Introduction

Heterocycles play a predominant role in all spheres including pharmaceuticals, natural resources, veterinary, analytical reagents, agriculture products, dyes (Sarvary and Maleki, 2015) and have become the new trend in the development of medicine and pharmaceuticals in modern days (Maddila et al., 2013a; Sarvary and Maleki, 2015). The new approaches for the synthesis of novel heterocycles substituted with unique functional groups form the basis for extensive research activity in synthetic organic chemistry (Roh et al., 2012). Many heterocyclic compounds are in use for industrial, biological and medicinal targets (Myznikov et al., 2007). As resistance to anticancer drugs is snowballing, there is an increasing demand for the novel structure leading that may be of use in designing new potent anticancer agents. Several heterocyclic moieties have been synthesized to develop new molecular entities with promising biological applications.

The tetrazole derivatives represent an important class of heterocyclic compounds, with nitrogen atoms in their five-membered ring. Several methods have been reported for their synthesis (Habich, 1992; Wittenberger, 1994; Koldobskii et al., 1981; Koldobskii, 2006). The chemistry of tetrazole derivatives has been the subject of intensive research due to their potential biological and pharmacological applications, such as antibacterial (Rostom et al., 2009), antifungal (Upadhayaya et al., 2004), antihistaminic (Giacomo et al., 1999), antiviral (Abdel-Aal et al., 2008), anti-inflammatory (Rajasekaran and Thampi, 2004), antioxidant (Pegklidou et al., 2010), antimalarial (Christophe et al., 2004), anticonvulsant (Abbott and Acheampong, 1988), antipyretic (Luigi et al., 1966), antiallergic (Wittenberger, 1994), antihypertensive and antianxiety agents (Hayao et al., 1967; Sarvary and Maleki 2015). Additionally, they are resistant to metabolic degradation as well as toward chemical oxidants (Yella et al., 2011). Recently, antiproliferative properties of tetrazole derivatives with antitumor applications have also been reported (Gundugola et al., 2010; Romagnoli et al., 2012). Furthermore, N-substituted tetrazoles will be advantageous, as these stable compounds can be used for any biological and pharmaceutical trials without the risk of undesirable decomposition (Maddila et al., 2015a). Possibly, the introduction of a substituent group at the N-1 position can further increase their antitumor activity.

Cancer is a worldwide health problem and the most frightening disease of humans and animals. In 2008, an estimated 1.38 million cancer-related deaths were reported (GLOBOCAN, 2008). Chemotherapy is often best applied alone or following surgery or radiation of tumors (Nuyttens et al., 2000). Many chemotherapeutic agents usually affect both tumor and normal tissue cells, like bone marrow, intestinal epithelium and hair follicles, alike (Dean et al., 2005). Hence, developing a chemotherapeutic agent with minimal toxicity to the normal cells would be rewarding. Recently, considerable attention has been dedicated to the design of new derivatives of tetrazole moieties due to their potential anticancer activities (Kumar et al., 2011; Kaplancikli et al., 2014). Among the aforementioned compounds, 2-(1-(4-substituted benzyl)-1H-tetrazol-5-yl)-3-phenylacrylonitrile derivatives were promising scaffolds for design of anticancer drugs.

In the recent past, we have reported the synthesis and activity evaluation of various heterocyclic derivatives, such as triazoles (Maddila et al., 2013a) and fused 1,2,4-triazolo-[3,4-b][1,3,4]-thiadiazole for anti-inflammatory activity (Maddila et al., 2013b), 1,2,4-triazolo-thieno[2,3-d]pyrimidine for antioxidant activity (Maddila et al., 2013c) and 1,3,4-thiadiazoles for antimicrobial activity (Maddila and Jonnagadda, 2013). In addition, using green and ecofriendly approaches, we have reported the one-pot synthesis of multisubstituted pyridines using Mg–V/CO3 hydrotalcite (Maddila et al., 2015b) or mesoporous ZrO2 (Pagadala et al., 2015a), multi-functionalized benzenes in water using Zn–VCO3 hydrotalcite (Pagadala et al., 2015b), selective oxidation of benzyl alcohol with Mn-based lacunary phosphotungstate supported mesoporous silica as catalyst (Rana et al., 2015) and dihydroquinoline derivatives in aqueous media using ultrasonication (Pagadala et al., 2014). Recently, we have also reported the synthesis of pyrrole-3-carbonitriles with water as solvent and supported by computational validation (Pagadala et al., 2015c). In this work, we focused on the synthesis of novel compounds by encompassing phenylacrylonitrile, substituted benzyl rings conjugated to tetrazole moieties, which could display good antitumor activity against different tumor cell lines.

