Triazine–benzimidazole conjugates: synthesis, spectroscopic and molecular modelling studies for interaction with calf thymus DNA

Abstract

Triazine–benzimidazole analogues with different substitutions of primary and secondary amines as well as aryl groups were synthesized and characterized by ¹H, ¹³C NMR and mass spectrometry. Interactions of these compounds with ct-DNA were explored by spectroscopic and viscometric techniques. These results and molecular modelling studies showed that compounds 7, 9–11, 16 and 21 interacted with ct-DNA through groove binding and not intercalation.

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Introduction

DNA is the molecule present in all cellular forms of life that contains the genetic information for biological development. DNA is a major drug target because of its vital role in the cell. New and more effective drugs have been designed which recognized a specific site or conformation of DNA. The ability to identify structural elements of a drug, responsible for the binding to DNA, improves the screening process for new drugs. Small molecules targeting DNA have attracted significant scientific interest due to their medicinal, biochemical and biological applications. These molecules are interacted with DNA through three different non-covalent modes such as intercalation,¹ groove binding,² and electrostatic interactions.³ These interactions are helpful for understanding the mechanism of DNA damage. Generally, drug binds to DNA, are stabilized by series of weak interactions such as π-stacking of aromatic heterocyclic groups between base pairs (intercalation), hydrogen bonding and van der Waals interactions of functional groups bound along the groove of the DNA helix.⁴ Hybrid or conjugate molecules have also been studied for their interactions with DNA.⁵

Triazine is a class of compound, well known for a long time, and still continues to be the object of considerable interest. This is mainly due to its biological activity⁶ including antiprotozoal,⁷ anticancer,⁸ antimalarial,⁹ antiviral,¹⁰ antimicrobial,¹¹ antileishmanial,¹² autoimmune disease¹³ etc. Benzimidazole ring system is also represents as an important pharmaceutical scaffold.¹⁴ Several benzimidazole derivatives have found use in nucleic acid recognition.¹⁵

These biological applications associated with triazine and benzimidazole are prompted us to investigate the corresponding structure as a potential molecular probe for interaction with calf thymus DNA. Herein, we have synthesized the triazine–benzimidazole analogues substituted with various primary and secondary amines as well as aryl groups for interaction with DNA. The binding interactions of triazine–benzimidazole conjugates with calf thymus DNA have been investigated by applications of UV-visible and fluorescence spectroscopy. In order to further confirm the mode of interaction between conjugates and DNA, thermal denaturation experiment, competitive binding study using ethidium bromide and Hoechst-33258, viscosity measurement, ionic strength effect and iodide quenching experiments were performed. Molecular modelling studies further revealed the interaction of compounds with DNA. These spectroscopic and viscometric results along with molecular modelling studies are helpful to understand the structural requirements of compounds for binding with ct-DNA. These would further be useful for design of new and potent drug molecules.

Results and discussion

2,4,6-Trisubstituted triazines (4–21) were prepared in moderate to good yields with nucleophilic substitution and Suzuki–Miyaura cross coupling reactions (Scheme 1). Cyanuric chloride was reacted with 4-fluoroaniline in the presence of 10% NaHCO₃ in THF at 0–5 °C for 6 h to obtain (4,6-dichloro-[1,3,5]triazin-2-yl)-(4-fluoro-phenyl)-amine (1) in 85% yield. Compound 1 was treated with 4-(1H-benzimidazol-2-yl)-phenylamine (2) (synthesized by the condensation of 4-aminobenzoic acid and o-phenylenediamine in the presence of polyphosphoric acid at 200 °C for 5 h) in the presence of 10% potassium carbonate and THF at room temperature for 24 h to provide N-[4-(1H-benzimidazol-2-yl)-phenyl]-6-chloro-N′-(4-fluoro-phenyl)-[1,3,5] triazine-2,4-diamine (3) in 82% yield. Further, treatment of 3 with different primary and secondary amines in the presence of potassium carbonate in 1,4-dioxane at 110 °C for 6–8 h to give compounds 4–15 in 66–78% yields. Compound 3 was also treated with five and six membered aryl boronic acids through Suzuki coupling, in the presence of 10 mol% of Pd(PPh₃)₄ and 1.5 equivalents of K₂CO₃ in 1,4-dioxane afforded compounds 16–21 in 65–71% yields as shown in Table 1. Structures of all new compounds were confirmed by ¹H and ¹³C NMR as well as mass spectrometry (ESI†).

