Synthesis, Characterisation, Biological Evaluation and In Silico Studies of Quinoline-1,2, 3-Triazole-Anilines as Potential Antitubercular and Anti-HIV Agents

1. Introduction

Africa and other developing countries have borne a substantial burden of mortality attributed to infectious diseases [1,2]. One of the most concerning infectious diseases, Tuberculosis (TB), is caused by Mycobacterium tuberculosis (Mtb), a gram-negative bacterium with a lipid-rich outer membrane that makes it resilient against various antibiotics [3,4,5]. To treat drug-susceptible TB, combinations of isoniazid, rifampicin, pyrazinamide, and ethambutol, termed first-line drugs, are administered [6,7,8]. Each of these drugs targets specific areas in Mtb with the shared objective of inhibiting the production of essential biological processes critical for bacterial replication and survival [6]. Over the years, due to the gene mutations, Mtb has evolved to more resistant isoforms, such as multi-drug resistant (MDR-TB), extensively drug-resistant (XDR-TB) and totally drug-resistant TB (TDR-TB) [9,10].

On the other hand, the human immunodeficiency virus (HIV) remains one of the most prevalent infectious diseases [11]. To date, 39.9 million people are living with HIV/AIDS (PLWH) worldwide [12]. Effective drug development over the years has saved many lives, with the discovery of the Highly active antiretroviral therapy (HAART) being the pivotal moment. The use of antiretroviral drugs has decreased the morbidity and mortality rate of PLWH by turning HIV-1 into a chronic and manageable disease [13,14]. Despite HAART’s early success, drug-resistant mutant development rendered it less effective over time. HIV is a complex virus with a high mutation rate, considering it replicates in several replication stages. Antiretroviral drugs target different stages of the HIV-1 life cycle: viral attachment, reverse transcription, integration, proteolysis and viral budding [15]. There are seven classes of antiretrovirals based on their molecular mechanisms and resistance profiles, namely: nucleoside-analogue reverse transcriptase inhibitors (NNRTIs), non–nucleoside reverse transcriptase inhibitors (NNRTIs), integrase inhibitors, protease inhibitors (PIs), fusion inhibitors, post-attachment inhibitor (PAI) and co-receptor antagonists [16,17,18,19]. These drug regimens constantly evolve, and new drugs are continually being developed in each class.

Various quinoline- and/or 1,2,3-triazole-containing compounds have been reported to show promising activities against either Mtb [20,21,22,23,24,25,26,27,28] or HIV infections [29,30,31,32], with some already in clinical settings, including the 1,2,3 triazole-pyrimidine hybrids that are now part of the second generation NNRTI’s [30]. Bedaquiline (or TMC-207) (Figure 1), a quinoline-based compound, is clinically utilised for treating multidrug-resistant TB [33]. This compound binds to the subunit C of mycobacterial ATP synthase, an essential enzyme for Mtb energy production and survival. However, bedaquiline suffers from several side effects due to its potent inhibition of the potassium ether-ago-go-related gene (hERG) that could potentially lead to cardiac arrest [34]. Thus, new quinoline-based anti-Mtb agents, devoid of bedaquiline’s shortcomings, are critically important. Due to the broad spectrum of activities, various quinoline-triazole hybrids have shown promise over the years. Thomas et al. [21] reported on a series of new 6-methoxyquinoline triazole amides (1) (MIC = 0.625 µg/ml), sulphonamides (2) (MIC = 0.625 µg/ml), and amidopiperazines (3) (MIC = 0.625 µg/ml) that exhibited promising antitubercular activities. Our group recently disclosed a series of 7-chloroquinoline-triazole-benzimidazole hybrids that demonstrated excellent Mtb activity, with isomeric mixture 4 showing MIC₉₀ of 1.49 µM [22]. Previously, Costa et al. [29] reported several quinoline-1,2,3-triazole hybrids, such as 5 (IC₅₀ = 800 nm), that showed promising activity against HIV reverse transcriptase. Maraviroc, the first licensed CCR5 co-receptor antagonist containing the triazole moiety, is used to treat HIV infections and is less prone to drug resistance than the presently used ([N]NRTI) [35,36,37].

Simultaneously targeting TB and HIV is of paramount importance, especially considering the high prevalence of co-infection, drug resistance and the potential for drug-drug interactions in patients receiving treatment for both diseases [38]. Combining quinoline and triazole scaffolds into hybrid structures may synergistically improve efficacy against both Mtb and HIV [39,40]. The structural analysis of some of the compounds shown in Figure 1 reveals common structural features, shown in blue in Figure 2. Could these structural features be responsible for these compounds combined anti-Mtb and anti-HIV activities? Thus, in this study, we report on the synthesis, biological evaluation and computational studies of a series of compounds containing these structural features, albeit with the substitution of the quinoline moiety limited to the 7-chloro only.

2. Results and Discussion

2.1. Chemistry

The target compounds were synthesised in two stages, as shown in Scheme 1. The first stage is the formation of the two key intermediates, namely the 7-chloroquinoline-4-azide 7 and the alkyne 10a-j, via steps 1 and 2. These intermediates are then subjected to “Click chemistry” [27] in step 3 to yield the final quinoline-1,2,3, triazole-aniline derivatives, 11a-j, in 43 to 92% yields (Table 1). 1D and 2D nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy were used to confirm the structures, while mass spectrometry confirmed the masses of the desired products (see supplementary information).

