2-Bromo-3-((1-(7-chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)-methoxy)-benzaldehyde

Abstract

The 1,2,3-triazole ring system can be easily obtained by copper-catalyzed click reaction of azides with alkynes. 1,2,3-Triazole exhibits a myriad of biological activities, including antimalarial, antibacterial, and antiviral activities. We herein reported the synthesis of quinoline-based [1,2,3]-triazole hybrid via Cu(I)-catalyzed click reaction of 4-azido-7-chloroquinoline with alkyne derivative of 2-bromobenzaldehyde. The compound was fully characterized by proton nuclear magnetic resonance (¹H-NMR), carbon-13 nuclear magnetic resonance (¹³C-NMR), heteronuclear single quantum coherence (HSQC), ultraviolet (UV), and high-resolution mass spectroscopies (HRMS). This compound was screened in vitro against two different normal cell lines. Preliminary studies attempted to evaluate its interaction with Delta RBD of spike protein of SARS-CoV-2 by bio-layer interferometry. Finally, the drug-likeness of the compound was also investigated by predicting its pharmacokinetic properties.

1. Introduction

In December 2019, a novel pneumonia emerged in Wuhan, caused by a previously unknown pathogen. This pathogen was later identified as a novel coronavirus (initially named 2019-nCoV and now called SARS-CoV-2, which has a similar infection to severe acute respiratory syndrome CoV (SARS-CoV-1) discovered in 2003 [1].

In early in vitro studies, antimalarial chloroquine (Figure 1a) was found to counteract COVID-19 infection at a low-micromolar concentration, with a half-maximal effective concentration (EC₅₀) of 1.13 μM and a half-cytotoxic concentration (CC₅₀) greater than 100 μM [2,3].

The 7-chloroquinoline moiety, a pharmacophore of several established antimalarial drugs, such as chloroquine (Figure 1a) [1], has been recently recognized as a potential anti-cancer agent, as well as a chemosensitizer, when used in combination with anti-cancer drugs [4].

Intensive drug discovery efforts for developing new antimalarial/antiviral drugs or modifying existing agents are ongoing (Figure 1b) [5]. Molecular hybridization is a well-established strategy that combines two compounds or their moieties into a new, single hybrid molecule [6].

Considering the physicochemical and pharmacological features of known scaffolds, it is possible to expand drug libraries of homologous molecular hybrids through the fusion (usually via a covalent linker) of two drugs [7].

Therefore, a well-known scaffold, 1,2,3-triazole [8], appears frequently in medicinal compounds, and many covalently linked triazole hybrids exhibit diverse biological activities, such as antibacterial [9], antitubercular [10], anti-viral [5], antimalarial [11], and anti-inflammatory effects (Figure 1c–f).

Previously, we reported the synthesis of a novel 4-amino-7-chloroquinoline-based 1,2,3-triazole hybrid f by Cu(I)-catalyzed azide-alkyne cycloaddition (click chemistry) [12]. Recently, a library of 1,2,3-triazoles 4-aminoquinoline-benzoxaborole derivatives was synthesized [13], and our recent interest in boron derivatives [14] advanced further studies on similar derivatives.

In this study, analog compound 5 was synthesized and characterized using NMR, MS and UV spectra. The cytotoxicity of 5 was also evaluated against different cell lines and evaluated for interaction of the RBD spike protein of SARS-CoV-2.

2. Results and Discussion

The synthetic route of the triazole hybrid compound, 2-bromo-4-((1-(7-chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)-methoxy)-benzaldehyde 5, started from the preparation of precursors, 4-azido-7-chloroquinoline 2 and O-acetylenic derivative 4 (Scheme 1).

Similar to the protocol reported by de Souza et al. [15], quinoline 2 was prepared by the reaction of 4,7-dichloroquinoline 1 with NaN₃ (2 equiv.) in anhydrous DMF at 65 °C for 6 h (Scheme 1a). The crude product was purified by recrystallization from CH₂Cl₂/hexane to afford 2 in 78% yield.

The other cycloaddition partner, acetylenic intermediate 4, was generated by O-alkylation of 2-bromo-3-hydroxybenzaldehyde 3 with propargyl bromide (1.5 equiv.) and anhydrous K₂CO₃ in anhydrous DMF with a good yield (82%) after recrystallization from CH₂Cl₂/hexane (Scheme 1b).

