Novel Thiosemicarbazone Derivatives from Furan-2-Carbaldehyde: Synthesis, Characterization, Crystal Structures, and Antibacterial, Antifungal, Antioxidant, and Antitumor Activities

, Candida albicans / Candida tropicalis fungi, and seven human tumor cell lines (HuTu80, H460, DU145, M-14, HT-29, MCF-7, and LNCaP), respectively. Te antioxidant activity was also studied by the DPPH assay. Compound 5 exhibited signifcant antibacterial activity against Staphylococcus aureus ATCC700699 (MIC=1 μ g/mL) compared to the nitrofurantoin and gentamicin reference drugs (MIC =1–25 and 10-> 100 μ g/mL, respectively). Compound 4 was ten times less active than amphotericin B (MIC= 5 μ g/mL) against Candida albicans (ATCC90028 and ATCC10231), while 1 exhibited a moderate efect of scavenging of DPPH radical (IC 50 =40.9 μ g/mL) in comparison to ascorbic acid reference compound (IC 50 =22.0 μ g/mL). Among all the studied thio-semicarbazones, 5 showed a higher cytotoxic activity (IC 50 =13.36–27.73 μ Μ ) in relation to the other tested compounds (IC 50 =34.84— > 372.34 μ Μ ) against all tested cell lines, except the LNCaP cell line, exhibiting its highest antiproliferative activity (IC 50 =13.36 μ Μ ) on the HuTu80 cell line. Besides, 8 and 9 exhibited high antitumor activity (IC 50 =13.31 and 7.69 μ Μ , respectively) against the LNCaP cells.


Introduction
Te interest in preparing new thiosemicarbazone derivatives of general formula R-CH�N-NHCSNHR 1 has attracted the attention of researchers in the area of organic and coordination chemistry, due to the ease of carrying out the reaction between diferent aldehyde groups (R-CHO) or ketones (R-CO-R′) with derivatives from thiosemicarbazide (NH 2 NHCSNHR 1 ) to obtain a broad spectrum of new thiosemicarbazone derivatives.Tese compounds which contain N and S atoms usually act as bidentate or multidentate chelating ligands and can coordinate to transition metal ions through the sulfur and hydrazine nitrogen atoms [1].Extensive investigations conducted by researchers have demonstrated that metal complexes with some thiosemicarbazone derivatives present a wide range of biological applications, such as antibacterial [2,3], antifungal [1,4], antiparasitic [5], antitubercular [6,7], antiviral [8,9], antioxidant [10,11], and antitumoral activities [12][13][14].
Numerous investigations have been carried out with α(N)-heterocyclic thiosemicarbazones because some of them act as potential antitumor agents.Te biochemical mechanism of action is due to the ability of these compounds in inhibiting the enzyme ribonucleoside diphosphate reductase (RDR) which is responsible for the synthesis of DNA precursors [15,16].Evidence of this fnding has been reported for Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbzone), a compound that inhibits the tumor cell growth at nanomolar concentrations and is being investigated in clinical trials (Phase 1 and Phase 2) for the treatment of cervical cancer [12].Besides, Triapine has chelating properties and forms coordination compounds with iron ions [17].Probably, the formation of redox-active iron complexes is a crucial step of the mechanism of action [18].
Due to the broad spectrum of biological applications of thiosemicarbazones containing heterocyclic groups, our research group presents the preparation and characterization of new compounds derived from R-furan-2-carbaldehyde thiosemicarbazone (R � H, CH 3 , CH 2 OH, CF 3 , NO 2 , Ph, F-Ph, CH 3 O-Ph, naphthoyl, and pyrazoyl) (chemical structure of these compounds are shown in Figure 1).Te in vitro antibacterial and antifungal activities of the synthesized thiosemicarbazone derivatives were evaluated against S. aureus and C. albicans, while the in vitro cytotoxic efect of these thiosemicarbazones was studied against several human tumor cell lines.Te antioxidant activity was also studied using the DPPH method.Relevant IC 50 and MIC values obtained from biological assays are also presented in Figure 1.

