Cytotoxic, DNA Cleavage and Pharmacokinetic Parameter Study of Substituted Novel Furan C-2 Quinoline Coupled 1, 2, 4-Triazole and Its Analogs

2.1. Materials and Methods

Chemicals used in the synthesis of compounds were purchased from Alfa Aesar, India and Spectrochem Pvt. Ltd. India. The solvents were of analytical grade. Melting points (M. Pt.) of the synthesized compounds were determined with the help of digital Raga digital melting point apparatus, Bengaluru, India and are uncorrected; Infrared data were recorded on a Bruker spectrophotometer using KBr pellets. ¹H and ¹³C NMR spectra were recorded on Bruker AVANCE II 400 and 100 MHz instruments using DMSO-d6/CDCl₃ as a solvent and TMS as an internal standard; chemical shifts are expressed as δ values (ppm). The J values are expressed in Hertz (Hz). Mass spectra (MS) were recorded in JEOL GCMATE II LC-Mass spectrometer using electron impact ionization (EI) technique. Analytical thin-layer chromatography (TLC) was performed on precoated TLC sheets of silica gel 60 F254 (Merck, Darmstadt, Germany), visualized by long and short wavelength UV lamps (356 and 254 nm). Chromatographic purifications were performed on silica gel (100-200 mesh, Merck, Germany).

2.2. Spectral Details

2.2.15. 15 3-{5-[6-Chloro-2-(Furan-2-yl) Quinolin-4-yl]-4H-1,2,4-Triazol-3-yl} Phenol (7o)

Yield: 81%. M. Pt. 264-266^oC; IR (KBr), cm^-1 3192.19 (N-H), 1654.92 (C=N), 1543.05 (C=C), 1311.59 (C-F), 748.38 (C-Cl); ¹H NMR (DMSO-d_6, 400 MHz, δ ppm): 12.25 (s, 1H), 9.69 (s, 1H), 8.29 (s, 1H), 8.20-8.21 (d, 1H, J=4 Hz), 8.09-8.12 (d, 1H, J=12 Hz), 7.966-7.969 (d, 1H, J=1.2 Hz), 7.850-7.855 (t, 1H, J=20 Hz), 7.51-7.52 (d, 1H, J=4 Hz), 7.24-7.25 (d, 2H), 7.14-7.16 (d, 1H, J=8 Hz), 6.884-6.889 (d, 1H, J=2 Hz), 6.77-6.78 (d, 1H, J=4 Hz); ¹³C NMR (DMSO-d_6, 100 MHz, δ ppm): 168.2, 162.4, 158.2, 157.9, 152.8, 149.8, 146.8, 146.0, 142.6, 140.4, 135.6, 132.2, 131.7, 130.4, 124.5, 119.5, 118.8, 117.9, 116.9, 113.5, 112.5; Calculated mass: 388.80g/mol; MS (m/z): 390.0 g/mol (M+H).

2.3. ADME-toxicity Prediction

The molecular descriptors of synthesized compounds 7(a-o) are predicted by pharmacokinetic parameters like absorption, distribution, metabolism, excretion and toxicity. The ADMET/SAR [22] helps to evaluate biologically active molecules and eliminate biologically poor molecules, active lead molecules which contain undesirable functional groups based on Lipinski rule. The statistical calculation for lead molecules includes surface area, geometry and fingerprint properties which help to understand biologically important end points. Aqueous Solubility (PlogS), Blood Brain Barrier Penetration (QlogBB), intestinal absorption (logHIA) [23] and hepatotoxicity, Caco-2 cell permeability (QPPCaco) also help to predict the toxicity of lead molecules with intraperitoneal, oral, intravenous and subcutaneous toxic effects. The in-silico study enables to decide the safety and efficacy of active molecules take up the molecule for in depth studies.

2.4. DNA Cleavage Study

The degree of DNA cleavage by the compounds was monitored by agarose gel electrophoresis method [24]. Agarose 0.25 gm was weighed and dissolved in 25 ml of tris acetate (TAE) buffer (50 mM, pH 8.0) and gel cassette was placed in the electrophoresis chamber inundated with TAE buffer. To this was loaded 20 µl of DNA sample along with bromophenol blue dye in 1:1 ratio with standard DNA marker. CT DNA was treated with analogs (40 µM, 2µl) followed by the dilution the buffer to a total volume of 20 µl. The samples after incubation at 37^oC were loaded onto the wells.