Confirmation of the 1,2,3-triazole formation was achieved by observing the chemical shift of the singlet triazole methine proton, which appears in the aromatic region around δ_H 8.7 ppm. Secondly, the methylene doublet, due to coupling with the neighbouring NH, appears as expected at around 4.47 ppm, while the NH appears as a triplet at 6.21 ppm. All the quinoline protons are appearing as expected. The ¹³C NMR ATP NMR analysis also confirmed the formation of the hybrid, with the critical methine and methylene resonances appearing at 125.68 and 38.95 ppm, respectively.

The heteronuclear multiple bond correlations (HMBC) experiment (Figure 3) further validated the triazole ring formation, with close correlations of H6-C13, H1-C13, H1-C6 and H6-C14 being observed and shown in the structure of 11a. The mass spectrometry confirmed the masses of the target compounds based on their main or major fragment as observed in the base peak. All the other compounds were characterized in a similar manner.

2.2. In Vitro Biological Activities

The synthesised quinoline-1,2,3-triazole anilines were evaluated in vitro against Mtb H37Rv (ATCC 27294) strain, against HIV-1 subtype B, and their cytotoxicity assessed using MTT assay on TZM-bl cell-line (Table 2). The Mtb activity is represented as the 90% minimum inhibitory concentration (MIC₉₀), while HIV and cytotoxicity are represented as the 50% inhibitory concentration (IC₅₀) and 50% cytotoxicity concentration (CC₅₀), respectively. In terms of anti-Mtb, the data reveals that all the synthesised hybrid compounds did not show any appreciable activity, with compounds 11b, 11c, 11i and 11j exhibiting poor activity (MIC₉₀ > 1000 μM), while 11a, 11d-g, and 11i fell within the range of 100-200 μM. Notably, compound 11h exhibited the most potent activity with MIC₉₀ of 88μM, distinguishing it as the most active among all these, albeit 9-fold less active than the reference drug, ethambutol.

Against HIV-1, initially, all the compounds showed > 50% inhibition in primary inhibition assay were progressed to determine their IC₅₀, and azidothymidine (AZT) was used as a control drug. Generally, all the compounds inhibited the growth of the HIV-1 subtype B virus, ranging from 58-100% inhibition potential (see percentage cell viability plots in the supplementary information). The IC₅₀ values for most of these compounds were moderate, with only three compounds exhibiting sub-micromolar activity, namely 11g, 11h and 11i, albeit not as active as AZT. Compound 11g (consisting of the trifluoromethyl substituent on the phenyl ring) (IC50 = 0.3883 µM) and 11i (the 3-nitrosubstituted) (IC₅₀ = 0.170 µM) were 4- and 1.8-fold less active than AZT, respectively. On the other hand, 11h (the 3-flourosubstituted) (IC₅₀ = 0.01032 µM) is 8.8 times more potent than AZT. Interestingly, compound 11h was also the most active against Mtb, highlighting its dual active potential. Cytotoxicity assessment revealed that five of the synthesised compounds, 11a, 11c-e and 11g, exhibited CC₅₀ >100 µM, with 11g showing the best cytotoxicity profile with CC₅₀ = 4414 µM and selectivity index (SI) of > 11300, which is not far off from that of AZT (SI = 12349). On the other hand, the CC₅₀ value of compound 11h was 25.52 µM and its SI > 2400, an indication of the less likely to be cytotoxic in vivo [41,42]. Thus, compound 11h is a potential “hit” compound for further optimisation as a potential anti-HIV agent based on its activity and cytotoxicity profile.

2.3. In Silico Studies

2.3.1. Molecular Docking

To validate the observed biological activities, all hybrid compounds underwent in silico docking simulations into the active site of the Mtb ATP synthase enzyme (PDB ID: 4VIF) [43] and the antiviral enzyme (PDB ID: 4MBS) [44] using the Maestro software in Schrödinger Suite [45]. Docking scores recorded in Table 3 ranged from -2.879 to -2.035 kcal/mol for the antimicrobial target and -7.371 to -4.815 kcal/mol for the antiviral target. Compound 11d had the best docking score, followed by 11e and 11h against the antiviral enzyme, while 11e had the best docking score against the antimicrobial ATP synthase, followed by 11g and 11h. Notably, the docking scores were lower for the TB target than for the HIV target, consistent with the obtained biological data. The two promising compounds in the anti-TB and anti-HIV biological screenings, and their interaction with the active sites of the proteins were observed, as depicted in Figure 4.

The 4V1F enzyme is known to be the target for compounds containing the quinoline moiety, such as mefloquine and bedaquiline, and has been used extensively in molecular docking of quinoline-based compounds [22,43]. Compound 11h (MIC₉₀ = 88.72 µM) displayed a docking score of -2.606 compared to 11a (MIC₉₀ = 186.52 µM), which had a docking score of -2.540. In Figure 4, compound 11a demonstrated hydrogen bond interactions with Glu65, Tyr68, and Phe69 and additional interactions with Ala66, Gly62, Val61, and Phe58 amino acid residues. On the other hand, 11h exhibited similar interactions with amino acid residues but without any visible hydrogen interaction. Other biological properties of 11h may contribute further to its biological activity.

