Design and synthesis of 1,2,3-triazole–etodolac hybrids as potent anticancer molecules

Abstract

A series of novel 1,2,3-triazole–etodolac hybrids (6a–l) were designed and synthesized as potent anti-cancer molecules. The synthesis strongly relied on Huisgen's 1,3-dipolar cycloaddition between etodolac azide 3 and substituted terminal alkynes 5a–l. The use of CH₂Cl₂ as a co-solvent with H₂O increased the reaction rate and provided the corresponding 1,2,3-triazole–etodolac hybrids (6a–l) in excellent yields compared to other organic co-solvent systems. All the compounds were screened for their in vitro anticancer activity against human A549 cell lines and compounds 6e, 6f, 6h, 6j, and 6l were found to be the best anti-cancer molecules as compared to the marketed drug doxorubicin.

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Cancer is a multi-factorial and dreadful disease characterized by uncontrolled growth of cells and spread of abnormal cells in the body.¹ It is the most common disease in the modern era and causes the most deaths (one in eight deaths) worldwide. Because of enduring concern across the globe, there is an ever-increasing requirement for safe and effective anti-tumour agents to fight against cancer.^2a–h

1,2,3-Triazole is one of the extensively used scaffolds in pharmaceuticals and exhibits a wide range of biological activities including significant anti-cancer profiles against many human cancer cell lines.^{3,4a–c,5a–f} These heterocycles are bestowed with special properties like moderate dipole character, hydrogen bonding capability, rigidity, stability and can be constructed through click chemistry approach in a facile manner.⁶ 1,2,3-Triazoles conjugated with other pharmacophores (triazole-hybrids) known to possess potent anti-cancer activities. In this context, Kamal et al. described a new class of triazole chalcone-pyrrolo[1,4]benzodiazepine (PBD) hybrids as inhibitors of cyclin D1 and NF-kB protein (Fig. 1) which showed significant G1 cell cycle arrest.³

Fig. 1 Few hybrid triazoles.

Liu et al. identified promising candidates with a broad spectrum of anti-cancer activities and low cytotoxicity against the normal cell lines HEK-293 by the synthesis of novel 1,2,3-triazole–dithiocarbamate hybrids and some of them were more potent than the well-known anti-cancer drug 5-fluorouracil^4a–c (Fig. 2).

Fig. 2 Hybrid anti-cancer molecules with triazole skeleton.

On the other hand nonsteroidal anti-inflammatory drugs (NSAIDs) are the cyclooxygenase (COX) inhibitors,^7a–f and recent findings advocate that they possess anti-cancer effects through a COX-2-independent mechanism.^7d Etodolac is a COX-2 inhibitor with analgesic and antipyretic properties and is approved by US FDA.^7f In recent years the antitumor activity of etodolac analogues against many types of cancer, such as urogenital system cancers, Burkitt's lymphoma, multiple myeloma, chronic lymphocytic leukemia, and prostate cancer has been described.^7e In view of the pharmacological significance of etodolac and triazole derivatives with reference to anticancer activity, we have attempted the synthesis of a new series of 1,2,3-triazole–etodolac hybrids and evaluated their anti-cancer properties (Fig. 3).

Fig. 3 Synthetic strategy.

Results and discussions

Chemistry

The synthesis of a series of 1,2,3-triazole–etodolac hybrids (6a–l) carried out by using the Cu(I)-catalyzed Huisgen's 1,3-dipolar cycloaddition reaction is presented in Scheme 1.

Scheme 1 Proposed Cu(I)-catalyzed Huisgen's 1,3-dipolar cycloaddition reaction.

The synthetic strategy to achieve the novel hybrid template was initiated from the reduction of acid functionality of commercially available etodolac 1 by using LAH (lithium aluminum hydride) to give the primary alcohol 2 in 92% yield.⁸ The newly formed hydroxyl was transformed into azide 3 using DPPA (diphenylphosphoryl azide), TPP (triphenylphosphine) and DIAD (diisopropyl azodicarboxylate) in THF. The reaction mixture was stirred for 12 h at room temperature. The whole reaction mixture was concentrated to get the residue, which was extracted with EtOAc. The organic layer dried over anhydrous Na₂SO₄ filtered and evaporated. The crude reaction mixture was purified by silica gel column chromatography using EtOAc/hexane (10%) as an eluent to provide the corresponding azide 3 in 86% yield.⁹ Finally, the synthesis of triazole–etodolac hybrid 6a was attempted by treating the azide 3 with the terminal alkyne 5a in the presence of CuSO₄·5H₂O and L-sodium ascorbate in ^tBuOH : H₂O (1 : 1) at room temperature and the desired compound 6a was formed in trace amount. The prolonged reaction time and high temperature also did not improve the outcome of the reaction. Later the reaction was performed in various organic co-solvents to improve the reaction outcome¹⁴ and the synthesis of 6a was successfully accomplished in 1 : 1 DCM : H₂O solvent system¹⁰ with excellent yield (86%) and the results are summarized in Table 1 (Scheme 2).

