition, Faculty of Pharmacy, “Iuliu Ha¸tieganu”University of Medicine and Pharmacy, 8 Victor Babes, Cluj-Napoca RO-400012, Romania 4 Department of Pharmacognosy Faculty of Pharmacy, “Iuliu Ha¸tieganu”University of Medicine and Pharmacy, 8 Victor Babes, Cluj-Napoca RO-400012, Romania; [email protected] * Correspondences: [email protected] (L.I.G.); lﬁ[email protected] (L.F.); Tel.: +40-264-593-833 (L.I.G.); Fax: +40-264-590-818 (L.I.G.)

Academic Editor: Wim Dehaen Received: 4 January 2017; Accepted: 7 February 2017; Published: 9 February 2017

Abstract: Newly synthesized 2-(2-((1H-indol-5yl)methylene)-hydrazinyl)-thiazole derivatives were evaluated for their in vitro cytotoxicity on two carcinoma cell lines A2780 and HeLa. Signiﬁcant cytotoxic activity for 2-(2-((1H-indol-5-yl)methylene)hydrazinyl)-4-methylthiazole (1) and 2-(2-((1H-indol-5-yl)methylene)hydrazinyl)-4-phenylthiazole (3), on both A2780 [IC50: 11.6 µM(1), and 12.4 µM(3)] and HeLa [IC50: 22.4 µM(1) and 19.4µM(3)] cell lines is reported. Their antioxidant potential was evaluated by spectrophotometric method, using DPPH radical or Fe (TPTZ)3+ complex, and EPR spectroscopy, therefore the compounds 1 and 3 showed remarkable antioxidant activity simultaneously with a cytotoxic effect on A2780 and HeLa cell lines. Furthermore, based on theoretical quantum chemical calculation, the present study analyzed the chemoselectivity of the hydrogen extraction from the indolyl-hydrazinil-thiazoles in reaction with free radicals.

Keywords: cytotoxic activity; antioxidant activity; DFT calculation; indol; thiazole

1. Introduction The heterocyclic derivatives with thiazole or indole units can be found in a variety of biologically active compounds that makes them some of the most extensively studied heterocycles. An example of a natural compound in which both indole and thiazole rings are linked is 3-thiazol-20-yl-indole known as Camalexin, a phytoalexin produced in different plant leaves [1,2] or synthetic derivatives based on thiazolyl-1H-indols with antiproliferative activity in peritoneal mesothelioma or MiaPaCa-2 cell line [3,4], HIV-1 RT inhibitors based on thiazolyl-hydrazinyl-1H-indol-2-one derivatives [5] and isomer analogs of our compounds acting as inhibitors for Mycobacterium tuberculosis [6]. The recent investigations are focused to develop new molecules based on indole scaffolds because of their biological potential, essential amino acid like tryptophan, psychoactive substances [7] like synthetic alkaloids, Sumatriptan for migraine treatment, Vincristine and other indole derivatives as antiproliferative agents [8], Delavirdine anti-HIV drug, Doleasetron antiemetic drug [9], and

Molecules 2017, 22, 260; doi:10.3390/molecules22020260 www.mdpi.com/journal/molecules Molecules 2017, 22, 260 2 of 12 Molecules 2017, 22, 260 2 of 11 antioxidantcompounds compounds[10] but the [10 examples] but the examplesof biologically of biologically active compounds active compounds bearing bearingindole indolering are ring in arecontinuous in continuous expansion. expansion. TheThe thiazole can be identifiedidentified in the structure of different drugs, epothilone a microbial product andand theirtheir syntheticsynthetic analogsanalogs havinghaving anti-canceranti-cancer properties,properties, thiaminethiamine isis vitaminvitamin B1,B1, sulfathiazolansulfathiazolan isis antimicrobialantimicrobial agent,agent, abafunginas is fungicide, tiazofurin an antineoplastic, ritonavir is anti-HIV, andand fanetizole,fanetizole, meloxicam,meloxicam, andand fentiazacarefentiazacare areare anti-inflammatoryanti-inflammatory agentsagents [[11,12].11,12]. ThiazolesThiazoles bearing bearing hydrazine hydrazine moieties moieties have have gained gained a special a special attention attention because becauseof their biological of their biologicaland pharmacological and pharmacological properties as properties inhibitors as on inhibitors human monoamine on human oxidase monoamine (hMAO) oxidase [13], (hMAO)antimicrobial [13], antimicrobialagents [14], histone agents [14acetyltransferase], histone acetyltransferase inhibitors [15], inhibitors anti-inflammatory [15], anti-inflammatory agents [16], agents as well [16], asas wellantiproliferative as antiproliferative activity activity highlighted highlighted by our byprevious our previous studies studies [17–19]. [17 –19]. ConsideringConsidering the the biological biological potential potential of heterocyclic of heterocyclic derivatives derivatives bearing bearing thiazole thiazole and indole and scaffolds, indole scaffolds,the objectives the objectivesof this study of this were study to identify were to ne identifyw possible new antiprol possibleiferative antiproliferative agents and agents the study and the of studytheir antioxidant of their antioxidant potential. potential.For a better For understand a better understandinging of the antioxidant of the antioxidant properties propertieson molecular on molecularlevel, the level,structure the structure of 2-(2-((1 of 2-(2-((1H-indol-5-yl)methylene)-hydrazinyl)-thiazoleH-indol-5-yl)methylene)-hydrazinyl)-thiazole derivatives derivatives and andthe thechemoselectivity chemoselectivity of the of the radical radical generation generation reactions reactions have have been been studied studied using using molecular modelingmodeling toolstools asas DensityDensity FunctionalFunctional TheoryTheory (DFT) using hybrid B3LYP, 6-31G*,6-31G*, andand Hartree-FockHartree-Fock 3-21G3-21G modelsmodels [[20–22]20–22] offeredoffered byby SpartanSpartan 0606 software.software.

2.2. Results and DiscussionDiscussions

2.1.2.1. Synthesis of the New 2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)thiazole2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)thiazole DerivativesDerivatives TheThe synthesissynthesis ofof newlynewly 2-(2-((12-(2-((1HH-indol-5-yl)methylene)-hydrazinyl)-thiazole-indol-5-yl)methylene)-hydrazinyl)-thiazole derivativesderivatives werewere performedperformed byby the the Hantzsch Hantzsch protocol protocol in twoin two steps, steps, the condensationthe condensation of 1H of-indole-5-carbaldehyde 1H-indole-5-carbaldehyde with hydrazinecarbothioamidewith hydrazinecarbothioamide yielding yielding (E)-2-[(1 (HE-indol-3-yl)methylene])-2-[(1H-indol-3-yl)methylene] thiosemicarbazone thiosemicarbazone [23] followed [23] byfollowed its cyclocondensation by its cyclocondensation with α-halocarbonyl with α-halocarbonyl derivatives, derivatives, Scheme1. Scheme 1.

Scheme 1. Synthesis of 2-(2-((1H-indol-5-yl)methylene)hydrazinyl)thiazole-indol-5-yl)methylene)hydrazinyl)thiazole derivatives.derivatives.

TheThe structure structure of indolyl-hydrazinyl-thiazole of indolyl-hydrazinyl-thiazole derivatives derivatives 1–6 were1 –confirmed6 were conﬁrmedby NMR, spectroscopy by NMR, 1 13 spectroscopy( H-NMR and (1H-NMRC-NMR, andCOSY,13 C-NMR,HMQC and COSY, HMBC) HMQC and andmass HMBC) spectrometry. and mass The spectrometry.characteristic Thesinglet characteristic for imine proton singlet (HC=N) for imine can proton be identified (HC=N) around can be 8.06–8.41 identiﬁed ppm, around while 8.06–8.41 the NH proton ppm, while from theindole NH ring proton appears from around indole ring10.6 appearsppm in acetone around 10.6but more ppm indeshielded, acetone but around more 11.5 deshielded, ppm, in arounda more 11.5polar ppm, solvent in a like more dimethyl polar solvent sulfoxide like. dimethyl sulfoxide.

2.2.2.2. Cytotoxic Activity TheThe new indolyl-hydrazinyl-thiazoles 1–6 were tested in vitrovitro,, by colorimetric method MTT, for theirtheir cytotoxiccytotoxic activityactivity onon twotwo tumortumor cellcell lines:lines: HeLa (human cervical cancer cells) and A2780 (human ovarianovarian cancercancer cells).cells). The The inhibitory inhibitory activity activity of of the the compounds compounds was was measured measured and and quantiﬁed quantified using using the the mathematic parameter IC50, (Figure 1). Cisplatin, a metal-based drug currently used in cancer therapy, was used as control (Figure 1). Molecules 2017, 22, 260 3 of 12

mathematic parameter IC50, (Figure1). Cisplatin, a metal-based drug currently used in cancer therapy, Moleculeswas used 2017 as, 22 control, 260 (Figure1). 3 of 11

IC50 (μM) Compounds HeLaIC50 (µ A2780M) Compounds Cisplatin 20.10HeLa ± 0.22 12.30 ± 0.22 A2780 Cisplatin1 20.1022.42± ± 0.220.24 11.65 12.30 ± 0.21± 0.22 1 2 22.4228.55± ± 0.240.25 26.49 11.65 ± 0.21± 0.21 2 3 28.5519.42± ± 0.250.22 12.99 26.49 ± 0.50± 0.21 3 19.42 ± 0.22 12.99 ± 0.50 4 33.98 ± 0.31 28.08 ± 0.23 4 33.98 ± 0.31 28.08 ± 0.23 5 5 19.84 ± ± 0.33 19.66 19.66 ± 0.50± 0.50 6 6 34.72 ± ± 0.25 20.15 20.15 ± 0.26± 0.26

Figure 1. Cytotoxic activity of in vitro of Compounds 1–6 against HeLa and A2780 tumor cells. * Data Figure 1. Cytotoxic activity of in vitro of Compounds 1–6 against HeLa and A2780 tumor cells. are reported as IC50 values determined by MTT assay after 24 h of continuous exposure to each compound. * Data are reported as IC50 values determined by MTT assay after 24 h of continuous exposure to Theeach values compound. were calculated The values using were sigmoidal calculated dose-r usingesponse sigmoidal relationship. dose-response The relationship.data represents The mean data valuesrepresents ± SEM mean (standard values error± SEM of (standardmean) of at error least of three mean) independent of at least three experiments. independent experiments.