Scheme 1 Synthesis of triazine–benzimidazole analogues.

Table 1 Physicochemical property of compounds 4–21

Entry	Product	Yield (%)	Mp (°C)	Entry	Product	Yield (%)	Mp (°C)
1		75	248–250	10		70	288–290
2		78	252–254	11		72	281–283
3		72	242–244	12		66	289–291
4		77	268–270	13		69	246–248
5		72	273–275	14		68	262–264
6		69	261–263	15		67	274–276
7		74	278–280	16		71	248–250
8		73	274–276	17		65	288–290
9		78	247–249	18		70	266–268

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Absorption¹⁶ and emission¹⁷ spectroscopy are extensively used as effective methods for monitoring complex formation between DNA and small molecules. Thus, these compounds were screened for their interaction with ct-DNA using UV-visible and fluorescence spectroscopic techniques. The interaction of compounds 4–21 (20 μM) towards ct-DNA were investigated by UV-visible spectroscopy in phosphate buffer (10 mM, pH 7.4). The electronic spectra of all the tested compounds showed intense band at the range of 300–335 nm due to transitions between π–π* energy levels. Addition of ct-DNA to the solution of compounds 7, 9–11, 16 and 21 in phosphate buffer resulted in gradual decrease in the absorption intensity. These compounds showed comparatively more hypochromicity in the respective percentage of 58.3%, 51%, 63%, 58.9%, 62.5% and 64.5% (Fig. 1) as compared to compounds 4–6, 8, 12, 14, 15, 18 and 20 which showed hypochromicity in the range of 21–49.3% (Fig. S41–S49†). Compounds 13, 17 and 19 did not show any significant change in the presence of ct-DNA. As there was no change in position of the absorption band (bathochromic or hypsochromic shift) of conjugates, it has been concluded that these compounds exhibited groove binding interactions to ct-DNA.¹⁸

Fig. 1 UV-vis spectral changes of compounds 7, 9–11, 16 and 21 (20 μM) with addition of ct-DNA (0–15 μM) in phosphate buffer (10 mM, pH 7.4).

In order to further explore the interactions of triazine–benzimidazole analogues, fluorescence titration experiments were performed. On exciting the compounds 7, 9–11, 16 and 21 at 305 nm, the emission spectra showed intense band between 375 nm and 473 nm. The fluorescence intensities of compounds were regularly decreased while the maximum emission wavelength did not apparently shift with increase in ct-DNA concentration. Addition of ct-DNA (0–17 μM), compounds 7, 9–11, 16 and 21 showed quenching in the percentage of 52.4%, 44.3%, 46.0%, 35.4%, 54.3% and 48.0% respectively (Fig. 2) while the emission of compounds 4–6, 8, 12, 14, 15, 18 and 20 were quenched in the range of 20–58% (Fig. S41–S49†). Compounds 13, 17 and 19 did not show any significant change in the presence of ct-DNA in emission spectroscopy. These changes in absorption and fluorescence intensities clearly indicated the interacting properties of compounds 7, 9–11, 16 and 21 with DNA. Inner filter effects need to be corrected since they deplete the fluorescence signal affecting the desired linear relationship between concentration of fluorophore and fluorescence intensity.¹⁹

Fig. 2 Fluorescence spectral changes of 7, 9–11, 16 and 21 (20 μM) with addition of ct-DNA (0–17 μM) in phosphate buffer (10 mM, pH 7.4).

The fluorescence intensity of samples was corrected using the method of Lakowicz.²⁰ The correction method of Lakowicz uses:

where F_corr and F_obs are corrected and uncorrected fluorescence intensities, A_ex is absorbance of solution at excitation wavelength and A_em is absorbance of solution at emission wavelength (Table S1†).