Table 1 Identification of co-solvent for the system of 6a

Entry	Substrate	Product	Solvent	Co-solvent	Yield (%)	Time (h)
1	5a	6a	H2O	CH3CN	46	12
2	5a	6a	H2O	DMSO	43	9
3	5a	6a	H2O	THF	54	10
4	5a	6a	H2O	t-BuOH	42	8
5	5a	6a	H2O	DMF	58	12
6	5a	6a	H2O	DCM	86	8

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Scheme 2 Synthesis of 1,2,3-triazole–etodolac hybrids 6a–l.

After optimizing the reaction conditions, we have preceded to synthesis the library of triazole–etodolac hybrids (6a–l) to prove the generality of the adopted synthetic procedure. For the purpose, we have synthesized desired alkynes (5a–l) by treating the commercially available phenols (4a–l) with propargyl bromide and K₂CO₃ (Scheme 2) in good yields.¹¹ Finally, the Huisgen's 1,3-dipolar cycloaddition^5c,12 reaction was employed to react the azide 3 and the alkynes 5a–l to furnish the hybrid molecules 6a–l in excellent yields and the results were summarized in Table 2.

Table 2 Yields for the Cu-catalyzed synthesis of 1,2,3-triazole–etodolac derivatives (6a–l)

S. No.	1-Alkynes (5a–l)	Product (6a–l)	Time	Yield (%)
1			8 h	86
2			6 h	92
3			8 h	87
4			6 h	92
5			6 h	89
6			6 h	93
7			8 h	92
8			6 h	93
9			6 h	90
10			6 h	94
11			7 h	92
12			7 h	94

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Biology

Cytotoxicity was measured using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay, against A549 lung cancer cell lines which is a colorimetric assay.^13a MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) is a water-soluble yellow color tetrazolium salt and is converted to an insoluble purple formazan by cleavage of the tetrazolium ring by succinate dehydrogenase which is present in the mitochondria. The formazan product is impermeable to the cell membranes and therefore it accumulates in healthy cells. Since the reduction of MTT can only occur in metabolically active cells, the intensity of the purple color is a measure of the viability of the cells. The intensity can be measured by spectrophotometer after dissolving formazan crystal in DMSO.^13b

The cells (5 × 10³) were seeded in each well containing 0.1 mL of medium in 96 well plates. After overnight incubation at 37 °C in 5% CO₂, the cells were treated with 100 μL of different test concentrations of test compounds at identical conditions with three replicates each. The cytotoxic effect of all the synthesized molecules was evaluated using doxorubicin a widely used anthracycline antibiotic and with IC₅₀ values of 3.3 μM on A549 as the standard reference at 25 μM doses. The cell viability was assessed after 48 h, by adding 10 μL of MTT (5 mg mL⁻¹) per well. The plates were incubated at 37 °C for additional 2–4 hours. The medium was discarded and the formazan violet crystals, which were coloured for the viable cells, were dissolved in 100 μL of DMSO. The rate of color formation was measured at 570 nm in a spectrophotometer (Spectra MAX Plus; Molecular Devices; supported by SOFT max PRO-5.4). The percent inhibition of cell viability was determined with reference to the control values (without test compound). The data were normalised with the lowest and highest values and subjected to non-linear regression analysis to obtain log (concentration) vs. normalised expression (variable slope) using graph pad prism 6. The IC₅₀ (inhibition of cell growth) concentrations were further obtained using the graph pad prism 6.

It is apparent from the results that compounds 6e, 6f, 6h, 6j, 6l are found to be potent on lung cancer cell lines than the other (Fig. 4, Table 3)

Fig. 4 Log concentration–response curve of the most active compounds on A549 cancer cell lines.

Table 3 Determination of IC₅₀ of selected compounds against cancer cell lines^a

IC50^b (μM)
Compound	6e	6f	6h	6j	6l
a Standard drug used for in vitro activity studies is doxorubicin and its IC50 3.3 μM.b IC50 (μM) is 50% inhibitory concentration and values are the means of three experiments each done in duplicate.
A549	4.50 ± 0.5	3.65 ± 0.4	5.38 ± 0.4	10.71 ± 0.3	3.29 ± 0.7

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The test compounds with >70% inhibition on cancer cell lines were chosen for the IC₅₀ prediction on lung cancers. It was interesting to note that compounds 6e, 6f, 6h, 6j and 6l found to possess more effect at the same dose compared to doxorubicin against A549 (lung) cancer cell line. Next, dose–response studies of active compounds were performed to calculate IC₅₀ value against the same cell lines. Among the whole series of title compounds (6a–l), compound 6l was found to be the most active compound with an IC₅₀ value of 3.29 ± 0.7 μM on lung cancer cell line (A549). The second-best compound identified in the series was 6f with IC₅₀ 3.65 ± 0.4 value. The IC₅₀ values of active compounds are summarized in Table 2.