We report a series of novel 2-(1-(4-substitutedbenzyl)-1H-tetrazol-5-yl)-3-phenylacrylonitrile were synthesized and their structures were confirmed by chemical and spectroscopic methods like FTIR, 1H NMR, 13C NMR spectroscopy and HRMS spectrometry. All the synthesized compounds were screened for their anticancer activity in vitro.

Results and discussion

Chemistry

The synthetic pathway for the newly synthesized compounds, 2-(1-(substitutedbenzyl)-1H-tetrazol-5-yl)-3-phenylacrylonitrile derivatives (6a–k) including the synthesis of intermediary (4), is outlined in Scheme 1. The 3-phenyl-2-(1H-tetrazol-5-yl)acrylonitrile (4), which assisted as an important intermediate, was synthesized starting from aromatic aldehyde, malononitrile and sodium azide in ethanol solvent under reflux condition for 2 h. The reaction of (4) with substituted benzyl halides in anhydrous acetone medium afforded 2-(1-(substitutedbenzyl)-1H-tetrazol-5-yl)-3-phenylacrylonitrile derivatives (6a–k), which on dehydrohalogenation in the presence of Cs2CO3 catalyst gave the final product. The purity of compounds was confirmed by TLC. All the structures of the novel synthesized compounds were proven by 1H NMR, 13C NMR, HRMS and FTIR spectral analysis. These instrumentation details are given in supporting information (S1).

Scheme 1
scheme 1

2-(1-(Substitutedbenzyl)-1H-tetrazol-5-yl)-3-phenylacrylonitrile derivatives (6a–k). Reagents and conditions (i) ethanol, reflux, 2 h, (ii) Cs2CO3, dry acetone, reflux, 3 h

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In infrared spectroscopic analysis of compound 4, the appearance of bands at 3462, 3017, 2209 and 1566 cm−1 correspond to NH, CH–Ar, CN and C=C stretching frequencies respectively. In case of 1H NMR spectrum, the broad singlet at 3.69 ppm corresponds to NH proton, sharp singlet at 8.45 ppm to CH proton and 7.12–7.95 ppm for protons in aromatic region. Formation of 2-(1-(substitutedbenzyl)-1H-tetrazol-5-yl)-3-phenylacrylonitrile derivatives (6a–k) is confirmed by the 1H NMR spectrum reflecting a singlet at δ 4.68–5.27 ppm for NCH2, another singlet at δ 7.77–8.20 ppm for =CH proton and δ 6.57–8.02 ppm due to aromatic region protons. The 13C NMR and HRMS spectral data of compounds 6a–k are given in “Experimental data” section.

Anticancer activity

In this study, we evaluated the anticancer activity of new tetrazole derivatives (6a–k) against four human cancer cell lines, breast (MCF-7), colon (CaCO2), cervical (HeLa) and (breast) SkBr3, in vitro, by applying the MTT assay. 5-Fluorouracil was used as a positive control, DMSO as a negative control. An observation of the biological activity results detailed in Table 1 indicate that 5-fluorouracil has actively inhibited the human cancer cell lines MCF-7, CaCO2, HeLa and SkBr3 and IC50 values of 12, 15, 10 and 26 μM respectively. The most promising compounds, 6b, 6h and 6j, inhibited proliferation of MCF-7, CaCO2, HeLa and SkBr3 cell lines with IC50 values of 30, 37, 29 and 35; 51, 49, 38 and 47; and 45, 50, 40 and 49 μM, respectively. In addition, with the exception of the compounds with hydroxy, nitro and simple (6d, 6e and 6i) substituents, all the other compounds had inactivity against the all MCF-7, CaCO2, HeLa and SkBr3 cell lines.