Reagents and Instruments.
All the chemicals were obtained from commercial suppliers and used as received.A Büchi melting point B-545 apparatus was employed for the determination of melting points.Infrared spectra (KBr discs) were recorded in the 4000−400 cm −1 region using a Nicolet iS10 FT-IR spectrometer.Mass spectra measurements of the prepared compounds in methanol solutions were performed with the assistance of the Waters-Quattro Premier XE ™ tandem quadrupole and MicrOTOF Bruker Daltonics mass spectrometers.Te NMR spectra were recorded at 500 or 400 or 300, 125.8 or 101 or 75.5, and 376 MHz for 1 H, 13 C, and 19 F acquisition, respectively, using dimethylsulfoxide-d 6 as solvent and tetramethylsilane 2 Journal of Chemistry (SiMe 4 ) as an internal reference, and employing the Bruker Avance III HD 500 or Varian Mercury Plus 400 or 300 spectrometers.Te chemical shifts expressed in ppm (δ) values for 1 H and 13 C spectra were referenced to residual 1 H and 13 C present in deuterated dimethylsulfoxide.Te NMR data are reported according to the atom numbers shown on the structural formulas for thiosemicarbazone derivatives as seen in Scheme 1.

General Procedure for the Synthesis of Compounds 1-10.
Compounds 1-10 were prepared according to the procedure described in the literature [22].Tiosemicarbazones were synthesized (Scheme 1) by refuxing the methanol solution (40 mL) of thiosemicarbazide (0.182 g, 2 mmol) with the furan-2-carbaldehyde derivative (2 mmol), dissolved in the same solvent (40-60 mL), for 3 h.Ten, the mixture was stirred for one day at room temperature.For compounds 2, 6, 7, and 8, good quality crystals were grown in methanol by slow evaporation of the solvent from their solutions.Tese crystals were selected and used for single crystal X-ray difraction studies.Te other prepared compounds were obtained as solids.Tese products were fltered, washed with ethanol (30 mL), dried, and recrystallized from hot acetone.  1  . 13

Gentamicin
Figure 1: General structure of the prepared X-furan-2-carbaldehyde thiosemicarbazone derivatives with some IC 50 and MIC values obtained from their biological assays.(9)