The in vitro results displayed a better cytotoxic activity of indolyl-hydrazinyl-thiazoles 1–6 on The in vitro results displayed a better cytotoxic activity of indolyl-hydrazinyl-thiazoles 1–6 on tumor line A2780, the IC50 values being lower compared with those obtained on HeLa tumor line. tumorCompounds line A2780, 1 theand IC 3 50displayedvalues being cytotoxic lower activity compared comparable with those to cisplatin obtained on on both HeLa tumor tumor cell line. lines 1 3 whileCompounds the cytotoxic andactivitydisplayed displayed cytotoxic by compounds activity comparable 4 and 6 appeared to cisplatin inferior on both to the tumor standard cell lines on 4 6 bothwhile tumor the cytotoxic cell lines. activity In comparison displayed bywith compounds cisplatin standard,and appeared compound inferior 5 has to a the similar standard activity on both on 5 HeLatumor cell cell lines, lines. but In it comparison proved to be with less cisplatin sensitive standard, on A2780 compound cell lines. has a similar activity on HeLa cell lines, but it proved to be less sensitive on A2780 cell lines. 2.3. Antioxidant Activity 2.3. Antioxidant Activity The spectrophotometric method based on free radical scavenging activity is widely used to The spectrophotometric method based on free radical scavenging activity is widely conveniently assess antioxidant potential in vitro. The scavenging potential of the indolyl-thiazole used to conveniently assess antioxidant potential in vitro. The scavenging potential of the derivatives, in terms of hydrogen donating ability, was evaluated using DPPH (1,1-Diphenyl-2- indolyl-thiazole derivatives, in terms of hydrogen donating ability, was evaluated using DPPH picrylhydrazyl) radicals. The scavenging potential of the indolyl-hydrazinyl-thiazole derivatives in (1,1-Diphenyl-2-picrylhydrazyl) radicals. The scavenging potential of the indolyl-hydrazinyl-thiazole the presence of DPPH decreases as follows 1 > 3 > 2 > 5 > 4 > 6, Table 1. The antioxidant potential of derivatives in the presence of DPPH decreases as follows 1 > 3 > 2 > 5 > 4 > 6, Table1. The antioxidant the compounds 1 (IC50 = 5.377 µg/mL) and 3 (IC50 = 9.131 µg/mL) is better than trolox-standard potential of the compounds 1 (IC50 = 5.377 µg/mL) and 3 (IC50 = 9.131 µg/mL) is better than (IC50 = 9.74 µg/mL) but lower than ascorbic acid (IC50 = 2.46 µg/mL) activity. trolox-standard (IC = 9.74 µg/mL) but lower than ascorbic acid (IC = 2.46 µg/mL) activity. The antioxidant50 potential of thiazols 1–6 towards free DPPH radicals50 was also investigated by The antioxidant potential of thiazols 1–6 towards free DPPH radicals was also investigated by Electron Paramagnetic Resonance (EPR) spectroscopy. The EPR spectra have shown that the integral Electron Paramagnetic Resonance (EPR) spectroscopy. The EPR spectra have shown that the integral intensity of DPPH radicals in the mixture with these antioxidant compounds decreases compared to intensity of DPPH radicals in the mixture with these antioxidant compounds decreases compared to free DPPH solution, (I = 252) and represents the oxido-reduction rate. The order of reactivity based free DPPH solution, (I = 252) and represents the oxido-reduction rate. The order of reactivity based on the calculated rates is identical to the one found by DPPH spectrophotometric method, as seen in on the calculated rates is identical to the one found by DPPH spectrophotometric method, as seen in Table 1. Table1.

Molecules 2017, 22, 260 4 of 12

Table 1. The antioxidant activity of indolyl-hydrazinyl-thiazoles 1–6 determined by different methods.

Methods Compounds DPPH FRAP EPR IC 50 (µg/mL) µM ET/g Integral Intensity Value 1 5.377 ± 0.52 9835.5518 ± 324 17 ± 0.14 2 11.850 ± 0.72 872.5288 ± 27 72 ± 0.23 3 9.131 ± 0.61 8176.5278 ± 298 24 ± 0.17 4 13.357 ± 0.68 6129.6298 ± 280 114 ± 0.3 5 12.974 ± 0.58 7431.0798 ± 263 82 ± 0.52 6 15.658 ± 0.63 3572.2890 ± 124 142 ± 0.34 Trolox 9.74 ± 0.24 - - Ac.ascorbic 2.46 ± 0.91 - -

The reducing capacity of indolyl-hydrazinyl-thiazoles 1–6 has been measured as ferric reducing antioxidant power (FRAP). The reduction of Fe3+ ion from tripyridyltriazine Fe (TPTZ)3+ complex to Fe2+ ion in the blue colored Fe (TPTZ)2+ complex depends on the electron donating capacity of the tested thiazoles 1–6 and was quantiﬁed by spectrophotometric method. The reducing ability of the compounds decreases as follows 1 > 3 > 5 > 4 > 6 > 2 (see Table1). According to the results obtained by spectrophotometric methods using DPPH radical, Fe (TPTZ)3+ complex and EPR spectroscopy, Compound 1 is the most active from the newly synthesized thiazole derivatives 1–6. These new indolyl-hydrazinyl-thiazoles displayed both antioxidant activity and cytotoxicity against human carcinoma cell lines A2780 and HeLa. Thus, they could be further evaluated as prophylactic agents usable in preventing the free radical induced chain reactions with repercussions in tumoral cell growth.

2.4. Computational Study The quantum chemical calculations for novel synthesized 2-(2-((1H-indol-5-yl)methylene)- hydrazinyl)-thiazole derivatives 1–6 were performed using Spartan 06 software (Wavefunction Inc., Irvine, CA, USA). The quantum molecular parameters such as energies corresponding to optimized geometries (E) and frontier molecular orbitals (EHOMO,ELUMO), were computed using Density Functional Theory (DFT), hybrid B3LYP, 6-31G*. Table2 summarizes representative computational results. The antioxidant activities of indolyl-hydrazinyl-thiazoles 1–6 were experimentally investigated by three method involving two different reaction pathways, the ﬁrst mechanism was based on hydrogen donation during the reaction with DPPH radicals and the second method was based on the electron donation to the Fe+3 ion in a reducing reaction known as FRAP method. For a better understanding of the radical scavenging abilities on a molecular level, the chemoselectivity of radical generation was investigated by theoretical quantum chemical calculations. During the reaction of DPPH radical with thiazoles 1–6, these are converted in radicals by losing one hydrogen atom. As the indolyl-hydrazinyl-thiazoles 1–6 contain two different NH units there are two possible ways to generate radicals; although the extraction of the hydrogen from the hydrazinyl bridge seems to have a higher probability, the possibility to extract hydrogen atom from the indole moiety can be taken into consideration. The analysis of this issue was performed using molecular mechanics and quantum chemical calculations. The HOMO orbital from indolyl-hydrazinyl-thiazoles 1–6. Table2, is mainly located on the hydrazinyl bridge and less in the indole group, favoring the electron or hydrogen atom extraction from the hydrazinyl moiety, Table3. Molecules 2017, 22, 260 5 of 12

Table 2. Quantum molecular parameter and frontier orbital density distribution for indolyl-hydrazinylthiazoles 1–6. Compounds EHOMO (eV) ELUMO (eV) EHOMO-LUMO (eV) HOMO Surface CompoundsCompoundsCompoundsCompounds EEEEHOMOHOMOHOMOHOMO (eV) (eV) (eV)(eV) E E E ELUMOLUMOLUMOLUMO (eV) (eV) (eV)(eV) E E E EHOMO-LUMOHOMO-LUMOHOMO-LUMOHOMO-LUMO (eV) (eV) (eV)(eV) HOMO HOMOHOMOHOMO Surface Surface SurfaceSurface

Compounds EHOMO (eV) ELUMO (eV) EHOMO-LUMO (eV) HOMO Surface Compounds EHOMO (eV) ELUMO (eV) EHOMO-LUMO (eV) HOMO Surface Compounds CompoundsEHOMO (eV) EHOMO E (eV)LUMO (eV) ELUMO EHOMO-LUMO (eV) (eV) EHOMO-LUMO (eV)HOMO Surface HOMO Surface 1 1 −−4.96 −−0.98 3.98 3.98 1111 −−−−4.964.964.964.96 −−−−0.980.980.980.98 3.3.983.3.989898 1 −4.96 −0.98 3.98 Compounds EHOMO (eV) ELUMO (eV) EHOMO-LUMO (eV) HOMO Surface 1 Compounds1 −4.96 EHOMO−4.96 (eV) −0.98 ELUMO−0.98 (eV)3. 98 EHOMO-LUMO3.98 (eV) HOMO Surface

Compounds EHOMO (eV) ELUMO (eV) EHOMO-LUMO (eV) HOMO Surface

Compounds EHOMO (eV) ELUMO (eV) EHOMO-LUMO (eV) HOMO Surface 2 2 −−5.291 2 −4.96 −−1.52−5.29 −0.98 −1.52 3.3.7798 3.77 3.77 2222 −−−−5.295.295.2925.29 21 −5.29 −−−−1.521.521.521.52−5.294.96 −1.52 −1.520.98 3.3.3.773.773.777777 3.7798 Compounds EHOMO (eV) ELUMO (eV) EHOMO-LUMO (eV) HOMO Surface 1 −4.96 −0.98 3.98

1 −4.96 −0.98 3.98 2 3 −5.29 −5.00 −1.52 −1.10 3.77 3.90 3 1 32 −5.00− 4.96 −5.005.29 −1.10−0.98 −1.101.52 3.3.9890 3.9077 3 −5.00 2 −1.10 −5.29 −1.523.90 3.77 3 333 −−5.00 −−1.101.10 3.90 3.90 3 −−−5.005.005.002 −5.29 −−−1.101.101.10 −1.52 3.3.773.3.909090 3 −5.00 −1.10 3.90 2 4 −5.29 −5.22 −1.52 −1.383.77 3.84 4 3 −5.22 −5.00 −1.38 −1.10 3.84 3.90 4 3 −5.22 −5.00 −1.38−1.10 3.843.90

3 −5.00 −1.10 3.90 4 3 −5.22−5.00 −1.38−1.10 3.3.9084 4 −5.22 5 −1.38−4.96 −0.96 3.84 4.00 4 4444 −−−−−5.225.225.2255.22 4 −4.96 −−−−−1.381.381.381.38−5.22 −0.96 −1.38 4.3.3.843. 3.84003.848484 3.84 5 4 −4.96 −5.22 −0.96−1.38 4.003.84 4 −5.22 −1.38 3.84 5 4 −4.96−5.22 −0.96−1.38 3.4.8400 6 −5.23 −1.20 4.03 6 −5.23 −1.20 4.03 65 5 −5.234.96 −4.96 −1.200.96−0.96 4.03004.00 5 −4.96 −0.96 4.00 5 −−4.965 −4.96 −−0.96 −0.96 4.4.0000 5 555 −−−4.964.964.966 5 −5.23−4.96 −−−0.960.960.96 −1.20−0.96 4.4.4.00034. 4.004.000000

6 6 −5.23 −5.23 −1.20−1.20 4.034.03

6 6 −5.23−5.23 −1.20−1.20 4.4.0303 6 −5.23 −1.20 4.03 6 6666 −−−−−5.235.235.235.23 −−−−−1.201.201.201.20 4.4.034. 4.034.030303