Table 1 In vitro cytotoxic effect of newly synthesized compounds (6a–k)
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From preliminary investigations of structure–activity relationships (SARs), the position of the substituent pattern of the N-benzyl fragment on the bearing tetrazole backbone was apparently crucial to antitumor activity. In general, compounds with substituents on the phenyl ring showed an enhanced antitumor activity. Compounds with fluoro group on benzyl (6b, 6h and 6j) exhibited noticeable antitumor potency because of the electron-withdrawing groups (reactive halogens) at the ortho, meta and para position and prove ideal for the cytotoxic activity. Interestingly, the introduction of an additional electron-withdrawing groups such as bromo, chloro (halogens) (6a, 6c and 6g) and electron donating groups such as methoxy groups (6f and 6k) led to an improvement in antitumor activity than the hydroxyl, nitro and simple substitution (6d, 6e and 6i). In addition, the position of a halogens and methoxy substituents clearly influenced the cytotoxic action. Introduction of hydroxyl, nitro and simple groups led to declining antitumor activity. Notably, the trifluoromethyl-substituted derivative 6b was very potent against MCF-7, CaCO2, HeLa and SkBr3 cell lines.

Computational analysis

During the past two decades, drug discovery has mainly concentrated on the single-target paradigm. Now it has shifted toward several target networks by substantial improvement in genomics, and finding the potential receptors for targeted drug has become challenging. In silico methods are generally very effective to examine the potential protein targets and to improve drug discovery of the molecules of interest within short periods and with cost-friendliness. PharmMapper is a web server, which is useful to find the potential target candidates for the given small molecules using pharmacophore mapping method. Of all the newly synthesized compounds in this study, the three best performed compounds (6b, 6h and 6j) were employed in silico screening for target protein identification on the PharmMapper (Liu et al., 2010). Among the different human proteins investigated, the MEK-1 was found to be potential protein target for 6b, 6h and 6j compounds with their fit scores of 4.43, 4.50 and 4.96, respectively. Figure 1 explains the nine pharmacophore features as spheres within the context of the interaction structure in compounds (6b, 6h, 6j). These are as follows: a hydrogen bond acceptor vector, hydrogen bond donor vector, aromatic plane, hydrophobic center, negatively charged center, positively charged center and metal interaction center. The tetrazole ring, benzyl rings, nitrile and fluoro groups were the important structural moieties, which aided interaction with receptor in those compounds.

Fig. 1
figure 1

Compounds a 6b, b 6h and c 6j showing different pharmacophore features (in spheres) identified using the PharmMapper server

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These observations further substantiate to validate the identified protein compounds that were subsequently docked into the binding site of the protein using the Autodock 4.2 software (Morris et al., 2009). Based on the Autodock score, the conformations were ranked keeping the criteria that lower the binding energy score higher the binding affinity. The binding energies of compounds 6b (−10.22), 6h (−10.43) and 6j (−10.10) show good binding affinities with the targeted protein. The intermol energy values for 6b, 6h and 6j were −11.72, −11.62 and −11.0, respectively.

The docked complexes of the compounds 6b, 6h and 6j with the protein were then visualized in Discovery studio to see a better understanding of the modes of interaction responsible for binding of ligands, which are diagrammatically represented in Fig. 2. The closer assessment of Fig. 2 shows that compound 6b exhibits both hydrogen bonding and hydrophobic interactions to the protein, specifically hydrogen bonds with PHE209: N with nitrile nitrogen and halogen fluorine interaction with oxygen of HIS188. Moreover, tetrazole ring shows the Pi–sigma interaction with ILE141 and Pi–alkyl interaction with LYS97. Compound 6h also interacted with the protein along with hydrophobic interactions with the VAL127, MET143 and LEU206, ASP208 amino acids of the protein. Compound 6j also interacted with protein with the LEU118, VAL127, MET143 and ASP208. Method for the computational study is shown in supporting information (S2).

Fig. 2
figure 2

Complexes of 6b, 6h and 6j with the MEK-1 protein (pdb code: 1S9J). Only interacting amino acid residues of the protein are shown for clarity purposes. Orange color shows the hydrogen bond interaction, and the light blue color (Pi–alkyl types) and the dark blue color (Pi–sigma types) show the hydrophobic interactions to the ligand (Color figure online)

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Hence, the active participation of the tetrazole rings, benzyl rings and fluoro substituents of these compounds was found to be very important for locking their geometries in the active site of the MEK-1 receptor.

Experimental data

General procedure for the synthesis of 3-phenyl-2-(1H-tetrazol-5-yl)acrylonitrile

To a mixture of benzaldehyde 1 (1 mmol), malononitrile (1.1 mmol) and sodium azide (1.2 mmol) in ethanol (10 mL) were added and the reaction mixture was stirred at 70 °C for 2 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was cooled to room temperature and poured into ice-cold water, and the solid separated was filtered, washed with water, dried and recrystallized from ethanol to obtain compound.