Similar scaffolds have been reported earlier for their potent anticancer activities.^14a–c This prompted us to investigate the possible mechanism of the synthesized compounds by performing the molecular docking studies into the binding pocket of the human topoisomerase-II ATPase co-crystallized with AMP–PNP complex (TPII) [PDB:1ZXM].¹⁵ The water molecules, substrate cofactors were removed from the PDB file and the missing hydrogen atoms were added to the system followed by energy minimization using OPLS 2005 force field with Polak–Ribiere Conjugate Gradient (PRCG) algorithm. The ligands were built using the builder feature in Maestro and each structure was prepared for docking using Ligprep 2.7 followed by molecular mechanics energy minimization using Macromodel 10.1. These geometrically optimized structures were used for Glide (grid-based ligand docking with energetics) docking using the “Extra Precision” (XP) mode of Glide 6.0.¹⁴ As depicted in (Fig. 5b), compound 6l showed hydrophobic interactions with active site residues Tyr 64, Ile 109, Pro 111, Val 236, Ala 237, Val 240, Pro 263, Leu 257 and interaction with other active site residues such as Asp 65, Glu 66, Asp 67, Lys 233, Lys 261 were also observed. Additionally, hydrogen bonding interactions with Tyr 244 and Asn 258 were noticed (Fig. 5c). These results also indicate that the compound 6l is bound well within the binding site pocket of doxorubicin and almost similar binding pattern was identified. The hydrogen bonding interaction with active site residues Tyr 244 and Asn 258 was in common (Fig. 5d).

Fig. 5 (a) Three-dimensional diagram displays the binding poses for compound 6l interactions at the active site of TP II. (b) Key residues within a 4.5 Å sphere of 6l in the binding pocket are shown. (c) Hydrogen bonding of 6l with Tyr 244 and Asn 258 in the active site. (d) Compound 6l overlaid with doxorubicin (orange). Yellow dotted lines represent H-bonding with amino acid backbone respectively.

Conclusion

A series of 1,2,3-triazole–etodolac hybrids (6a–l) have been designed and synthesized by employing Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction. The in vitro anti-cancer activity of all the synthesized compounds against human A549 cancer cell lines was evaluated and it is found that the compounds 6e, 6f, 6h, 6j, and 6l have shown promising results in comparison with the marketed drug doxorubicin. In the series, 6l has shown very good anticancer activity with an IC₅₀ value of 3.29 ± 0.7 μM and second-best compound was 6f with an IC₅₀ value of 3.65 ± 0.4 μM. Therefore, these compounds can serve as a promising lead candidate for further study.

Experimental

General information

The reagents and materials including etodolac were of the highest commercially available purity grade and were used without any further purification. Flash column chromatography was performed on Merck silica gel 60–120 mesh. Compounds were visualized with UV light. ¹H NMR spectra were recorded on Bruker Avance 400 MHz spectrometer and ¹³C NMR spectra were recorded on Bruker Avance 100 MHz spectrometer in CDCl₃ as a solvent, with TMS as an internal standard. Multiplicities were denoted by s (singlet), brs (broad singlet), d (doublet), t (triplet), q (quartet), dd (double doublet) and m (multiplet). IR spectra were recorded on Perkin Elmer spectrophotometer by using KBr pellets. Melting points were determined in open capillary tubes. Reactions were monitored by thin-layer chromatographic (TLC) technique using silica gel plates. Phosphate-buffered saline (PBS), antibiotic mixture, Roswell Park Memorial Institute (RPMI) 1640 medium, F-12K medium and new born calf serum (NBCS) were obtained from Hi Media Laboratories Pvt. Ltd. MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide) was procured from Sigma-Aldrich (Bangalore, India). Cell line that was used for testing in vitro cytotoxicity included A549 derived from human lung epithelial cells (ATCC No. CCL-185), which was procured from American Type Culture Collection, Manassas, VA, USA.

2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl)ethanol (2)

A stirred solution of LiAlH₄ (0.38 g, 9.93 mmol) in dry THF (35 mL), was added to a solution of 2-(1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl)acetic acid 1 (1.42 g, 4.96 mmol) in dry THF (14 mL) at 0 °C. The mixture was stirred overnight at room temperature. Then reaction mixture was quenched with saturated Na₂SO₄ solution at 0 °C, the mixture was filtered through Celite, and washed with ethyl acetate. The solvent was removed to yield pure product 2 (0.33 g, 92%).

