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Original article
12 (
8
); 3183-3192
doi:
10.1016/j.arabjc.2015.08.013

Synthesis, structural elucidation and cytotoxicity of new thiosemicarbazone derivatives

, , , ,
a School of Chemical Sciences, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia
b School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia
c University of Basrah, College of Science, Department of Chemistry, Basra 61004, Iraq
d School of Chemistry, Minhaj University Lahore, Pakistan

⁎Corresponding author. Tel.: (604) 6533888x3578. sgteoh@usm.my (Teoh Siang Guan)

Received: , Accepted: ,
© The Author(s) 2015
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Syntheses and characterization (1H & 13C NMR and CHN) of four new thiosemicarbazone derivatives (1–4) are reported. The molecular structures of compounds 1–4 were determined using single crystal X-ray crystallographic technique. Cytotoxic effect of all the compounds was determined against three cancerous (PANC-1, HCT 116 and MCF-7) and one non-cancerous (NIH/3T3) cell lines. All the compounds remained safe against non-cancerous cells (IC50 > 500 μM) but induced significant cytotoxicity in the cancerous cell lines. Importantly, compounds 2 and 4 showed excellent cytotoxicity (IC50 = 10.0 and 0.7 μM against PANC-1; 14.9 and 9.4 μM against HCT 116; 14.3 and 15.8 μM against MCF-7 for 2 and 4 respectively). Based on the results it can be concluded that the thiosemicarbazone derivatives (2 and 4) can be further studied in detail for the purpose of chemotherapeutic drug development.

Keywords

Thiosemicarbazones
Crystal structures
Antitumor agents
Bioorganic chemistry
Cytotoxicity
1

1 Introduction

Thiosemicarbazones are a class of compounds having general structure R1R2C⚌N—NH—CS—NH2. These compounds can be synthesized by condensation of thiosemicarbazide with suitable aldehydes and ketones (Casas et al., 2000). Furthermore, suitable variations (substitutions) on either thioamide nitrogen atom, sulfur atom, or hydrazine nitrogen atoms provide interesting derivatives (Fig. 1).

Possible derivatives of thiosemicarbzides which can be obtained by substituting at, (a) thioamide nitrogen atom labeled as N4, (b) sulfur atom, and (c) hydrazine nitrogen atom.
Figure 1 Possible derivatives of thiosemicarbzides which can be obtained by substituting at, (a) thioamide nitrogen atom labeled as N4, (b) sulfur atom, and (c) hydrazine nitrogen atom.

Thiosemicarbazone molecules react with a wide range of metal cations to form metal complexes. The exploration of the antiviral, antibacterial, and antitumor properties of the thiosemicarbazones and their metal complexes has urged numerous investigations related to the catalysis and biological importance (Ferrari et al., 2004; Afrasiabi et al., 2004; Hu et al., 2006; Đilović et al., 2008; Kalaivani et al., 2012a,b; Yousef et al., 2013; Khandani et al., 2013; Demoro et al., 2013; Matesanz et al., 2013; Chandra et al., 2013; Sankaraperumal et al., 2013; Kunos et al., 2013; Moradi-Shoeili et al., 2013; Lukmantara et al., 2013; Su et al., 2013; Ermut et al., 2013; Marković et al., 2013; Kovala-Demertzi et al., 2004; Pingaew et al., 2013; Hussein et al., 2014). A number of thiosemicarbazone derivatives were synthesized and reported as potential anticarcinogenic (Lukmantara et al., 2013; Ermut et al., 2013; Pingaew et al., 2013). For example, Lukmantara and co-workers synthesized a series of thiosemicarbazone derivatives, mainly by varying the substitutions at thioamide nitrogen (Fig. 2A). All the synthesized compounds were screened against human neuroblastoma (SK-N-MC) and human feta lung fibroblast (MRC-5) cell lines which showed significant anticancer activity (Lukmantara et al., 2013). Ermut et al. (2013) synthesized a number of thiosemicarbazone derivatives (Fig. 2B), also by varying substitutions at thioamide nitrogen. All the compounds were screened against cervical cancer cell line (HeLa). Pingaew et al (2013) reported the syntheses of a series novel thiosemicarbazone derivatives (Fig. 2C) and their anticancer properties against four human cancerous cell lines namely cholangiocarcinoma (HuCCA-1), liver carcinoma (HepG2), lung carcinoma (A549) and acute lymphoblastic leukemia (MOLT-3). The significant anticancer properties were reported. Triapine is among the thiosemicarbazones which has been studied in detail for its anticancer properties (Ishiguro et al., 2015; Altintop et al., 2015; Rand et al., 2015; Trondl et al., 2014; Kunos and Sherertz, 2014; Merlot et al., 2015; Jansson et al., 2015).