Finally, the hybrid compound 5 was then furnished by using a modified protocol of click reaction reported by Fokin et al. [16]. Equimolars of quinoline 2 and acetylene 4 in tBuOH/water (1:1) were subjected to sequential additions of sodium ascorbate (0.4 equiv.) and CuSO₄ (20 mol%) (Scheme 1b). After stirring at 65 °C for 24 h, compound 5 was isolated by column chromatography in a high yield (77%).

The structure of 5 was determined by ¹H and ¹³C-NMR spectra (Supplementary Materials, Figures S3 and S4). The ¹H-NMR spectrum showed a singlet at 5.53 ppm corresponding to C13-methylene group and a singlet at 10.30 ppm corresponding to the C20-aldehydic proton.

For the ¹³C-NMR signals, the disappearance of the characteristic peaks of acetylenic group at 77.4 and 76.7 ppm, and the appearance of the C-13 methylene signal at 61.6 ppm and C-11 vinylic signal at 126.6 ppm were the proposed relevant characteristics to recognize the synthesis of a triazole moiety.

Heteronuclear single quantum coherence spectroscopy (HSQC) was also used to assign ¹³C signals of compound 5, as shown in Table S1 (see Supplementary Materials for 2D spectra, Figures S5 and S6).

The UV spectrum of compound 5 showed an absorption peak at 233 nm [11,12,13] and a broad absorption peak from 275 nm to 325 nm (n→π transition) (Supplementary Materials, Figure S7).

The HRMS of 5 was also obtained for further characterization, validating the proposed structure determined by NMR spectra (Supplementary Materials, Figure S8).

On the other hand, target compound 5 showed a low cytotoxicity by evaluating its in vitro cytotoxic activities against liver and lung normal cells (LO₂ and BEAS-2B) (IC₅₀ values > 100 and 55 μM, respectively, Supplementary Materials, Figure S9).

In addition, compound 5 was assessed by predicting its physicochemical properties and oral bioavailability. From the calculated physicochemical properties (Supplementary Materials, Table S2), compound 5 did not violate any Lipinski’s rules [17], indicating its drug-like character and a good chance for oral administration.

This finding corroborates the results of gastrointestinal absorption from SwissADME in which compound was predicted by BOILED-Egg model with high probability to passively permeate through the blood–brain barrier absorption [18] (Figure S10). Compound 5 was predicted to be orally bioavailable and absorbed in human intestine from admetSAR 2 [19].

The binding of compound 5 with Delta RBD of spike protein of SARS-CoV-2 was then tested in vitro by bio-layer interferometry (BLI), a label-free technology for measuring biomolecular interactions. This experiment is based on the immobilization of the target protein on the biosensor exposed to different ligand concentrations, thus monitoring the real-time association and dissociation events. BLI also provides the calculation of kinetic parameters, such as association rate (k_on) and dissociation rate (k_dis), as well as of dissociation constant (K_D). This technique was recently adopted to screen RBD ligands in combination with virtual screening [20]. As shown in Figure S11 of the Supplementary Materials, the results of BLI conducted on synthesized compound demonstrated a poor affinity with RBD spike of SARS-CoV-2 (delta variant), with a modest correlation value.

3. Materials and Methods

3.1. Chemistry

Silica gel (FCP 230–400 mesh) was used for column chromatography. Thin-layer chromatography was carried out on E. Merck precoated silica gel 60 F₂₅₄ plates and visualized with phosphomolybdic acid, iodine, or a UV-visible lamp.

All chemicals were purchased from Bide Pharmatech., Ltd. (Shanghai, China) and J & K scientific (Hong Kong, China). ¹H-NMR and ¹³C-NMR spectra were collected in CDCl₃ at 25 °C on a Bruker Ascend^®-600 NMR spectrometer (600 MHz for ¹H and 150 MHz for ¹³C). All chemical shifts were reported in the standard δ notation of parts per million using the peak of residual proton signals of CDCl₃ or DMSO-d₆ as an internal reference (CDCl₃, δ_C 77.2 ppm, δ_H 7.26 ppm; DMSO-d₆, δ_C 39.5 ppm, δ_H 2.50 ppm). High resolution mass spectra (HRMS) were measured using electrospray ionization (ESI). The measurements were done in a positive ion mode (interface capillary voltage 4500 V); the mass ratio was from m/z 50 to 3000 Da; external/internal calibration was done with electrospray calibration solution.