The selected CCR5 chemokine receptor acts as a co-receptor for HIV-1 viral entry and its associated enzyme (4MBS) is a known target for maraviroc, a triazole-containing antiretroviral [36]. Furthermore, Singh et al. [46] and Ibrahim et al. [47] reported quinoline-based compounds as chemokine receptor CCR5 inhibitors. Compound 11h (IC₅₀ = 0.01032 µM) demonstrated a significantly higher docking score of -7.362 than 11a (IC₅₀ = 3.013 µM). Hydrogen bonding interactions were observed with various amino acid residues in the active sites; the quinoline and aniline moieties interacted with Phe112, Tyr108, Glu283, and Trp86 for 11h and 11a. Further interactions with Tyr89 and Trp248 amino acid residues are observed for both compounds.

2.3.2. Density Functional Theory Studies

The stability of a compound is a consequence of its orbital energies [48]. Table 4 shows the stability and/or reactivity indices of 11h at DFT/B3LYP/6-311++G(d,p) level of theory. These reactivity indices are vital for molecular reactivity. The ionisation potential (I) and the electron affinity (A) of 11h were 5.89 eV and 2.64 eV, respectively. A low I value suggests that a molecule can give up electrons readily [49]. A high value of A implies a good electron-accepting potential for the molecules [49]. The energy gap (Eg) is derived as a difference between the frontier orbital energies. The Eg of 11h was 3.25 eV, while its chemical hardness was 1.63 eV. The global softness was 0.615 eV-1. The ability of a molecule to attract an electron is related to the electronegativity, χ. The χ of 11h was 4.27 eV, while its electrophilicity was 5.59 eV. The values of the reactivity descriptors in 11h here are close to reported bioactive compounds at the same level of theory [50]. Compound 11h can give up electrons easily and also accept electrons from the values of its ionisation potential and electron affinity.

HOMO, LUMO and ESP surface maps of 11h

The optimised structure, HOMO, LUMO and electrostatic potential maps of 11h are shown in Figure 5. The HOMO map of the compound was spread across the entire fluoroaniline moiety, while the LUMO was delocalised over the other side of the compound (quinoline and triazole rings). This indicates that the compound has the ability to act as an electron donor as well as an acceptor of electrons.

The electrostatic potential (ESP) map (Figure 6) accounts for the regions in a molecule prone to nucleophilic and electrophilic attacks. While the red- and yellow-mapped regions show negative electrostatic potential and are prone to attack by an electrophile, the blue- and/or green-mapped regions indicate positive electrostatic potential and are prone to nucleophilic attack.

The sites prone to electrophilic attack in 11h are the fluorine and chlorine atoms, as well as the quinoline nitrogen. The sites prone to nucleophilic attack are the triazole’s C19, C23, N21, N22 and H30 atoms, extending to N18 through C24 and their hydrogen atoms; some faint blue/green maps were seen on other parts of the quinoline rings. The charge separation between these two opposite electrostatic potential sites could facilitate intramolecular charge transfer [51].

2.3.3. ADMET Predictions

Adsorption, distribution, metabolism, excretion and toxicity (ADMET) properties describe a drug's absorption, distribution, metabolism, excretion and toxicity within living organisms [52]. ADMET predictions of the synthesised 1,2,3-triazole-quinoline-aniline compounds were calculated using the QikProp utility in the Schrödinger Suite [45]. The human serum binding ability coefficient (QPlogKhsa) ranged from 0.500 to 0.779, suggesting likely favourable bioavailability and less likely to be protein-bound. The predicted aqueous solubility (QPlogS) values for all but one compound, 11g (-6.979), were within the acceptable range, suggesting good intestinal absorption.

The predicted percentage of human oral absorption for most compounds was 100%, except for 11i, which has ~88%, indicating potential excellent oral bioavailability, with >80% being categorised as high. Furthermore, the predicted brain/blood coefficient (QPlogBB) values were also in the accepted range (-0.023 to -1.346), while these compounds were predicted to be likely inactive in the central nervous system (CNS) (< +2). The likely number of metabolic reactions from the cytochrome P450 enzyme was predicted to be less than 7, indicating a favourable outcome. Lastly, most of these compounds adhered to Lipinski's criteria for molecular weight, octanol-water coefficient, and the number of hydrogen bond donors and acceptors, except 11c and 11g, which violated this guideline due to their high lipophilicity (clogP) of greater than 5.

3. Materials and Methods

3.1. Chemistry

All the Chemical reagents used in the synthesis were purchased from Merck South Africa/Sigma Aldrich and I&A Chemicals, with purity ranging from 97-100%. HPLC grade and crude solvents were used. The reaction progress was monitored using a thin layer chromatography (TLC) analysis on aluminium-backed TLC plates (Kiese gel 60 F254 plates, Merck South Africa) and visualised under ultraviolet light (254 nm wavelength) All the synthesised final compounds and some intermediates were purified using the flash-column chromatography on silica gel (0.063-0.200mm) and various solvent systems. Melting point analysis was conducted using an electrothermal IA9100 melting point apparatus on the solid compounds using glass capillary tubes; melting points are recorded in $\mathrm{℃}$ and are uncorrected. Final compounds and intermediates’ functional groups were analysed and confirmed by Fourier Transform Infrared (FTIR) spectroscopy on the Perkin Elmer 100 spectrophotometer with Universal ATR sampling accessory; wavenumbers (ʋ) on the spectra expressed in cm-1.