Thiosemicarbazone derivatives reported recently as anticarcinogenic compounds.
Figure 2 Thiosemicarbazone derivatives reported recently as anticarcinogenic compounds.

The structure–activity relationship studies have shown that thiosemicarbazone derivatives containing phenolic groups have interesting anticancer properties (Baldini et al., 2003; Bisceglie et al., 2007; Ramachandran et al., 2012; Sampath et al., 2013). It has been observed that various substitutions (including nitro, methoxy, phenolic, etc.) at the thioamide nitrogen atoms, N(4), have significant effect on the biological efficacy of these compounds (Kowol et al., 2007; Mendes et al., 2009; Bernhardt et al., 2008). For example, the heterocyclic-N(4)-thiosemicarbazones have shown promising activity against breast cancer (Zeglis et al., 2011) as well as the presence of aromatic group at N(4) of isatin-β thiosemicarbazones derivatives has shown increased cytotoxicity against the parental KB-3-1 cell lines and the P-glycoprotein-expressing cell line KB-V1 (Hall et al., 2011). Herein, we describe the synthesis of some new thiosemicarbazone derivatives with various substitutions at terminal nitrogen (R3) as well as at the benzene ring (R1 and R2) as shown in Scheme 1, to further study this phenomenon.

Syntheses of thiosemicarbazone derivatives (1–4) by refluxing different hydroxybenzaldehydes (A) and thiosemicarbazide (B) in ethanol.
Scheme 1 Syntheses of thiosemicarbazone derivatives (1–4) by refluxing different hydroxybenzaldehydes (A) and thiosemicarbazide (B) in ethanol.

2

2 Results and discussion

2.1

2.1 Synthesis

New thiosemicarbazones (1–4) were prepared by condensation of thiosemicarbazide, 4-phenylthiosemicarbazide, 4-dimethylthiosemicarbazide and 4-ethylthiosemicarbazide with corresponding aldehyde derivatives (Vrdoljak et al., 2005). The substitutions (NO2, OCH3, CH2⚌CHCH2) were used to observe their effects on the biological efficacy. In the reaction medium compounds appeared either as white or yellow powderous material and were collected after filtration. Each of the products so obtained was washed with the fresh and cooled ethanol to remove the unreacted material and recrystalized. Each of the compounds was found to be easily soluble in DMF and DMSO. The assigned structures were confirmed by 1H NMR, 13C NMR and elemental analysis.

2.2

2.2 Characterization by NMR

In general, for all the synthesized compounds, the aromatic C—H chemical shifts appeared in the range 6.78–8.37 δ ppm. This is in accordance with the previous reports having similar structural features (Hu et al., 2006; Đilović et al., 2008). Importantly, compound 1 showed aromatic chemical shifts in the downfield region (7.05–8.37 δ ppm) which might be due to electron withdrawing effect of nitro group (R2 = NO2) attached to the benzene ring. In rest of the three compounds (2–4) these ranges remained almost comparable (6.78–7.55 δ ppm). Furthermore, the chemical shifts for —CH⚌N— group in all the synthesized compounds appeared at 8.60 ± 0.20 δ ppm which is in accordance with the reports involving thiosemicarbazones (Pingaew et al., 2013; Đilović et al., 2008). The —N—NH— proton peaks appeared at comparable region (11.44 ± 0.04 δ ppm), except this chemical shift in 4 which appeared at 11.77 δ ppm. This might be due to the presence of a phenyl group in 4 (R3 = C6H5) which might have induced electron withdrawing inductive effect that ultimately shifted the labeled peak toward downfield region compared to the other compounds of its class. The chemical shifts for rest of the substitutions (methoxy, allyl and alkyl) appeared in the range below 6.00 δ ppm.