1-(2-Azidoethyl)-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole (3)

To a stirred solution of 2-(1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl)ethanol 2 (0.5 g, 1.75 mmol) in THF (10 mL), were DPPA (diphenylphosphoryl azide) (3.50 mmol), DIAD (di isopropyl azodicarboxylate) (5.26 mmol) and triphenylphosphine (5.26 mmol) added at 0 °C. The reaction mixture was stirred overnight under nitrogen saturated atmosphere. After completion of reaction. The reaction mixture was poured in 100 mL water and thoroughly extracted with ethyl acetate (3 × 50 mL). The organic layer was dried over Na₂SO₄, filtered and evaporation under reduced pressure. The resulting residue was purified by flash chromatography using petroleum ether : ethyl acetate (90 : 10) as the eluent, to afford the 1-(2-azidoethyl)-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole 3 (0.45 g, 86%) as a white solid. Chemical formula: C₁₇H₂₂N₄O.

1,4-Dimethyl-2-(prop-2-yn-1-yloxy)benzene (5a)

A mixture of 2,5-dimethylphenol (0.5 g, 4.09 mmol), propargyl bromide (0.44 mL, 4.91 mmol) and K₂CO₃ (0.84 g, 6.13 mmol) was stirred in dimethylformamide (20 mL) at room temperature. After completion of the reaction (TLC monitoring), the reaction mixture was diluted with EtOAc (100 mL) and washed with water (3 × 50 mL) times. The organic phase was dried over with anhydrous Na₂SO₄ and concentrated to dryness in vacuo. The obtained crude crystalline was purified by column chromatography to obtain a pure white solid 5a.

1-(2-(4-((2,5-Dimethylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole (6a)

To a solution of 1,4-dimethyl-2-(prop-2-yn-1-yloxy)benzene (54 mg, 0.33 mmol) 5a and 1-(2-azidoethyl)-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole 3 (100 mg, 0.33 mmol) in CH₂Cl₂ (0.7 mL) and H₂O (0.7 mL) were added CuSO₄·5H₂O (0.08 mg, 0.0033 mmol) and sodium ascorbate (6.6 mg, 0.033 mmol). The resulting solution was stirred for 8 h at room temperature. The reaction mixture was diluted with CH₂Cl₂ (5 mL) and H₂O (5 mL). The organic layer was separated, dried over Na₂SO₄, and concentrated. The residue was purified by flash column chromatography (hexane/EtOAc, 2 : 1) to give 1,2,3-triazole 6a as a pale green solid. Yield 86%; mp 140 °C; chemical formula: C₂₈H₃₄N₄O₂; ¹H NMR (400 MHz, CDCl₃): δ 0.92 (t, J = 7.09, 3H), 1.25 (t, J = 7.3, 3H), 1.76–1.86 (m, 1H), 1.92–2.04 (m, 1H), 2.07 (s, 3H), 2.26 (s, 3H), 2.45–255 (m, 1H), 2.56–2.65 (m, 1H), 2.74–2.88 (m, 4H), 3.95–4.05 (m, 1H), 4.08–4.20 (m, 1H), 4.20–4.35 (m, 1H), 4.43–4.52 (m, 1H), 4.72 (d, J = 12.22 Hz, 1H), 4.88 (d, J = 11.98 Hz, 1H), 6.61–6.74 (m, 2H), 6.88–7.18 (m, 2H), 7.22–7.40 (m, 3H), 8.22 (s, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 7.85, 13.68, 15.83, 21.41, 21.96, 23.91, 31.53, 38.04, 46.17, 60.47, 61.82, 75.44, 108.88, 112.44, 115.89, 119.91, 120.57, 121.41, 123.74, 126.21, 126.66, 129.79, 130.46, 135.00, 135.30, 136.57, 144.35, 156.23; HRMS: m/z [M + H]⁺ calcd for C₂₈H₃₅N₄O₂ 459.2760, found: 459.2786; IR (KBr, cm⁻¹): 3243, 2958, 2914, 2870, 1714, 1593, 1451, 1259, 1128, 1051, 958, 810, 739.