Synthesis of 3-phenyl-2-(1H-tetrazol-5-yl)acrylonitrile (4)

Light yellow solid, M.P. 170–171 °C; 1H NMR (400 MHz, DMSO-d6) δ = 3.69 (br s, 1H, NH), 7.14 (t, J = 7.20 Hz, 1H, ArH), 7.22 (d, J = 7.45 Hz, 1H, ArH), 7.65 (t, J = 7.80 Hz, 2H, ArH), 7.95 (d, J = 7.45 Hz, 1H, ArH), 8.45 (s, 1H, CH); 13C NMR (100 MHz, CDCl3): δ 94.8, 114.9, 128.7, 130.1, 132.8, 133.2, 147.9, 156.1; IR (KBr, cm−1): 3461, 3017, 2209, 1566, 1255; HRMS of [C10H7N5 + H]+ (m/z): 197.1109; Calcd.: 197.1105.

General procedure for the synthesis of 2-(1-(substitutedbenzyl)-1H-tetrazol-5-yl)-3-phenylacrylonitrile (6a-k)

A mixture of 3-phenyl-2-(1H-tetrazol-5-yl)acrylonitrile (1 mmol), substituted benzyl halides (1 mmol) and cesium carbonate (2 mmol) in dry acetone (10 mL) was refluxed for 3 h. The reaction mixture was monitored by TLC. After completion of the reaction, the solvent was evaporated. The solid was filtered and washed with ice-cold water. Finally, the crude product was purified by recrystallization in ethanol. All the compound details are showed in supplementary information (S3).

2-(1-(4-Bromobenzyl)-1H-tetrazol-5-yl)-3-phenylacrylonitrile (6a)

White solid, M.P. 168–169 °C; 1H NMR (400 MHz, DMSO-d6) δ = 5.01 (s, 2H, CH2), 6.75 (t, J = 7.30 Hz, 1H, ArH), 7.06 (d, J = 8.00, 2H, ArH), 7.22 (t, J = 7.80, 2H, ArH), 7.55 (d, J = 8.60, 2H, ArH), 7.59 (d, J = 8.68, 2H, ArH), 7.81 (s, 1H, CH); 13C NMR (100 MHz, CDCl3): δ 49.98, 112.04, 118.96, 120.62, 127.40, 129.10, 131.52, 134.13, 135.11, 145.13; IR (KBr, cm−1): 3033, 2225, 1576, 1261, 1069; HRMS of [C17H12BrN5 + Na]+ (m/z): 388.1161; Calcd.: 388.1148.

2-(1-(4-(Trifluoromethyl)benzyl)-1H-tetrazol-5-yl)-3-phenylacrylonitrile (6b)

Yellow solid, M.P. 210–212 °C; 1H NMR (400 MHz, DMSO-d6) δ = 5.26 (s, 2H, CH2), 6.78 (t, J = 7.30 Hz, 1H, ArH), 7.10 (d, J = 8.10, 2H, ArH), 7.24 (t, J = 7.80 2H, ArH), 7.70 (d, J = 8.10, 2H, ArH), 7.83 (d, J = 8.10 Hz, 2H, ArH), 7.90 (s, 1H, CH); 13C NMR (100 MHz, CDCl3): δ 49.92, 112.24, 119.36, 122.98, 125.44, 125.48, 125.88, 127.61, 127.61, 129.14, 134.37, 139.89, 144.77; IR (KBr, cm−1): 3047, 2220, 1578, 1249, 1021; HRMS of [C18H12F3N5 + Na]+ (m/z): 378.2042; Calcd.: 378.2036.

2-(1-(2-Chlorobenzyl)-1H-tetrazol-5-yl)-3-phenylacrylonitrile (6c)

Yellow solid, M.P. 232–233 °C; 1H NMR (400 MHz, DMSO-d6) δ = 4.75 (s, 2H, CH2), 6.77 (t, J = 7.60 Hz, 1H, ArH), 7.09 (d, J = 8.30, 2H, ArH), 7.21–7.33 (m, 3H, ArH), 7.36 (t, J = 8.10, 1H, ArH), 7.44 (d, J = 8.00 Hz, 1H, ArH), 8.02 (d, J = 8.80 Hz, 1H, ArH), 8.20 (s, 1H, CH); 13C NMR (100 MHz, CDCl3): δ 49.90, 112.16, 119.28, 125.78, 127.33, 129.05, 129.15, 129.67, 131.15, 131.91, 132.90, 144.80; IR (KBr, cm−1): 3028, 2220, 1568, 1276, 1019; HRMS of [C17H12ClN5 + Na]+ (m/z): 344.1141; Calcd.: 344.1141.