Single Crystal X-Ray Difraction Studies.
For compounds 2 and 7, crystallographic data were collected at 200 K using a STOE IPDS-2T imaging plate difractometer equipped with a sealed X-ray tube (Mo Kα radiation, λ = 0.71073 Å), while for compounds 6 and 8, data collection was performed at 180 K with a STOE StadiVari difractometer equipped with a microfocus X-ray tube (Cu Kα radiation, λ = 1.54186Å) and a Dectris Pilatus 300k detector.Unit cell parameters were determined and intensity data were integrated using Stoe X-Area software [27].A numerical absorption correction was applied, and for microsource data, also, a scaling correction was performed.Te data were corrected for Lorentz and Polarization efects [27].Direct methods were applied to solve the structures using ShelxT [28], and the structures were refned with ShelxL [29].For all nonhydrogen atoms, anisotropic thermal parameters were used.In the structure analysis of 2, hydrogen atoms were localized and refned with isotropic thermal parameters, and for 6, 7 and the C-H groups in 8, the hydrogen atoms were included on idealized positions applying the riding model.Te N-H hydrogen atoms in 8 were refned with isotropic thermal parameters.Diagrams of the molecular structures were obtained using Diamond software [30].Crystal data, experimental details, and refnement results are listed in Table 1.
(2) In Vitro Antitumor Assay.For a performed experiment, the cells were inoculated into 96-well plates at plating densities ranging from 3000 to 5000 cells per well and kept in humidifed 5% CO 2 incubator for 24 h at 37 °C, prior to the addition of the compounds.After 24 h, a plate containing the diferent cell lines was fxed with TCA in order to obtain the basal amount of cells.Te solutions of the prepared compounds in DMSO (1.0, 4.0, 16.0, and 63.0 μg/mL) and 5-fuorouracyl in DMSO (0.20, 0.70, 2.90, and 11.70 μg/mL) were transferred to the plates containing the diferent cell lines and incubated for an additional 48 h at 37 °C.Appropriate solvent controls were tested for comparison.After the 48 h, each plate was treated with TCA, washed, and air dried, and then it was stained with 0.4% sulforhodamine B dissolved in 1% acetic acid for 20 minutes at room temperature.After staining, unbound dye was discarded by washing with 1% acetic acid.A 10 mM Tris bufer solution (pH 10.5) was used with the purpose of extracting the proteinbound dye and then proceeded to read the absorbance at 510 nm on an automated microplate reader.Te IC 50 value corresponds to the concentration of the compound that inhibits cell growth by 50% and was determined graphically from percentage inhibition results-log concentrations curves.All compounds were tested in duplicate.
(3) Antibacterial Tests.Te in vitro antibacterial activity of the furan-2-carbaldehyde thiosemicarbazone derivatives 1-10 was studied against some of Gram-positive bacteria: Staphylococcus aureus ATCC43300, Staphylococcus aureus ATCC29213, Staphylococcus aureus ATCC25923, and Staphylococcus aureus ATCC700699, using the standard microdilution method and employing M07-A8 protocols CLSI [32].Nitrofurantoin and Gentamicin were tested and used as standard reference drugs.Briefy, inoculum was prepared with fresh bacterial cultures (suspended for 18 h) in NaCl 0.8% until to get an optical density in the range of 0.08-0.1 at 620 nm, which was diluted again at 1 : 20 with Müller-Hinton broth II (MHBII, Difco).Te prepared compounds were dissolved in 100% dimethyl sulfoxide (DMSO) to obtain 50 μg/μL Journal of Chemistry stock solutions and then were diluted with MHBII to get a fnal concentration of 12.5, 25, 50, 100, 150, and 200 μg/mL.Te wells were flled with these dilutions (100 μL/well) and 10 μL of inoculum suspension.Controls of growth and contamination were added.All the tests were performed in triplicate.After incubation at 35 °C for 24 h, 10 μL of tetrazolium violet 0.1% (Sigma T0138) was used as a growth indicator.Tese microplates were incubated again at 35 °C for 4 h and the minimum inhibitory concentration (MIC) value was determined considering the lowest sample concentration (without violet precipitate) that inhibits bacterial growth.
(4) Antifungal Activity.Te in vitro antifungal activity of the furan-2-carbaldehyde thiosemicarbazone derivatives 1-10 was studied against yeast strains: Candida albicans ATCC90028, Candida albicans ATCC10231 and Candida tropicalis ATCC750, using the standard microdilution method.Te M27-2A (CLSI) protocol was applied with few modifcations [32].Amphotericin B and Nystatin (commercial suspension) were tested and used as standard reference drugs.Briefy, inoculum was prepared with fresh cultures (suspended for 18 h) in NaCl (0.8%) adjusting at 0.08-0.1 of optical density (λ � 620 nm) followed by two dilutions (1 : 20 and 1 : 50) with RPMI medium in 2% of dextrose (RPMI-D).Te stock solutions of the studied compounds (50 μg/μL) were serially diluted in RPMI-D to get various fnal concentrations: 0.1, 0.2, 0.3, and 0.4 mg/mL.Tese diluted solutions were added into 96-well plates (50 μL per well) and inoculated with 50 μL of yeast inoculum prepared before.Te tetrazolium salt was used as a growth.Controls of growth and contamination were prepared, and three replicates were performed.Plates were incubated at 35 °C for 72 h.Te MIC value was determined similarly to that for antibacterial activity.
(5) Antioxidant Activity.Te antioxidant assay by DPPH radical scavenging was performed by the microdilution method [32].Tiosemicarbazone derivatives (50 mg/L) were serially diluted with methanol to obtain various concentrations in the region 25-2000 μg/mL.Before mixing, 100 μL of each compound was dispensed inside the microplate and immediately 10 μL of a fresh solution of DPPH (10 mg/L in methanol) was added.Plates were placed for 30 minutes in dark at 25 °C.Ten, the absorbance values were read at 517 nm.Ascorbic acid (reference antioxidant) was used as a positive control while methanol was used a negative control.Also, three replicates for each sample were performed.Te 50% antioxidant activity (IC 50 ) was calculated from the regression curve (% I vs. sample concentration) of each tested compound or ascorbic acid [32].

