Azaindole-1,2,3-triazole conjugate in a tripod for selective sensing of Cl⁻, H₂PO₄⁻ and ATP under different conditions

Abstract

A new tripodal sensor 1 has been designed and synthesized. The cavity of the tripod selectively recognizes Cl⁻ and H₂PO₄⁻ over a series of other anions in CH₃CN containing 0.01% DMSO by exhibiting a significant change in emission. Between H₂PO₄⁻ and Cl⁻ ions, H₂PO₄⁻ is distinguished by 1 through a ratiometric change in emission. In comparison, the indole-based tripod 2 did not show any binding-selectivity with the same anions. Compound 1, furthermore, selectivity recognizes phosphate based biomolecule ATP over ADP and AMP in semi aqueous solvent (CH₃CN containing 0.01% DMSO : H₂O, 1 : 1, v/v) at pH 7.3. The tripod 1 is cell permeable and detects ATP by showing quenching of emission.

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Introduction

Anion complexation by design-based synthetic receptors has become an important topic in supramolecular chemistry due to the amazing impact of anions in many chemical and biological processes.¹ Among various design-based synthetic receptors, tripodal receptors are of special interest for their preorganized C₃-symmetry anion-binding geometry. The tripodal molecular platform with three arms allows the rational control of binding properties such as complex stability and selectivity. Compared to a rigid cyclic system, they can show rapid complexation/decomplexation kinetics and may undergo significant conformational changes upon binding. Therefore, the tripodal receptors are hypothesized to be between cyclic and acyclic ligands with regard to preorganization and are thus believed to be able to complex an ion more effectively than analogous acyclic one.² A number of synthetic receptors of this class with different functional groups are known in the literature.³ In this context, the use of 7-azaindole as an anion binder around the tripodal core is completely unknown. Recently, for the first time, we have used 7-azaindole in collaboration with a 1,2,3-triazole motif in the construction of an anion binding receptor.⁴ The promising results have inspired us further to undertake the design of a tripodal-shaped receptor 1 that recognizes different anions such as dihydrogenphosphate, chloride and ATP under different conditions by exhibiting a considerable change in emission in different modes. To establish the binding role of the ring nitrogen atom in 1, a model compound 2 was prepared. Structure 2 did not give any measurable selectivity under identical conditions.

It is worth mentioning that the recognition and sensing of inorganic phosphates (Pi), dihydrogenphosphate (H₂PO₄⁻) and phosphate-based biomolecules such as ATP, ADP and AMP draws attention. Till now, a plethora of receptors that are capable of fluorimetric sensing of H₂PO₄⁻ anions are known in the literature.⁵ Among the nucleotides, ATP is very important as it is deeply involved in intercellular energy transfer, DNA duplication and transcription.⁶ Its concentration in the cell is indicative of cell metabolic rate. So, its selective detection by synthetic receptors is desirable. There are only a few synthetic receptors that are found to recognize these phosphate-based biomolecules.⁷

On the other hand, chloride ion recognition by an abiotic host is important due to its biological significance. It is associated with a chloride channel that is critically linked to respiration with the exchange of Cl⁻ for HCO₃⁻ ion in erythrocytes. Disorder of the chloride channel causes the genetic disease cystic fibrosis.⁸ Therefore, synthetic receptors for chloride recognition is also crucial. It is mentionable that a single structure that recognizes multiple anions by exhibiting different photo physical features is demanding and along this direction the receptor structure 1 in this report draws attention.

Results and discussion

Compounds 1 and 2 were obtained according to Scheme 1. For 1, the tris azide 3 obtained from 1,3,5-tris(bromomethyl)-2,4,6-trimethylbenzene was treated with 5 equiv. 2-alkynyl-7-azaindole 8a⁴ to couple via click reaction. Compound 8a was obtained from 7-azaindole after performing a series of reactions as mentioned in Scheme 1b. In Scheme 1b, the intermediate compound 6a was synthesized according to the literature procedure.⁹ Indole-based compound 2 was synthesized following the same reaction pathway as maintained for the synthesis of 1. Use of 5 equiv. 8b⁴ in the click reaction with 3 afforded compound 2 in appreciable yield. Like 8a, compound 8b was synthesized from indole according to Scheme 1b. In this regard, the intermediate compound 6b, used for the synthesis of 2, was prepared according to the literature procedure.⁹ Both compounds 1 and 2 were fully characterized spectroscopically.

