1H-1,2,3-Triazole Tethered Nitroimidazole-Isatin Conjugates: Synthesis, Docking, and Anti-Proliferative Evaluation against Breast Cancer.

1H-1,2,3-Triazole tethered imidazole-isatin and imidazole-isatin-thiosemicarbazone conjugates were synthesized and evaluated against MCF-7 and MDA-MB-231 cell lines. Antiproliferative activities of the synthesized conjugates revealed an optimum combination of longer alkyl chain length as spacer and a halogen-substituent on the isatin ring as a pre-requisite for good activity. The compound 6g with an optimum combination of chloro-substituent at C-5 position of isatin ring and a butyl chain length proved to be most active and noncytotoxic with IC50s of 54.25 and 26.12 μM against MCF-7 and MDA-MB-231 cell lines, respectively.

Introduction

Cancer is responsible for approximately 8.2 million deaths and remains as the most difficult disease to treat worldwide.[1] Rapidly dividing cells such as breast, skin, and uterine cells are at higher risk of mutations compared with cells which do not divide and thus are more prone to develop cancer. Breast cancer (BC) is one of the most frequently occurring cancers leading to significant morbidity and mortality among women.[2] An insight into cancer facts and figures for the year 2016–2017 revealed an estimated number of deaths of 41 070 through BC among women and men in US.[3] The situation is worse in India where BC accounts 25–32% of all cancer cases. According to Indian Council of Medical Research (ICMR) survey report 2016, an estimated new number of cancer patients were 14.5 million in 2016 which will likely to reach around 17.3 million by 2020.[4]

One of the significant approaches for the treatment of BC comprises the selectively inhibiting the binding of estrogen with estrogen receptors (ERα and ERβ) and resulted in the identification of selective estrogen modulators, SERMs. Estrogen has been reported to play a vital role in the growth and development of mammary glands. Interaction of estrogen with estrogen receptors (ERα and ERβ) stimulates the proliferation of mammary cells. MCF-7 cells, being ERα dependent, are found to be sensitive to SERMs while MDA-MB-231 cells which are ER-β dependent are characterized by the absence of immune-histochemical expression of estrogen, progesterone, and HER2 receptors.[5−8] Triarylethylenes derivatives like raloxifene, tamoxifen (TAM), and toremifene are approved SERMs by US Food and Drug Administration (FDA) with significant anti-BC profile.[9] TAM, an antagonist, is the first-line drug employed for the treatment of BC. However, only 70% of ERα cases respond to TAM while 30–40% patients with adjuvant TAM therapy eventually relapse.[10−12] Furthermore, TAM has an adverse effect on endometrium causing endometrial cancer while raloxifene causes hot flashes, insomnia, dizziness, and melancholy.[13]

Imidazole and its derivatives are of great interest in medicinal chemistry because of their versatile pharmacological properties such as antibacterial, antifungal, anti-tuberculosis, and anticancer activities.[14−17] Natural imidazolium halides for example, lepidiline A and lepidiline B, are isolated from the roots of Lepidiummeyenii and showed potent cytotoxic activity against the human cancer cell lines.[18] Further studies on molecular mechanism confirmed that the imidazolium salt hybrids can induce G1 phase cell cycle arrest and apoptosis in tumor cells.[19]

Another important scaffold isatin possess a wide variety of activities such as anticancer, antidepressant, anticonvulsant, antifungal, anti-HIV, and anti-inflammatory properties.[20−25] BIBF1120 II, an isatin-based triple angio-kinase inhibitor for the diagnosis of nonsmall cell lung cancer, is in phase III clinical trials.[26] Sunitinib III (Sutent) is another isatin based scaffold approved recently by FDA for treating gastrointestinal stromal tumors and advanced renal cell carcinoma.[27]

A recent report from our laboratory has shown the synthesis and antiproliferative evaluation of a series of 1H-1,2,3-triazole linked ospemifene–isatin conjugates against MCF-7 and MDA-MB-231 cell lines.[28] In continuation,[29] the present work comprises the synthesis, antiproliferative evaluation, and docking studies of 1H-1,2,3-triazole tethered imidazole–isatin and imidazole–isatin–thiosemicarbazone conjugates against MCF-7 and MDA-MB-231 cell lines. The promising scaffolds were also evaluated to inspect their cytotoxicity against normal HEK-293 (human embryonic kidney) cell line.