1,8-Diethyl-1-(2-(4-((naphthalen-2-yloxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-1,3,4,9-tetrahydropyrano[3,4-b]indole (6b)

Pale brown solid; yield 92%; mp 190 °C; chemical formula: C₃₀H₃₂N₄O₂; ¹H NMR (400 MHz, CDCl₃): δ 0.96 (t, J = 7.09, 3H), 1.3 (t, J = 7.3, 3H), 1.82–1.89 (m, 1H), 1.9–2.1 (m, 1H), 2.45–2.68 (m, 2H), 2.75–3.0 (m, 4H), 3.9–4.15 (m, 2H), 4.3–4.45 (m, 2H), 4.85 (d, J = 12.22 Hz, 1H), 5.2 (d, J = 11.98 Hz, 1H), 6.84–7.15 (m, 1H), 7.18–7.29 (m, 2H), 7.3–7.6 (m, 6H), 7.75–7.95 (m, 2H), 8.2 (s, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 7.79, 13.64, 22.40, 23.85, 31.43, 37.96, 46.11, 60.38, 61.43, 75.34, 107.06, 108.81, 115.84, 118.72, 119.84, 120.46, 123.58, 123.78, 126.14, 126.37, 126.62, 126.81, 127.56, 129.05, 129.41, 129.75, 134.32, 134.95, 135.27, 156.01; HRMS: m/z [M + H]⁺ calcd for C₃₀H₃₃N₄O₂ 481.2603, found: 481.2604; IR (KBr, cm⁻¹): 3254, 2958, 2898, 2870, 1709, 1632, 1511, 1462, 1259, 1215, 1177, 1078, 1051, 832, 744.

1-(2-(4-((2,4-Dibromo-6-nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole (6c)

Brown colour solid; yield 87%; mp 141 °C; chemical formula: C₂₆H₂₇Br₂N₅O₄; ¹H NMR (400 MHz, CDCl₃): δ 0.95 (t, J = 7.09, 3H), 1.3 (t, J = 7.3, 3H), 1.75–1.9 (m, 1H), 1.95–2.15 (m, 1H), 2.45–2.68 (m, 2H), 2.75–2.85 (m, 3H), 2.87–3.02 (m, 1H), 3.95–4.05 (m, 1H), 3.95–4.15 (m, 2H), 4.25–4.35 (m, 1H), 4.44–4.55 (m, 1H), 4.85 (d, J = 12.22 Hz, 1H), 5.15 (d, J = 11.98 Hz, 1H), 7.08–7.15 (m, 2H), 7.25–7.4 (m, 2H), 7.75–7.95 (m, 2H); ¹³C NMR (100 MHz, CDCl₃): δ 7.88, 13.25, 21.96, 23.87, 31.55, 38.11, 46.25, 60.48, 67.72, 75.40, 108.99, 115.90, 117.26, 119.88, 120.52, 121.17, 124.80, 126.22, 126.60, 127.29, 134.98, 135.25, 140.21, 141.98, 145.61, 148.22; HRMS: m/z [M + H]⁺ calcd for C₂₆H₂₈Br₂N₅O₄ 632.0508, found: 632.0508; IR (KBr, cm⁻¹): 3249, 3073, 2964, 2920, 1692, 1528, 1445, 1352, 1254, 1139, 1045, 969, 870, 810, 744, 728.

1,8-Diethyl-1-(2-(4-((naphthalen-1-yloxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-1,3,4,9-tetrahydropyrano[3,4-b]indole (6d)

Pale yellow solid; yield 92%; mp 161 °C; chemical formula: C₃₀H₃₂N₄O₂; ¹H NMR (400 MHz, CDCl₃): δ 0.96 (t, J = 7.09, 3H), 1.3 (t, J = 7.3, 3H), 1.80–1.90 (m, 1H), 1.91–2.15 (m, 1H), 2.45–2.58 (m, 1H), 2.59–2.68 (m, 1H), 2.71–2.86 (m, 3H), 2.87–3.01 (m, 1H), 3.9–4.15 (m, 2H), 4.25–4.35 (m, 1H), 4.45–4.6 (m, 1H), 4.85 (d, J = 12.22 Hz, 1H), 5.2 (d, J = 11.98 Hz, 1H), 6.84–6.95 (m, 1H), 7.15–7.20 (m, 2H), 7.3–7.6 (m, 6H), 7.82 (m, 1H), 8.2 (s, 2H); ¹³C NMR (100 MHz, CDCl₃): δ 7.8, 13.62, 21.90, 23.85, 31.49, 37.99, 46.12, 60.40, 61.85, 70.02, 105.13, 108.87, 115.86, 119.89, 120.49, 120.68, 121.95, 123.47, 125.15, 125.54, 125.72, 126.16, 126.38, 126.57, 127.40, 134.45, 134.95, 135.24, 153.84; HRMS: m/z [M + H]⁺ calcd for C₃₀H₃₃N₄O₂ 481.2603, found: 481.2600; IR (KBr, cm⁻¹): 3276, 2953, 2898, 2870, 1741, 1681, 1577, 1456, 1259, 1095, 788, 771.