Similarly, in 13C NMR spectral features, the aromatic carbon chemical shifts appeared in the range 111.0–144.3 δ ppm (Đilović et al., 2008; Hu et al., 2006). Noticeably, in 13C NMR, compound 1 showed aromatic carbon chemical shifts in the upfield region (111.0–37.6 δ ppm) compared to the other members of its class (2–4). It is important to highlight that in 1H NMR (as stated above) the respective aromatic C—H chemical shifts appeared downfield compared to the other members of its class whereas in 13C NMR these appeared in the upfield region. The exact reason of this observation is not clear however; the observed phenomenon has been reported to be noticed. Furthermore, the —CH⚌N— chemical shift for Cl and NO2 substituted compounds (1–2) appeared at comparable region (155.5 ± 0.1 δ ppm) whereas for allyl substituted compounds (3–4) at comparatively upfield region (147.8 δ ppm). The C⚌S chemical shifts appeared in the range 176.3–177.7 δ ppm and were found comparable with the literature (Pingaew et al., 2013; Afrasiabi et al., 2004; Chen et al., 2004). The chemical shifts for rest of the moieties (alkyl and methoxy groups) appeared in the range 14.4–55.8 δ ppm.

2.3

2.3 Crystallographic study

The crystallographic section describes the crystal structures of thiosemicarbazones (1–4). The crystallographic data have been provided in Table 1. The selected bond lengths and angles have been listed in Table 2 and their molecular structures have been shown in Figs. 3 and 4. It can be noticed that although both the compounds (1 and 3) have different substitutions at R1, R2 and R3 positions (Scheme 1), the compounds crystallized in the same crystal system (Triclinic) and the same space group (P i ¯ ). However, their asymmetric unit cells contain different number of molecules (2 molecules in the unit cell of 1 and 4 in case of 3). Furthermore, it can be noticed that the asymmetric units of 1 and 3 contain two crystallographically independent molecules (Rubčić et al., 2008; Hassan et al., 2014; Seidel et al., 2013). This characteristic was not found in rest of the compounds (2 and 4). Unlike 1 and 3, the compounds 2 and 4 crystallized in the monoclinic crystal system but in different space groups (P21/c for 2 and C2/c for 4). Among the crystallized compounds, maximum number of molecules were found in the asymmetric unit of 4 (8 molecules) with the maximum unit cell volume of 3353.8(2) Å3. The compound 4 has the highest melting point (228 °C) among the test compounds which might be due to the strong intermolecular interactions and molecular packing as discussed below.