Result and Discussion

Synthetic methodology involved sodium hydride-promoted N-alkylation of 1a–c and subsequent treatment with sodium azide afforded the N-alkylazido-isatins 3a–c (Scheme ). Another precursor viz. 2-methyl-5-nitro-1-(prop-2-yn-1-yl)-1H-imidazole 5 was prepared via K2CO3-promoted N-propargylation of 4 in anhyd DMF.

Scheme 1

Synthesis of Substituted N-Alkyl-azido-isatins

The target 1H-1,2,3-triazole-tethered-2-methyl-5-nitroimidazole–isatin conjugates 6a–l were obtained via Cu-promoted 1,3-cycloaddition reaction between appropriate precursors’ 5 and 3 as depicted in Scheme .

Scheme 2

Synthesis of 2-Methyl-5-nitroimidazole–Isatin Conjugates and 2-Methyl-5-nitroimidazole–Isatin–Thiosemicarbazones

The synthesized conjugates 6a–l were further reacted with thiosemicarbazide in refluxing ethanol to yield the corresponding 2-methyl-5-nitroimidazole–isatin–thiosemicarbazones 7a–l. The structure assigned to the synthesized conjugates was based on spectral data and analytical evidence. Compound 6b, for example, characterized as 1-(3-(4-((2-methyl-5nitro-1H-imidazole-1-yl)-1H-1,2,3-triazol-1-yl)propyl)indoline-2,3-dione showed a molecular ion peak at m/z 396.1389 [M + H]+ in its high-resolution mass spectrum (HRMS). The salient features of its proton nuclear magnetic resonance (1H NMR) spectrum encompassed the appearance of distinguishing peaks at δ 2.17, 3.72, 4.46, and 5.34 conforming to different methylenes and a singlet at δ 2.39 corresponding to methyl at C-2 position of the imidazole ring. Its carbon nuclear magnetic resonance (13C NMR) spectrum, apart from requisite number of carbons, exhibited characteristic absorptions corresponding to isatin carbonyls at δ 158.7 and 183.7, further corroborating the assigned structure.

The synthesized library of 2-methyl-5-nitroimidazole–isatin and 2-methyl-5-nitroimidazole–isatin–thiosemicarbazones were assessed for their antiproliferative profile against MCF-7 (ERα) and MDA-MB-231 (ERβ) human BC cell lines using an MTT assay. The growth inhibitions of synthesized compounds against MCF-7 and MDA-MB-231 cells at variable concentrations have been summarized in Figure using plumbagin as the positive control. IC50 value, which is the concentration required to inhibit 50% of cell viability by the test compounds, has been summarized in Table .

Figure 1

Representative graph comparing the percentage growth inhibition of MCF-7 and MDA-MB 231 cells at selected concentrations of test compounds 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j, 6l, 7a, 7b, 7c, 7d, 7e, 7f, 7g, 6k, 7i, and 7k. Plumbagin (40 μM) was used as a positive control. Data are mean ± standard deviation S.D. (n = 3) from raw data, where *p < 0.05, **p < 0.01 and ***p < 0.001 significant difference to untreated control.

Table 1

IC50 Values of Synthesized Conjugates against MCF-7 and MDA-MB-231 Cell Lines along with % Yield

As evident, the activities of synthesized conjugates were found to depend upon the nature of substituent at C-5 position of isatin ring, the alkyl chain length introduced as spacer, and the nature of functionality at C-2 position of isatin ring. Analyzing structure–activity relationship (SAR) among conjugates 6a–d (R=H); revealed that the compounds were more active against MCF-7 than MDA-MB-231 cells at higher concentrations. Among conjugates 6e–h (R=Cl); the activity tends to increase with the increase in chain length, with compounds being more cytotoxic against MDA-MB-231 than MCF-7 cell lines. The conjugate 6g (R=Cl, n = 3); exhibited comparable IC50 value to that of standard drug TAM against MCF-7 while ∼3 times more activity has been observed against MDA-MB-231 cell line. Similarly, 6h (R=Cl, n = 4) was found to be ∼2 times more potent in MCF-7 and ∼5 times in MDA-MB-231 compared with TAM (Figure ).

Similar effects were observed by replacing chloro with fluoro at C-5 position of isatin ring. The conjugate 6k (n = 3) and 7l (n = 4) showed comparable activities to TAM against both cell lines (Figure ). The carbonyl group at C-3 position of isatin ring was converted to the corresponding thiosemicarbazone with a view of enhancing their cytotoxic activities as evidenced by literary rationale.[30] The resulting isatin–nitroimidazole–thiosemicarbazones were then evaluated against both cell lines viz. MCF-7 and MDA-MB-231. Interestingly, the introduction of thiosemicarbazone functionality improved the activity profiles as evident by 7d which exhibited an IC50 value of ∼70 μM against both cell lines while the parent compounds 6a–d were inactive (Figure ).