1-(2-(4-((4-Bromophenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole (6e)

Off white solid; yield 89%; mp 132 °C; chemical formula: C₂₆H₂₉BrN₄O₂; ¹H NMR (400 MHz, CDCl₃): δ 0.95 (t, J = 7.09, 3H), 1.3 (t, J = 7.3, 3H), 1.74–1.89 (m, 1H), 1.91–2.15 (m, 1H), 2.40–2.15 (m, 1H), 2.52–2.67 (m, 1H), 2.70–2.85 (m, 3H), 2.85–3.01 (m, 1H), 3.95–4.15 (m, 2H), 4.3–4.45 (m, 2H), 4.55 (d, J = 12.22 Hz, 1H), 4.85 (d, J = 11.98 Hz, 1H), 6.78 (m, 2H), 7.0–7.2 (m, 1H), 7.25–7.45 (m, 5H), 8.3 (s, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 7.89, 13.19, 20.92, 23.53, 31.42, 37.55, 46.26, 61.36, 62.67, 75.34, 108.95, 113.63, 116.65, 117.82, 120.02, 121.93, 123.20, 125.63, 126.67, 130.05, 133.13, 135.27, 135.83, 157.62; HRMS: m/z [M + H]⁺ calcd for C₂₆H₃₀BrN₄O₂ 509.1552, found: 509.1553; IR (KBr, cm⁻¹): 3276, 2953, 2931, 2892, 1709, 1593, 1489, 1456, 1237, 1193, 963, 826, 782, 689, 585 cm⁻¹.

1,8-Diethyl-1-(2-(4-(phenoxymethyl)-1H-1,2,3-triazol-1-yl)ethyl)-1,3,4,9-tetrahydropyrano[3,4-b]indole (6f)

Pale yellow solid. Yield 93%; mp 84 °C; chemical formula: C₂₆H₃₀N₄O₂, ¹H NMR (400 MHz, CDCl₃): δ 0.93 (t, J = 7.3, 3H), 1.36 (t, J = 7.3, 3H), 1.83–1.95 (m, 1H), 1.99–2.13 (m, 1H), 2.38–2.94 (m, 1H), 2.61–2.68 (m, 1H), 2.72–2.91 (m, 3H), 2.89–2.99 (m, 1H), 3.98–4.05 (m, 1H), 4.06–4.14 (m, 1H), 4.15–4.26 (m, 1H), 4.35–4.42 (m, 1H) 4.61 (d, J = 12.22 Hz, 1H), 4.93 (d, J = 11.98 Hz, 1H), 6.85–7.05 (m, 2H), 7.12–7.34 (m, 2H), 7.32–7.41 (m, 5H), 8.52 (s, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 7.83, 13.70, 22.50, 23.88, 29.73, 31.42, 37.87, 46.17, 60.47, 61.29, 75.41, 108.61, 114.70, 115.82, 119.81, 120.41, 121.60, 123.72, 126.18, 129.82, 135.06, 135.49, 143.62, 150.08, 158.16; HRMS: m/z [M + H]⁺ calcd for C₂₆H₃₁N₄O₂ 431.2447, found: 431.2446; IR (KBr, cm⁻¹): 3248, 2953, 2925, 1711, 1586, 1493, 1460, 1186, 968, 754, 694.

1,8-Diethyl-1-(2-(4-((m-tolyloxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-1,3,4,9-tetrahydropyrano[3,4-b]indole (6g)

Yellow solid; yield 92%; mp 129 °C; chemical formula: C₂₇H₃₂N₄O₂: ¹H NMR (400 MHz, CDCl₃): δ 0.96 (t, J = 7.08, 3H), 1.36 (t, J = 7.32, 3H), 1.81–1.90 (m, 1H), 1.97–2.07 (m, 1H), 2.18 (s, 3H), 2.46–2.52 (m, 1H), 2.62–2.69 (m, 1H), 2.75–2.82 (m, 3H), 2.89–2.96 (m, 1H), 3.98–4.04 (m, 1H), 4.09–4.12 (m, 1H), 4.22–4.29 (m, 1H), 4.39–4.46 (m, 1H), 4.79 (d, J = 12.22 Hz, 1H), 5.01 (d, J = 12.22 Hz, 1H), 6.80 (d, J = 8.07 Hz, 1H), 6.86–6.90 (m, 1H), 7.01–7.35 (m, 4H), 7.36–7.45 (m, 2H), 8.56 (s, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 7.73, 13.61, 16.14, 21.85, 23.82, 31.33, 37.93, 46.13, 60.38, 61.68, 75.40, 108.64, 111.25, 115.72, 119.74, 120.36, 120.76, 123.27, 126.12, 126.64, 126.69, 126.82, 130.65, 134.94, 135.31, 144.05, 156.24; HRMS: m/z [M + H]⁺ calcd for C₂₇H₃₃N₄O₂ 445.2603, found: 445.2603; IR (KBr, cm⁻¹): 3215, 2958, 2920, 2893, 2871, 1739, 1689, 1591, 1498, 1460, 1378, 1241, 1050, 858, 793, 742.