EHOMO ELUMO Spin Density EHOMO ELUMO Spin DensitySpin Density SOMO Orbital Spin Density SOMO5 Orbital Radical E (kcal/mol) HOMO(eV) LUMO(eV) 5 on C for Radical E (kcal/mol) (eV) (eV)E E Map for Honradical C for Spin Density for Hradical radical Spin Density radical SOMO Orbital Table 3. The quantum molecular parameter andHradical spin densityMapHradical for H distribution for H typeHradicalfor 5 radicals(eV) H * generated Radical E (kcal/mol)HEradicalHOMO HEradicalLUMO(eV) (eV) SpinHradical Density (eV) * on C for Spin Density radical SOMO Orbital radical Map for H 5 for H Radical E (kcal/mol) (eV) (eV)Hradical Hradical on C for Hradical (eV) * from indolyl-hydrazinylthiazoles 1–6. Map for Hradical for Hradical H −695,328.05 EHOMO ELUMO Spin Density H −695,328.05 Hradical Hradical SpinH Densityradical (eV) * SOMO Orbital Radical EEHOMO (kcal/mol) ELUMO (eV) (eV) Spin Density on C5 for 1 H −695,328.05 EHOMO−7.94 ELUMO3.09Spin Density SOMOSpin0.035 Density Orbital 1 Radical E (kcal/mol) −E7.94HOMO(eV) 3.09ELUMO(eV) SpinMap DensitySpin onfor C H50.035 Density forradical SOMOfor H Orbitalradical H Radical−695,328.05 E (kcal/mol) (eV) H radical (eV)MapSpinH for radicalDensity H radical foronSOMOHradical HCradical5 for(eV) Orbital * Hradical Hradical Hradical (eV)5 * 5 E RadicalI EHOMO −695,310.801E (kcal/mol)(eV)I −695,310.80E(eV)LUMO (eV) (eV)−7.94 Spin3.09 Density MapMap for HonSpinradical C for Density on0.035 C SOMOfor OrbitalHradical for Radical 1 −7.94 3.09Hradical HMapradical for Hradical 0.035 Hradicalfor (eV) Hradical * (kcal/mol) H H HradicalH−695,328.05 Hradical for H Hradicalfor (eV) H * (eV) * H Hradical −695,328.05I −695,310.80 radical radical radical radical I −695,310.80 H 1 −695,328.05 −7.94 3.09 0.035 H −695,328.05 H 1H −790,033.74−695,328.05H −790,033.74−7.94 3.09 0.035 1 −7.94 I −3.09695,310.80 0.035 1 I 1 2 −695,310.80H −790,033.74 −7.94 3.09−7.94− 8.08 3.092.54 0.035 0.0350.036 I −695,310.802 H −790,033.74 −8.08 2.54 0.036 I −695,310.80I −695,310.80 2 I −790,016.362 I −790,016.36−8.08 2.54−8.08 2.54 0.036 0.036 H −790,033.74 H −790,033.74H −790,033.74 E−HOMO I −E790,016.36LUMO Spin Density 2 I EHOMOHOMOHOMO8.08−790,016.36 ELUMOLUMOLUMO2.54 Spin0.036 Density 2H EEEHOMO−790,033.74 2 EEELUMO−8.08 2.54 −8.08Spin Density2.54 0.036Spin SpinSpin Density Density Density0.036 SOMO Orbital RadicalI E− (kcal/mol)790,016.36 H (eV)−814,214.89 HH −790,033.74−(eV)814,214.89 SpinSpinSpinSpin Density Density DensityDensity on C5 for SOMOSOMOSOMOSOMO Orbital Orbital OrbitalOrbital Radical E (kcal/mol) (eV)I − 790,016.36 (eV) on C555 5for RadicalRadicalRadicalRadical EEEE (kcal/mol) (kcal/mol) (kcal/mol)(kcal/mol)2 (eV)(eV)(eV)(eV) I (eV)(eV)(eV)(eV)− 790,016.36 Map for Hradicalradical onononon C C CC for for forfor for Hradicalradical 2 −8.08 2.54 Map for Hradical 0.036 for Hradical H −814,214.893 H Hradicalradical−3814,214.89 H −H814,214.89−radical7.98radical 2.91−8.08− 7.98 MapMapMapMap2.542.91 for for forfor H H HHradicalradicalradical 0.037H radicalradical (eV)0.0360.037 * forforforfor H H HHradicalradicalradical 3 I HHH−Hradicalradicalradical7.98radical−790,016.36 HHHHradicalradicalradicalradical2.91 HHHHradicalradicalradicalradical0.037 (eV) (eV) (eV)(eV) * * ** 3 I H−814,185.61 3 −814,214.89I I −790,016.36 −814,185.61−7.98 2.91−7.98 2.91 0.037 0.037 I −814,185.61 H −814,214.89 3I −814,185.61 −7.98 2.91 0.037 H −695,328.05 I 3 −814,185.61 HHH −−−695,328.05695,328.05695,328.05 H −814,214.89 −7.98 2.91 0.037 H −695,328.05 H −I 861,114.17−814,185.61HH −814,214.89 −861,114.17 H −861,114.17 3 I −7.98−814,185.61 2.91 0.037 1 H −7.94−861,114.17 3.09 0.0360.035 41111 4 −−−−7.947.947.948.067.943 4 H −861,114.173.09−3.093.098.063.09 2.59 2.59−7.98− 8.06 2.912.59 0.036 0.0350.0350.0350.035 0.037 0.036 I H −814,185.61−861,114.17 4 H −8.06−861,108.82 2.59 0.036 I I −−695,310.80861,098.95 I H −861,098.954 −861,108.82I I − H814,185.61− 861,098.95−861,114.17 − 8.06 2.59 0.036 I −695,310.80 4 −8.06 2.59 0.036 II I −−−695,310.80695,310.80695,310.80 I −861,098.95 H −861,108.8254 H −861,108.82 −−7.978.06 2.982.59 0.0380.036 H −861,108.82 5 H I −861,114.17−861,098.95I −861,098.95 −7.97 2.98 0.038

5 5 −7.97 5 I −7.97−861,085.822.98 2.98 −7.97 2.98 0.038 0.038 0.038 4 I −861,085.82H − I861,114.17 −8.06−861,098.95 2.59 0.036

I −861,085.82 I −861,085.82 I −861,085.82 H −790,033.74 I 4− 861,098.95 −8.06 2.59 0.036 HHH −−−790,033.74790,033.74790,033.74 H −836,749.36 1 1 H −836,749.36 H −836,749.36I − 861,098.95 1 62 −−8.088.07 2.54 2.20 1 0.0370.036 222 6 H −−−8.088.088.08−836,749.36 6 H 2.542.542.54−8.07−836,749.36 2.20 −8.07 2.20 1 0.037 0.0360.0360.036 0.037 2 I 836,728.71 −8.08 2.54 1 0.036 I −790,016.36 6 I 836,728.716 I −8.07836,728.71 2.20 −8.07 2.20 0.037 0.037 I −790,016.36 5 5 * TheII I spin− density−−790,016.36790,016.36790,016.36 for radical I H on836,728.71 C atom I (indole-C836,728.71 H=N-NH-thiazole)1 represent the value at the cursor point and

the blue color on the surface indicates maximum electron density on that1 atoms.

H −814,214.89 HHHH −−−−814,214.89814,214.89814,214.89814,214.89 3 This supposition was−7.98 conﬁrmed 2.91 by the values of the energies corresponding0.037 to optimized 3333 −−−−7.987.987.987.98 2.912.912.912.91 0.0370.0370.0370.037 geometriesI for−814,185.61 both possible radical H ad I types, as seen in Table3 and Scheme2. III I −−−−814,185.61814,185.61814,185.61814,185.61 The radicals belonging to the thiazoles 1–6 were generate from optimized geometries, previously calculatedH by−861,114.17 DFT B3LYP, 6-31G* and computed using MMFT molecular mechanics to establish HHHH −−−−861,114.17861,114.17861,114.17861,114.17 equilibrium4 conformation. The−8.06 semi-empirical 2.59 AM1 model was used to establish0.036 equilibrium geometry 444 −−−8.068.068.06 2.592.592.59 0.0360.0360.036 and4 Hartree-Fock 3-21G model−8.06 for a basis2.59 of graphical calculation. 0.036 I −861,098.95 III I −−−−861,098.95861,098.95861,098.95861,098.95

1 1111

2 2 2 Molecules 2017, 22, 260 6 of 12

Comparing the calculated energies (E) for both type of radicals I and H we found that the H type radicals have a lower energy than I type radicals, Table3. The energy gap (E I–EH) between radicals H and I was around 15–29 kcal/mol, enough to generate predominantly H radicals from all thiazoles 1–6. The H type radicals can be stabilized by internal conjugation with indole and thiazole ring and the favored pathway for spin delocalization is on indolyl-methylene moiety, in respect with SOMO orbital,Molecules Table2017, 223., 260 6 of 11

Scheme 2. TheThe structure structure of of two two possible possible radicals radicals of of 2-(2-((1 2-(2-((1HH-indol-5-yl)methylene)-hydrazinyl)-thiazole-indol-5-yl)methylene)-hydrazinyl)-thiazole 1 1locatedlocated on on hydrazinyl hydrazinyl (H), (H respective), respective on indolyl on indolyl (I) moiety (I) moiety and spin and density spin density map for map radicals for radicals H and HI type.and I type. In the spin density map the blue color on the surface indicates maximum electron density that decreasesIn the from spin blue density to red map color. the As blue expected, color on in the each surface case the indicates indolyl-methylene maximum electron unit contains density a high that 5 decreaseselectron density from blue located to red on color. the methylene As expected, atom in C each. case the indolyl-methylene unit contains a high electron density located on the methylene atom C5. 3. Experimental Section 3. Experimental Section 3.1. Materials and Methods 3.1. Materials and Methods For the chemical synthesis the reagents and solvents were bought from chemical suppliers and usedFor without the chemical further puri synthesisfication. the 2,4,6-Tripyridyl-s-triazine reagents and solvents were (TPTZ), bought iron from (III) chemical chloride suppliers(FeCl3·6H and2O), used2,2-diphenyl-1-picryl without further purification.hydrazyl (DPPH) 2,4,6-Tripyridyl-s-triazine and standards as 6-hydroxy-2,5,7,8-thetramethylchroman-2- (TPTZ), iron (III) chloride (FeCl3·6H2O), 2,2-diphenyl-1-picrylcarboxilyc acid (Trolox) hydrazyl were purchased (DPPH) and from standards Sigma-Aldrich as 6-hydroxy-2,5,7,8-thetramethylchroman-2- Chemie Gmbh (Munich, Germany). carboxilycThe tumor acid (Trolox)cells were were grown purchased in the presence from Sigma-Aldrich of RPMI 1640 Chemie (Sigma-Aldrich), Gmbh (Munich, fetal calf Germany). serum (FCS, Sigma-Aldrich,The tumor cells5%), wereglutamine grown (Sigma-Aldrich, in the presence of0.1%), RPMI and 1640 antibiotics (Sigma-Aldrich), penicillin–streptomycin fetal calf serum (FCS,ActavisSindanPharma Sigma-Aldrich, 5%), (Bucharest, glutamine Romania). (Sigma-Aldrich, 0.1%), and antibiotics penicillin–streptomycin ActavisSindanPharmaThe 1H-NMR and (Bucharest,13C-NMR spectra Romania). were recorded in acetone-d6, or DMSO-d6 (locked to Me4Si) 1 13 usingThe a 400MHzH-NMR Bruker and AvanceC-NMR NMR spectra spectrometer were recorded produced in acetone- by Brukerd6, or BioSpin DMSO- GmbHd6 (locked (Rheinstetten, to Me4Si) usingGermany). a 400MHz The mass Bruker spectra Avance were NMR recorded spectrometer on Thermo produced Scientific by BrukerLTQ Orbitrap BioSpin XL GmbH mass (Rheinstetten,spectrometer Germany).produced by The ThermoFisher mass spectra wereScientific recorded (Bremen, on Thermo Germany). Scientific Melting LTQ points Orbitrap were XL measured mass spectrometer with an producedElectrothermal by ThermoFisher IA 9200 apparatus Scientific produced (Bremen, by Co Germany).le-Parmer Melting(Staffordshire, points UK), were and measured are uncorrected with an Electrothermalvalues. Colorimetric IA 9200 measurements apparatus produced were recorded by Cole-Parmer with Biotek (Staffordshire, Synergy 2 UK), Multi-Mode and areuncorrected Microplate values.Reader with Colorimetric SQ Xenon measurements Flash light source were Produced recorded by with BioTek Biotek (Winooski, Synergy VT, 2 Multi-Mode USA). The absorbance Microplate Readerhas been with measured SQ Xenon using Flash a Jasco light V-530 source UV-Vis Produced spectrophotometer by BioTek (Winooski, produced VT, USA).by Jasco The International absorbance hasCo. beenLtd. measured(Tokyo, Japan). using aElectron Jasco V-530 paramagnetic UV-Vis spectrophotometer resonance measurements produced were by Jasco performed International on a Co.BrukerElexsys Ltd. (Tokyo, E500 Japan). spectrometer Electron (Bruker, paramagnetic Billerica,resonance MA, USA) measurements operating in X wereband performed(~9.4 GHz) onwith a BrukerElexsys100 kHz modulation E500 spectrometerfrequency; the (Bruker,parameters Billerica, of scanning MA,USA) are: center operating field, in3360 X bandG; sweep (~9.4 width, GHz) 60 with G; 100power, kHz 2 mW; modulation receiver frequency;gain, 1 × 103; the modulation parameters amplitude, of scanning 2 G; are:time centerof conversion, field, 3360 15 ms; G; sweeptime constant, width, 6030.72 G; power,ms; sweep 2 mW; time receiver 60 s. gain, 1 × 103; modulation amplitude, 2 G; time of conversion, 15 ms; time constant, 30.72 ms; sweep time 60 s. 3.2. Chemistry