Figure 2

Representative graph showing percentage growth inhibition of HEK-293 cells at selected concentrations of test compounds 6g, 6h, 6l, and 7d. Plumbagin (40 μM) was used as a positive control. Data are mean ± standard deviation S.D. (n = 3) from raw data, where *p < 0.05, **p < 0.01 and ***p < 0.001 significant difference to untreated control.

Furthermore, Cl-substituted-isatin–imidazole–thiosemicarbazones were found to be more active against MDA-MB-231 cells than in ER + MCF-7 cells but did not cause 50% growth inhibition in either of the cell line (Figure ). Especially compounds 7e–7g (Figure ) inhibited growth of more than 30% of cells at low concentrations (1, 5 and 10 μM), whereas less than 50% cell death was observed in MCF-7 cells even at higher concentrations. The compounds, 7b–7d showed similar patterns indicating that these compounds are not necessarily reliant on the presence of the ER. A further trend has also been observed where 50 μM concentrations proved to be most effective in inhibiting cell growth, which could be attributed to the development of resistance at higher concentrations.

Because 6g, 6h, 6l, and 7d showed significant growth inhibition in both MCF-7 and MDA-MB-231 cells, we further tested them on a normal cell line HEK-293 to confirm their potential as anticancer agents. The percentage growth inhibition of the promising compounds along with their IC50 values is provided in Figure and Table , respectively. As evident, except for 6h, the other three tested compounds exhibited IC50 values greater than 100 μM, indicative of the fact this molecular framework holds plausible promise for the design of new antiproliferative agents against BC.

Table 2

IC50 Values of Synthesized Conjugates against HEK-293 Cell Lines

entry	HEK-293 (μM)
6g	>100
6h	12.11
6I	>100
7d	>100

Molecular Docking Studies

Molecular docking of the active ligands in the binding site of insulin growth factor-1 (IGF-1) was performed to determine the crucial binding interactions responsible for the inhibitory effect observed. IGF-1 is a transmembrane tyrosine kinase which plays a significant role in the development and function of many tissues, including the mammary gland. It is implicated in malignant BC as it is over-expressed in tumors. It is responsible for mediating cell-proliferation and providing protection against cell apoptosis. The signaling of the IGF pathway has been noted to directly affect the propensity for invasion and metastasis by influencing tumor cell motility and adhesion.[31]

The native ligand in the crystal structure of IGF-1 (PDB ID: 1JQH) was an adenylyl imidodiphosphate which inhibited the receptor by binding to the kinase domain. The binding interaction profile of the ligand–protein complex largely comprised hydrogen bonds with residues Ser26, Lys22, Glu97, Met99, and Lys50, as well as the formation of a water bridge with Lys22. Compound 6h (Figure ) was measured to be the most potent inhibitor against MCF-7 and MDA-MB-231, IC50 = 20.76 and 16.06 μM, respectively (lowest docking score = −7.8 kcal/mol, Table ). The binding landscape of the ligand–receptor complex incorporates hydrophobic interactions between residue Val30 with the carbons of six-membered ring of the indoline moiety (bond distance = 3.88 and 3.85 Å) as well as residue Val80 and alkyl linker (bond distance = 3.80 Å).

Figure 3

Three-dimensional illustration of docked ligands in IGF-1 receptor kinase domain.

Table 3

Docking of the Most Active Compounds in IGF-1 Receptor

ligand identifier	docking score/(kcal/mol)	rmsd i.b.	rmsd u.b.
6g	–7.3	1.968	2.325
6h	–7.8	2.452	3.354
6l	–7.6	2.790	3.639
7b	–7.2	3.738	8.021
7d	–6.7	3.006	4.077
native ligand	–7.2	1.721	2.587

This is accompanied by two hydrogen bonds between the hydrogen bond donors Lys50 and Met99 with the sp2-hybridized nitrogen of the imidazole and carbonyl of the indoline-2,3-dione, respectively (bond distance = 2.00 and 1.90 Å; bond angle = 157.21° and 157.05°, respectively). A salt bridge facilitates an electron shuttle between the sp3-hybridized nitrogen of the triazole which becomes protonated and residue Asp165, bond distance = 4.12 Å). The interaction network of the second most active inhibitor, compound 6g, is similar to compound 6h (Figure ); however, it comprises fewer hydrophobic interactions, which is compensated by an increased number of hydrogen bonds. The two van der Waals interactions exist between residue Val80 with the alkyl linker and Asp103 with the methyl group of the imidazole ring (bond distance = 3.78 Å).