Table 1 Crystallographic data of compounds 1–4.
Parameter 1 2 3 4
Empirical formula C10H12N4O3S,CH4O C10H12ClN3OS C12H15N3O2S C18H19N3O2S
Formula weight 297.24 257.75 265.34 341.43
Temperature (K) 100 100 100 100
Radiation/wavelength Mo Kα/0.71073 Å Mo Kα/0.71073 Å Mo Kα/0.71073 Å Mo Kα/0.71073 Å
Crystal morphology and color Blocks/yellow Needles/brown Plates/yellow Blocks/yellow
Crystal system, space group Triclinic, P i ¯ Monoclinic, P21/c Triclinic, P i ¯ Monoclinic, C2/c
Unit cell dimensions a = 7.0270(2) Å a = 13.6811(12)Å a = 10.5445(2)Å a = 30.9436(11)Å
b = 12.4004(4) Å b = 6.9785(6)Å b = 11.2670(2)Å b = 6.1094(2)Å
c = 16.3121(5) Å c = 12.3973(11)Å c = 11.4921(2)Å c = 18.0439(7))Å
α = 69.310(2)° α = 90° α = 78.678(1)° α = 90°
β = 85.745(2)° β = 102.324(2)° β = 80.444(1)° β = 100.518(2)°
γ = 85.452(2)° γ = 90° γ = 72.488(1)° γ = 90°
Volume (Å3) 1323.91(7) 1156.34(18) 1268.33(4) 3353.8(2)
Z, Calculated density (Mg/m3) 2, 1.426 4, 1.480 4, 1.390 8, 1.352
F(0 0 0) 596 536 560 1440
Crystal size (mm3) 0.12 × 0.29 × 0.61 0.07 × 0.25 × 0.49 0.08 × 0.21 × 0.46 0.12 × 0.16 × 0.56
θ range for data (°) 1.8–30.0 3.0–30.6 1.8–30.2 2.3–30.5
Limiting indices −9 ⩽ h ⩽ 9, −17 ⩽ k ⩽ 17, −22 ⩽ l ⩽ 22 −19 ⩽ h ⩽ 17, −9 ⩽ k ⩽ 9, −17 ⩽ l ⩽ 17 −14 ⩽ h ⩽ 14, −15 ⩽ k ⩽ 15, −15 ⩽ l ⩽ 16 −43 ⩽ h ⩽ 44, −8 ⩽ k ⩽ 8, −25 ⩽ l ⩽ 25
Reflections collected/unique 23,890/7643 11,491/3530 27,320/7398 19,809/5095
R (int) 0.043 0.047 0.054 0.042
Goodness of fit (S) 1.1 1.04 1.04 1.07
R [F2 > 2σ(F2)], wR(F2) 0.0572, 0.1297 0.0410, 0.1038 0.0513, 0.1131 0.0519, 0.1376
Observed data [I > 2σ(I)] 5842 2480 4782 3813
Table 2 Selected bond length (Å) and bond angles (°) for compounds 1–4.
Bonds 1 A, B 2 3 A, B 4
Bond lengths
S1—C8 [1.695(2), 1.707(2)] 1.6818(18) [1.6969(19), 1.6958(19)] 1.6863(19)
N1—N2 [1.373(3), 1.380(3)] 1.364(2) [1.382(2), 1.374(2)] 1.3747(18)
N1—C7 [1.283(3), 1.285(3)] 1.281(2) [1.284(2), 1.289(2)] 1.285(2)
N2—C8 [1.365(3), 1.354(3)] 1.368(2) [1.344(2), 1.351(2)] 1.359(2)
N3—C8 [1.325(3), 1.322(3)] 1.342(2) [1.329(2), 1.324(2)] 1.433(2)
N3—C9 [1.456(3), 1.459(3)] 1.468(2) 1.433(2)
C6—C7 [1.468(3), 1.461(3)] 1.457(3) [1.465(3), 1.462(3)] 1.464(3)
Bond angles
N2—N1—C7 [116.9(2), 114.37(19)] 119.04(15) [115.99(15), 117.94(15)] 115.43(15)
N1—N2—C8 [118.1(2), 120.18(19)] 118.35(14) [118.94(15), 120.15(15)] 1.3747(18)
C8—N3—C9 [125.9(2), 125.5(2)] 121.69(15) - 125.19(17)
S1—C8—N2 [118.49(17), 18.11(16)] 120.83(14) [120.12(14), 119.69(14)] 119.61(13)
S1—C8—N3 [125.29(19), 24.46(18)] 123.85(13) [122.67(15), 122.28(14)] 125.18(13)
N2—C8—N3 [116.2(2), 117.4(2)] 115.31(15) [117.21(17), 118.03(17)] 115.21(16)

Note: The assignments A and B for compounds 1 and 3 represent the bond angles and bond lengths of crystallographically different units (A and B) of same compound.

(A and B): (A) Molecular structure of compound 1 drawn at 50% probability, having two crystallographically independent molecules interacted by π–π stacking interactions. Both the units have slightly different geometric parameters. A methanol molecule was found to be packed along with the molecules of 1. (B) Molecular structure of 3 drawn at 50% probability, having two crystallographically independent molecules. Both the units have slightly different geometric parameters but same in all the chemical characteristics.
Figure 3 (A and B): (A) Molecular structure of compound 1 drawn at 50% probability, having two crystallographically independent molecules interacted by π–π stacking interactions. Both the units have slightly different geometric parameters. A methanol molecule was found to be packed along with the molecules of 1. (B) Molecular structure of 3 drawn at 50% probability, having two crystallographically independent molecules. Both the units have slightly different geometric parameters but same in all the chemical characteristics.
Molecular structures of 2 (A) and 4 (B) drawn at 50% probability.
Figure 4 Molecular structures of 2 (A) and 4 (B) drawn at 50% probability.