Scheme 1 (a) (i) Acetone–water (4 : 1, v/v) NaN₃, reflux, 3 h; (ii) 8a (5 equiv.), sodium ascorbate, CuSO₄, EtOH–H₂O (1 : 1, v/v); (iii) 8b (5 equiv.), sodium ascorbate, CuSO₄, EtOH–H₂O (1 : 1, v/v); (iv) PhSO₂Cl, K₂CO₃, dry acetone, reflux, 16 h; (v) LDA, TMEDA, I₂, THF, −30 °C; (vi) NaO^tBu, dioxane, sealed tube, 80 °C, 3 h; (vii) TMS–acetylene, Pd(PPh₃)₂Cl₂, CuI, Et₃N, THF, rt; (viii) TBAF, THF, rt.

Anion recognition studies

Before investigating the anion binding behavior, the absorption and emission spectra of both 1 (Fig. 1) and 2 (ESI†) were recorded in DMSO and CH₃CN containing 0.01% DMSO. We were unable to record the spectra in other solvents due to insolubility. As can be seen from Fig. 1, the emission of 1 is much changed in DMSO rather than CH₃CN. This is attributed to the coordination role of DMSO with the azaindole-1,2,3-triazole conjugate. This is in accordance with our previous observation on dipodal structure.⁴

Fig. 1 Absorbance spectra (a) and emission spectra (b) of 1 (c = 5.08 × 10⁻⁵ M) in different solvents.

The anion binding ability (F⁻, Cl⁻, Br⁻, I⁻, AcO⁻, NO₃⁻, HSO₄⁻, H₂PO₄⁻ and HP₂O₇³⁻ as their tetrabutylammonium salts) of 1 was evaluated by fluorescence titration in CH₃CN containing 0.01% DMSO. In fluorescence, the emission of 1 (c = 5.02 × 10⁻⁵ M; λ_exc = 310 nm) at 363 nm for the azaindole motif underwent change to different extents in the presence of the anions (ESI†). Fig. 2 highlights the change in fluorescence ratio of 1 in the presence of 15 equiv. amounts of a particular anion.

Fig. 2 Change in fluorescence ratio of 1 (c = 5.02 × 10⁻⁵ M) at 369 nm upon addition of 15 equiv. amounts of different anions in CH₃CN containing 0.01% DMSO.

Among the anions, the Cl⁻ ion brought about a considerable increase in monomer emission of 1 without producing any other change in the spectrum (Fig. 3). The preorganised scaffold of the tripodal core with the triazole ring leads to the formation of a perfect cavity for accommodating the Cl⁻ ion. On the other hand, a ratiometric change in emission with H₂PO₄⁻, which was convenient to distinguish it from the other tested anions, was observed (Fig. 4). This finding is in accordance with our previously reported dipodal receptor.⁴ In comparison to the dipodal receptor,⁴ the tripodal receptor 1 in the present report is much more attractive in its ratiometric behavior. In relation to this, the change in emission at the two different wavelengths 369 nm and 475 nm is presented in the inset of Fig. 4. Careful scrutiny reveals that ratiometric chemosensors for H₂PO₄⁻ are rare in the literature.^10,11 The ratiometric chemosensors offer advantages over the usual monitoring of fluorescence intensity at a single wavelength. A dual emission system can minimize the measurement errors because of factors such as phototransformation, receptor concentrations, and environmental effects.¹⁰

Fig. 3 Change in emission of 1 (c = 5.02 × 10⁻⁵ M) upon addition of 15 equiv. Cl⁻ (c = 1 × 10⁻³ M) in CH₃CN containing 0.01% DMSO.