Four distinct hydrogen bonds which confer inhibition occur between the proton donor residues Ser26 and the oxygen of the nitro functional group, Lys50, and the sp2-hybridized nitrogen of the triazole ring, Met99, and the oxygen of the indoline moiety as well as the proton acceptor residue Asp165 and the sp2-hybridized nitrogen of the triazole ring (bond distance = 3.23, 2.27, 1.89 and 2.39 Å; bond angle = 136.17°, 138.48°, 154.26° and 125.62°, respectively). A salt bridge exists between the sp3-hybridized nitrogen of the triazole which becomes protonated and residue Asp165, (bond distance = 4.32 Å) like compound 6h.

Two compounds were identified as potential candidates as anti-BC agents in both the MCF-7 and MDA-MB-231 cell-lines with the aid of further derivatisation, these include conjugates 6l and 7d. The complexes of these two ligands share a similar binding profile with contributions specifically from hydrophobic interactions and hydrogen bonds. There are five notable van der Waals interactions between: Leu22/Val30/Ala48/Met99 and the six-membered ring of the indoline moiety, as well as Phe166 with the methyl group of the imidazole ring (bond distance = 3.99, 3.71/3.87, 3.97 and 3.90 Å, respectively). A well-defined network of hydrogen bonds is observed between hydrogen bond donors: Ser26/Lys176 with oxygen of the nitroimidazole moiety, Lys50/Gly167 with nitrogen of the imidazole ring, and Met99 with the carbonyl oxygen adjacent to the amine of the indoline pharmacophore (bond distance = 2.03, 2.03, 2.06, 3.07, and 2.62 Å; bond angle = 163.38°, 173.54°, 158.68°, 130.33°, and 145.78°, respectively).

Compound 7d exhibited an additional hydrogen bond interaction between the donor Leu22 and the terminal amine of the hydrazinecarbothioamide (bond distance = 1.95 and 2.15; bond angle = 146.25 and 139.07). A pertinent difference in the binding profile of compound 6l over compound 7d in the form of a salt bridge, which facilitates the transfer of electrons between Asp165 and the sp3-hybridized nitrogen of the triazole, may be directly responsible for the improved inhibition highlighted against the MDA-MB-231 cell-line (IC50 = 56.18 (6l) and 70.40 (7d) μM).

It is further surmised that the length of the alkyl chain can be no shorter that four/five carbons long, if any shorter the salt bridge may be disrupted through space by the electron-withdrawing effect of chloride and nitro groups. This effect is negated in the imidazole–isatin–thiosemicarbazones, whereby the presence of the hydrazinecarbothioamide has an electron-donating effect to compensate thus improving the inhibitory activity of alkyl chain lengths of two/three carbons.

Table highlights the physical properties of the designed inhibitors. It is evident that in order for the isatin conjugate to be active against both MCF-7 and MDA-MB-231 cell lines, the ligand must maintain an overall more hydrophobic nature (TPSA = 131.73 Å2 for both compounds 6g and 6h). The log P further supports this, as compounds 6g, 6h, and 6l have a higher partition co-efficient which indicates improved lipophilicity. This study has revealed high value scaffolds in the development of anti-BC chemotherapies capable of multi-targeting different types of BCs. This work provides a platform on to which further development of the selective inhibitors can be pursued as selectivity toward a triple negative BC cell-line is rarely observed with imidazole derivatives.

Table 4

Physical Properties of the Docked Compounds

					pharmacophore features
compound ID	log P	TPSA/Å2	rotatable bonds	molecular weight/g mol–1	acceptors	donors	ionizable groups/charge
6g	1.55	131.73	8	443.84	7	0	0
6h	1.98	131.73	9	457.87	7	0	0
6l	1.77	131.73	9	441.42	8	0	0
7b	0.82	197.16	8	454.47	7	2	1/+
7d	1.50	197.16	11	496.55	7	2	1/+
native ligand	–7.21	307.09	8	514.26	18	11	3/–

On the basis of the above evaluation studies, the developed SAR can be summarized as elucidated in Figure .

Figure 4

Generalized SAR of the synthesized compounds against BC.