The crystallized molecules packed through the H-bondings and close π–π stacking interactions. For example, in compound 1 the molecules were found to have intermolecular H-bondings between phenolic and nitro groups (1.976 Å) as well as between sulfur atom of the molecule and hydrogen atom of solvent methanol (2.482 Å), see Fig. S9. Similarly, compound 2 has H-bonding between sulfur atom and methylenehydrazine group hydrogen atoms (2.729 Å) in a “V” shape and also between the oxygen atom of phenol group and aromatic ring hydrogen atom (Fig. S10). Compound 3 has intramolecular H-bondings in addition to the intermolecular H-bondings (Fig. S11).The hydrazine nitrogen atoms have intramolecular H-bonding with the phenolic oxygen atom (2.623 Å) and the intermolecular H-bonding between methoxy oxygen atom and hydrogen atoms of alkyl chains (Fig. S11). Compound 4 has similar but dense intra and intermolecular interactions (Fig. S12) compared to the other molecules (1–3). This is perhaps the reason that compound 4 has the highest melting point among the synthesized compounds. The molecular structures of 2 and 4 were found to have single firm units (Fig. 4).

In general, the molecular structures of 1, 3 and 4 showed anti orientation of N1 in relation to S, and syn orientation in relation to N3 (Fig. 5B). The terminal C9 atom of 1 and 4 is anti in relation to N2, and syn in relation to S. However, the compound 2 showed different orientations than compared to 1, 3 and 4. The nitrogen N1 is syn in relation to S, and anti in relation to N3 (Fig. 5A). The terminal carbon (C10) showed the syn geometry in relation to S, and the anti in relation to N2, whereas the other terminal C9 showed the contradictory orientation in relation to S and N2. Fig. 5 highlights selected geometrical orientations as described above.

Showing the syn- and anti- relation of N1 to S and N3 in compounds 1–4. All the orientations have been assigned according to the crystallographic data collected for these compounds.
Figure 5 Showing the syn- and anti- relation of N1 to S and N3 in compounds 1–4. All the orientations have been assigned according to the crystallographic data collected for these compounds.

Furthermore, the asymmetric unit of 3 contains two independent molecules (A and B), see Fig. 6. These molecules showed different conformations, the salicylaldimine plane of B, containing O1, N1 and C1—C7, rotates approximately 180° around C7—N1 bond compared to that of unit A, leads to change the anti geometry between C1 and N1 atoms in A to syn geometry in B. The S—C8 bond distance is longer than the N2—C8 bond distance, which indicates the compounds are present in the thione form.

Showing different confirmations of compound 3 based on crystallographic elucidation.
Figure 6 Showing different confirmations of compound 3 based on crystallographic elucidation.

2.4

2.4 In vitro anticancer study

All the synthesized compounds were tested for their inhibitory potential on the proliferation of three cancerous cell lines, namely pancreas cancer (PANC-1), breast cancer (MCF-7) and human colon cancer (HCT-116) as well as on the normal mouse fibroblasts NIH/3T3 cells using MTT assay. The median inhibitory concentrations (IC50) of test compounds are given in Table 3 whereas the photomicrographs of the treated cells are presented in Fig. 7. The compounds showed considerable cytotoxic effect on the three cancerous cell lines; however, none of the test compounds was found cytotoxic in normal mouse fibroblasts. PANC-1 was the most sensitive cell line to the cytotoxic potential of the test compounds where the IC50 of 1–4 was between 0.73 and 13.7 μM. Interestingly, the cytotoxic effect of compounds 2 and 4 was found to be stronger than that of 1 and 3. Although the cytotoxic effect of the test compounds was not as much stronger in MCF-7 and HCT-116 cells as it was in PANC-1 cells, 2 and 4 showed considerable cytotoxic effect in the two mentioned cell lines as well (IC50 < 16 μM). This relative increase in the cytotoxic potential of 2 and 4 may be attributed to the presence of R3 (C6H5) and R2 (Cl) groups in 2 and 4 respectively, as the activity reduced by the substitution of these groups by H and NO2 in 1 and 3 respectively. Furthermore, the cytotoxic potential of 1 and 4 may also be attributed to the presence of the terminal methyl and phenyl groups. These groups may help in interactive properties of a compound which may lead to these molecules to cross the cell wall barriers and let them interact with the DNA. Moreover, the presence of chlorine group in 1 may have enhanced its biological properties. In the recent year, a number of thiosemicarbazones have been synthesized and screened against various cell lines (Vandresen et al., 2014), where the thiosemicarbazones having halide showed noticeable activity. The effect of functional groups on the biological efficacy has always been significant (Wang et al., 2015; Duan and Zhang, 2011).