Fig. 4 Emission titration spectra of 1 (c = 5.02 × 10⁻⁵ M) in CH₃CN containing 0.01% DMSO upon addition of 15 equiv. H₂PO₄⁻. Inset: change in emission at the monomer and excimer wavelengths with guest concentration.

Dihydrogenphosphate binding induced quenching of the monomer emission is explained to be due to the perturbation of nπ* state of the azaindole moiety in a stabilizing manner, for which presumably the lowest energy singlet excited state (nπ*) comes closer to the ground singlet state.¹¹ Detailed information supported by theoretical calculation in this aspect has been given in our earlier report on a dipodal system.⁴ The growth of the emission at the longer wavelength upon complexation is supposed to be due to excimer formation by closely spaced azaindole rings or a strong conjugation established between the azaindole and 1,2,3-triazole motifs. DFT calculation¹² using the b3lyp function and 6-31(g) basis set on the complex of 1 with H₂PO₄⁻ as displayed in Fig. 5A clearly interprets the hydrogen bonding features of the pseudo cavity and the orientation of the azaindole motifs around the tripodal core. In 1, C–H, N–H and ring nitrogens are found to participate actively in making a strong complex with H₂PO₄⁻. DFT calculation on the complex 1 with Cl⁻ ion was also performed. The hydrogen bonding arrangements of the complex are depicted in Fig. 5B. A lower number of hydrogen bonds are noted in the complex and thus corroborates a weak complex.

Fig. 5 DFT optimized geometries of the complexes of 1 with (i) H₂PO₄⁻ (a = 1.70 Å, b = 1.64 Å, c = 1.58 Å, d = 2.82 Å, e = 2.45 Å, f = 2.03 Å, g = 1.54 Å, h = 2.87 Å and i = 1.84 Å) and (ii) Cl⁻ (a = 1.83 Å, b = 2.32 Å, c = 3.01 Å, d = 2.88 Å) ions.

Fig. 6 corroborates the comparative views on the emission changes upon addition of 15 equiv. F⁻, Cl⁻, Br⁻, I⁻, NO₃⁻, AcO⁻, ClO₄⁻, HSO₄⁻ and H₂PO₄⁻ to the solution of 1 in CH₃CN containing 0.01% DMSO. The stoichiometries of the complexes of 1 with Cl⁻ and H₂PO₄⁻ were determined to be 1 : 1 as confirmed by the Job plot¹³ (ESI†). Non linear curve fitting gave binding constant values¹⁴ (ESI†) of (1.12 ± 0.14) × 10⁴ M⁻¹ with H₂PO₄⁻ and (2.04 ± 0.29) × 10³ M⁻¹ with Cl⁻ ions. Under similar conditions, gradual addition of different anions to the solution of 2 in CH₃CN containing 0.01% DMSO brought no selectivity in emission. This is easily understandable from Fig. 7. This observation is also similar to the case of our earlier reported dipodal receptor.⁴

Fig. 6 Change in emission of 1 (c = 5.02 × 10⁻⁵ M) upon addition of 15 equiv. amounts of different guests (c = 1 × 10⁻³ M) in CH₃CN containing 0.01% DMSO.

Fig. 7 Change in fluorescence ratio of 2 (c = 5.07 × 10⁻⁵ M) at 369 nm upon addition of 15 equiv. amounts of different guests in CH₃CN containing 0.01% DMSO.

To be acquainted with the selectivity of the functional receptor 1 towards Cl⁻ and H₂PO₄⁻ ions, the fluorescence sensing of a particular ion by 1 was understood by recording the emission spectra of 1 in the presence and absence of other anions in CH₃CN containing 0.01% DMSO. As shown in Fig. 8, the anions except H₂PO₄⁻ in the study show negligible interference in the binding of the Cl⁻ ion. On the other hand, Fig. 9 displays the selectivity profile where the interference of other anions in the selective sensing of H₂PO₄⁻ is noted to be negligible. Even the Cl⁻ ion did not interfere (Fig. 9). This is in contrast to the case of Fig. 8. This happens due to a stronger affinity of the H₂PO₄⁻ ion than Cl⁻ ion.