2-(1-Benzyl-1H-tetrazol-5-yl)-3-phenylacrylonitrile (6d)

White solid, M.P. 192–194 °C; 1H NMR (400 MHz, DMSO-d6) δ = 4.68 (s, 2H, CH2), 6.74 (t, J = 7.30 Hz, 1H, ArH), 7.07 (d, J = 7.95, 2H, ArH), 7.21 (t, J = 7.84, 2H, ArH), 7.28 (t, J = 7.30, 1H, ArH), 7.38 (t, J = 7.70 Hz, 2H, ArH), 7.65 (d, J = 8.10 Hz, 2H, ArH), 7.86 (s, 1H, CH); 13C NMR (100 MHz, CDCl3): δ 49.90, 111.95, 118.70, 125.58, 127.87, 128.60, 129.07, 135.80, 136.38, 145.26; IR (KBr, cm−1): 3116, 2231, 1582, 1281, 1031; HRMS of [C17H13N5 + H]+ (m/z): 288.1040; Calcd.: 288.1039.

2-(1-(4-Hydroxybenzyl)-1H-tetrazol-5-yl)-3-phenylacrylonitrile (6e)

Yellow solid, M.P. 185–186 °C; 1H NMR (400 MHz, DMSO-d6) δ = 4.91 (s, 2H, CH2), 6.70 (t, J = 7.28 Hz, 1H, ArH), 6.78 (d, J = 8.60 2H, ArH), 7.01 (d, J = 7.70, 2H, ArH), 7.18 (t, J = 7.90, 2H, ArH), 7.46 (d, J = 8.58 Hz, 2H, ArH), 7.77 (s, 1H, CH), 10.02 (s, 1H, OH); 13C NMR (100 MHz, CDCl3): δ 50.02, 111.69, 112.05, 115.51, 118.10, 126.87, 127.13, 137.09, 145.66, 157.65; IR (KBr, cm−1): 3050, 2224, 1585, 1282, 1038; HRMS of [C17H13N5O + H]+ (m/z): 304.0766; Calcd.: 304.0763.

2-(1-(4-Methoxybenzyl)-1H-tetrazol-5-yl)-3-phenylacrylonitrile (6f)

White solid, M.P. 208–209 °C; 1H NMR (400 MHz, DMSO-d6) δ = 3.77 (s, 3H, OCH3), 4.95 (s, 2H, CH2), 6.71 (t, J = 7.24 Hz, 1H, ArH), 6.95 (d, J = 8.80, 2H, ArH), 7.03 (d, J = 8.10, 2H, ArH), 7.19 (t, J = 7.85, 2H, ArH), 7.57 (d, J = 8.80 Hz, 2H, ArH), 7.81 (s, 1H, CH); 13C NMR (100 MHz, CDCl3): δ 50.02, 55.14, 111.77, 114.14, 118.29, 126.99, 128.47, 129.02, 133.35, 136.54, 145.54, 159.27; IR (KBr, cm−1): 3019, 2224, 1568, 1271, 1072; HRMS of [C18H15N5O + H]+ (m/z): 318.1057; Calcd.: 318.1059.

2-(1-(2-Bromobenzyl)-1H-tetrazol-5-yl)-3-phenylacrylonitrile (6g)

White solid, M.P. 184–186 °C; 1H NMR (400 MHz, DMSO-d6) δ = 5.27 (s, 2H, CH2), 6.77 (t, J = 7.00 Hz, 1H, ArH), 7.09 (d, J = 7.70, 2H, ArH), 7.21–7.25 (m, 3H, ArH), 7.38 (t, J = 7.70 Hz, 1H, ArH), 7.60 (d, 1H, J = 7.40 Hz, ArH), 7.99 (d, 1H, J = 7.40 Hz, ArH), 8.16 (s, 1H, CH); 13C NMR (100 MHz, CDCl3): δ 49.98, 112.16, 119.28, 121.65, 126.15, 127.83, 128.80, 129.15, 129.38, 132.90, 134.30, 134.33, 144.80; IR (KBr, cm−1): 3033, 2215, 1566, 1255, 1040; HRMS of [C17H12BrN5 + Na]+ (m/z): 388.0124; Calcd.: 388.0113.