Nuclear magnetic resonance (NMR) analysis was conducted on the Bruker Avance III 600 Hz spectrometer using deuterated chloroform (CDCl₃) and dimethylsulfoxide (DMSO-d6) and solvents. Topspin was used for spectra analysis; coupling constants (J) were reported in Hertz (Hz) and the chemical shifts in parts per million (ppm) using tetramethyl silane (TMS) peak as reference. The splitting patterns are reported as singlet (s), doublet (d), multiplet (m), triplet (t), quartet (q), doublet of doublets (dd), doublet of triplets (dt), or triplet of doublets (td). The solvent peaks were referenced at 2.50 (¹H) and δ 39.5 (¹³C) for DMSO-d6, and 7.26 (¹H) and 77.0 (¹³C) for CDCl₃, while residual water was observed at 3.35 and 1.56 ppm, respectively.

Preparation of N-[1-(7-chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl]anilines (11a-j)

100 mg (0,77 mmol) of the respective N-(prop-2-ny-l-yl)anilines (10a-j) and 189 mg (0.92mmol) 7-chloroquinoline-4-azide (7) were dissolved in 10mL DCM in a 100 mL round bottom flask. Thereafter, 22% sodium ascorbate, 10% copper sulphate and 10mL of water were added, and the reaction mixture was vigorously stirred at room temperature until completion (24 hrs). On completion, based on TLC, 100mL of water was added, followed by 5 x 40mL DCM extraction, and the combined extracts evaporated in vacuo to afford crude compounds, which were purified by column chromatography (DCM: MeOH; 95:5%):

1-(7-Chloro-4-quinolinyl)-1H-1,2,3,-triazole-4-methanamine (11a): As a cream white solid, yield 88%, mp 15-152℃, IR (cm^-1) C-H 2900-3000, ¹H-NMR (DMSO-d6, 600 MHz, ppm) δ_H 4.47 (2H, d, J = 5.7 Hz, H-1), 6.21 (1H, t, J = 5.7 Hz, H-2), 6.58 (1H, t, J = 7.5 Hz, H-5), 6.72 (2H, d, J = 7.9 Hz, H-3 and H-3’), 7.11 (2H, dd, J₁ = 8.3 Hz, J₂ = 7,4 Hz, H-4 and H-4’), 7.78 (1H, dd, J₁=7.0 Hz, J₂= 2 Hz, H-10), 7.8 (1H, d, J = 4.5 Hz, H-7), 8.02 (1H, d, J = 9.1 Hz, H-11), 8.28 (1H, d, J = 2.0 Hz, H-9), 8.74 (1H, s, H-6), 9.14 (1H, d, J = 4.5 Hz, H-8). ¹³C-NMR (DMSO-d6, 150 MHz, ppm) δ_C 39.9 (C-1), 112.9 (C-3), 116.7 (C-5), 117.3 (C-7), 120.7 (C-15a), 125.7 (C-11), 125.9 (C-6), 128.6 (C-9), 129.4 (C-4,10), 135.8 (C-16), 140.9 (C-14), 147.1 (C-13), 148.7 (C-12), 149.9 (C-15b), 152.8 (C-8). TOFF MS ES-: (m/z) 306.0968 (100%) [(M-H) - N₂]^- (Calculated for C₁₈H₁₃ClN₃^-(306.0803)].

[1-(7-Chloro-4-quinolinyl)-1H-1,2,3-triazole-4-methyl]-4-bromoaniline (11b): As a light brown solid, yield 87%, mp 187-190$\mathrm{℃}$, IR (cm^-1) C-H 2900-3000; ¹H-NMR (DMSO-d6, 600 MHz, ppm) δ_H 4.48 (2H, d, J = 5.2 Hz, H-1), 6.4 (1H, s, H-2), 6.71 (2H, d, J = 8.7 Hz, H-4), 7.25 (2H, d, J =8.7 Hz, H-3), 7.76 (1H, dd, J₁ = 9.0, J₂ = 1.5 Hz, H-10), 7.81 (1H, d, J = 4.6 Hz, H-7), 8.01 (1H, d, J = 9.0 Hz, H-11), 8.26 (1H, d, J = 1.5 Hz, H-9), 8.74 (1H, s, H-6), 9.14 (1H, d, J = 4.5 Hz, H-8). ¹³C-NMR (DMSO-d6, 150 MHz, ppm) δ_C 38.0 (C-1)114.0 (C-4), 131.0 (C-3), 117.3 (C-7), 120.7 (C-15a), 125.6 (C-6), 125.8 (C-11), 128.6 (C-9), 129.4 (C-10), 135.8 (C-16), 140.9 (C-14), 148.0 (C-12), 147.0 (C-13), 149.8 (C-15b), 152.0 (C-8), 107.0 (C-5). TOF MSMS ES-: (m/z) 451.4776 [(M^- + HCl]^- [Calculated for C₁₈H₁₉BrCl₂N₅ (451.1490)].