Fig. 8 Change in fluorescence ratio of 1 (c = 5.08 × 10⁻⁵ M) upon addition of 15 equiv. Cl⁻ to the receptor solution containing other anions in CH₃CN containing 0.01% DMSO.

Fig. 9 Change in fluorescence ratio of 1 (c = 5.08 × 10⁻⁵ M) upon addition of 15 equiv. H₂PO₄⁻ to the receptor solution containing other anions in CH₃CN containing 0.01% DMSO.

The anion binding of the tripod 1 in the ground state was understood from UV-vis titration. In all cases the absorption centered at 306 nm in 1 decreased upon complexation (ESI†). The change was only appreciable for 1 with H₂PO₄⁻ and Cl⁻ ions (Fig. 10).

Fig. 10 UV-vis titration spectra for 1 (c = 2.51 × 10⁻⁵ M) with (a) Cl⁻ and (b) H₂PO₄⁻ (c = 1 × 10⁻³ M) in CH₃CN containing 0.01% DMSO.

Without having much information in UV, we performed NMR study to realize the actual binding features in the ground state. ¹H NMR of 1 in the presence of 1 equiv. Cl⁻ was recorded in CD₃CN containing 2% d₆-DMSO (Fig. 11A). Upon interaction, while the NH proton of the azaindole motif moved downfield slightly (Δδ = 0.07 ppm), the signal for the CH proton of the 1,2,3-triazole ring suffered much downfield chemical shift (Δδ = 0.31 ppm) and thereby confirmed the participation of both the azaindole and triazole motifs in complexation. Unfortunately, we failed to record the ¹H NMR of 1 with H₂PO₄⁻ in the same solvent system due to precipitation. Thus we had to check the interaction of H₂PO₄⁻ with 1 in d₆-DMSO (Fig. 11B). In this solvent, the signal for the triazole ring proton (Fig. 11B) moved downfield by 0.12 ppm upon interaction with H₂PO₄⁻. The NH proton that appeared at 12.10 ppm gave a downfield chemical shift (Δδ = 0.18 ppm). The upfield chemical shift of the rest aromatic protons is assumed to be due to partial displacement of DMSO by the H₂PO₄⁻ ion from the interfering zone of 1. DMSO is a hydrogen bond interfering solvent in such systems, as established in the earlier case.⁴ It severely interferes with the 7-azaindole-1,2,3-triazole conjugate and blocks the cavity from allowing complexation of anions. Comparison of ¹H NMR of 1 in CD₃CN containing 2% d₆-DMSO and pure d₆-DMSO (ESI†) indicated the change in chemical shifts of the signals and thereby corroborated the interaction role of DMSO. Indeed, the emission of 1 in pure DMSO was also checked with the addition of all anions considered in the study. No characteristic selectivity in the sensing process was observed (ESI†).

Fig. 11 Partial ¹H NMR (400 MHz) of 1 (c = 3.27 × 10⁻³ M) in the absence (a) and presence of 1 equiv. (b) tetrabutylammonium chloride in CD₃CN containing 2% d₆-DMSO; (B) partial ¹H NMR (400 MHz) of 1 (c = 5.83 × 10⁻³ M) in the absence (a) and presence of 1 equiv. (b) tetrabutylammonium dihydrogenphosphate in d₆-DMSO (for labeling of protons see structure 1).

Furthermore, ³¹P NMR was used to check the interaction. The signal for the P-atom in H₂PO₄⁻ that appeared at 1.93 ppm suffered an upfield chemical shift by 0.5 ppm in the presence of 1 in CD₃CN containing 4% d₆-DMSO (ESI†) and indicated a measurable interaction. In d₆-DMSO the shift was noted to be less (Δδ = 0.1 ppm). This occurred due to less interaction of H₂PO₄⁻ for the severe interference of DMSO.