2-(1-(3,5-Difluorobenzyl)-1H-tetrazol-5-yl)-3-phenylacrylonitrile (6h)

Yellow solid, M.P. 221–223 °C; 1H NMR (400 MHz, DMSO-d6) δ = 5.03 (s, 2H, CH2), 6.63 (d, 1H, J = 7.10 Hz, ArH), 6.72 (t, J = 7.30 Hz, 1H, ArH), 6.88 (d, J = 8.10 Hz, 2H, ArH), 7.01 (s, 1H, ArH), 7.19–7.28 (m, 3H, ArH), 8.03 (s, 1H, CH); 13C NMR (100 MHz, CDCl3): δ 49.87, 102.45, 107.43, 111.41, 111.94, 118.48, 129.21, 139.20, 144.97, 156.82, 157.48, 158.76, 158.93, 160.29, 160.33; IR (KBr, cm−1): 3011, 2210, 1582, 1224, 1052; HRMS of [C17H11N5F2 + H]+ (m/z): 324.1397; Calcd.: 324.1396.

2-(1-(2-Hydroxy-5-nitrobenzyl)-1H-tetrazol-5-yl)-3-phenylacrylonitrile (6i)

Yellow solid, M.P. 189–191 °C; 1H NMR (400 MHz, DMSO-d6) δ = 4.95 (s, 2H, CH2), 6.57–6.60 (m, 1H, ArH), 6.69 (d, J = 8.80 Hz, 1H, ArH), 6.75 (t, J = 7.40 Hz, 1H, ArH), 6.95–6.97 (m, 3H, ArH), 7.22 (t, J = 7.80, 2H, ArH), 8.06 (s, 1H, CH), 9.72 (s, 1H, OH); 13C NMR (100 MHz, CDCl3): δ 49.96, 111.65, 112.20, 116.47, 116.54, 118.78, 120.87, 129.21, 136.72, 144.87, 148.43, 149.91; IR (KBr, cm−1): 3037, 2223, 1584, 1221, 1057; HRMS of [C17H12N6O3 + Na]+ (m/z): 371.0389; Calcd.: 371.0395.

2-(1-(2,4-Difluorobenzyl)-1H-tetrazol-5-yl)-3-phenylacrylonitrile (6j)

White solid, M.P. 215–216 °C; 1H NMR (400 MHz, DMSO-d6) δ = 5.07 (s, 2H, CH2), 6.68–6.74 (m, 1H, ArH), 7.03–7.05 (m, 3H, ArH), 7.10 (s, 1H, ArH), 7.15–7.23 (m, 3H, ArH), 7.77 (s, 1H, CH); 13C NMR (100 MHz, CDCl3): δ 50.02, 111.55, 111.90, 115.28, 117.16, 118.65, 129.08, 129.60, 136.61, 137.08, 145.29, 157.53; IR (KBr, cm−1): 3033, 2225, 1580, 1215, 1094; HRMS of [C17H11N5F2 + H]+ (m/z): 324.0929; Calcd.: 324.0937.

2-(1-(2,3-Dimethoxybenzyl)-1H-tetrazol-5-yl)-3-phenylacrylonitrile (6k)

White solid, M.P. 175–177 °C; 1H NMR (400 MHz, DMSO-d6) δ = 3.76 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 5.26 (s, 2H, CH2), 6.73 (t, J = 7.30 Hz, 1H, ArH), 6.96 (d, J = 8.10 Hz, 1H, ArH), 7.03–7.06 (m, 3H, ArH), 7.20 (t, J = 7.80 Hz, 2H, ArH), 7.47 (d, J = 7.30 Hz, 1H, ArH), 8.11 (s, 1H, CH); 13C NMR (100 MHz, CDCl3): δ 50.22, 55.63, 60.82, 111.89, 112.00, 116.32, 118.71, 124.15, 129.08, 129.19, 131.77, 145.20, 146.33, 152.65; IR (KBr, cm−1): 3037, 2230, 1572, 1241, 1060; HRMS of [C19H17N5O2 + H]+ (m/z): 348.1057; Calcd.: 348.1052.