Electrophoretic mobility was achieved by supplying 50 V of electricity for about 45 min. in TAE buffer. The gel along with platform was stained with 100 ml ethidium bromide (10 µg/ml) in sterile distilled water. Ethidium bromide binds to double stranded DNA by intercalation, because of steric hindrance for intercalation in covalently closed circular DNAs; it binds less than linear and open circles. After about 10-15 min. the platform and the gel were rinsed with distilled water and gel was gently placed onto the UV transilluminator. The DNA bands appeared on the gel determined the cleavage by the entitled molecules tested.

2.5. Cell Lines and Cell Culture

The human melanoma cell line, A375 and human breast cancer cell line MDA-MB231 were purchased from the National Centre For Cell Science (NCCS Pune, India), cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 2 mmol/l glutamine, 1% antibiotics solution (100 U/ml penicillin G and 100 mg/ml streptomycin). Cells were grown in 25 cm² tissue culture flasks and maintained at 37^oC in a humidified incubator with 5% CO₂. All the consumables and plastic-wares were procured from Himedia, India.

3.1. Chemistry

Substituted furan quinoline carboxylic acids (3) were prepared by coupling substituted isatins and 2-acetylfuran (Scheme 1). Furan quinoline carboxylic acids were converted to their corresponding carboxylates (4) using methanol and catalytic amount of sulphuric acid. The carboxylates upon treatment with hydrazine hydrate resulted in carbohydrazide (5), which upon intermolecular cyclization with substituted benzaldehydes and heterocyclic aldehydes 6(a-o) in the presence of glacial acetic acid afforded novel 2-(1-furan-2-yl)-4-(5-phenyl-4H-1,2,4-triazol-3-yl) quinoline and its analogs 7(a-o) (Scheme 1). IR, ¹H NMR, ¹³C NMR and mass spectral data are in well agreement with the proposed structures of the synthesized compounds. Formation of products was confirmed by spectroscopic analysis as in case compound 2-(1-furan-2-yl)-4-(5-phenyl-4H-1,2,4-triazol-3-yl) quinoline 7(a-o), the IR spectra indicated the absence of band around 3200 cm^-1 due to the absence of -NH₂ and observed band at 3405 cm^-1 due to the presence of -NH group in triazole ring system.

Formation of product was further established by ¹H NMR spectrum, here we can easily characterize the molecule by peak observed at 12.31 ppm (-NH). Furan, quinoline and phenyl protons resonated between the ranges of 8.21-7.23 ppm. Disappearance of NH₂ proton of 7a in proton NMR proved the ring closer by the formation of 1,2,4 triazole ring from 6a.

This was further substantiated by the ¹³C NMR data of 7a, exhibited signals at 162.1 and 152.2 ppm due to the C₂ and C₅ carbons of triazole. The aromatic carbons resonated signals in between 149.3-112.2 ppm which are in agreement with the expected values. Mass spectrum of 7a displayed molecular ion peak at m/z 375.20 (M+2) g/mol, confirming its molecular weight. Rest of the compounds gave satisfactory spectroscopic data, which has been in accordance with their assigned structures.

3.2. In Silico ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) Profile

The pharmacokinetic studies indicated that the compounds with poor bioavailability show less effectiveness against disease. To overcome this problem, predicting bioavailability properties will be of great advantage for drug development. Hence, using computer based methods like ADMET and SAR tools the molecular descriptors and drug likeliness properties were studied. The toxicity of the substituted 2-(furan-2-yl)-4-(5-phenyl-4H-1,2,4-triazol-3-yl) quinoline was predicted based on lethal dosage and functional ranges in different tissues. The mouse LD₅₀ and probability of health effects were predicted using ACD / I-Lab 2.0 (guest). LD₅₀ of potential compounds detect the cumulative potential of acute toxicity administered through oral, subcutaneous, intraperitoneal, intravenous and subcutaneous routes on mouse models. The analysis with test compounds on oral, subcutaneous, intraperitoneal and intravenous is lower when compared to reference molecule. The toxicity results suggest that the compounds 7a-o have less toxic effect in internal tissue and with no side effect (Table 1).

The interpretation of tested compounds 7a-o was in acceptable range and hence, they can be used to make an oral formulation for absorption. The intestinal absorption (log_HIA) and Caco-2 cell permeability (PCaco-2) are within the range of -2 poor absorption and +2 more absorption show that the compounds are more permeable in intestine and help in for transporting good drug metabolic compounds.