Results and Discussion
3.1.Synthesis.Te condensation reaction of furan-2-carbaldehyde derivatives with thiosemicarbazide in a 1 : 1 molar ratio in methanol solution resulted in the formation of the corresponding thiosemicarbazone derivatives 1-10 [22,33,34].Tis reaction condition led to good yields (63-90%) (Scheme 1).All the novel prepared thiosemicarbazones were characterized by mass spectrometry, IR, and 1 H, 13 C, and 19 F NMR spectroscopy.Te synthesized compounds are soluble in MeOH, CH 2 Cl 2 , CHCl 3 , CH 3 COCH 3 , DMF, and DMSO.Te mass and spectroscopic data confrmed the proposed structures and this information is given as Supplementary Material.2 confrm the proposed structural formulas of the synthesized compounds.

NMR Spectral Analysis.
Te characterization of the thiosemicarbazone derivatives 1-10 was carried out by using 1 H and 13 C NMR spectra, recorded in DMSO-d 6 solution.Te 1 H and 13 C NMR and 2D NMR spectra rendered the chemical shifts (δ), which were assigned to the corresponding groups.As referential evidence of this characterization, multinuclear ( 1 H, 13 C, and 19 F) NMR spectra for compound 7 are presented in Figures 2-4.
Te 1 H NMR spectra of the prepared thiosemicarbazones give singlets in the δ 11.28-11.85region assignable to the protons of the functional group = N-NH [41].Tese chemical shifts are similar to those reported for other thiosemicarbazones with fragments phenyl, chromone, and pyridine, where these compounds exist in the E or Z isomeric forms [42][43][44].Te -CH=N protons appeared as singlets in the region δ 7.94-8.07[41].Te furan ring protons were located at δ 6.43-7.80[45].Te chemical shifts of the = N-NH protons were afected by the presence of the methyl, hydroxymethyl, trifuoromethyl, nitro, and naphthyl substituents on the C-2 (in 2) and C-5 (in 3, 4, 5, and 9, respectively) carbons of the furan ring.For 2 and 3, the signals of the C-2 and C-5 protons are shifted upfeld (0.13 and 0.10 ppm, respectively), while for 4, 5, and 9, the signals of the C-5 protons are shifted downfeld (0.23, 0.44, and 0.13 ppm) in relation to the furan ring without substituent groups in 1 [45].For 3, 4, 5, 6, 9, and 10, the signals for the aromatic furan protons were afected due to the presence of the hydroxymethyl, trifuoromethyl, nitro, phenyl, naphthyl, and pyrazoyl substituents at the C-5 carbon of the furan ring.Tese protons are shifted, compared to the unsubstituted furan moiety, 0.18 ppm upfeld in the position C-4 for 3 and also showed a shift to downfeld for the C-3 (0.21, 0.41 and 0.16 ppm for 4, 5, and 9, respectively) and C-4 (0.73, 1.17, 0.5, 0.61 and 0.22 ppm for 4, 5, 6, 9, and 10, respectively) positions.Moreover, the signals for the phenyl ring protons were afected due to the presence of the fuoro and methoxy substituents (in 7 and 8, respectively).Tese signals are shifted, in relation to the unsubstituted phenyl group in 6, 0.16 ppm upfeld for the proton at the C3′carbon (for 7) and 0.46 ppm downfeld for the protons at the C3′and C5′carbons (for 8).
Te -NH 2 protons in compounds 1-10 show two broad singlets at δ 7.35-8.52,due to the delocalization of electron density on the C-N bond [20,35].
In the 13 C NMR spectra of the synthesized thiosemicarbazones 1-10, the signal at δ 130.24-134.07corresponds to the imine carbon (C=N).Tese chemical shifts are similar to those found for the pyrazole and pyridine thiosemicarbazone derivatives [4,22].Te signals due to the thiocarbonyl group (C=S) for all the synthesized compounds appear in the δ 177.39-178.89region [4,46].Te carbon chemical shifts found on the furan ring were observed at δ 108.11-157.54[45].Te presence of the diferent substituents groups on the furan ring produce slight changes in the chemical shifts of these aromatic carbons.For 5, the electron withdrawing efect of the NO 2 group was confrmed by the downfeld chemical shifts observed for C-1 (2.16 ppm), C-4 (2.80 ppm), and C-5 (7.65 ppm) of the furan ring with respect to the unsubstituted furan fragment.For 7 and 8, the phenyl ring carbon signals were afected due to the presence of the fuoro and methoxy substituents at the C-2 and C-4 carbons, respectively.Tese signals are shifted at δ 11.97-12.71and δ 14.53 upfeld at the C1′, C3′ and C3′, C5′ carbons for 7 and 8, respectively, compared to the unsubstituted phenyl fragment present in 6 [4].
On the other hand, the presence of the 2-fuorophenyl group in 7 was detected by using the 19 F NMR spectrum (Figure 4).A multiplet observed at δ −114.46 was due to the coupling of the fuorine atom with the aromatic protons.Tis chemical shift is in agreement with that found for 6-(4fuorophenyl)-pyridine-3-carbaldehyde thiosemicarbazone (δ −114.54)[22].