For the quantitative investigation of the binding strength, the binding constants of these compounds with ct-DNA were determined with UV-visible and fluorescence titrations data using Benesi–Hildebrand equation²¹ (Fig. S50 and S51†). It has been observed that the binding constant of ligand which bind to DNA usually fall in the range of 10⁴ to 10⁶ M⁻¹. Ethidium bromide,²² which is an excellent DNA intercalator, binds with ct-DNA having binding constant value of 4.3 × 10⁵ while the magnitude of binding constant of groove-binding drugs such as 2-imidazolidinethione and levetiracetam are 1.4 × 10³ M⁻¹ and 4.9 × 10³ M⁻¹, respectively.²³ The K values of triazine–benzimidazole analogues have been shown in the range of 10³ to 10⁴ that indicated their binding interaction with ct-DNA through groove mode as shown in Table 2.

Table 2 Photophysical properties and binding constants (K) of the compounds 7, 9–11, 16 and 21 with ct-DNA

Compds	Hypochromicity (%) in absorption	Emission quenching (%)	K (absorption) (M^−1)	K (emission) (M^−1)
7	58.3	52.4	1.7 × 10^4	2.3 × 10^3
9	51.0	44.3	5.4 × 10^3	9.7 × 10^4
10	63.0	46.0	1.8 × 10^4	2.8 × 10^4
11	58.9	35.4	1.3 × 10^4	2.7 × 10^4
16	62.5	54.3	4.1 × 10^4	4.8 × 10^4
21	64.5	48.0	2.3 × 10^4	2.8 × 10^4

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To evaluate the possible mode of binding of compounds 7, 9–11, 16 and 21 with DNA, thermal denaturation studies were performed. Significant stabilization of double helix is commonly observed for intercalating ligand, whereas groove binder leads to stabilization or destabilization or negligible change in the melting temperature (T_m) of double standard DNA.²⁴ The T_m value for free ct-DNA has been found to be 65.6 °C whereas complex of compounds 7, 9–11, 16 and 21 with DNA, the T_m was measured in the range of 57.0–64.4 °C. It has been found that triazine–benzimidazole analogues destabilize DNA double helix by 1.2–8.6 °C (Table 3). This indicated that the binding modes of compounds are not due to intercalation but some other interactions are responsible for their binding to DNA.

Table 3 Average T_m and ΔT_m for ct-DNA in the absence and presence of compounds 7, 9–11, 16 and 21

Compounds	Tm (°C)	ΔTm (°C)
ct-DNA	65.6	0.0
7	60.1	−5.5
9	57.0	−8.6
10	58.8	−6.8
11	62.4	−3.2
16	64.4	−1.2
21	59.3	−6.3

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In order to rationalize the binding mode, competitive binding experiments were performed with compounds 7, 9–11, 16 and 21 and DNA in the presence of a minor groove binder (Hoechst 33258) and an intercalator (ethidium bromide).²⁵ DNA (0–30 μM) was added to Hoechst 33258 (20 μM) solution and the fluorescence emission spectrum was recorded when saturation achieved. In this case, the emission intensity was gradually increased and reached a maximum value (saturation). With increase in concentration of compounds 7, 9–11 and 16, the emission intensities of Hoechst·DNA complex were enhanced to significant levels while in case of compound 21, the emission intensity of Hoechst·DNA complex was quenched (Fig. 3). This indicated that there is replacement of Hoechst dye with compounds 7, 9–11, 16 and 21 from the minor groove of DNA. A similar experiment was performed with addition of DNA to a solution of ethidium bromide (EB) that provided an increase in emission intensity of ethidium bromide to saturation. Ethidium bromide is one of the most sensitive fluorescent probes with a planar structure that binds to DNA via intercalative mode. In EB displacement assay, any molecule that binds to DNA via the same mode as EB will replace EB from the DNA helix and results in a decrease in the fluorescent intensity of EB·DNA system. The extent of fluorescence quenching of EB·DNA system are used to determine the extent of intercalation between the molecule and DNA.²⁶

Fig. 3 Gradual addition of ligands 7, 9–11, 16 and 21 (2–30 μm) result in significant changes in the fluorescence intensities of Hoechst bound ct-DNA.