For the application of the tripod 1 in an aqueous system, we studied the complexation of 1 with different phosphate salts, ATP, ADP, AMP and Cl⁻ as its potassium salt in CH₃CN containing 0.01% DMSO : H₂O (1 : 1, v/v) at pH 7.3 containing 10 mM HEPES buffer. The change in emission of 1 under physiological condition was found to be considerable in the presence of ATP (Fig. 12a) only. Other inorganic phosphate salts including sodium triphosphate brought about minor change in emission (Fig. 12b). Receptor 1 showed 1 : 1 binding with ATP and gave a binding constant value¹⁴ of (8.27 ± 1.89) × 10⁴ M⁻¹ (ESI†). The preferential binding of ATP over ADP and AMP is presumably attributed to the pseudo cavity of 1 that accommodates ATP comfortably in comparison to our previously reported dipodal receptor⁴ involving a greater number of hydrogen bonds. The selective recognition of ATP by 1 in a semi aqueous system was ascertained in the presence and absence of all the phosphate salts undertaken in the study. Fig. 13A represents this feature.

Fig. 12 (a) Change in emission of 1 (c = 4.25 × 10⁻⁵ M) upon addition of 15 equiv. ATP in CH₃CN containing 0.01% DMSO : H₂O (1 : 1, v/v) at pH 7.3 containing 10 mM HEPES buffer; (b) change in fluorescence ratio of 1 (c = 4.25 × 10⁻⁵ M) upon addition of 15 equiv. amounts of different guests in CH₃CN containing 0.01% DMSO : H₂O (1 : 1, v/v) at pH 7.3 containing 10 mM HEPES buffer.

Fig. 13 (A) Change in fluorescence ratio of 1 (c = 4.25 × 10⁻⁵ M) at 369 nm upon addition of 15 equiv. ATP in the presence and absence of other anions in CH₃CN : H₂O (1 : 1, v/v, 10 mM HEPES buffer) at pH 7.3; (B) partial ³¹P NMR (400 MHz) of 1 (c = 5.02 × 10⁻³ M) in the absence (a) and presence (b) of 1 equiv. ATP in CD₃CN containing 0.01% d₆-DMSO : D₂O (1 : 1, v/v).

The substantial interaction of 1 with ATP was confirmed by recording ³¹P NMR of 1 in the presence and absence of ATP in CD₃CN containing 0.01% d₆-DMSO : D₂O (1 : 1, v/v). Considerable shift of the different P-atoms of the phosphate chain in ATP was observed (Fig. 13B) upon interaction. In ¹H NMR (taken in d₆-DMSO : D₂O (1 : 1, v/v)) upon interaction the signals of aromatic protons were too broad to calculate the chemical shift values exactly (ESI†).

Due to good anion sensing properties of 1 with H₂PO₄⁻ ion in CH₃CN containing 0.01% DMSO and ATP in aq. CH₃CN containing 0.01% DMSO (CH₃CN containing 0.01% DMSO : H₂O = 1 : 1, v/v, pH 7.3 using 10 mM HEPES buffer), we further applied the compound 1 to evaluate its ATP sensing potential in cancer cell lines.

Human cervical cancer cells (HeLa) were incubated with different concentrations of 1 (10.0 mM and 20.0 mM in H₂O–CH₃CN containing 0.01% DMSO (100 : 1, v/v; buffered with HEPES, pH 7.0) in a DMEM medium for 20 min at 37 °C and washed with a phosphate-buffered saline (PBS) (pH = 7.4) to remove excess of receptor 1. Microscopic images showed mild fluorescence due to the accumulation of 1 within the cells (Fig. 14b). Compound 1 was non-toxic as confirmed by MTT assay (ESI†). However, the subsequent incubation of cells pre-incubated cells with 1 exhibited no fluorescence after further incubation with ATP (5 mM) for 1 h at 37 °C. These results suggest that receptor 1 is highly cell membrane permeable and can be used as a bio-sensor probe to detect/sensitize the intracellular ATP concentration in living cells. ADP and AMP remained silent in the similar study. Further we investigated the potentiality/ability of this biosensor to detect the intercellular cleavage of ATP by ALP. The experiment was carried out with cells pre-incubated with 1 and then further incubated with an equimolar mixture of ATP and ALP. The cells were then observed under microscope after 3 h. Un-interruption of the fluorescence intensity were observed similar to that of 1 with ADP and AMP which confirms the selective detection of ALP mediated cleavage of ATP with 1.