X-Ray Difraction
Studies of 2, 6, 7 and 8. Crystals of 2, 6, 7, and 8 suitable for single crystal structure analyses were grown in methanol by slow evaporation at room temperature from their respective concentrated methanol solutions.For the other thiosemicarbazone derivatives, except 1, only solids were obtained from acetone.Crystal data, data collection, and refnement details are summarized in Table 1.Besides, selected bond distances and bond/torsion angles are Journal of Chemistry given in Tables 3 and 4, respectively.Hydrogen-bonding interactions for 2, 6, 7, and 8 are summarized in Table 5. Te ORTEP diagrams of compounds 2, 6, 7, and 8 are shown in Figures 5-8.
As reported in Table 1, 2 and 7 belong to the monoclinic system, space group P2 1 /c, while 6 crystallizes in the orthorhombic space group Pbca and 8 in the triclinic space group P1, with one molecule in each asymmetric unit.
Te packing diagram of compound 2 (Figure 9) reveals that two parallel molecules, related by inversion symmetry, located in the central part of the unit cell and ten molecules located around the cell are stabilized by intermolecular N-H. ..O and N-H. ..S hydrogen bonds (Table 5).
On the other hand, the unit cell of 8 contains two molecules parallel to each other, related by inversion symmetry.For clarity, only one of them is shown in Figure 12.Te diagram of compound 8 shows that a molecule inside the unit cell and two molecules located on the same side of the outer part of the cell are stabilized by intermolecular N3-H. ..N1 and N3-H. ..S1 hydrogen bonds (Table 5).Besides, neighboring molecules outside the cell are linked by intermolecular N3-H. ..S1 hydrogen bonds.Figure 13 shows that molecules of 8 are packed as layers where it is evidenced by the closest distance between atoms of neighbouring layers: C2 (phenyl group) of one layer has a distance of 3.351(2) Å to the furan ring O atom (O2) of the next layer.