3.2.1. General Procedure for (E)-2-[(1H-Indol-3-yl) methylene]thiosemicarbazone To a solution of 1H-indole-5-carbaldehyde (14.5 g, 0.1 mol) in ethanol (100 mL) was added a solution of hydrazine carbothioamide (9.1 g, 0.1mol) in water (30 mL). The solution was stirred under reflux for 5 h. The reaction progress was monitored by TLC (toluene/AcOEt 2/1 v/v; silica plates), after the reaction completed the solid phase was filtered and recrystallized from ethanol obtaining a light brown powder of thiosemicarbazone (15 g, 78%, m.p. 217 °C, m.p. 217–218 °C lit. [23]).

3.2.2. General Procedure for Compounds 1–6 A mixture of (E)-2-[(1H-indol-3-yl)methylene]thiosemicarbazone (0.01 mol) and α-halogenocarbonyl derivative (0.01 mol) in ethanol/acetone/acetic acid (20 mL, 1/0.5/0.1 v/v/v) was stirred at room temperature Molecules 2017, 22, 260 7 of 12

3.2. Chemistry

3.2.1. General Procedure for (E)-2-[(1H-Indol-3-yl) methylene]thiosemicarbazone To a solution of 1H-indole-5-carbaldehyde (14.5 g, 0.1 mol) in ethanol (100 mL) was added a solution of hydrazine carbothioamide (9.1 g, 0.1mol) in water (30 mL). The solution was stirred under reﬂux for 5 h. The reaction progress was monitored by TLC (toluene/AcOEt 2/1 v/v; silica plates), after the reaction completed the solid phase was ﬁltered and recrystallized from ethanol obtaining a light brown powder of thiosemicarbazone (15 g, 78%, m.p. 217 ◦C, m.p. 217–218 ◦C lit. [23]).

3.2.2. General Procedure for Compounds 1–6 A mixture of (E)-2-[(1H-indol-3-yl)methylene]thiosemicarbazone (0.01 mol) and α-halogenocarbonyl Moleculesderivative 2017 (0.01, 22, 260 mol) in ethanol/acetone/acetic acid (20 mL, 1/0.5/0.1 v/v/v) was stirred at room7 of 11

Moleculestemperature 2017, 22 for, 260 24 h. The reaction progress was monitored by TLC (toluene/AcOEt 2/1 v/v;7 silica of 11 for 24 h. The reaction progress was monitored by TLC (toluene/AcOEt 2/1 v/v; silica plates), after the plates), after the reaction completed, the mixture was neutralized at pH 7 with NaHCO3 aqueous reaction completed, the mixture was neutralized at pH 7 with NaHCO3 aqueous solution (10%). The forsolutionMolecules 24 h. 2017 The (10%)., 22 reaction, 260 The precipitate progress was was monitored ﬁltered and by recrystallized TLC (toluene/AcOEt from ethanol, 2/1 v/ derivativesv; silica plates),4 and after6 7were of the 11 precipitate was filtered and recrystallized from ethanol, derivatives 4 and 6 were purified by column puriﬁedreaction bycompleted, column chromatographythe mixture was (silicagelneutralized 60, at eluent pH 7 toluene/acetonewith NaHCO3 aqueous 4/1 v/ vsolution). (10%). The chromatographyfor 24 h. The reaction (silicagel progress 60, eluent was monitored toluene/acetone by TLC 4/1 (toluene/AcOEt v/v). 2/1 v/v; silica plates), after the 2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)-4-methylthiazoleprecipitate was filtered and recrystallized from ethanol, derivatives(1) 4 and 6 were purified by column 2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)-4-methylthiazolechromatographyreaction completed, (silicagel the mixture 60, eluent was toluene/acetoneneutralized at pH 4/1 7 (vwith1/v) ). NaHCO3 aqueous solution (10%). The precipitate was filtered and recrystallized from ethanol, derivatives 4 and 6 were purified by column 2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)-4-methylthiazolechromatography (silicagel 60, eluent toluene/acetone 4/1 (v1/)v ).

2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)-4-methylthiazole (1) Light gray powder, yield 2.0 g, 79%, m.p. 182 °C, crystallized from ethanol; 1H-NMR (DMSO-d6, ◦ 1 400 MHz)Light grayδ (ppm): powder, 2.16 yield (s, 3H), 2.0 g, 6.33 79%, (s, m.p. 1H), 182 6.48–6.49C, crystallized (m, 1H), from 7.37–7.38 ethanol; (m,H-NMR 1H), 7.42 (DMSO- (d, 1H,d6, Light gray powder, yield 2.0 g, 79%, m.p. 182 °C, crystallized from ethanol; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 2.16 (s, 3H), 6.33 (s, 1H), 6.48–6.49 (m, 1H), 7.37–7.38 (m, 1H), 7.42 (d, 1H, J = 8.5Hz), J400 = 8.5Hz), MHz) 7.49δ (ppm): (dd, 1H,2.16 J =(s, 1.3 3H), Hz, 6.33J = 8.5 (s, Hz),1H), 7.37 6.48–6.49 (s, 1H), (m, 8.06 1H), (s, 1H),7.37–7.38 11.27 (s,(m, 1H), 1H), 11.56 7.42 (br, (d, 1H);1H, 13 137.49 (dd, 1H, J = 1.36 Hz, J = 8.5 Hz), 7.37 (s, 1H), 8.06 (s, 1H), 11.27 (s, 1H), 11.561 (br, 1H); C-NMR J C-NMR= 8.5Hz),Light (DMSO- gray7.49 (dd,powder,d ,1H, 100 Jyield MHz)= 1.3 2.0 Hz, δ g,(ppm): J 79%, = 8.5 m. 17.6,Hz),p. 182 102.2,7.37 °C, (s, 104.0,crystallized 1H), 8.06112.4, (s, from119.2, 1H), ethanol; 11.27120.3, (s, 126.2q, H-NMR1H), 11.56 126.7, (DMSO- (br, 128.1q, 1H);d6, (DMSO-136.9q, 143.8,d6, 100 155.3q, MHz) 168.6q.δ (ppm): HRMS 17.6, 102.2,(APCI) 104.0, calcd. 112.4, for C 119.2,13H13N 120.3,4S [M 126.2q,+ H]: 257.0861 126.7, 128.1q, found 136.9q, 257.0812. 143.8, 13400C-NMR MHz) (DMSO- δ (ppm):d6, 2.16100 MHz)(s, 3H), δ (ppm):6.33 (s, 17.6, 1H), 102.2, 6.48–6.49 104.0, (m, 112.4, 1H), 119.2, 7.37–7.38 120.3, (m, 126.2q, 1H), 126.7, 7.42 (d,128.1q, 1H, 155.3q, 168.6q. HRMS (APCI) calcd. for C13H13N4S [M + H]: 257.0861 found 257.0812. 1-(2-(2-((1H-Indol-5-yl)m136.9q,J = 8.5Hz), 143.8, 7.49 155.3q, (dd, 1H, 168.6q.ethylene)hydrazinyl)-4-m J = 1.3 HRMS Hz, J(APCI) = 8.5 Hz), calcd. 7.37ethylthiazol-5-yl)ethanone for (s, C 1H),13H13 N8.064S [M(s, +1H), H]: ( 11.272257.0861) (s, 1H), found 11.56 257.0812. (br, 1H); 13 1-(2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)-4-methylthiazol-5-yl)ethanoneC-NMR (DMSO-d6, 100 MHz) δ (ppm): 17.6, 102.2, 104.0, 112.4, 119.2,( 2120.3,) 126.2q, 126.7, 128.1q, 1-(2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)-4-methylthiazol-5-yl)ethanone (2) 136.9q, 143.8, 155.3q, 168.6q. HRMS (APCI) calcd. for C13H13N4S [M + H]: 257.0861 found 257.0812. 1-(2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)-4-methylthiazol-5-yl)ethanone (2)

Sand-colored powder, yield 2.4 g, 82%, m.p. 256 °C, crystallized from ethanol; 1H-NMR (acetone-d6,