However, in this case, on subsequent addition of compounds 7, 9–11, 16 and 21 to the ethidium bromide·DNA complex, there was no significant change in emission intensity of ethidium bromide·DNA complex, suggesting that triazine–benzimidazole binds to DNA in non-intercalative mode (Fig. S52†). These results further supported the binding of compounds 7, 9–11, 16 and 21 through groove mode.

Further, viscosity titrations were performed to confirm the binding modes between triazine–benzimidazoles and ct-DNA. The intercalation of compound into DNA causes a significant increase in viscosity due to increase in separation of base pairs at the intercalation site of the DNA helix as the base pairs are pushed apart to accommodate the binding molecule. On the other hand, groove or electrostatic binding leads to a minor or no increment in the DNA viscosity.²⁷ A viscosity plot of (η/η_o)^1/3 versus ligand–DNA ratio was obtained to study any change in the viscosity of ct-DNA solution in the presence of compound. As shown in Fig. 4, with continuous addition of compounds 7, 9–11, 16 and 21 to ct-DNA solution, small increase in viscosity has been obtained which was not as pronounced as observed for classical intercalators. The results revealed that these compounds interact with minor groove of DNA and rule out the possibility of intercalation.

Fig. 4 Relative specific viscosity of aqueous solution of ct-DNA in the presence of ligands 7, 9–11, 16 and 21.

The effect of ionic strength was also determined to evaluate the electrostatic binding between triazine–benzimidazole and ct-DNA. The presence of NaCl weaken the surface-binding interaction because positively charged sodium ions binds to negatively charged ct-DNA phosphate group via electrostatic attraction, and then neutralize the negative charge of the helix backbone to ct-DNA. If there is no electrostatic binding, there is no effect on the ratio of fluorescence quenching.²⁸ An increase in NaCl concentration did not significantly changes in the slopes of F_o/F of the free compounds (Fig. 5a) and compound·DNA complexes (Fig. 5b), indicating that triazine–benzimidazoles do not bind to ct-DNA by electrostatic binding (Fig. S53†).

Fig. 5 Fluorescence quenching plots of compounds 7, 9–11, 16 and 21 and compound·DNA complexes by NaCl (a) and (b), and KI (c) and (d).

To further explore the binding mode between compounds and ct-DNA, iodine quenching experiments were performed. When small molecule intercalate into DNA base pairs, the bound molecule is protected by the double helix of DNA, thus it is difficult for the anionic quencher to come into contact with small molecule. Therefore, the quenching constant of compound·DNA complex should be smaller than that for free small molecule.²⁹ Potassium iodide is highly negative charged quencher, which is often used to determine the binding mode of compound with DNA.^25a The ratio of fluorescent intensities of compounds 7, 9–11, 16 and 21 and DNA complexes did not show any significant changes upon successive addition of iodide that further eliminated the possibilities of intercalations (Fig. S54†). These results also signified the interaction of compounds 7, 9–11, 16 and 21 with DNA through groove binding mode (Fig. 5c–d).

Docking experiments were performed to find the most stable and favourable orientation as blind dockings (blind docking refers to the use of a grid box that is large enough to encompass any possible ligand receptor complex) using Argus lab.³⁰ The structures of the ligands (7, 9–11, 16 and 21) were optimized to true minima on the potential energy surface. The structural coordinates of DNA were obtained from the protein data bank (PDB ID: 1BNA).³¹ The 100 docking runs were conducted on the basis of principle of optimal energy. Based on the energies and the numbers of occurrence, the conformation with highest number of occurrence and most optimal energy of clusters were chosen for the final binding orientation analysis (Fig. 6). In the docking analysis, the binding site was assigned across the entire groove of the DNA molecule. The docking studies of ligands clearly showed the importance of the mode of action through the interaction between the triazine derivatives and DNA. Moreover, hydrogen bonds were formed between compounds (7, 9–11, 16 and 21) with DNA base pairs (Table 4, Fig. S55†). Due to differences in the functional groups, there were differences in their hydrogen bonding interaction with ct-DNA, and thus differences in the values of binding constants. The molecular modelling studies showed that all the six ligands bind DNA preferably through the groove mode. Charges obtained from the PM3 method of compounds 7, 9–11, 16 and 21 were also compared with charges of AM and HF methods of Gaussian 09 (Tables S2–S4†).