In Vitro Antitumor Evaluation.
Te cytotoxicity of all the studied compounds 1-10 in were assayed for their antiproliferative activities in vitro against diferent human tumor cells: H460, DU145, HuTu80, MCF-7, M-14, HT-29, and LNCap, as well as these prepared compounds were evaluated against BALB/3T3 normal cells using the sulforhodamine B colorimetric method [31].Te 5-fuorouracile (5-FU) reference drug was assayed as positive control.Te results are summarized as IC 50 values in μM in Table 6.
Te obtained results indicate that 5 (X = 5-NO 2 ) showed the greatest cytotoxic efect (IC 50 = 13.36-23.98μM) in relation to the other tested compounds (IC 50 = 34.84> 372.34 μM) against all cell lines assayed, except for the prostate carcinoma cell line (LNCaP), where 9 showed an excellent antiproliferative activity with IC 50 value of 7.69 μM against these cells.Tese results evidence that the cytotoxicity of 5 and 9 is enhanced when the nitro and naphthoyl substituent groups are attached to the furan ring at C-5 position, respectively [50,51].With respect to compound 5, the efect of the nitro group in the increase of the antiproliferative activity is probably related to the generation of morphological cell changes associated with apoptosis [52].Although it was shown that compound 5 exerted a great cytotoxic efect against several tested tumor cell lines, 5 showed a moderate toxicity (IC 50 = 11.67 μM) than 5-fuorouracile (IC 50 = 7.21 μM) against the 3T3 normal cells.On the other hand, substituting the hydrogen atom at the C-5 of the furan ring with the 4-methoxyphenyl group increased the cytotoxicity of 8 (IC 50 � 13.31 μΜ) in comparison to the other tested thiosemicarbazones, except for 9 against the LNCaP cell line.With respect to the 5fuorouracil (5-FU) antitumor drug (IC 50 � 11.97 μM) assayed against LNCaP cell line, 8 exhibited similar cytotoxic activiy.

Antifungal Activity.
Te in vitro antifungal activities of 1-10 were studied along with those of the standard Amphotericin and Nystatin.Te microorganisms used in this study included 3 yeasts from Candida species, namely, Candida albicans ATCC 90028, Candida albicans ATCC 10231, and Candida tropicalis ATCC 750.Te results of the in vitro antifungal activities for 1-10, and the reference drugs, are listed in Table 7.

Conclusion
In this work, ten furan-2-carbaldehyde thiosemicarbazone derivatives with diferent substituents were synthesized and characterized by mass spectrometry and FT-IR and NMR spectroscopic techniques.According to single crystal structure analyses, the crystal structures of 2, 6, and 7 exhibit E conformation around the N1-C6, N1-C11, and N1-C11 bonds, while the crystal structure of 8 shows Z conformation around the N1-C12 bond, involving an intramolecular N2-H. ..O2 hydrogen bond.
Te antibacterial and antifungal data revealed that 5 exhibited good activity against the Staphylococcus aureus ATCC700699 compared to the Nitrofurantoin and Gentamicin reference drugs.On the other hand, 1 exhibited moderate efect of scavenging of DPPH radical with respect to the ascorbic acid reference compound.Te new synthesized thiosemicarbazone derivatives were tested as candidate cytotoxic agents against diferent human tumor cells.Compound 5 showed moderate cytotoxic activity towards all tested cell lines, but it was particularly active at low micromolar concentrations against H460, HuTu80, MCF-7, and HT-29 cells, whereas compound 9 exhibited high cytotoxicity in comparison to the 5-fuorouracyl anticancer drug against the LNCaP cells and was more innocuous against 3T3 normal cells.

Journal of Chemistry
Our above mentioned results demonstrate that 5 is a promising candidate as antibacterial agent, while 5 and 9 can be considered as novel cytotoxic agents.Tese valuable fndings should have a signifcant impact on the antibacterial and antitumor drugs development feld.

Figure 14 :
Figure 14: Structures of the comparative thiosemicarbazone derivatives I (a-e).

Table 6 :
In vitro antiproliferative activity (IC 50 values a in μΜ) of the furan-2-carbaldehyde thiosemicarbazone derivatives (1-10) against the 3T3 nontumor cells and the diferent human tumor cell lines.Concentration of the compound (μΜ) that causes a 50% cell growth inhibition after 48 h of compound exposure.Te IC 50 values are averages of two independent assays.Te values are mean ± standard deviation of two independent experiments.b Mouse embryonic fbroblast cells.c 5-fuorouracile.Bold values represent a comparison of the best obtained results of the tested compounds with those obtained for the antitumoral drug for clinical use, 5-FU.

Table 7 :
Biological activity of the thiosemicarbazone derivatives 1-10 against Gram-positive bacteria and fungi strains determinated on the basis of MIC values, and scavenging DPPH radical activities determined on the basis of IC