400 MHz) δ (ppm): 2.44 (s, 3H), 2.53 (s, 3H), 3.66 (br, 1H), 6.57–6.58 (m, 1H), 7.39–7.41 (m, 1H), 7.25 Sand-colored powder, yield 2.4 g, 82%, m.p. 256 °C, crystallized from ethanol; 1H-NMR (acetone-d6, 13 (d,400 1H, MHz)Sand-colored J = 8.6δ (ppm): Hz), 7.66 powder,2.44 (d, (s, 1H, 3H), yield J = 2.53 8.6 2.4 Hz),(s, g, 3H), 7.88 82%, 3.66 (s, m.p. 1H), (br, 8.271H), 256 (s, ◦6.57–6.58C, 1H), crystallized 10.60 (m, (br, 1H), 1H); from 7.39–7.41 C-NMR ethanol; (m, (acetone- 11H),H-NMR 7.25d6, 1 100 MHz)Sand-colored δ (ppm): powder, 19.6, 29.6, yield 101.1,2.4 g, 82%,104.0, m. 111.7,p. 256 119.°C, crystallized6, 120.9, 126.0, from 128.3q, ethanol; 130.5q, 13H-NMR 132.4q, (acetone- 146.9,d6, (acetone-(d, 1H, J =d 68.6, 400 Hz), MHz) 7.66 δ(d,(ppm): 1H, J = 2.44 8.6 (s,Hz), 3H), 7.88 2.53 (s, (s,1H), 3H), 8.27 3.66 (s, 1H), (br, 1H), 10.60 6.57–6.58 (br, 1H); (m,C-NMR 1H), 7.39–7.41 (acetone- (m,d6, 1H),156.0q,100400 MHz) 7.25 171q, (d, δδ (ppm): 1H,(ppm):189.4q.J = 8.62.44HRMS19.6, Hz), (s, 29.6, 3H),(APCI) 7.66 101.1, 2.53 (d, calcd. 1H, (s, 104.0, 3H),J =for 8.6 111.7, 3.66C Hz),15H (br,15 119.N 7.88 41H),OS6, (s, [M120.9, 6.57–6.58 1H), + H]: 8.27126.0, 299.0967, (s,(m, 128.3q, 1H), 1H), 10.60found 7.39–7.41 130.5q, (br, 299.0911. 1H); 132.4q,(m,13 1H), C-NMR 146.9, 7.25 (d, 1H, J = 8.6 Hz), 7.66 (d, 1H, J = 8.6 Hz), 7.88 (s, 1H), 8.27 (s, 1H), 10.60 (br, 1H); 13C-NMR (acetone-d6, (acetone-2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)-4-phenylthiazole156.0q, 171q,d6, 100 189.4q. MHz) HRMSδ (ppm): (APCI) 19.6, calcd. 29.6, 101.1,for C15 104.0,H15N4 111.7,OS ( 3[M) 119.6, + H]: 120.9,299.0967, 126.0, found 128.3q, 299.0911. 130.5q, 132.4q, 146.9,100 MHz) 156.0q, δ (ppm): 171q, 189.4q. 19.6, 29.6, HRMS 101.1, (APCI) 104.0, calcd. 111.7, for 119. C H6, 120.9,N OS 126.0, [M + H]:128.3q, 299.0967, 130.5q, found 132.4q, 299.0911. 146.9, 2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)-4-phenylthiazole15 (153) 4 156.0q, 171q, 189.4q. HRMS (APCI) calcd. for C15H15N4OS [M + H]: 299.0967, found 299.0911. 2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)-4-phenylthiazole (3) 2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)-4-phenylthiazole (3)

Light brown powder yield 2.3 g, 75%, m.p. 246 °C, crystallized from ethanol; 1H-NMR (DMSO-d6,

400 MHz) δ (ppm): 6.52 (s, 1H), 7.34 (s, 2H), 7.41–7.45 (m, 3H), 7.51 (d, 1H, J = 8.5 Hz), 7.60 (d, 1H, Light brown powder yield 2.3 g, 75%, m.p. 246 °C, crystallized from ethanol; 1H-NMR (DMSO-d6, 13 J400 = 8.5 MHz) Hz), δ7.74 (ppm): (d, 2H, 6.52 J = (s,7.4), 1H), 7.86 7.34 (s, 1H),(s, 2H), 8.41 7.41–7.45(s, 1H), 11.47 (m, (s,3H), 1H), 7.51 12.01 (d, (br, 1H, 1H); J = 8.5C-NMR Hz), 7.60 (DMSO- (d, 1H,d6, 100 MHz) δ (ppm): 102.6, 104.5, 112.6, 119.7, 121.6, 125.1, 126.4q, 126.5 (2C), 127.0q, 128.1,1 129.2 (2C), 130.2q,6 J = 8.5Light Hz), brown7.74 (d, powder2H, J = 7.4), yield 7.86 2.3 (s, g, 1H),75%, 8.41 m.p. (s, 246 1H), °C, 11.47 crystallized (s, 1H), 12.01from ethanol;(br, 1H); 13H-NMRC-NMR (DMSO-(DMSO-d6, ◦ 1 137.5,100400 MHz)MHz)Light 148.5, δ brownδ (ppm):154.6q, (ppm): powder 102.6, 168.8q. 6.52 104.5, (s, yieldHRMS 1H), 112.6, 2.3 7.34(APCI) g, 119.7, 75%,(s, calcd.2H), 121.6, m.p. 7.41–7.45 for 246125.1, C18C,H 126.4q,15 crystallized(m,N4S 3H), [M126.5 + 7.51H]: (2C), from 319.1017, (d, 127.0q, ethanol;1H, J found128.1, = 8.5H-NMR 129.2Hz),319.0959. (2C),7.60 (DMSO- (d,130.2q, 1H,d6, 400 MHz) δ (ppm): 6.52 (s, 1H), 7.34 (s, 2H), 7.41–7.45 (m, 3H), 7.51 (d, 1H, J13= 8.5 Hz), 7.60 (d,6 Ethyl137.5,J = 8.5 2-(2-((1H-Indol-5-yl)methylene)h 148.5,Hz), 7.74 154.6q, (d, 2H,168.8q. J = 7.4), HRMS 7.86 (APCI) (s,ydrazinyl)-4-methylthi 1H), calcd. 8.41 (s,for 1H), C18H 11.4715Nazole-5-carboxylate4S (s, [M 1H), + H]: 12.01 319.1017, (br, (1H);4 )found C-NMR 319.0959. (DMSO- d , 1H,100 MHz)J = 8.5 δHz (ppm):), 7.74 102.6, (d, 2H,104.5,J =112.6, 7.4), 119.7, 7.86 (s,121.6, 1H), 125.1, 8.41 126.4q, (s, 1H), 126.5 11.47 (2C), (s, 1H),127.0q, 12.01 128.1, (br, 129.2 1H); (2C),13C-NMR 130.2q, Ethyl 2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)-4-methylthiazole-5-carboxylate (4) (DMSO-137.5, 148.5,d6, 100154.6q, MHz) 168.8q.δ (ppm): HRMS 102.6, (APCI) 104.5, calcd. 112.6, for C 119.7,18H15N 121.6,4S [M 125.1,+ H]: 319.1017, 126.4q, 126.5 found (2C), 319.0959. 127.0q, 128.1,

Ethyl 2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)-4-methylthiazole-5-carboxylate (4) Light gray powder, purified by column chromatography, yield 1.9 g, 60%, m.p. 265 °C, from 1 toluene/acetone;Light gray powder,H-NMR purified(DMSO- byd6, column400 MHz) chroma δ (ppm):1.27tography, (t, yield 3H, 1.9J = g,7.1Hz), 60%, m.p.2.47 265(s, 3H),°C, from4.22 (q, 2H, 7.1), 6.50–6.51 (m, 1H), 7.39–7.40 (m, 1H), 7.45 (d, 1H, J = 8.5 Hz), 7.54 (d, 1H, J = 8.5 Hz), 7.80 toluene/acetone; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm):1.27 (t, 3H, J = 7.1Hz), 2.47 (s, 3H), 4.22 13 (s,(q, 1H),2H,Light 7.1),8.16 gray 6.50–6.51(s, 1H), powder, 11.33 (m, 1H),purified(s, 1H), 7.39–7.40 12.28by column (br, (m, 1H).1H), chroma 7.45C-RMN: (d,tography, 1H, (DMSO- J = 8.5yield dHz),6 , 1.9100 7.54 g, MHz) 60%, (d, 1H,δ m.p. (ppm): J = 2658.5 14.8, Hz),°C, from17.4, 7.80 60.5, 102.4, 108.9Cq,1 112.5, 119.5, 121.1,6 125.6Cq, 126.7, 128.1Cq, C137.3Cq, 146.9, 158.1Cq, 162.9Cq, (s,toluene/acetone; 1H), 8.16 (s, 1H), H-NMR 11.33 (s,(DMSO- 1H), 12.28d , 400 (br, MHz) 1H). 13δC-RMN: (ppm):1.27 (DMSO- (t, 3H,d6, 100J = MHz)7.1Hz), δ 2.47(ppm): (s, 14.8,3H), 17.4,4.22 169.7Cq.60.5,(q, 2H, 102.4, 7.1), HRMS 108.9Cq,6.50–6.51 (APCI) 112.5, (m, calcd. 1H), 119.5, for7.39–7.40 C121.1,16H17 N125.6Cq,(m,4O 21H),S [M 7.45126.7, + H]: (d, 329.1072,128.1Cq, 1H, J = 8.5 C137.3Cq,found Hz), 329.1013. 7.54 146.9, (d, 1H, 158.1Cq, J = 8.5 Hz),162.9Cq, 7.80 13 Ethyl169.7Cq.(s, 1H), 2-(2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)thiazol-4-yl)acetate 8.16 HRMS (s, 1H), (APCI) 11.33 calcd. (s, 1H), for C12.2816H17 (br,N4O 1H).2S [M +C-RMN: H]: 329.1072, (DMSO- found (5d)6 , 100 329.1013. MHz) δ (ppm): 14.8, 17.4, 60.5, 102.4, 108.9Cq, 112.5, 119.5, 121.1, 125.6Cq, 126.7, 128.1Cq, C137.3Cq, 146.9, 158.1Cq, 162.9Cq, Ethyl 2-(2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)thiazol-4-yl)acetate (5) 169.7Cq. HRMS (APCI) calcd. for C16H17N4O2S [M + H]: 329.1072, found 329.1013. Ethyl 2-(2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)thiazol-4-yl)acetate (5)

Light brown powder yield 1.9 g, 60%, m.p. 191 °C, crystallized from ethanol; 1H-NMR (acetone-d6,