1,8-Diethyl-1-(2-(4-((o-tolyloxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-1,3,4,9-tetrahydropyrano[3,4-b]indole (6h)

Yellow solid; yield 93%; mp 130 °C; chemical formula: C₂₇H₃₂N₄O₂: ¹H NMR (400 MHz, CDCl₃): δ 0.95 (t, J = 7.08, 3H), 1.29 (t, J = 7.3, 3H), 1.79–1.88 (m, 1H), 1.95–2.03 (m, 1H), 2.18 (s, 3H), 2.44–2.52 (m, 1H), 2.60–2.68 (m, 1H), 2.74–2.82 (m, 3H), 2.88–2.96 (m, 1H), 3.97–4.03 (m, 1H), 4.08–4.12 (m, 1H), 4.21–4.28 (m, 1H), 4.38–4.45 (m, 1H), 4.75 (d, J = 12.22 Hz, 1H), 4.98 (d, J = 12.22 Hz, 1H), 6.81 (d, J = 7.82 Hz, 1H), 6.86–6.90 (m, 1H), 7.01–7.09 (m, 2H), 7.13 (d, J = 7.33 Hz, 2H), 7.26–7.37 (m, 2H), 8.42 (s, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 7.76, 13.60, 16.16, 21.87, 23.83, 31.39, 37.93, 46.12, 60.39, 61.70, 75.39, 108.71, 111.29, 115.76, 119.79, 120.40, 120.77, 123.28, 126.13, 126.62, 126.71, 126.84, 130.66, 134.95, 135.29, 144.09, 156.26; HRMS: m/z [M + H]⁺ calcd for C₂₇H₃₃N₄O₂ 445.2603, found: 445.2602; IR (KBr, cm⁻¹): 3248, 2964, 2925, 2871, 1744, 1684, 1591, 1493, 1454, 1372, 1236, 1055, 858, 787, 738.

1,8-Diethyl-1-(2-(4-((p-tolyloxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-1,3,4,9-tetrahydropyrano[3,4-b]indole (6i)

Pale yellow solid; yield 90%; mp 140 °C; chemical formula: C₂₇H₃₂N₄O₂; ¹H NMR (400 MHz, CDCl₃): δ 0.95 (t, J = 7.08, 3H), 1.35 (t, J = 7.3, 3H), 1.77–1.86 (m, 1H), 1.94–2.01 (m, 1H), 2.28 (s, 3H), 2.42–2.48 (m, 1H), 2.56–2.64 (m, 1H), 2.74–2.80 (m, 3H), 2.88–2.95 (m, 1H), 3.96–4.02 (m, 1H), 4.07–4.13 (m, 1H), 4.16–4.27 (m, 1H), 4.36–4.45 (m, 1H), 4.72 (d, J = 12.22 Hz, 1H), 4.91 (d, J = 12.22 Hz, 1H), 6.78 (d, J = 8.3 Hz, 2H), 7.01–7.10 (m, 3H), 7.26–7.33 (m, 2H), 7.35 (d, J = 7.82 Hz, 1H), 8.35 (s, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 7.75, 13.60, 20.39, 22.39, 23.80, 31.37, 37.89, 46.07, 60.37, 61.45, 75.34, 108.68, 114.48 (2C), 115.75, 119.77, 120.38, 126.64, 129.71, 129.83 (2C), 130.31, 134.94, 135.30, 143.75, 155.96; HRMS: m/z [M + H]⁺ calcd for C₂₇H₃₃N₄O₂ 445.2603, found: 445.2605; IR (KBr, cm⁻¹): 3254, 2953, 2909, 1722, 1596, 1503, 1312, 1225, 1055, 951, 825, 787, 749, 650, 508.

1,8-Diethyl-1-(2-(4-((4-iodophenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-1,3,4,9-tetrahydropyrano[3,4-b]indole (6j)