Table 3 IC50 values (μM) of compounds 1–4 tested against cancerous and non-cancerous cell lines.
Carcinoma cell lines Normal cell line
Cell lines PANC-1 HCT 116 MCF-7 NIH.3T3
Compounds 1 13.7 71.2 54.2 >500
2 10.0 14.9 14.3 >500
3 1.7 212.8 127.7 >500
4 0.7 9.4 15.8 >500
Standards 5-Flourouracil 5-Flourouracil Betulinic acid Betulinic acid
96.8 21.0 44.1 28.4
Photomicrographs of PANC-1, MCF-7, HCT-116 and NIH/3T3 cells treated with compounds 1–4. The images of cells were taken by a phase-contrast microscope (20× magnification) at the 48th hour of treatment with test drugs.
Figure 7 Photomicrographs of PANC-1, MCF-7, HCT-116 and NIH/3T3 cells treated with compounds 1–4. The images of cells were taken by a phase-contrast microscope (20× magnification) at the 48th hour of treatment with test drugs.

3

3 Experimental

3.1

3.1 Materials and instrumentation

Melting points were determined using a Stuart Scientific SMP1 melting point apparatus. NMR (1H and 13C) spectra were recorded on a Bruker spectrophotometer at 500 and 400 MHz using tetramethylsilane as an internal standard and DMSO-d6 as the solvent. Elemental analysis was performed using a Perkin Elmer 2400 series-11 CHN analyzer. X-ray crystallographic experiments were performed on a Bruker SMART APEXII CCD area-detector diffractometer using graphite monochromated Mo Kα radiation (λ = 0.71073 Å) at 100 K. The data were collected and reduced using APEX2 and SAINT programs. The structural features of all compounds were solved using the SHELXS-97 program package, and refined using the SHELXL-97 program (Sheldrick, 2008). All non-hydrogen atoms were anisotropically refined. The molecular graphics were created using SHELXTL. The single crystals of synthesized compounds (1–4) were obtained by slow evaporation method. According to this method, a saturated solution of each of the compounds in a suitable solvent (DMF or methanol) was sealed in a beaker using parafilm and then small holes were made into it using needle to let the solvent evaporate at slower rate. The crystals appeared and suitable crystals were selected for crystallography. All chemicals, including thiosemicarbazide, aldehydes, and solvents, were purchased from Sigma–Aldrich.

3.2

3.2 Synthesis

Compounds (1–4) were prepared by reacting equimolar ethanolic solutions of respective aldehyde derivative and the corresponding thiosemicarbazide. The reaction mixture was refluxed with stirring for 2 h. The product was filtered, washed with ethanol, recrystalized and air-dried. The single crystals of 2, 3 and 4, suitable for X-ray crystallographic analysis, were obtained by the slow evaporation of DMF solution at room temperature. The crystals of 1 were obtained by slow evaporation of methanol solution at room temperature.