400 MHz) δ (ppm): 1.33 (t, 3H, J = 7.1 Hz), 3.05 (br, 1H), 3.58 (s, 2H), 4.22 (q, 2H, J = 7.1 Hz), 6.54–6.55 Light brown powder yield 1.9 g, 60%, m.p. 191 °C, crystallized from ethanol; 1H-NMR (acetone-d6, (m, 1H), 6.59 (s, 1H), 7.37–7.38 (m, 1H), 7.49 (d, 1H, J = 8.5 Hz), 7.63 (dd, 1H, J = 8.5Hz, J = 1.4 Hz), 8.19 400 MHz) δ (ppm): 1.33 (t, 3H, J = 7.1 Hz), 3.05 (br, 1H), 3.58 (s, 2H), 4.22 (q, 2H, J = 7.1 Hz), 6.54–6.55 (m, 1H),Light 6.59 brown (s, 1H), powder 7.37–7.38 yield (m, 1.9 1H),g, 60%, 7.49 m.p. (d, 1H,191 °C,J = 8.5crystallized Hz), 7.63 from(dd, 1H,ethanol; J = 8.5Hz, 1H-NMR J = 1.4 (acetone- Hz), 8.19d6, 400 MHz) δ (ppm): 1.33 (t, 3H, J = 7.1 Hz), 3.05 (br, 1H), 3.58 (s, 2H), 4.22 (q, 2H, J = 7.1 Hz), 6.54–6.55 (m, 1H), 6.59 (s, 1H), 7.37–7.38 (m, 1H), 7.49 (d, 1H, J = 8.5 Hz), 7.63 (dd, 1H, J = 8.5Hz, J = 1.4 Hz), 8.19 Molecules 2017, 22, 260 7 of 11 forMolecules 24 h. 2017 The, 22 reaction, 260 progress was monitored by TLC (toluene/AcOEt 2/1 v/v; silica plates), after7 of the 11 reaction completed, the mixture was neutralized at pH 7 with NaHCO3 aqueous solution (10%). The precipitatefor 24 h. The was reaction filtered progress and recrysta was monitoredllized from by ethanol, TLC (toluene/AcOEt derivatives 4 and 2/1 6 v were/v; silica purified plates), by after column the chromatographyreaction completed, (silicagel the mixture 60, eluent was toluene/acetoneneutralized at pH 4/1 7 vwith/v). NaHCO3 aqueous solution (10%). The precipitate was filtered and recrystallized from ethanol, derivatives 4 and 6 were purified by column 2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)-4-methylthiazolechromatography (silicagel 60, eluent toluene/acetone 4/1 v(1/v) ). 2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)-4-methylthiazole (1)

Light gray powder, yield 2.0 g, 79%, m.p. 182 °C, crystallized from ethanol; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 2.16 (s, 3H), 6.33 (s, 1H), 6.48–6.49 (m, 1H), 7.37–7.38 (m, 1H), 7.42 (d, 1H, J = 8.5Hz),Light gray7.49 (dd,powder, 1H, Jyield = 1.3 2.0 Hz, g, J79%, = 8.5 m. Hz),p. 182 7.37 °C, (s, crystallized 1H), 8.06 (s, from 1H), ethanol; 11.27 (s, 1 H-NMR1H), 11.56 (DMSO- (br, 1H);d6, 13400C-NMR MHz) (DMSO- δ (ppm):d6, 2.16100 MHz)(s, 3H), δ (ppm):6.33 (s, 17.6, 1H), 102.2, 6.48–6.49 104.0, (m, 112.4, 1H), 119.2, 7.37–7.38 120.3, (m, 126.2q, 1H), 126.7, 7.42 (d,128.1q, 1H, 136.9q,J = 8.5Hz), 143.8, 7.49 155.3q, (dd, 1H, 168.6q. J = 1.3 HRMS Hz, J(APCI) = 8.5 Hz), calcd. 7.37 for (s, C 1H),13H13 N8.064S [M(s, 1H),+ H]: 11.27257.0861 (s, 1H), found 11.56 257.0812. (br, 1H); 13C-NMR (DMSO-d6, 100 MHz) δ (ppm): 17.6, 102.2, 104.0, 112.4, 119.2, 120.3, 126.2q, 126.7, 128.1q, 1-(2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)-4-methylthiazol-5-yl)ethanone (2) 136.9q, 143.8, 155.3q, 168.6q. HRMS (APCI) calcd. for C13H13N4S [M + H]: 257.0861 found 257.0812. 1-(2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)-4-methylthiazol-5-yl)ethanone (2)

Sand-colored powder, yield 2.4 g, 82%, m.p. 256 °C, crystallized from ethanol; 1H-NMR (acetone-d6, 400 MHz) δ (ppm): 2.44 (s, 3H), 2.53 (s, 3H), 3.66 (br, 1H), 6.57–6.58 (m, 1H), 7.39–7.41 (m, 1H), 7.25 1 6 (d, 1H,Sand-colored J = 8.6 Hz), powder,7.66 (d, 1H, yield J = 2.4 8.6 g, Hz), 82%, 7.88 m. p.(s, 256 1H), °C, 8.27 crystallized (s, 1H), 10.60 from (br, ethanol; 1H); 13H-NMRC-NMR (acetone-d6, 100400 MHz) δδ (ppm):(ppm): 2.44 19.6, (s, 29.6, 3H), 101.1, 2.53 (s, 104.0, 3H), 111.7,3.66 (br, 119. 1H),6, 120.9, 6.57–6.58 126.0, (m, 128.3q, 1H), 7.39–7.41 130.5q, 132.4q,(m, 1H), 146.9, 7.25 13 6 156.0q,(d, 1H, J171q, = 8.6 189.4q.Hz), 7.66 HRMS (d, 1H, (APCI) J = 8.6 calcd. Hz), 7.88 for C(s,15 H1H),15N 8.274OS [M(s, 1H), + H]: 10.60 299.0967, (br, 1H); found C-NMR 299.0911. (acetone- d , 100 MHz) δ (ppm): 19.6, 29.6, 101.1, 104.0, 111.7, 119.6, 120.9, 126.0, 128.3q, 130.5q, 132.4q, 146.9, 2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)-4-phenylthiazole (3) 156.0q, 171q, 189.4q. HRMS (APCI) calcd. for C15H15N4OS [M + H]: 299.0967, found 299.0911. 2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)-4-phenylthiazole (3)

Molecules 2017, 22, 260 8 of 12 Light brown powder yield 2.3 g, 75%, m.p. 246 °C, crystallized from ethanol; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 6.52 (s, 1H), 7.34 (s, 2H), 7.41–7.45 (m, 3H), 7.51 (d, 1H, J = 8.5 Hz), 7.60 (d, 1H, 1 Light brown powder yield 2.3 g, 75%, m.p. 246 °C, crystallized from ethanol; 13H-NMR (DMSO-d6, 129.2J = 8.5 (2C),Hz), 130.2q,7.74 (d, 137.5,2H, J = 148.5, 7.4), 7.86 154.6q, (s, 1H), 168.8q. 8.41 HRMS(s, 1H), (APCI)11.47 (s, calcd. 1H), 12.01 for C (br,H 1H);N SC-NMR [M + H]: (DMSO- 319.1017,d6, 400 MHz) δ (ppm): 6.52 (s, 1H), 7.34 (s, 2H), 7.41–7.45 (m, 3H), 7.51 (d, 181H,15 J = 48.5 Hz), 7.60 (d, 1H, found100 MHz) 319.0959. δ (ppm): 102.6, 104.5, 112.6, 119.7, 121.6, 125.1, 126.4q, 126.5 (2C), 127.0q, 128.1, 129.2 (2C), 130.2q, 13 6 137.5,J = 8.5 148.5,Hz), 7.74 154.6q, (d, 2H, 168.8q. J = 7.4), HRMS 7.86 (APCI) (s, 1H), calcd. 8.41 (s,for 1H), C18H 11.4715N4S (s, [M 1H), + H]: 12.01 319.1017, (br, 1H); found C-NMR 319.0959. (DMSO- d , Ethyl100 MHz) 2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)-4-methylthiazole-5-carboxylate δ (ppm): 102.6, 104.5, 112.6, 119.7, 121.6, 125.1, 126.4q, 126.5 (2C), 127.0q,(4 )128.1, 129.2 (2C), 130.2q, Ethyl 2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)-4-methylthiazole-5-carboxylate (4) 137.5, 148.5, 154.6q, 168.8q. HRMS (APCI) calcd. for C18H15N4S [M + H]: 319.1017, found 319.0959. Ethyl 2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)-4-methylthiazole-5-carboxylate (4)

Light gray powder, purified by column chromatography, yield 1.9 g, 60%, m.p. 265 °C, from ◦ toluene/acetone;Light gray powder,1H-NMR puriﬁed (DMSO- byd6, column400 MHz) chromatography, δ (ppm):1.27 (t, yield 3H,1.9 J = g, 7.1Hz), 60%, m.p. 2.47 265(s, 3H),C, from4.22 Light gray powder,1 purified by column chromatography, yield 1.9 g, 60%, m.p. 265 °C, from toluene/acetone;(q, 2H, 7.1), 6.50–6.51H-NMR (m, 1H), (DMSO- 7.39–7.40d6, 400 (m, MHz) 1H), 7.45δ (ppm):1.27 (d, 1H, J = (t, 8.5 3H, Hz),J = 7.54 7.1Hz), (d, 1H, 2.47 J (s,= 8.5 3H), Hz), 4.22 7.80 (q, 1 6 2H,(s,toluene/acetone; 1H), 7.1), 8.16 6.50–6.51 (s, 1H), H-NMR (m, 11.33 1H), (s,(DMSO- 7.39–7.40 1H), 12.28d , (m, 400 (br, 1H), MHz) 1H). 7.45 13δC-RMN:(d, (ppm):1.27 1H, J (DMSO-= 8.5 (t, Hz), 3H,d6,7.54 100J = (d,MHz)7.1Hz), 1H, δJ 2.47(ppm):= 8.5 (s, Hz), 14.8,3H), 7.80 17.4,4.22 (s, (q, 2H, 7.1), 6.50–6.51 (m, 1H), 7.39–7.40 (m, 1H),13 7.45 (d, 1H, J = 8.5 Hz), 7.54 (d, 1H, J = 8.5 Hz), 7.80 1H),60.5, 8.16102.4, (s, 108.9Cq, 1H), 11.33 112.5, (s, 1H), 119.5, 12.28 121.1, (br, 1H).125.6Cq,C-RMN: 126.7, (DMSO-128.1Cq,d 6C137.3Cq,, 100 MHz) 146.9,δ (ppm): 158.1Cq, 14.8, 17.4,162.9Cq, 60.5, 13 6 102.4,169.7Cq.(s, 1H), 108.9Cq, 8.16 HRMS (s, 112.5,1H), (APCI) 11.33 119.5, calcd. (s, 121.1, 1H), for 125.6Cq,C12.2816H17 (br,N4 126.7,O 1H).2S [M 128.1Cq, +C-RMN: H]: 329.1072, C137.3Cq, (DMSO- foundd 146.9,, 100 329.1013. 158.1Cq,MHz) δ (ppm): 162.9Cq, 14.8, 169.7Cq. 17.4, HRMS60.5, 102.4, (APCI) 108.9Cq, calcd. 112.5, for C 119.5,H N 121.1,O S [M 125.6Cq, + H]: 329.1072, 126.7, 128.1Cq, found 329.1013.C137.3Cq, 146.9, 158.1Cq, 162.9Cq, Ethyl 2-(2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)thiazol-4-yl)acetate16 17 4 2 (5) 169.7Cq. HRMS (APCI) calcd. for C16H17N4O2S [M + H]: 329.1072, found 329.1013. Ethyl 2-(2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)thiazol-4-yl)acetate (5) Ethyl 2-(2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)thiazol-4-yl)acetate (5)