Fig. 14 Bright field and fluorescence microscopic images of HeLa cell: (a) bright field image of HeLa cells incubated with receptor 1 (10 mM) for 30 min and subsequently until 3 h; (b) fluorescence image of HeLa cells incubated with receptor 1 (10 mM) for 30 min and subsequently until 3 h; (c) fluorescence image of HeLa cells incubated with receptor 1 (10 mM) for 30 min and subsequently treated with 5 mM ATP for 3 h; (d) fluorescence image of HeLa cells incubated with receptor 1 (10 mM) for 30 min and subsequently treated with 5 mM ADP for 3 h; (e) fluorescence image of HeLa cells incubated with receptor 1 (10 mM) for 30 min and subsequently treated with 5 mM AMP for 3 h; (f) fluorescence image of HeLa cells incubated with receptor 1 (10 mM) for 30 min and after addition of ALP and ATP subsequently until 3 h.

Conclusions

In continuation of our recently reported dipodal 7-azaindole-1,2,3-triazole conjugate,⁴ the tripodal compound 1 in the present account shows a marked affinity for a variety of anions under different conditions. While the tripod 1 prefers to detect H₂PO₄⁻ in fluorescence via a sharp ratiometric fashion, Cl⁻ ions are sensed through chelation induced fluorescence enhancement in CH₃CN containing 0.01% DMSO. On the contrary, the tripod in a semi-aqueous environment [CH₃CN containing 0.01% DMSO : H₂O (1 : 1, v/v), pH = 7.3, 10 mM HEPES buffer] exhibits a significant preference for ATP over ADP, AMP and other inorganic phosphate salts. The dimension of the cleft and the hydrogen bonding features of 7-azaindole-1,2,3-triazole conjugate play a pivotal role in the recognition process as supported by theoretical observation also. Experimentation on the model structure 2 gave no meaningful result in emission and thus proved the role of the ring nitrogens at the 7-positions of azaindole motifs of 1 in the recognition process. The tripod 1 is cell permeable and can sense ATP inside the cell. ALP mediated cleavage of ATP can also be assessed by 1 in the cell line.

Experimental section

For the syntheses of compounds 1 and 2, the intermediates 7–8 were obtained according to our recently reported methods.⁴ The detailed procedures have been cited in the report.

2-((Trimethylsilyl)ethynyl)-1H-pyrrolo[2,3-b]pyridine (7a)

¹H NMR (400 MHz, CDCl₃) δ 10.14 (1H, s), 8.34 (1H, d, J = 4 Hz), 7.88 (1H, d, J = 8 Hz), 7.08 (1H, dd, J₁ = 8 Hz, J₂ = 4 Hz), 6.70 (1H, s), 0.28 (9H, s); ¹³C NMR (100 MHz, CDCl₃) δ 148.3, 143.2, 129.4, 120.6, 120.0, 116.2, 106.5, 99.1, 97.0, −0.09; mass (LCMS): 215.0 (M + 1)⁺.

2-Ethynyl-1H-pyrrolo[2, 3-b]pyridine (8a)

¹H NMR (400 MHz, CDCl₃) δ 10.2 (1H, s), 8.37 (1H, d, J = 4 Hz), 7.90 (1H, d, J = 8 Hz), 7.09 (1H, dd, J₁ = 8 Hz, J₂ = 4 Hz), 6.75 (1H, s), 3.38 (1H, s); FT-IR: ν cm⁻¹ (KBr): 3436, 3286, 2897, 2817, 2355, 1694, 1583; mass (LCMS): 144.2 (M + 2)⁺, 142.8 (M)⁺.