Conclusion

In conclusion, a series of 1H-1,2,3-triazole tethered imidazole–isatin conjugates and imidazole–isatin–thiosemicarbazones were synthesized and evaluated against MCF-7 and MDA-MB-231 cell lines. Although the compounds were not as active as the standard compound plumbagin, some of the conjugates showed comparable activity to that of TAM. The conjugate 6h, with an optimum combination of pentyl chain length as spacer and a chloro-substituent on the isatin ring, exhibited IC50s of 20.76 and 16.06 μM against MCF-7 and MDA-MB-231 cell lines, respectively, but also seemed to inhibit the growth of the control cells HEK-293 with an IC50 value of 12.11 μM. However, compounds 6g, 6l, and 7d with promising IC50s against the MCF-7 and MDA-MB-231 cell lines proved to be noncytotoxic to HEK-293 and can be taken further as possible anti-BC agents.

Experimental Section

General Information

1H NMR spectra were recorded in CDCl3 and DMSO-d6 with a JEOL 400 (400 MHz) and Bruker 500 (500 MHz) spectrometer using TMS as an internal standard. Chemical shift values are expressed as parts per million downfield from TMS and J values are expressed in hertz. Splitting patterns in the obtained NMR’s are indicated as s: singlet, d: doublet, t: triplet, m: multiplet, dd: double doublet, ddd: doublet of a doublet of a doublet, and br: broad peak. 13C NMR spectra were recorded on a JEOL 400 (100 MHz) and Bruker 500 (125 MHz) spectrometer in CDCl3 and DMSO-d6 using TMS as an internal standard. HRMS were recorded on a Bruker-micrOTOF-Q II spectrometer.

Materials and Methods

The cytotoxicity studies of the synthesized conjugates viz. 6a–l and 7a–l was tested on two different BC cell lines, namely, ER positive (ER+) MCF-7 cells and triple negative MDA-MB-231 (ER−) using MTT (3-(4,5-dimethylthiazol-z-yl)-2,5-diphenyltetrazolium bromide) assay.[28]

Cell Culturing

MCF-7 cells were cultured in Dulbecco’s media Eagle’s medium (DMEM) media with 10% fetal bovine serum (FBS), and 1% penicillin–streptomycin and MDA-MB 231 cells were cultured in DMEM supplemented with 5% FBS and 1% penicillin–streptomycin, both incubated at 37 °C and 5% carbon dioxide.

MTT Assay

Cells were seeded in 96-well plates at a density of 5000 cells per well in triplicate in media. After 24 h, the test compounds, diluted in complete DMEM were added to each well. Cells were treated with a range of different concentrations of drug (1, 5, 10, 20, 50, 100 μM) for 24 h at 37 °C and 5% carbon dioxide.[28] Subsequently, sterile 5 μL of 5 mg/mL MTT (Sigma-Aldrich) dissolved in phosphate-buffered saline was added to each well and incubated with cells for 2 h. Solubilization solution (10% sodium dodecyl sulfate, 10 mM HCl) of equal volume to the wells was then added to each well, which was incubated with cells for 16 h at 37 °C. The optical density of each well was read at 570 nm using a microtiter plate reader (Thermo Fisher Scientific Multiskan GO Microplate Reader, SkanIt software).[32]

Statistical Analysis

The statistical analysis was performed using Excel, and IC50 values were estimated using Graph pad Prism5 software (Hearne Scientific Software). The experiments were performed in duplicate and the statistical significance was calculated using Student’s t-test.[29a] A p-value of less than 0.05 was used to estimate the significance of the observations. A Z-factor was calculated for each 96-well plate and assays having Z-factor above >0.5 were included in the statistical analysis.[33]

Docking Methods

The X-ray crystal structures of the IGF-1 receptor (PDB ID: 1JQH) was obtained from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). Using the UCSF Chimera package the ligand and receptor complexes were prepared for docking.[34] Chem3D Ultra was used to construct a three-dimensional (3D) structure of the native ligand and most active isatin conjugates. The 3D structure of the compounds was further optimized using the force field, MMFF94s in the Avogadro package (V 1.2.0). AutoDock Tools (V 1.5.6) was used to establish the grid box parameters. All nonpolar hydrogens were merged, Gasteiger partial atomic charges added, and rotatable bonds assigned. The grid box of the complex had a grid spacing of 0.375 Å. The parameter of the grid points are given in Table S1 (Supporting Information). Autodock Vina was used to perform the docking of each ligand into the IGF-1 receptor in triplicate.[35] The docking systems were validated by redocking the ligand of the protein crystal structures into the binding site. The protein–ligand interaction profiler was used to analyze the nonpolar interactions between the protein and ligand in complex.[36]