3.2.1

3.2.1 N-ethyl-2-(5-nitro-2-hydroxybenzylidene) hydrazine carbothioamide (1)

A solution of 5-nitro-2-hydroxybenzaldehyde (0.70 g, 4.19 mmol) in ethanol (20 ml) was added to a solution of 4-ethyl-3-thiosemicarbazide (0.5 g, 4.19 mmol) in ethanol (20 ml). The resulting yellow solution was refluxed with stirring for 2 h. A yellow product formed when the solution cooled down to room temperature, then filtered, washed with fresh ethanol, recrystalized and air-dried. Needle yellow crystals were obtained by slow evaporation of its saturated dimethylformamide (DMF) solution at room temperature. m.p: 221–223 °C, (0.83 g, 79%). Anal. Calc. for C10H12N4O3S: C, 47.55; H, 4.75; N, 22.19. Found: C, 47.89; H, 4.68; N, 22.21%; 1H NMR (DMSO-d6, δ ppm): 1.03 (t, CH3), 3.41 (dd, CH2), 7.05, 8.13, 8.37 (d, d, s, H-aromatic), 8.73 (s, CS—NH), 8.81 (s, CH⚌N), 11.48 (s, N-NH); 13C NMR (DMSO-d6, δ ppm): 14.4 (CH3), 30.5 (CH2), 111.0–137.6 (C-aromatic), 155.4 (C⚌N), 176.3 (C⚌S).

3.2.2

3.2.2 N-dimethyl-2-(5-chloro-2-hydroxybenzylidene) hydrazine carbothioamide (2)

A solution of 5-chloro-2-hydroxybenzaldehyde (0.65 g, 4.19 mmol) in ethanol (20 ml) was added slowly to a solution of 4-dimethyl-3-thiosemicarbazide (0.5 g, 4.19 mmol) in ethanol (20 ml). The resulting yellow solution was refluxed with stirring for 2 h. White precipitates appeared when the solution cooled down to room temperature, then filtered and washed with fresh ethanol. The resulting material was recrystalized and air-dried. Colorless plates like crystals were obtained by slow evaporation of a saturated DMF solution at room temperature. m.p: 177–179 °C, (0.82 g, 76%). Anal. Calc. for C10H12ClN3OS: C, 46.55; H, 4.65; N, 16.29. Found: C, 47.00; H, 4.68; N, 16.52%; 1H NMR (DMSO-d6, δ ppm): 3.30 (CH3), 6.91, 7.28, 7.47 (H-aromatic), 8.43 (CH⚌N), 11.40 (N—NH); 13C NMR (DMSO-d6, ppm): 41.0 (CH3), 118.4–144.6 (C-aromatic), 155.6 (C⚌N), 179.1 (C⚌S).

3.2.3

3.2.3 2-(5-allyl-3-methoxy-2-hydroxybenzylidene) hydrazine carbothioamide (3)

A solution of 5-allyl-3-methoxy-2-hydroxybenzaldehyde (1.05 g, 5.48 mmol) in ethanol (20 ml) was added to a solution of thiosemicarbazide (0.5 g, 5.48 mmol) in ethanol (20 ml). The resulting colorless solution was refluxed with stirring for 2 h. A white fluffy product appeared when the solution cooled down to the room temperature, which was then filtered and washed with fresh ethanol. The resulting material was recrystalized and air-dried. Yellow plates like crystals were obtained by slow evaporation of a saturated DMF solution at room temperature. m.p: 189–191 °C, (1.17 g, 81%). Anal. Calc. for C12H15N3O2S: C, 54.26; H, 5.65; N, 15.82. Found: C, 54.53; H, 5.40; N, 16.05%; 1H NMR (DMSO-d6, δ ppm): 3.28 (CH2-Ph), 3.76 (OCH3), 5.02 (CH2⚌), 5.98 (⚌CH), 6.78, 7.34 (H-aromatic), 7.87, 8.08 (NH2), 8.37 (CS—NH), 9.13 (CH⚌N), 11.40 (N—NH); 13C NMR (DMSO-d6, δ ppm): 55.8 (O—CH3), 113.2 (CH2-Ph), 115.5 (CH2⚌), 117.4 (⚌CH), 120.1–144.3 (C-aromatic), 147.8 (C⚌N), 177.8 (C⚌S).