HRMS analyses were performed using an Agilent 6230 electrospray ionization (ESI) time-of-flight (TOF) mass spectrometer with Agilent C18 column (4.6 mm × 150 mm, 3.5 μm). The mobile phase was isocratic (water + 0.01% TFA; CH₃CN) at a flow rate of 0.35 mL/min. The peaks were determined at 254 nm under UV.

UV analysis was performed by a Shimadzu UV–2600 with 1 cm quartz cell and a slit width of 2.0 nm. The analysis was carried out using wavelength in the range of 200–400 nm.

3.1.1. Synthesis of 4-Azido-7-chloroquinoline (2)

4,7-Dichloroquinoline 1 (1.98 g, 10 mmol) was dissolved in 5 mL anhydrous DMF. NaN₃ (1.3 g, 20 mmol) was then added in one portion, and the resulting mixture was stirred at 65 °C for 6 h, whereupon TLC indicated the completion of the reaction. The reaction mixture was then allowed to cool to room temperature, after which it was diluted with 100 mL CH₂Cl₂, washed with water (3 × 30 mL), dried over anhydrous Na₂SO₄, and evaporated to dryness. The resulting residue was recrystallized from a 1:1 mixture of CH₂Cl₂/hexane to yield the final pure product 2 as a colorless, needle-like crystal in 78% yield.

R_f (CH₂Cl₂) 0.5; δ_H (600 MHz, CDCl₃) 8.82 (1H, d, J = 4.9 Hz, H-2), 8.09 (1H, d, J = 2.4 Hz, H-8), 8.01 (1H, d, J 9.3, H-5), 7.49 (1H, dd, J 2.4 and 9.3, H-6) 7.12 (1H, d, J 4.9, H-3) ppm; δ_C (150 MHz, CDCl₃) 150.9, 149.1, 146.8, 136.9, 127.9, 123.8, 119.9, 108.7 ppm. The spectral characteristics are consistent with those of 2 in the literature [20].

3.1.2. Synthesis of 2-Bromo-3-(prop-2-yn-1-yloxy) benzaldehyde (4)

2-Bromo-3-hydroxybenzaldehyde 3 (2.61 g, 13 mmol) was dissolved in 10 mL of anhydrous DMF. Anhydrous K₂CO₃ (2.7 g, 19.5 mmol) was then added to the solution, and the mixture was stirred at 30 °C for 30 min. Propargyl bromide (3-bromopropyne, 2.2 mL, 19.5 mmol) was then added slowly to the reaction mixture, and subsequently stirred at 30 °C for 6 h upon which TLC indicated completion of the reaction. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (3 × 50 mL). These extracts were then combined, washed with water (2 × 50 mL), dried over anhydrous Na₂SO₄ and evaporated in vacuo to yield the product residue that was then recrystallized from CH₂Cl₂/hexane 1:1 mixture to yield the compound 4 in 82% yield (2.54 g). Mp 90 °C; R_f (CH₂Cl₂) 0.9; δ_H (600 MHz, CDCl₃) 2.58 (H, s), 4.84 (2H, s, CH₂), 7.30 (H, dd, J = 1.33 and 8.24 Hz, H_Ar), 7.40 (1H, t, H_Ar), 7.58 (1H, dd, J = 1.4 and 7.9 Hz, H_Ar), 10.44 (CHO, s) ppm; δ_C (150 MHz, CDCl₃) 57.4, 76.7, 77.4, 117.8, 119.2, 122.5, 128.1, 135.0, 154.4, 192.4 ppm; HRMS-ESI m/z 236.9556 [M–H]⁻ (calcd. for C₁₀H₇BrO₂, m/z 236.9557)