2-Iodo-7-azaindole (6a)

Compound 6a^9a,b was prepared according to the reported procedure. ¹H NMR (400 MHz, d₆-DMSO) δ 12.20 (1H, s), 8.13 (1H, d, J = 4 Hz), 7.85 (1H, d, J = 8 Hz), 7.01 (1H, dd, J₁ = 8 Hz, J₂ = 4 Hz), 6.69 (1H, s); mass (LCMS): 245.0 (M + 2)⁺.

2-((Trimethylsilyl)ethynyl)-1H-indole (7b)

¹H NMR (400 MHz, CDCl₃) δ 8.16 (1H, s), 7.56 (1H, d, J = 8 Hz), 7.28 (1H, d, J = 8 Hz), 7.21 (1H, t, J = 8 Hz), 7.10 (1H, t, J = 8 Hz), 6.76 (1H, s), 0.26 (9H, s); ¹³C NMR (100 MHz, CDCl₃) δ 135.9, 127.5, 123.7, 121.0, 120.5, 118.6, 110.7, 109.3, 98.5, 97.0, −0.09; mass (LCMS): 213.0 (M)⁺.

2-Ethynyl-1H-indole (8b)

¹H NMR (400 MHz, d₆-DMSO) δ 11.6 (1H, s), 7.51 (1H, d, J = 8 Hz), 7.31 (1H, d, J = 8 Hz), 7.15 (1H, t, J = 8 Hz), 7.02 (1H, t, J = 8 Hz), 6.73 (1H, s), 4.46 (1H, s); FT-IR: ν cm⁻¹ (KBr): 3387, 3268, 2923, 2857, 2365, 1607; mass (LCMS): 141.0 (M)⁺, 140.0 (M − 1)⁺.

2-Iodoindole (6b)

Compound 6b^9c was prepared according to the reported procedure. ¹H NMR (400 MHz, CDCl₃) δ 8.06 (1H, s), 7.52 (1H, d, J = 8 Hz), 7.32 (1H, d, J = 8 Hz), 7.14–7.05 (2H, m), 6.71 (1H, s); mass (LCMS): 243.0 (M)⁺.

1,3,5-Tris(azidomethyl)-2,4,6-trimethylbenzene (3)

A mixture of 1,3,5-tris(bromomethyl)-2,4,6-trimethylbenzene (1.5 g, 3.76 mmol) and NaN₃ (1.22 g, 18.80 mmol) in acetone–water (4 : 1 v/v, 30 mL) was refluxed for 3 h. Acetone was evaporated and the mass was extracted with ethyl acetate (20 mL × 3). Evaporation of ethyl acetate afforded compound 3 as a white solid (1 g, yield: 93%). ¹H NMR (400 MHz, CDCl₃) δ 7.40 (1H, t, J = 8 Hz), 7.29–7.27 (3H, m), 4.36 (4H, s).

Compound 1

To a solution of 3 (300 mg, 1.05 mmol) in ethanol–water (1 : 1 v/v, 15 mL), alkyne 8a (747 mg, 5.26 mmol) was added. Then a fresh solution of sodium ascorbate (126 mg, 0.6 mmol) and CuSO₄·5H₂O (78 mg, 0.3 mmol) in distilled water (3 mL) was added and the mixture was stirred under air for 16 h. After completion of the reaction, ethanol was evaporated; the brown solid was washed with distilled water and recrystallized with ethyl acetate to afford product 1 (600 mg, yield: 80%, decomposition temp. 270 °C).

¹H NMR (400 MHz, d₆-DMSO) δ 12.11 (3H, s), 8.36 (6H, brs), 7.91 (3H, d, J = 8 Hz), 7.07 (3H, brt), 6.84 (3H, s), 5.86 (6H, s), 2.56 (9H, s); ¹³C NMR (100 MHz, d₆-DMSO) δ 148.6, 142.4, 140.5, 140.1, 139.1, 131.3, 130.3, 128.2 (2C unresolved), 121.7, 97.4, 49.1, 16.9; HRMS (TOF MS ES⁺): calcd for C₃₉H₃₃N₁₅ (M + 1)⁺ 712.3043: found 712.3094; FT-IR: ν cm⁻¹ (KBr): 3270, 1612, 1489, 1453, 1320, 1220; anal. calcd for C₃₉H₃₃N₁₅: C, 65.81; H, 4.67; N, 29.52; found: C, 65.83; H, 4.70; N, 29.50%.