3.2.4

3.2.4 N-phenyl-2-(5-allyl-3-methoxy-2-hydroxybenzylidene) hydrazine carbothioamide (4)

A solution of 5-allyl-3-methoxy-2-hydroxybenzaldehyde (0.57 g, 2.98 mmol) in ethanol (20 ml) was added to a solution of 4-phenyl-3-thiosemicarbazide (0.5 g, 2.98 mmol) in ethanol (20 ml). The resulting colorless solution was refluxed with stirring for 2 h. A white fluffy product appeared in the reaction mixture when the solution cooled down to room temperature, which was then filtered, washed with ethanol, recrystalized and air-dried. Yellow blocks were obtained by slow evaporation of a saturated DMF solution at room temperature. m.p: 228–230 °C, (0.87 g, 86%). Anal. Calc. for C18H19N3O2S: C, 63.26; H, 5.56; N, 12.30. Found: C, 63.72; H, 5.97; N, 12.56%; 1H NMR (DMSO-d6, δ ppm): 3.30 (CH2-Ph), 3.79 (OCH3), 5.03 (CH2⚌), 5.99 (⚌CH), 6.80, 7.22, 7.37, 7.45, 7.55 (H-aromatic), 8.50 (CH⚌N), 9.98 (NH-Ph), 11.77 (N—NH); 13C NMR (DMSO-d6, δ ppm): 55.8 (O—CH3), 113.3 (CH2-Ph), 115.4 (CH2⚌), 117.7 (⚌CH), 120.0–144.5 (C-aromatic), 147.8 (C⚌N), 177.7 (C⚌S).

3.3

3.3 In Vitro anticancer studies

3.3.1

3.3.1 Cell lines

Human colorectal carcinoma cells (HCT-116), Human breast cancer cells (MCF-7), Human pancreatic epithlioidal carcinoma cells (PANC-1) and normal mouse fibroblasts NIH/3T3 cells were purchased from American-type culture collection (Rockvill, USA). HCT-116 cells were maintained in RPMI 1640 medium (Gibco, USA) whereas PANC-1, MCF-7 and NIH/3T3 cells were maintained in DMEM medium (Gibco, USA).

3.3.2

3.3.2 Anti-proliferative assay

The effect of compounds 1–4 on cell proliferation was evaluated by MTT assay as described earlier (Umar et al., 2014). The cells were harvested by trypsinization, re-suspended in 5 ml of the fresh medium and seeded thereafter in 96-well culture plates (1 × 105/ml). After an overnight incubation, the cells were treated with graded doses (200, 100, 50, 25, 12.5, 6.25 μM) of compounds 1–4, 5-Fluorouracil (reference drug for PANC-1 and HCT-116 cells) and betulinic acid (reference drug for NIH/3T3 and MCF-7 cells). The cells were incubated at 37 °C and 5% CO2 for 48 h. Images of cells were taken by using AMG EVOS fI inverted microscope at 20× magnification. 20 μL of MTT reagent (5 mg/ml) was added in each well and the cells were incubated further for 3 h at 37 °C and 5% CO2. The media were removed from each well and the crystals of formazan were dissolved by adding 150 μL of DMSO in each well and the absorbance was measured at 570 nm wavelength by using microplate reader (Multi-mode microplate reader Model Infinite 200, Tecan, Mannedorf, Switzerland). % Inhibition was calculated using the formula: % Inhibition of Cell Viability = 1 - ( absorbance of sample - blank ) / ( absorbance of negative control - blank ) × 100

4

4 Conclusions

Four new thiosemicarbazone derivatives were synthesized and structurally elucidated. All the tested compounds remained safe against normal cells whereas they showed significant cytotoxicity in cancerous cells. Furthermore, the test compounds, specifically 2 and 4, induced cytotoxicity several times better than the standard drugs (5-Flourouracil and Betulinic acid). The results showed that substitution of phenolic groups at terminal thioamide nitrogen may enhance the biological significance of thiosemicarbazones. Conclusively, the synthesized thiosemicarbazone derivatives (1–4) have worth to be further studied.

Acknowledgments

We thank the Malaysian Government for a Research University Grant which partly supported this work. Dr. Muhammad Adnan Iqbal is the recipient of USM-Postdoctoral Fellowship in research.

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Appendix A

Supplementary material

CCDC 935334, 960261, 953519 and 953518 contain the supplementary data for 1, 2, 3 and 4, respectively.

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.arabjc.2015.08.013.

Appendix A

Supplementary material

Supplementary data 1

Supplementary data 1


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