[1-(7-Chloro-4-quinolinyl)-1H-1,2,3-triazole-4-methyl]-4-iodoaniline (11c): As a brown solid, yield 92%, mp 195-199 $\mathrm{℃}$, IR (cm^-1) C-H 2900-; ¹H-NMR (DMSO-d6, 600 MHz, ppm) δ_H 4.45 (2H, d, J =5.5 Hz, H-1), 6.47 (1H, t, J = 5.5 Hz, H-2), 6.58 (2H, d, J = 8.7 Hz, H-4), 7.37 (2H, d, J = 8.7 Hz, H-3), 7.78 (1H, dd, J₁ = 9.0, J₂ = 1.9 Hz, H-10), 7.81 (1H, d, J = 4.5 Hz, H-7), 7.99 (1H, d, J = 8.8 Hz, H-11), 8.28 (1H, s, H-9), 8.72 (1H, s, H-6), 9.15 (1H, s, H-8). ¹³C-NMR (DMSO-d6, 150 MHz, ppm) δ_C 39.0 (C-1), 137.6 (C-3),117.3 (C-7), 77.4 (C-5), 120.7 (C-15a), 125.6 (C-6), 125.9 (C-11), 128.5 (C-9), 129.3 (C-10), 115.8 (C-4), 135.7 (C-16), 140.9 (C-14), 137.9 (C-12), 147.1 (C-13), 149.8 (C-15b), 152.8 (C-8). TOFF MS ES-: (m/z) [(M + Cl]^- 495.9821 (100%) (Calculated for C₁₈H₁₃Cl₂N₅^-(495.9593)].

[1-(7-Chloro-4-quinolinyl)-1H-1,2,3-triazole-4-methyl]-4-flouroaniline (11d): As a light grey solid, yield 79%, mp-145-148, IR (cm^-1) C-H 2900-3000, ¹H-NMR (DMSO-d6, 600 MHz, ppm) δ_H 4.44 (2H, d, J = 5.2 Hz, H-1), 6.15 (1H, t, J = 5.6 Hz, H-2), 6.71 (2H, dd, J1 = 9.1 Hz, J2 = 4.4 Hz, H-4), 6.96 (2H, t, J = 9.1 Hz, H-3), 7.79 (1H, dd, J₁ = 9.0 Hz, J₂ = 1.5 Hz, H-10), 7.82 (1H, d, J = 4.5 Hz, H-7), 8.01 (1H, d, J = 9.0, H-11), 8.29 (1H, d, J = 1.5 Hz, H-9), 8.73 (1H, s, H-6), 9.15 (1H, d, J = 4.5 Hz, H-8). ¹³C-NMR (DMSO-d6, 150 MHz, ppm) δ_C 39.0 (C-1), 113.7 (C-4), 115,8 (C-3), 117.3 (C-7), 120.7 (C-15a), 125.6 (C-6), 125.8 (C-11), 128.6 (C-9), 129.4 (C-10), 135.8 (C-16), 140.9 (C-14), 145.4 (C-12), 147.0 (C-13), 149.8 (C-15b), 152 (C-8), 154.3/155.8 (C-5). TOFF MS ES^-: (m/z) [(M-H) - N₂]- 324.0866 (100%) (Calculated for C₁₈H₁₂ClFN₃^-(324.0709)].

[1-(7-Chloro-4-quinolinyl)-1H-1,2,3-triazole-4-methyl]-3-chloroaniline (11e): As a yellow solid, yield 69%, mp 169-172 $\mathrm{℃}$, IR (cm^-1) C-H 2900-3000, ¹H-NMR (DMSO-d6, 600 MHz, ppm) δ_H 4.48 (2H, d, J = 5.6 Hz, H-1), 6.53 (1H, t, J=Hz, H-2), 6.66 (H, t, J = 8.0 Hz, H-5), 6.74 (1H, s, H-3’), 6.74 (1H, dd, J₁ = 3 Hz, J₂ = 2.4 Hz H-4’), 7.10 (1H, dd, J₁ = 9.2 Hz, J₂ =1.9 Hz, H-3), 7.12 (1H, s, H-3), 7.80-7.79 (2H, m, H-7 and H-10), 8.01 (1H, d, J = 9.0 Hz, H-11), 8.27 (1H, d, J = 1.6 Hz, H-9), 8.72 (1H, s, H-6), 9.13 (1H, d, J = 4.5 Hz, H-8). ¹³C-NMR (DMSO-d6, 150 MHz, ppm) δ_C 38.6 (C-1), 112.1 (C-3), 111.5(C-3’), 117,3 (C-7), 130.8 (C-5), 120.7 (C-15a), 125.7 (C-6), 125.8 (C-11), 128.6 (C-9), 129.3 (C-10), 124.7 (C-4’), 135.8 (C-16), 134.1 (C-4), 140.9 (C-14), 150.2 (C-12), 147.0 (C-13), 149.8 (C-15b), 152.8 (C-8). TOFF MS ES^-: (m/z) [(M-H) - N₂]^- 340.0580 (100%) (Calculated for C₁₈H₁₂Cl₂N₃^-(340.0414)].