3.1.3. Synthesis of 2-Bromo-3-((1-(7-chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)-methoxy)-benzaldehyde (5)

The O-acetylenic derivative 4 (239 mg, 1 mmol) and azide 2 (204 mg, 1 mmol) were dissolved in 5 mL tBuOH/water (1:1) and, while stirring at 65 °C, 1 M sodium ascorbate (0.4 mL, 0.4 mmol) and 1 M CuSO₄ (0.2 mL, 20 mol%) were added sequentially, in that order. The reaction mixture was then stirred at 65 °C for 24 h. The crude product was then precipitated out by slowly adding cold water to the reaction mixture, after which it was filtered, washed with water, air dried and purified by silica column chromatography (eluents: from EtOAc/Hex 3:7 to 5% MeOH in EtOAc) in yield 77% (341 mg). Mp 140 °C; R_f (CH₂Cl₂) 0.2; δ_H (600 MHz, DMSO-d₆) 5.53 (2H, s, CH₂-O), 7.49 (1H, dd, J = 1.3 and 7.6 Hz, H-17), 7.58 (1H, t, J = 9 Hz, H-18), 7.75 (1H, dd, J = 1.3 and 8.2 Hz, H-19), 7.81 (1H, dd, J = 2.3 and 9.1 Hz, H-6), 7.90 (1H, d, J = 4.6 Hz, H-3), 7.99 (1H, d, J = 9.1 Hz, H-5), 8.31 (1H, d, J = 2.1 H-8), 9.02 (1H, s, H-11), 9.18 (1H, d, J = 4.6 Hz, H-2), 10.30 (1H, s, CHO) ppm; δ_C (150 MHz, DMSO-d₆) 61.6 (C-13), 115.4 (C-15), 116.6 (C-3), 119.1, 119.7, 121.3, 124.7 (C-5), 126.6 (C-11), 127.5, 128.3 (C-6), 128.5, 133.9 (C-16), 134.8 (C-4), 139.7, 142.3, 148.8 (C-9), 151.7 (C-2), 154.2 (C-14), 191.3 (CO) ppm; HRMS-ESI m/z 442.9917 [M + H]⁺ (calcd. for C₁₉H₁₂BrClN₄O₂, m/z 442.9905); UV (CH₂Cl₂) peaks 221, 233, 284 and 312 nm.

3.2. Biological Studies

3.2.1. Cytotoxicity Drug Assay

Compound 5 was dissolved in DMSO at a final concentration of 100 mM and stored at −20 °C before use. Cytotoxicity was assessed by using the 3-(4,5-dimethylthiazole-2yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/mL) assay as previously described [21]. Briefly, 4 × 10³ cells per well were seeded in 96-well plates before drug treatments. After overnight cell culture, the cells were then exposed to different concentration of selected compounds (0.8–200 μM) for 48 h. Cells without drug treatment were used as control. Subsequently, 10 μL of 5 mg/mL MTT solution was added to each well and incubated at 37 °C for 4 h followed by addition of 100 μL solubilization buffer (10 mM HCl in solution of 10% of SDS) and overnight incubation. A₅₇₀ nm was then determined in each well on the next day. The percentage of cell viability was calculated using the following formula: Cell viability (%) = A_treated/A_control × 100. A representative graph of at least three independent experiments was shown in the Supplementary Materials, Figure S9.

3.2.2. Biolayer Interferometry Assay

Purified SARS-CoV-2 RBD peptide (Sino Biological, Beijing, China) were conjugated with biotin using EZ-Link^™ Sulfo-NHS-Biotin (Genemore, Suzhou, China) according to the manufacturer’s protocol. Then, the biotinylated SARS-CoV-2 RBD peptide were immobilized onto Super Streptavidin (SSA) biosensors (Fortebio, San Francisco, CA, USA). SARS-CoV-2 RBD peptide (Sino Biological, Beijing, China) was immobilized onto the biosensor coated with nickel-nitrilotriacetic acid (Ni-NTA, Fortebio, San Francisco United States). After the setup of the baseline with PBS containing 2% DMSO (Sigma-aldrich, St. Louis, MA, USA), biosensor tips were immersed into the wells containing serial dilutions of SARS-CoV-2 RBD peptide for 180 s of association, followed by a dissociation step of 180 s. The KD value was calculated by using a 1:1 binding model in Data Analysis Software 9.0 (Fortebio, San Francisco, CA, USA). Compounds were serially diluted from 200 μM to 6.25 μM with PBS (Figure S11 of the Supplementary Materials).

4. Conclusions

The synthesis of a triazole-based quinoline was presented. The synthesized compound was characterized by using NMR, mass and UV spectrometry. The cytotoxicity and drug-likeness of the compound were also determined by MTT assay and computations respectively. Finally, the compound was investigated for its interaction with Delta RBD of spike protein of SARS-CoV-2 by bio-layer interferometry.