In vitro pharmacological studies

Chemicals

Eagle’s minimum essential medium (EMEM) with l-glutamine (4.5 g L−1), antibiotics (100×) containing penicillin (10,000 U mL−1), streptomycin (10,000 µg mL−1) and amphotericin B (25 µg mL−1), and trypsin–versene mixture was purchased from Lonza BioWhittaker (Verviers, Liège, Belgium). Gamma-irradiated fetal calf serum (FCS) was purchased from Highveld Biological (Pty) Ltd (Lyndhurst, Gauteng, RSA). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO, cryoprotective medium) and phosphate-buffered saline (PBS) tablets were acquired from Merck (Darmstadt, Hesse, Germany). All tissue culture plastic ware, consumables and cryogenic storage vials were attained from Corning Incorporated (New York City, NY, USA). All other chemicals and reagents were of analytical purity grade or higher and purchased commercially. Ultrapure deionized 18 MΩ water (Milli-Q50) was used throughout.

Cell lines

MCF-7 (human breast adenocarcinoma), CaCO2 (human epithelial colorectal adenocarcinoma), HeLa (human cervical tumor cells) and SkBr3 (human breast adenocarcinoma) cell lines were routinely propagated in gas-permeable 25-cm2 culture flasks containing 5 mL of EMEM supplemented with 10 % (v/v) gamma-irradiated FCS and antibiotics (100 U mL−1 penicillin, 100 µg mL−1 streptomycin, 0.25 µg mL−1 amphotericin B) at 37 °C under a humidified atmosphere with 5 % CO2 (Thermo Electron Corp. Steri-Cult CO2 incubator, HEPA Class 100). At semi- to full confluence (contact inhibition), cells were split at a 1:3–1:5 ratio every 3–4 days. Cells were freshly cultured and plated 24 h prior to each experiment to maintain the correct pH balance and to eliminate waste product. All preparation of complete culture media, mammalian cell line propagation or maintenance and routine cell culture procedures were performed in a class II Airvolution biological safety cabinet [United Scientific (Pty) Ltd].

Procedure

Anticancer activity screening of the drugs against four cell lines was quantified in vitro by the MTT reduction assay (van Meerloo et al., 2011), according to reported standard procedure. Briefly, cells were separately trypsinized and seeded into 96-well plates at a density of 2.1–2.5 × 104 cells/well and cultured overnight in 0.1 mL complete medium under the stated conditions to favor cell attachment and to achieve ∼80 % cell confluence. Cells were then introduced to different concentrations of the target compounds (25–200 µg mL−1; diluent = DMSO @ 1 mg mL−1 stock concentrations) and further incubated for 48 h. Following the treatment, cells were then incubated for 4 h with equal volume (0.1 mL) of fresh medium and MTT reagent (5 mg mL−1 in sterile PBS, pH 7.4) to allow the formation of formazan by the action of succinate–tetrazolium reductase in living metabolically active cells. Thereafter, cells were rinsed with 0.1 mL of PBS and formazan crystals were solubilized in 0.1 mL DMSO. Absorbance was recorded at 570 nm (detection λ) and 630 nm (reference λ) using a Vacutec MR-96A microplate photometer (Vacutec, Hamburg, Germany). Triplicate wells (n = 3) were prepared for each individual dose. Control wells containing only cells unexposed to drug were run concurrently. Results were compared to the antiproliferative effects of fluorouracil (5-Fu) as a reference drug. The relationship between surviving ratio and drug concentration was plotted to obtain the survival curve of each cell line as follows: [(OD570 Treated − OD630 Treated)/(OD570 Control − OD630 Control)] × 100 (Sylvester, 2011). IC50 response parameter was then deduced which corresponds to the concentration of the individual compound eliciting 50 % mortality in net cells (Ghora et al., 2010).

Conclusion

In summary, we synthesized a series of phenylacrylonitrile, benzyl substitutes bearing tetrazole derivatives and discovered their antitumor activity. Exclusively, compounds 6b, 6 h and 6j exhibited good anticancer activity and also compounds 6a, 6c, 6g, 6f and 6k showed moderate anticancer activity against four human cancer cell lines, MCF-7, CaCO2, HeLa and SkBr3, in the MTT assay. SAR analysis revealed that trifluoro, difluoro substituents at positions ortho, meta and para on the benzyl ring promoted antitumor activity. Among the tested molecules, compound 6b, 6h and 6j exhibited very good binding affinities and it shows potential target for anticancer therapy and it can be modified to increase their anticancer activity. In conclusion, the substituent group at the N-1 position proved to enhance antitumor activity of the parent compound.