[1-(7-Chloro-4-quinolinyl)-1H-1,2,3-triazole-4-methyl]-2-methoxyaniline (11f): As a brown liquid, yield 43%, IR (cm^-1) C-H 2900-3000 ¹H-NMR (DMSO-d6, 600 MHz, ppm) δ_H 3.79 (3H, s, H-3’’), 4.52 (2H, d, J = 6 Hz, H-1), 5.46 (1H, t, J = 6 Hz, H-2), 6.59 (1H, td, J₁ = 7.7 Hz, J₂ = 1.4 Hz, H-5), 6.72 (1H, dd, J₁ = 7.7 Hz, J₂ = 1.4, H-3’), 6.78 (1H, d, J = 8.0 Hz, H-4), 6.83 (1H, td, J₁ = 7.7 Hz, J₂ = 0.6 Hz, H-4’), 7.76 (1H, dd, J₁ = 9.1 Hz, J₂ = 2.1 Hz, H-10), 7.81 (1H, d, J = 4.6 Hz, H-7), 8.01 (1H, d, J = 9.1 Hz, H-11), 8.26 (1H, d, J = 2.0 Hz, H-9), 8.69 (1H, s, H-6), 9.12 (1H, d, J = 4.6 Hz, H-8). ¹³C-NMR (DMSO-d6, 150 MHz, ppm) δ_C 39.0 (C-1), 110.2 (C-3’), 138.0 (C-3), 117,3 (C-7), 116.7 (C-5), 120.7 (C-15a), 125.6 (C-6), 125.9 (C-11), 128.5 (C-9), 129.3 (C-10), 110.3 (C-4), 135.7 (C-16), 121.4 (C-4’), 140.9 (C-14), 137.9 (C-12), 147.1 (C-13), 149.8 (C-15b), 152.8 (C-8), 55.7 (C-3’’). TOF MSMS ES⁺: (m/z) 388.1096 (M+Na)⁺ [Calculated for C19H16ClN5NaO (388.0941)].

[1-(7-Chloro-4-quinolinyl)-1H-1,2,3-triazole-4-methyl]-2-triflouromethylaniline (11g): As a yellow liquid, yield 48%, IR (cm^-1) C-H 2900-3000 ¹H-NMR(DMSO-d6, 600 MHz, ppm) δ_H 4.65 (2H, d, J = 5.7 Hz, H-1), 6.11 (1H, s, H-2), 6.73 (1H, t, J = 7.5 Hz, H-5), 7.01 (1H, d, J = 8.4 Hz, H-3’), 7.43 (1H, m, H-4,4’), 7.76 (1H, dd, J₁ = 9.0 Hz, J₂ = 2.0 Hz, H-10), 7.80 (1H, d, J = 4.6 Hz, H-7), 7.96 (1H, d, J = 9.0 Hz, H-11), 8.25 (1H, d, J = 2.0, H-9), 8.65 (1H, s, H-6), 9.11 (1H, d, J = 4.6 Hz, H-8). ¹³C-NMR (DMSO-d6, 150 MHz, ppm) δ_C 39.0 (C-1), 145.4 (C-3), 112.9 (C-3’), 117.4 (C-7), 116.2 (C-5), 120.7 (C-15a), 126.1 (C-6), 125.8 (C-11), 128.5 (C-9), 129.3 (C-10), 126.7 (C-4’), 135.8 (C-16), 134.0 (C-4), 140.9 (C-14), 146.5 (C-12), 146.5 (C-13), 149.8 (C-15b), 152.8 (C-8),126 (C-3’’).

[1-(7-Chloro-4-quinolinyl)-1H-1,2,3-triazole-4-methyl]-3-flouroaniline (11h): As a crem white solid, yield 85%, mp 145-148 $\mathrm{℃}$, IR (cm^-1) C-H 2900 ¹H-NMR (DMSO-d6, 600 MHz, ppm) δH 4.47 (2H, d, J = 5.7 Hz, H-1), 5.40 (1H, s, H-2), 6.35 (1H, td, J₁ = 8.6, J₂= 2 Hz, H-4), 6.49 – 6.55 (3H, m, H-3, H-3’ and H5), 7.76 (1H, dd, J1 = 9.0 Hz, J2 = 1.9 Hz, H-10), 7.80 (1H, d, J = 4.7 Hz, H-7), 8.00 (1H, d, J = 9.2 Hz, H-11), 8.28 (1H, s, Hz, H-9), 8.72 1(H, s, H-6), 9.13 (1H, d, J = 4.6 Hz, H-8). ¹³C-NMR (DMSO-d6, 150 MHz, ppm) δ_C 38.7 (C-1), 99.2 (C-3), 109.2 (C-3’), 117.3 (C-7), 130.7 (C-5), 143.5 (C-4’) 120.7 (C-15a), 125.6 (C-6), 125.9 (C-11), 128.5 (C-9), 129.4 (C-10), 135.8 (C-16), 165.1(C-4), 140.9 (C-14), 153.5 (C-12), 146.6 (C-13), 149.8 (C-15b), 152.8 (C-8). TOFF MS ES^-: (m/z) [(M-H) - N₂]- 324.0868 (100%) (Calculated for C₁₈H₁₂ClFN₃^-(324.0709)].