Light brown powder yield 1.9 g, 60%, m.p. 191 °C, crystallized from ethanol; 1H-NMR (acetone-d6, 400 MHz) δ (ppm): 1.33 (t, 3H, J = 7.1 Hz), 3.05 (br, 1H), 3.58 (s, 2H), 4.22 (q, 2H, J = 7.1 Hz), 6.54–6.55 (m, 1H),Light 6.59 brown (s, 1H), powder 7.37–7.38 yield (m, 1.9 1H),g, 60%, 7.49 m.p. (d, 1H,191 °C,J = 8.5crystallized Hz), 7.63 from(dd, 1H,ethanol; J = 8.5Hz, 1H-NMR J = 1.4 (acetone- Hz), 8.19d6, ◦ 1 400 MHz)Light δ brown (ppm): powder 1.33 (t, yield 3H, J 1.9 = 7.1 g, 60%, Hz), m.p.3.05 191(br, 1H),C, crystallized 3.58 (s, 2H), from 4.22 ethanol; (q, 2H, JH-NMR = 7.1 Hz), (acetone- 6.54–6.55d6, 400(m, 1H), MHz) 6.59δ (ppm): (s, 1H), 1.33 7.37–7.38 (t, 3H, (m,J = 7.11H), Hz), 7.49 3.05 (d, 1H, (br, 1H),J = 8.5 3.58 Hz), (s, 7.63 2H), (dd, 4.22 1H, (q, J 2H, = 8.5Hz,J = 7.1 J Hz),= 1.4 6.54–6.55Hz), 8.19 (m,Molecules 1H), 2017 6.59, 22 (s,, 260 1H), 7.37–7.38 (m, 1H), 7.49 (d, 1H, J = 8.5 Hz), 7.63 (dd, 1H, J = 8.5Hz, J = 1.48 Hz),of 11 13 8.19 (s, 1H), 10.61 (br, 1H); C-NMR (acetone-d6, 100 MHz) δ (ppm): 13.5, 37.0, 60.1, 102.1, 104.9, 111.8,(s, 1H), 119.3, 10.61 120.2, (br, 1H); 125.8, 13C-NMR 126.4q, 128.2q,(acetone- 137.0q,d6, 100 143.6, MHz) 145.6q, δ (ppm): 170q, 13.5, 189.4q. 37.0, HRMS60.1, 102.1, (APCI) 104.9, calcd. 111.8, for 16 17 4 2 C119.3,16H17 120.2,N4O 2125.8,S [M 126.4q, + H]: 329.1072, 128.2q, 137.0q, found 143.6, 329.1013. 145.6q, 170q, 189.4q. HRMS (APCI) calcd. for C H N O S [M + H]: 329.1072, found 329.1013. (E)-Ethyl 2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)thiazole-4-carboxylate (6) (E)-Ethyl 2-(2-((1H-Indol-5-yl)methylene)hydrazinyl)thiazole-4-carboxylate (6)

Light brown powder, purified by column chromatography, yield 1.4 g, 45%, m.p. 210 °C, from ◦ toluene/acetone;Light brown 1H-NMR powder, (DMSO- puriﬁedd6 by, 400 column MHz) chromatography,δ (ppm):1.29 (t, 3H, yield J = 6.8 1.4 Hz), g, 45%, 4.24 m.p.(q, 2H, 210 J = C,6.8 fromHz), 1 toluene/acetone;6.49–6.50 (m, 1H), H-NMR7.38–7.39 (DMSO- (m, 1H),d6 ,7.45 400 (d, MHz) 1H,δ J(ppm):1.29 = 8.8 Hz), 7.52 (t, 3H, (d,J 1H,= 6.8 J Hz),= 8.8 4.24Hz), (q, 7.72 2H, (s,J =1H), 6.8 Hz),7.77 6.49–6.50(s, 1H), 8.08 (m, (s, 1H), 1H), 7.38–7.39 11.30 (s, (m, 1H), 1H), 12.06 7.45 (br, (d, 1H,1H).J 13=C-NMR: 8.8 Hz), 7.52(DMSO- (d, 1H,d6, J100= 8.8 MHz) Hz), δ 7.72 (ppm): (s, 1H), 14.8, 7.77 60.5, (s, 13 1H),102.3, 8.08 108.5Cq, (s, 1H), 112.5, 11.30 119.2, (s, 1H), 120.6, 12.06 125.8Cq, (br, 1H). 126.8,C-NMR: 128.1Cq, (DMSO- C137.1Cqd6, 100, MHz)144.6, δ161.5,(ppm): 164.1Cq, 14.8, 60.5, 168.7Cq. 102.3, 108.5Cq,HRMS (APCI) 112.5, calcd. 119.2, for 120.6, C15H 125.8Cq,15N4O2S 126.8, [M + H]: 128.1Cq, 315.0916, C137.1Cq, found 144.6,315.0965. 161.5, 164.1Cq, 168.7Cq. HRMS (APCI) calcd. for C15H15N4O2S [M + H]: 315.0916, found 315.0965. 3.3. Biology 3.3. Biology 3.3.1. Cell Cultures 3.3.1. Cell Cultures The human cervical cancer cells (HeLa) and the human ovarian cancer cells (A2780) were obtained fromThe the humanEuropean cervical Centre cancer of Cell cells Cultures (HeLa) and(ECA theCC—Sigma human ovarian Aldrich, cancer Munich, cells (A2780) Germany). were These obtained cell fromlines were the European cultivated Centre under steril of Celle conditions Cultures (ECACC—Sigmaby using RPMI 1640 Aldrich, growth Munich,media supplemented Germany). These with fetal cell lines were cultivated under sterile conditions by using RPMI 1640 growth media supplemented calf serum (FCS), glutamine, and antibiotics (penicillin—streptomycin, 0.1%) at 37 °C and CO2 (5%) with fetal calf serum (FCS), glutamine, and antibiotics (penicillin—streptomycin, 0.1%) at 37 ◦C and COCell2 Proliferation(5%) Inhibition The MTT assay was used in order to determine the cytotoxicity. The cells were seeded in 96-well plates with 100 µL of cell solution (ca. 10.000 cells/well) and incubated for 24 h. The compounds 1–6 were initially dissolved in DMSO, followed by a series of successive dilutions using RPMI 1460 media, so that the final concentration of DMSO was under 0.1%. The cells were treated with indolyl-hydrazinyl-thiazoles derivatives (concentrations between 0.1 µM–100 µM) for 24 h. After this treatment, we removed the culture media and we added MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) Hanks media solution in each well. Then, the plates were incubated for 2 h. The formazan crystals that were formed by the mitochondrial dehydrogenase activity of vital cells were dissolved in DMSO. The optical density, an indicator of cellular viability, was quantified by colorimetric measurements and it is directly proportional to the amount of formazan crystals formed in the cells. A multimode microplate reader was used to measure the plates and the absorbance was detected at 570 nm. Note: As a positive control in the experiment, we used cisplatin drugs, in the same concentrations of the studied compounds. All the experiments were performed in triplicate.

Statistical Analysis Values are given as the mean ± SEM. The data, performed in triplicate, are represented as averages of independent experiments. Graph Pad Prism 5 biostatistics software was used in order to process the experimental data.

3.3.2. Antioxidant Activity

DPPH Based Free Radical Scavenging Activity The antioxidant potential of the indolyl-hydrazinyl-thiazoles 1–6 was measured in reaction with DPPH radical and quantified using a spectrophotometric method. The color change of DPPH radical solution from purple to yellow in the presence of indolyl-hydrazinyl-thiazoles 1–6, generated a decrease of absorbance intensity at 517 nm. Molecules 2017, 22, 260 9 of 12

Cell Proliferation Inhibition The MTT assay was used in order to determine the cytotoxicity. The cells were seeded in 96-well plates with 100 µL of cell solution (ca. 10.000 cells/well) and incubated for 24 h. The compounds 1–6 were initially dissolved in DMSO, followed by a series of successive dilutions using RPMI 1460 media, so that the ﬁnal concentration of DMSO was under 0.1%. The cells were treated with indolyl-hydrazinyl-thiazoles derivatives (concentrations between 0.1 µM–100 µM) for 24 h. After this treatment, we removed the culture media and we added MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) Hanks media solution in each well. Then, the plates were incubated for 2 h. The formazan crystals that were formed by the mitochondrial dehydrogenase activity of vital cells were dissolved in DMSO. The optical density, an indicator of cellular viability, was quantiﬁed by colorimetric measurements and it is directly proportional to the amount of formazan crystals formed in the cells. A multimode microplate reader was used to measure the plates and the absorbance was detected at 570 nm. Note: As a positive control in the experiment, we used cisplatin drugs, in the same concentrations of the studied compounds. All the experiments were performed in triplicate.

3.3.2. Antioxidant Activity

DPPH Based Free Radical Scavenging Activity The antioxidant potential of the indolyl-hydrazinyl-thiazoles 1–6 was measured in reaction with DPPH radical and quantiﬁed using a spectrophotometric method. The color change of DPPH radical solution from purple to yellow in the presence of indolyl-hydrazinyl-thiazoles 1–6, generated a decrease of absorbance intensity at 517 nm. A DPPH methanolic solution (200 µL, 0.1 g/L) was mixed with a methanolic solution of one of the tested compounds 1–6 as follows 1 (1.6 µg/mL), 2 (1.8 µg/mL), 3 (1.9 µg/mL), 4 (2 µg/mL), 5 (2 µg/mL), and 6 (2.4 µg/mL) and then each sample volume was adjusted up to 10 mL with methanol. The absorbance was measured at 517 nm after the samples were incubated for 30 min at 40 ◦C[24]. The radical scavenging ability for the tested indolyl-hydrazinyl-thiazoles 1–6 was calculated as a percentage of absorbance intensity: radical scavenging ability (%) = [(Ac − As)/Ac] × 100, where Ac is the absorbance of DPPH radical solution and As is the absorbance of the solution containing DPPH radical mixed with tested compound. The scavenging activity of Trolox and ascorbic acid were measured and compared with the one of the tested compounds. Antiradical activity has been expressed as IC50, the concentration (µg/mL) of the tested compounds required for a 50% DPPH inhibition.

Reducing Power by Ferric Reducing Antioxidant Power (FRAP) Test The solutions of the tested indolyl-hydrazinyl-thiazoles 1–6 have been prepared in the range of 1.35–1.64 mg/mL concentration. The samples were prepared by mixing of “FRAP” reagent see lit [25]. (6 mL) with H2O (0.4 mL) and indolyl-hydrazinyl-thiazole (0.4 mL). After 10 min the absorbance has been measured at 593 nm and the antioxidant capacity has been expressed by µM Trolox equivalent/g.

Electron Paramagnetic Resonance (EPR) Spectroscopy Method In EPR quartz capillary is inserted a mixture of DPPH methanolic solution (10 µL, 4.5 mmol) and indolyl-hydrazinyl-thiazole methanolic solution (10 µL, 5mmol) and EPR spectra were recorded at room temperature. Molecules 2017, 22, 260 10 of 12

The relative concentration of paramagnetic species were determined by a double integration of experimental spectra using XEPR Bruker software [26,27].