Yellow solid; yield 94%; mp 119 °C; chemical formula: C₂₆H₂₉IN₄O₂; ¹H NMR (400 MHz, CDCl₃): δ 0.95 (t, J = 7.09 Hz, 3H), 1.30 (t, J = 7.2 Hz, 3H), 1.76–1.85 (m, 1H), 1.93–2.01 (m, 1H), 2.43–2.50 (m, 1H), 2.55–2.63 (m, 1H), 2.72–2.79 (m, 3H), 2.87–2.94 (m, 1H), 3.94–4.00 (m, 1H), 4.07–4.11 (m, 1H), 4.20–4.27 (m, 1H), 4.37–4.44 (m, 1H), 4.64 (d, J = 12.22 Hz, 1H), 4.95 (d, J = 12.22 Hz, 1H), 6.64 (d, J = 8.8 Hz, 2H), 7.01 (d, J = 7.0 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 7.1 (s, 1H), 7.35 (d, J = 7.5 Hz, 1H), 7.5 (d, J = 8.80 Hz, 2H), 8.03 (s, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 7.78, 13.57, 21.89, 23.78, 31.49, 37.91, 46.03, 60.37, 61.37, 75.26, 108.83, 115.84, 117.07, 119.88, 120.47, 123.59, 126.09, 126.52, 134.90, 135.19, 138.17, 143.09, 157.94; HRMS: m/z [M + H]⁺ calcd for C₂₆H₃₀IN₄O₂ 547.1413, found: 547.1415; IR (KBr, cm⁻¹): 3271, 2958, 2926, 2893, 1736, 1687, 1583, 1523, 1484, 1369, 1249, 1227, 1106, 1046, 805, 745, 635, 580.

4-((1-(2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)-3-methoxybenzaldehyde (6k)

Pale yellow solid. Yield 92%; mp 193 °C; chemical formula: C₂₈H₃₂N₄O₄; ¹H NMR (400 MHz, CDCl₃): δ 0.96 (t, J = 7.07 Hz, 3H), 1.32 (t, J = 7.3 Hz, 3H), 1.77–1.82 (m, 1H), 1.92–1.99 (m, 1H), 2.44–2.58 (m, 2H), 2.75–2.80 (m, 3H), 2.86–2.96 (m, 1H), 3.88 (s, 3H), 3.42–4.05 (m, 1H), 4.06–4.18 (m, 1H), 4.21–4.33 (m, 1H), 4.37–4.48 (m, 1H), 4.75 (d, J = 12.42 Hz, 1H), 5.08 (d, J = 12.71 Hz, 1H), 7.01–7.12 (m, 3H), 7.20 (s, 1H), 7.34–7.47 (m, 3H), 7.55 (brs, 1H), 9.83 (s, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 7.83, 13.54, 22.39, 23.79, 31.66, 37.93, 46.00, 55.95, 60.36, 62.26, 75.17, 109.09, 109.13, 112.32, 115.99, 120.05, 120.60, 124.01, 126.12, 126.37, 126.68, 130.42, 134.90, 135.09, 142.69, 149.82, 153.01, 190.88; HRMS: m/z [M + H]⁺ calcd for C₂₈H₃₃N₄O₄ 489.2501, found: 489.2502; IR (KBr, cm⁻¹): 3271, 2958, 2909, 2904, 2854, 1687, 1583, 1512, 1463, 1265, 1172, 1128, 1013, 854, 810, 750.

4-((1-(2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)benzaldehyde (6l)

Brown solid; yield 94%; mp 123 °C; chemical formula: C₂₇H₃₀N₄O₃: ¹H NMR (400 MHz, CDCl₃): δ 0.95 (t, J = 7.06 Hz, 3H), 1.30 (t, J = 7.2 Hz, 3H), 1.78–1.90 (m, 1H), 1.95–2.09 (m, 1H), 2.44–2.55 (m, 1H), 2.56–2.67 (m, 1H), 2.70–2.85 (m, 3H), 2.86–3.01 (m, 1H), 3.92–4.04 (m, 1H), 4.05–4.19 (m, 1H), 4.20–4.34 (m, 1H), 4.37–4.49 (m, 1H), 4.70 (d, J = 12.22 Hz, 1H), 4.95 (d, J = 12.22 Hz, 1H), 6.95–7.09 (m, 4H), 7.16 (s, 1H), 7.36 (d, J = 7.33 Hz, 1H), 7.80 (d, J = 8.80 Hz, 2H), 8.24 (brs, 1H), 9.86 (s, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 7.78, 13.58, 22.39, 23.78, 31.44, 37.85, 46.06, 60.35, 61.47, 75.21, 108.80, 114.92 (2C), 115.86, 119.87, 120.47, 123.77, 126.09, 126.50, 130.12, 131.92 (2C), 134.90, 135.25, 142.50, 163.07, 190.82; HRMS: m/z [M + H]⁺ calcd for C₂₇H₃₁N₄O₃ 459.2396, found: 459.2395; IR (KBr, cm⁻¹): 3183, 2953, 2926, 2854, 1698, 1600, 1512, 1463, 1315, 1260, 1167, 1073, 1013, 865, 827, 750.

Acknowledgements

Bhaskar Kummari thankful to CSIR for awarding Senior Research Fellowship. Thankful to TEQIP II for laboratory development and PS3 LABORATORIES LLP for HPLC analysis.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra28525b

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