The reference range of -5 (poor) to +1 (good) and substrate inhibitor from 0 to 1 in which the reference and test compounds 7a-o show good activity with human intestinal absorption and metabolism. The aqueous solubility of compounds lies in the range of 0 (poor) to 2 (good) showing that all the molecules had good solubility. While the reference compound as well as test compounds came within acceptable range (Table 2).

3.3. DNA Cleavage Study

DNA has been the drug target for drug as it regulates many biochemical reactions occuring in the cellular system. Literature studies revealed that the clinical efficacies of many drugs correlate with their ability to induce enzyme mediated DNA cleavage. The loci present in the DNA are involved in various regulatory processes, such as gene expression, gene transcription, mutagenesis, and carcinogenesis [25] etc. In particular, designing of the compound having the ability to cleave DNA is utmost important not only from the primary biological point of view but also in terms of photodynamic therapeutic approach to develop potent drugs [26]. The compounds which were found to be active in CT-DNA cleavage were screened for their antiproliferative study. The DNA cleavage of furan quinoline triazole hybrids were studied by agarose gel electrophoresis method. The cleavage potential of the tested compounds were examined by comparing the bands appeared in control and test compounds at 100 µg concentration. The photograph (Fig. 1) clearly demonstrates that compounds 7(a-o) did cleave the DNA completely, as no traces of DNA fragments were found. However, the ability of reactive intermediates involved in the DNA cleavage by the compounds has not been cleared. Control experiment does not reveal any significant cleavage of DNA even after prolong exposure of substrate.

3.4. In Vitro Cytotoxic Activity

The oxygen and nitrogen coupled heterocyclic compounds, prepared in this study, were evaluated according to standard protocols for their in vitro anticancer activity against two human cancer cell lines including cells derived from melanoma cell line, A375 and breast cancer cell line MDA-MB-231 [23]. All the IC₅₀ values in micro gram/mililiter (µg/ml) are listed in Table 3 and Fig. (2). For comparison cisplatin was used as a standard cytotoxic drug, that showed the IC₅₀ values of 1.3 and 5.1 µg/ml against melanoma cell line and breast cancer cell line, respectively. Four of the tested compounds: 7a (A375) 7b-c (MDA-MB-231) and 7k exhibited good cytotoxic effects with IC₅₀ values of 2.9, 4.0, 7.8 and 5.1 µg/ml against A375; and 6.2, 9.5, 11.3 and 7.3 µg/ml against, MDA-MB 231, respectively. While rest of the compounds exhibited moderate to less cytotoxic against the cancer cell lines studied. The results revealed that, 7a, 7b, 7c and 7k can be taken up for future investigation as these molecules can be taken up as possible hits.

3.5. Structural Activity Relationship Study

There are few key features regarding structural requirements for these FQT hybrids 7(a-o) to exert their anticancer activity. Our initial strategy was to identify the key sub unit required for activity such as, furan (antibiotic properties of drugs), quinoline (nitrogen heterocycles -Anti cancer agents) and 1,2,4 triazole (active pharmacophore, which allows its derivatives to readily interact with diversity of enzymes and receptors in organisms). Further essential substituents like, [-N(C₂H₅)₂, -N(CH₃)₂, -CH₃ and -NH₂ (electron donating), -OCH₃ (electron releasing), -F, -Cl (halogen) and -NO₂, -OH,(electron withdrawing)] groups were varied at ortho, meta and para-position of the phenyl ring to acquire the optimum results.

The results demonstrated the following assumptions about the structural activity relationship, it is evident, that in a group of compounds having -H-, 4-CH₃, 4-OH, 3-OCH₃, indole, quinoline, 2-CF₃, 3-OH, 4-OCH₃ and 3,4,5 tri-OCH₃ substituents on phenyl ring (7d-f and 7g-7o) were essentially influencing the anticancer activity significantly, particularly, the compounds substituted with halogens on phenyl ring was (7a and 7b-c) were found to be the most active compounds in vitro cytotoxicity. The phenyl ring substituted apart from halogen groups slightly lowers the activity. Lower activity was observed even when the phenyl ring substituted with 3,4,5 tri-OCH₃ (7i). Whereas electron releasing in groups substituted on phenyl ring were found to be less active against cancers cell lines.

Overall, it can be hypothesized that compounds bearing halogens substituent on phenyl ring have shown potent anticancer activity. In fact, the ortho substituents were found to reduce or diminish the overall activity of the compound. Hence, it can also be hypothesized that steric hindrance might have also played an important role in influencing the activity of the compound. The results from the preliminary structure activity analysis have led to the determination of some key structural requirements for the FQT hybrids to exert their anticancer activity, providing insights into further structural modifications.