4. Conclusions A new series of 2-(2-((1H-Indol-5-yl) methylene)-hydrazinyl)-thiazole derivatives, 1–6, has been synthesized in moderate to good yields by a Hantzsch protocol and their structures were conﬁrmed by NMR spectroscopy and mass spectrometry. The in vitro cytotoxicity was evaluated for all indolyl-hydrazinyl-thiazoles 1–6 on human cervical cancer cells (HeLa) and human ovarian cancer cells (A2780); Compounds 1 and 3 had a good inhibition on both A2780 and HeLa while derivative 5 has been active only on HeLa cell lines. The antioxidant potential of newly synthesized thiazoles 1–6 was studied by three different methods, in two of these the radical scavenging properties were tested and one was based on their capacity to act as reducing agent. According to these results, derivatives 1 and 3 were found to be promising antioxidant agents. The chemoselectivity of radicals formation in reaction of 2-(2-((1H-indol-5-yl)methylene)- hydrazinyl)-thiazoles 1–6 with free DPPH radicals was investigated using theoretical quantum chemical calculation; based on this study the hydrogen atom is extracted from hydrazinyl bridge rather than from indole moiety.

Acknowledgments: Financial support from the Romanian Ministry of Education and Research (PN-II-ID-PCE-2012-4-0488) is greatly acknowledged. This work was supported also by the Swiss Enlargement Contribution in the framework of the Romanian-Swiss Research Program, project number IZERZO-142198/1. (A.Grozav). This paper was published under the frame of European Social Found, Human Resources Development Operational Programme 2007–2013, project no. POSDRU/159/1.5/S/136893” (A. Grozav). Author Contributions: Adriana Grozav conceived and designed the experiments; Adriana Grozav and Luiza Ioana Gaina performed the experiments and analyzed the data; Luiza Ioana G˘ainaand Ioan-Dan Porumb performed the quantum chemical calculations. The antiproliferative and antioxidant experiments have been made by Lorena Filip, Daniela Hanganu. Adriana Grozav, Luiza Ioana Gaina, Lorena Filip, Daniela Hanganu, Ioan-Dan Porumb wrote or contributed to the writing of the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

1. Browne, L.M.; Conn, K.L.; Ayert, W.A.; Tewari, J.P. The camalexins: New phytoalexins produced in the leaves of camelinasativa (cruciferae). Tetrahedron 1991, 47, 3909–3914. [CrossRef] 2. Glawischnig, E. Camalexin. Phytochemistry 2007, 68, 401–406. [CrossRef][PubMed] 3. Carbone, A.; Pennati, M.; Parrino, B.; Lopergolo, A.; Barraja, P.; Montalbno, A.; Spanò, V.; Sbarra, S.; Doldi, V.; de Cesare, M.; et al. Novel 1H-pyrrolo[2,3-b]pyridine derivatives nortopsentin analogues: Synthesis and antitumor activity in peritoneal mesothelioma experimental models. J. Med. Chem. 2013, 56, 7060–7072. [CrossRef][PubMed] 4. Diana, P.; Carbone, A.; Barraja, P.; Montalbano, A.; Parrino, B.; Lopergolo, A.; Pennati, M.; Zaffaroni, N.; Cirrincione, G. Synthesis and antitumor activity of 3-(2-phenyl-1,3-thiazol-4-yl)-1H-indoles and 3-(2-phenyl-1,3-thiazol-4-yl)-1H-7-azaindoles. ChemMedChem 2011, 6, 1300–1309. [CrossRef][PubMed] 5. Meleddu, R.; Distinto, S.; Corona, A.; Bianco, G.; Cannas, V.; Esposito, F.; Artese, A.; Alcaro, S.; Matyus, P.; Bogdan, D.; et al. (3Z)-3-(2-[4-(aryl)-1,3-thiazol-2-yl]hydrazin-1-ylidene)-2,3-dihydro-1H-indol-2-one derivatives as dual inhibitors of HIV-1 reverse transcriptase. Eur. J. Med. Chem. 2015, 93, 452–460. [CrossRef] [PubMed] 6. Makam, P.; Kankanala, R.; Prakash, A.; Kannan, T. 2-(2-Hydrazinyl)thiazole derivatives: Design, synthesis and in vitro antimycobacterial studies. Eur. J. Med. Chem. 2013, 69, 564–576. [CrossRef][PubMed] 7. Kondrasenko, A.A.; Goncharov, E.V.; Dugaev, K.P.; Rubaylo, A.I. CBL-2201. Report on a new designer drug: Napht-1-yl 1-(5-ﬂuoropentyl)-1H-indole-3-carboxylate. Forensic Sci. Int. 2015, 257, 209–213. [CrossRef] [PubMed] Molecules 2017, 22, 260 11 of 12

8. Cappadone, C.; Stefanelli, C.; Malucelli, E.; Zini, M.; Onofrillo, C.; Locatelli, A.; Rambaldi, M.; Sargenti, A.; Merolle, L.; Farruggia, G.; et al. p53-dependent and p53-independent anticancer activity of a new indole derivative in human osteosarcoma cells. Biochem. Biophys. Res. Commun. 2015, 467, 348–353. [CrossRef] [PubMed] 9. Kaushik, N.K.; Kaushik, N.; Attri, P.; Kumar, N.; Kim, C.H.; Verma, A.K.; Choi, E.H. Biomedical Importance of Indoles. Molecules 2013, 18, 6620–6662. [CrossRef][PubMed] 10. Estevão, M.S.; Carvalho, L.C.; Ribeiro, D.; Couto, D.; Freitas, M.; Gomes, A.; Fernandes, L.M.F.E.; Marques, M.M.B. Antioxidant activity of unexplored indole derivatives: Synthesis and screening. Eur. J. Med. Chem. 2010, 45, 4869–4878. [CrossRef][PubMed] 11. Ayati, A.; Emami, S.; Asadipour, A.; Shafiee, A.; Foroumadi, A. Recent applications of 1,3-thiazole core structure in the identification of new lead compounds and drug discovery. Eur. J. Med. Chem. 2015, 97, 699–718. [CrossRef][PubMed] 12. Rouf, A.; Tanyeli, C. Bioactive thiazole and benzothiazole derivatives. Eur. J. Med. Chem. 2015, 97, 911–927. [CrossRef][PubMed] 13. Chimenti, F.; Bolasco, A.; Secci, D.; Chimenti, A.; Granese, A.; Carradori, S.; Yáñez, M.; Orallo, F.; Ortuso, F.; Alcaro, S. Investigations on the 2-thiazolylhydrazyne scaffold: Synthesis and molecular modeling of selective human monoamine oxidase inhibitors. Bioorg. Med. Chem. 2010, 18, 5715–5723. [CrossRef][PubMed] 14. Nosrat, O.; Mahmoodi, N.O.; Khalili, B.; Rezaeianzade, O.; Ghavidast, A. One-pot multicomponent synthesis of indol-3-ylhydrazinyl thiazoles as antimicrobial agents. Res. Chem. Intermed. 2016, 42, 6531–6542. 15. Carradori, S.; Rotili, D.; de Monte, C.; Lenoci, A.; D’Ascenzio, M.; Rodriguez, V.; Filetici, P.; Miceli, M.; Nebbioso, A.; Altucci, L.; et al. Evaluation of a large library of (thiazol-2-yl)hydrazones and analogues as histone acetyltransferase inhibitors: Enzyme and cellular studies. Eur. J. Med. Chem. 2014, 80, 569–578. [CrossRef][PubMed] 16. Moldovan, C.M.; Oniga, O.; Parvu, A.; Tiperciuc, B.; Verite, P.; Pirnau, A.; Crisan, O.; Bojita, M.; Pop, P. Synthesis and anti-inflammatory evaluation of some new acyl-hydrazones bearing 2-aryl-thiazole. Eur. J. Med. Chem. 2011, 46, 526–534. [CrossRef][PubMed] 17. Grozav, A.; Gaina, L.I.; Pileczki, V.; Crisan, O.; Silaghi-Dumitrescu, L.; Therrien, B.; Zaharia, V.; Berindan-Neagoe, I. The Synthesis and Antiproliferative Activities of New Arylidene-Hydrazinyl-Thiazole Derivatives. Int. J. Mol. Sci. 2014, 15, 22059–22072. [CrossRef][PubMed] 18. Ignat, A.; Lovasz, T.; Vasilescu, M.; Fischer-Fodor, E.; Tatomir, C.B.; Cristea, C.; Silaghi-Dumitrescu, L.; Zaharia, V. Heterocycles 27. Microwave Assisted Synthesis and Antitumour Activity of Novel Phenothiazinyl-Thiazolyl-Hydrazine Derivatives. Arch. Pharm. Chem. Life Sci. 2012, 345, 574–583. [CrossRef] [PubMed] 19. Zaharia, V.; Ignat, A.; Palibroda, N.; Ngameni, B.; Kuete, V.; Fokunang, C.N.; Moungang, M.L.; Ngadjui, B.T. Synthesis of some p-toluenesulfonyl-hydrazinothiazoles and hydrazino-bisthiazoles and their anticancer activity. Eur. J. Med. Chem. 2010, 45, 5080–5085. [CrossRef] 20. Spartan’06. Wavefunction, Inc.: Irvine, CA, USA. Available online: http://www.wavefun.com/ (accessed on 20 September 2016). 21. Hehre, W.J. A Guide to Molecular Mechanics and Quantum Chemical Calculations; Wavefunction, Inc.: Irvine, CA, USA, 2003. 22. Stephens, P.J.; Devlin, F.J.; Chabulowski, C.F.; Frisch, M. Ab initio calculation of vibrational absorbtion and circular dichroism spectra using density functional force fields. J. Phys. Chem. 1994, 98, 11623. [CrossRef] 23. Yi, W.; Dubois, C.; Yahiaoui, S.; Haudecoeur, R.; Belle, C.; Song, H.; Hardre, R.; Reglier, M.; Boumendjel, A. Refinement of arylthiosemicarbazone pharmacophore in inhibition of mushroom tyrosinase. Eur. J. Med. Chem. 2011, 46, 4330–4335. [CrossRef] 24. Nastas˘a,C.; Tiperciuc, B.; Duma, M.; Benedec, D.; Oniga, O. New hydrazones bearing thiazole scaffold: Synthesis, characterization, antimicrobial, and antioxidant investigation. Molecules 2015, 20, 17325–17338. 25. Stratil, P.; Klejdus, B.; Kuban, V. Determination of total content of phenolic compounds and their antioxidant activity in vegetables-evaluation of spectrophotometric methods. J. Agric. Food Chem. 2006, 54, 607–616. [CrossRef][PubMed] 26. Espinoza, M.; Olea-Azar, C.; Speisky, H.; Rodríguez, J. Determination of reactions between free radicals and selected Chilean wines and transition metals by ESR and UV–vis technique. J. Spectrochim. Acta A Mol. Biomol. Spectrosc. 2009, 71, 1638–1643. [CrossRef][PubMed] Molecules 2017, 22, 260 12 of 12

27. Mocan, A.; Crisan, G.; Vlase, L.; Crisan, O.; Vodnar, D.C.; Raita, O.; Gheldiu, A.-M.; Toiu, A.; Oprean, R.; Tilea, I. Comparative Studies on Polyphenolic Composition, Antioxidant and Antimicrobial Activities of Schisandrachinensis Leaves and Fruits. Molecules 2014, 19, 15162–15179. [CrossRef][PubMed]

Sample Availability: Samples of the compounds 1–6 are available from the authors A.G. anl L.I.G.

© 2017 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).