Fig. 6 Docked poses of compounds (7, 9–11, 16 and 21) with DNA.

Table 4 H-bonding of compounds 7, 9–11, 16 and 21 with DNA base pairs

Compds	NH of fluoroaniline	NH of phenyl linker	NH of imidazole ring of benzimidazole	Variable substituent	No. of H-bonding
7	7DT (2.26 Å), 6DA (1.86 Å), 7DA (1.74 Å)	18DA (3.35 Å), 18DA (1.69 Å)	17DA (2.78 Å), 8DT (2.89 Å), 16DG (2.16 Å), 9DC (2.38 Å)	—	9
9	22DG (2.28 Å), 23DC (2.75 Å)	5DA (1.46 Å), 5DA (1.38 Å)	22DG (0.76 Å), 3DC (2.95 Å), 22DG (2.81 Å)	NH of ethanolamine with DC (1.17 Å), DG (2.09 Å), 24DG (2.82 Å)	12
				OH of ethanolamine with 2DG (2.09 Å), 1DC (1.17 Å)
10	6DA (1.66 Å)	6DA (2.36 Å), 7DT (2.30 Å), 19DT (2.75 Å), 18DA (2.10 Å)	16DG (2.13 Å)	NH of aminoethylmorpholine with 5DA (2.66 Å), 6DA (2.40 Å), 5DA (1.21 Å)	9
11	5DA (2.58 Å), 5DA (1.51 Å), 5DC (2.87 Å)	20DT (2.38 Å), 5DA (2.61 Å)	18DA (1.54 Å), 7DT (1.73 Å), 5DA (2.65 Å)	OH of hydroxyethylpiperazine with 24DG (2.74 Å), 2DG (1.84 Å), 2DG (2.41 Å)	11
16	7DT (1.38 Å)	17DA (2.22 Å), 18DA (2.03 Å)	10DG (2.95 Å), 15DC (2.63 Å), 9DC (2.74 Å), 16DG (2.34 Å)	—	7
21	8DT (2.57 Å), 7DT (2.30 Å)	17DA (1.67 Å), 17DA (2.99 Å)	10DG (2.48 Å), 9DC (2.97 Å)	—	6

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Conclusions

Through the analysis of UV-visible and fluorescence spectra, formations of triazine–benzimidazole·DNA complexes were confirmed. Competitively displacement assay with ethidium bromide and Hoechst 33258 revealed that compounds interacted with DNA through groove binding. Thermal denaturation experiments and viscosity techniques also supported the interactions of these compounds through groove binding mode. The effect of ionic strength and iodide quenching again confirmed the involvement of groove binding and not intercalation. Molecular docking experiments further confirmed that triazine analogue binds to groove of DNA. Thus, the structure features essential for the groove binding activity of triazine–benzimidazole analogues as determined by the overall outcome of the results revealed that: (i) the triazine and benzimidazole rings are satisfactory backbone for binding with DNA, (ii) the presence of functional groups enhanced specificity of these analogues due to additional hydrogen bonding to the floor of groove, (iii) chair conformation of piperazine and morpholine is directed towards the base pairs on the groove, increased stability of the complex and, (iv) flexibility of the side chains of triazine directly affects the groove width. These results provided valuable information regarding drug–DNA interactions, which may be useful for the greater clinical efficacy of rational drug design.

Acknowledgements

KP thanks the Department of Science and Technology, New Delhi (EMR/2014/000669). PS is grateful for a DST/Inspire Fellowship (Fellow Code-IF110542).

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Footnote

† Electronic supplementary information (ESI) available: Experimental section and final compounds characterization data; materials and methods associated with this article. See DOI: 10.1039/c5ra24001h

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