[1-(7-Chloro-4-quinolinyl)-1H-1,2,3-triazole-4-methyl]-3-nitroaniline (11i): As a orange solid, yield 79%, mp 192-197 $\mathrm{℃}$, IR (cm^-1) C-H 2900-3000. ¹H-NMR(DMSO-d6, 600 MHz, ppm) δ_H 4.58 (2H, d, J = 5.7 Hz, H-1), 7.00 (H, s, H-2), 7.15 (1H, d, J = 7.74 Hz, H-3’), 7.37 (2H, m, H-4’ and H-5), 7.52 (1H, d, J = 7.7 Hz, H-3), 7.77 (1H, t, J = 2.0 Hz, H-10), 7.78 (1H, dd, J₁ = 8.7 Hz, J₂ = 2, H-7), 7.81 (H, dd, J₁ = 9.0 Hz, J₂ = 1.5 Hz, H-11), 8.29 (1H, d, J = 2 Hz, H-9), 8.77 (1H, s, H-6), 9.14 (1H, d, J = 4.5 Hz, H-8). ¹³C-NMR (DMSO-d6, 150 MHz, ppm) δ_C 39.1 (C-1), 130 (C-4’), 106 (C-3’), 117.4 (C-7), 119.1 (C-3) 120.8 (C-15a), 125.8 (C-6), 125.8 (C-11), 128.6 (C-9), 129.4 (C-10), 135.8 (C-16), 140.9 (C-14), 145.4 (C-12), 146.2 (C-13), 149.81 (C-4), 149.8 (C-15b), 152 (C-8), 111.0 (C-5). TOF MSMS ES⁺: (m/z) 381.2533 (M+1)^- [Calculated for C₁₈H₁₄ClN₆O₂ (381.7920)].

[1-(7-Chloro-4-quinolinyl)-1H-1,2,3-triazole-4-methyl]-4-methoxyaniline (11j): As a brown solid, yield 86%, mp-xx, IR (cm^-1) C-H 2900-3000. ¹H-NMR(DMSO-d6, 600 MHz, ppm) δ_H 3.64 (3H, s, H-5’), 4.42 (2H, d, J = 5.8 Hz, H-1), 5.76 (1H, t, J = 5.8 Hz, H-2), 6.68 (2H, d, J = 8.8 Hz, H-4), 6.74 (2H, d, J = 8.8 Hz, H-3), 7.75 (1H, dd, J₁ = 9.0 Hz, J₂ = 1.8 Hz, H-10), 7.79 (1H, d, J = 4.6 Hz, H-7), 8.01 (1H, d, J = 9.0 Hz, H-11), 8.25 (1H, d, J = 1.7 Hz, H-9), 8.68 (1H, s, H-6), 9.11 (1H, d, J = 4.6 Hz, H-8). ¹³C-NMR (DMSO-d6, 150 MHz, ppm) δ_C 40.4 (C-1), 115.07 (C-3), 117.2 (C-7), 151.6 (C-5), 55.7 (C-5’), 120.7 (C-15a), 125.6 (C-6), 125.9 (C-11), 128.5 (C-9), 129.3 (C-10), 135.7 (C-16), 114.1 (C-4), 140.9 (C-14), 142.9 (C-12), 147.1 (C-13), 149.8 (C-15b), 152.8 (C-8). TOFF MS ES^-: (m/z) [(M-H) - N₂]- 336.1097 (100%) [Calculated for C₁₉H₁₅ClN₃O^- (336.0909)].

3.2. Biology

3.2.1. Antimycobacterial Evaluation

In vitro antimycobacterial evaluation assay was performed against the H37Rv strain using the previously reported procedure [22]. Cultured H37Rv (ATCC 27294) in Middlebrook 7H9 (Difco) broth supplemented with 0.1 % glycerol (Merck) and 10 % oleic acid-albumin-dextrose-catalase (OADC) (Becton-Dickenson) were aerobically grown at 37 °C until an optical density (OD)600nm of 1 was attained. This was equivalent to approximately 3 x 108 bacilli/mL.

The antimicrobial activity of the various compounds was tested in triplicates using micro broth dilution assays in 96 well plates. These plates were sealed and incubated at 37 °C for 7 days and microbial growth was measured by observing the resazurin colour change from blue to pink. The minimum inhibitory concentration (MIC) was interpreted as the lowest concentration inhibiting a colour change from blue to pink.

3.2.2. MTT Cytotoxicity Evaluation

In vitro cytotoxicity evaluation assay was performed on the TZM-bl cell line, a HeLa cell line clone, as previously reported [22]. The cell were seeded at a density of 25000 (DEAE dextran 44 µl/10ml) cells + 150µl DMEM/well in a 96-well microtiter plate, in duplicates and incubated overnight for attachment (37℃, 5% CO₂). Treatments (2.5mg/ml) were prepared and following incubation, the supernatant (treatment medium) was removed, and 120 µl of MTT solution comprising 100µl fresh CCM and 20µl of MTT (5mg/ml MTT salt in 0.1M PBS) was added to each well. The plate was then incubated for 4 hours (37℃, 5% CO₂). The optical density of each sample was measured at 450 using a microplate reader (Perkin Elmer). The maximum inhibitory concentration resulting in 50% cytotoxicity concentration (CC₅₀) was obtained using GraphPad Prism version 5.01 by plotting a dose-response curve (concentration versus the percentage cell viability of the samples). (see cytotoxicity dose-response curved in the supplementary information).