Compound 2

Following the same procedure as mentioned for compound 1, compound 2 was prepared. The amounts taken were: compound 3 (300 mg, 1.05 mmol), alkyne 8b (742 mg, 5.26 mmol), sodium ascorbate (126 mg, 0.6 mmol), CuSO₄·5H₂O (78 mg, 0.3 mmol). After completion of reaction, ethanol was evaporated, water was added, extracted with CH₂Cl₂, organic part was washed with brine solution, and dried over Na₂SO₄. The solvent was removed under vacuum and the product 2 (610 mg, yield: 81%, mp 165 °C) was isolated by column chromatography using 5% MeOH in CH₂Cl₂ as eluent.

¹H NMR (400 MHz, d₆-DMSO) δ 11.51 (3H, s), 8.29 (3H, s), 7.48 (3H, d, J = 8 Hz), 7.36 (3H, d, J = 8 Hz), 7.06 (3H, t, J = 8 Hz), 6.97 (3H, t, J = 8 Hz), 6.78 (3H, s), 5.84 (6H, s), 2.54 (9H, s); ¹³C NMR (100 MHz, d₆-DMSO) δ 141.1, 140.1, 136.9, 131.3, 129.6, 128.6, 121.9, 121.2, 120.4, 119.8, 111.8, 99.1, 49.1, 16.9; LCMS (EI⁺): 709.2 (M + H)⁺; FT-IR: ν cm⁻¹ (KBr): 3398, 3305, 1660, 1616, 1453, 1316; anal. calcd for C₄₂H₃₆N₁₂: C, 71.17; H, 5.12; N, 23.71; found: C, 71.21; H, 5.10; N, 23.68%.

Method for MTT assay

Reagents. MTT [3-(4,5-dimethyl-thiazol-2-yl)-2,S-diphenyltetrazolium bromide] and all other reagents were purchased from Sigma-Aldrich Inc. (St-Louis, MO, USA); Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, penicillin, streptomycin, neomycin (PSN) antibiotics were purchased from Gibco BRL (Grand Island, NY, USA).

Cell culture. Human cervical cancer cell line HeLa was procured from NCCS Pune, India. The cells were cultured at 5 × 10⁵ cells per mL in DMEM supplemented with 10% fetal bovine serum and 1% PSN antibiotic at 37 °C, 5% CO₂ for experimental purpose.

Assessment of percentage of viable cells. The percentage viability of HeLa cells, after being exposed to the receptors, was evaluated by MTT assay.¹⁵ The cells were incubated in 96-well microplates for 24 hours along with the receptors at different concentrations. A series of normal cells (without any exposure) and a series of positive control cells (exposed to similar concentrations of acetonitrile, the “vehicle solvent” of the receptors as that of the exposure of the receptors) were taken. The intracellular formazan crystals formed were solubilized with dimethyl sulfoxide (DMSO) and the absorbance of the solution was measured at 595 nm by using a microplate reader (Thermo scientific, Multiskan ELISA, USA). The calculated cell survival rate = percentage of MTT inhibition as follows: percentage of survival = (mean experimental absorbance/mean control absorbance) × 100%.

Acknowledgements

D.K. thanks CSIR, New Delhi, India for fellowship.

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Footnote

† Electronic supplementary information (ESI) available: Figures showing the fluorescence and UV-vis titrations of receptor 1 with various anions, absorption and emission spectra of 2, Job plot, binding curves, fluorescence titration of 1 in DMSO, ¹H and ³¹P NMR, other spectral data for characterization. See DOI: 10.1039/c3ra45018j

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