3.2.3. Luciferase-Based Antiviral Assay Evaluating Human Immunodeficiency Virus

Maintenance of Cell Lines

In sterile 75 cm2 culture flasks, the TZM-bl cell lines (NIH AIDS Research and Reference Reagents Programme) were cultured as a monolayer using Dulbecco's Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; heat-inactivated and gamma irradiated) (LTC Biosciences, Gainesville, FL, USA), 25 mM of HEPES (Thermo Fisher Scientific, Waltham, MA, USA), and 50 μL/mL of gentamicin (Thermo Fisher Scientific, Waltham, MA, USA). A HeLa cell line clone, the TZM-bl cell line is altered to produce CD4 and CCR5, enabling HIV-1 infection and firefly luciferase regulated by the HIV-1 long-terminal repeat (LTR) [53].

Antiviral Assay

The HIV-1 inhibition of synthesised drugs was evaluated using a Luciferace-based antiviral assay [54]. Initially, 96-well cell culture plates were filled with 150 uL, 100 uL, and 140 uL of DMEM; test compound, viral control, and cell control were added, respectively. Briefly, 96-cell culture well plates (Corning Costar, New York, NY, USA) were filled with 11 uL of AZT drug (positive control) and test compounds. The plates were then diluted three times in 140 uL of DMEM supplemented with 10% FBS, 25% HEPES buffer, and 1% penicillin-streptomycin. 10,000 TZM-bl cell lines were infected with 50 uL NL4.3 virus (subtype B) in 96 well culture plates. Experimental controls included infected (virus control) and uninfected (cell control) TZM-bl cell line, which was incubated for an hour. After adding 10,000 cells to each 96-well plate, the cells were grown for 48 hours at 37 °C, 5% CO₂, 95% humidity, and 37.5 ug/mL of DEAE-dextran. 150 ul of medium was taken out and replaced with 100 uL of the Bright-Glo TM luciferase reagent without light exposure following a 48-hour incubation period. After aspirating the supernatant, 150 uL of the mixture containing the Bright-Glo TM luciferase reagent was put into a Corning Costar 96-well black plate. It was measured right away at 540 nm in the Victor Nivo microplate reader (PerkinElmer, Waltham, USA). Then, the percentage of viral inhibition was calculated as follows:

% HIV inhibition = (average sample–average control)/(1 − (average viral control–average control) × 100

(1)

The results of the absorbance-based quantification of the viral cell were through the inhibitory concentration at 50% was obtained by plotting the dose-response curve (log concentration versus % HIV inhibition) (see HIV assay dose-response curved in the supplementary information).

3.3. In Silico Studies

3.3.1. Molecular Docking

The compounds and proteins were prepared using the ligand preparation (LigPrep) and protein preparation wizard modules [55] (on Maestro software in Schrödinger Suite [45]). The compounds were docked at the active sites of the proteins.

3.3.2. DFT Studies

The structure of 11h was optimised at the DFT/B3LYP/6-311++G(d,p) level of theory in the gas phase. Frequency calculations of the optimised structure were performed to ensure that the geometry conforms to minima. The ionisation potential and electron affinity were calculated from the frontier molecular orbital energies (EHOMO) and (ELUMO), respectively [56]. Other reactivity indices such as the energy gap (Eg), chemical hardness and softness (η and S, respectively), electronegativity (χ) and electrophilicity (ω) were all calculated [57]; see equations 1-8. The distribution of the molecular orbitals over the molecular surface was visualised via the HOMO and LUMO maps [51]. An electrostatic potential (ESP) map was used to visualise the selective reactive sites of interaction of 11h with an electron donating or withdrawing neighbour [58].

$$I={-E}_{HOMO}$$

(2)

$$A={-E}_{LUMO}$$

(3)

$$E_g=E_{LUMO}-E_{HOMO}$$

(4)

$$\eta ={\displaystyle \frac{I-A}{2}}$$

(5)

$$S={\displaystyle \frac{1}{\eta }}$$

(6)

$$\chi ={\displaystyle \frac{I+A}{2}}$$

(7)

$$\mathsf{\omega }={\displaystyle \frac{{(I+A)}^2}{8\eta }}={\displaystyle \frac{{\chi }^2}{2\eta }}$$

(8)

4. Conclusions

Applying molecular hybridisation, new quinoline-1,2,3-triazole-anilines were successfully synthesised in moderate to excellent yields. Their structures were confirmed using spectroscopic and spectrometric techniques. The synthesised compounds demonstrated moderate to negligible activity against Mtb in vitro. However, notable anti-HIV activity was observed in compounds 11g, 11h and 11i, with their IC₅₀ being 0.3883 µM, 0.0103 µM and 0.167 µM, respectively, with 11h exhibiting the best activity against both Mtb and HIV. Furthermore, 11h showed 9-fold superior activity than the reference drug, AZT (0.0909 µM).

Additionally, the presence of fluoride in certain compounds appears to have improved antiviral activity. Cytotoxicity assessments generally revealed low toxicity except for a few compounds. Selective indices (SI) of 11g and 11h are 11367 and 2472.87, respectively, suggesting that these compounds would pose less cytotoxic effects in vivo. Molecular docking studies revealed that 11h interacted with some of the essential amino acid residues in the active site of the HIV-1 co-receptor entry enzyme. The DFT studies on 11h revealed reactivity and reactive sites in the compound, while the predicted ADMET parameters for most compounds indicated drug-like molecules. Thus, 11h is a potential hit for further optimisation studies against HIV-1.