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Home > New indole and indazole derivatives as potential antimycobacterial agents
New indole and indazole derivatives as potential antimycobacterial agents
Received:21October2018/Accepted:18January2019/Publishedonline:8February2019 ©SpringerScience+BusinessMedia,LLC,partofSpringerNature2019
Introduction
Inspired by the antimycobacterial activity of the reported indole and indazole derivatives, we expand further this scaffold concept towards hydrazide–hydrazone derivatives. Structural modification efforts are directed to the identifi-cation of novel indole and indazole derivatives with hydrazide–hydrazone moiety as potent antimycobacterial agents.
The search for effective and nontoxic chemotherapeutic agents for tuberculosis (TB) treatment is a very important issue worldwide. It has been intensified by the striving to overcome the increasing problem in most of the countries (da Silva et al. 2017) related to the occurrence of MDR-TB (multidrug resistant TB) and associated with tuberculosis viral infections (human immunodeficiency virus (HIV) infection) which cause a number of inadequate effects of the first- and second-line anti-tuberculosis drugs. According to the World Health Organization (WHO), tuberculosis affects one-third of the world’s population, with 10.4 million new cases in 2016 (10% among HIV co-infected individuals), 1.67 million deaths and 490,000 MDR plus an additional 110,000 rifampicin-resistant cases (World Health Organization 2018). The urgent improvement of new potent antitubercular drugs has four promising targets: FASII enoylacyl carrier protein reductase (InhA), transmembrane transport protein large (MmpL3), decaprenylphospho-betadribofuranose 2-oxidase (DprE1), and ubiquinolcytochrome C reductase (QcrB) (Campaniço et al. 2018).
A new and more efficient approach in the fight against tuberculosis is the development of compounds targeting directly InhA which do not require activation by KatG. In this aspect, the hydrazone derivatives turned to be effective and less hepatotoxic agents (Elhakeem et al. 2015). Furthermore, hydrazone derivatives are present in many bioactive molecules and display a wide variety of biological activities, such as anticancer (Peng et al. 2018; Parlar et al. 2018; Rudavath et al. 2018) anti-inflammatories (Poma et al. 2012), antimicrobial (Peng et al. 2018; Popiołek an Biernasiuk 2017; Popiołek et al. 2018), anticonvulsant (Dehestani et al. 2018; Ragavendran et al. 2007), antiviral (Şenkardeş et al. 2016), and antiprotozoal (Inam et al. 2014). Actually, among all the biological properties of the hydrazide–hydrazones, their antimycobacterial activity is the most frequently discussed in the scientific literature (Velezheva et al. 2016; Coelho et al. 2012; Cihan-Üstündağ and Çapan 2012; John et al. 2016; Angelova et al. 2017b).
The indole ring system has been another scaffold among the most-studied pharmacophore groups in drug research studies (North and Jackson 2018). It is present in the structure of many pharmacologically active products that exhibit antimycobacterial activity (Ashok et al. 2018; Negatu et al. 2017; Nyantakyi et al. 2018; Shirinzadeh et al. 2011; Yang et al. 2017; Yurttaş et al. 2017). Also, some indazole derivatives have been identified as attractive new classes of drug candidates against Mycobacterium tuberculosis (Park et al. 2016, Malapati and Dharmarajan 2018, Vidyacharan et al. 2017, Faidallah et al. 2013).
Materials and methods
Chemistry
General
The melting points were determined using a Buchi 535 apparatus and Melting point meter M5000 apparatus. The Fourier-transform infrared spectroscopy (FTIR) spectra were recorded on a Nicolet IS10 FT-IR Spectrometer from Thermo Scientific (USA) using an attenuated total reflection (ATR) technique and FTIR spectrometer Bruker-Tenzor 27. All nuclear magnetic resonance (NMR) experiments were
carried out on a BrukerAvance spectrometer II+600 MHz at 20 °C in dimethyl sulfoxide (DMSO)-d6 as a solvent, using tetramethylsilane as an internal standard. The precise assignment of the 1 H and 13C NMR spectra was accomplished by measurement of two-dimensional (2D) homonuclear correlation (correlation spectroscopy (COSY)), DEPT-135, and 2D inverse detected heteronuclear (C–H) correlations (heteronuclear single-quantum correlation spectroscopy (HMQC) and heteronuclear multiple-bond correlation spectroscopy (HMBC)). Mass spectra were measured on a Q Exactive Plus mass spectrometer (ThermoFisher Scientific) equipped with a heated electrospray ionization (HESI-II) probe (Thermo Scientific). All chemicals as well as compounds 1 and 2a–k used for the synthesis were commercial products and used without further purification. The purity of the new compounds was checked by thin-layer chromatography (TLC) on silica gel 60 GF254 Merck pre-coated aluminum sheets, eluted by hexane–chloroform–acetone–methanol 4:3:2:1 (vol. parts); the spots were visualized under ultraviolet irradiation (λ = 254 nm). General procedure for the synthesis of compounds 3a–k, 5l–m To a solution of appropriate hydrazides 2a–m (2.0 mmol) in absolute (abs.) ethanol a stirred solution of 5-metoxyindole-3-carbaldehyde (2.0 mmol) 1a, 5-bromoindole-3-carbaldehyde (2.0 mmol) 1b, 5-benzyloxyindole-3-carbaldehyde (2.0 mmol) 1c, or 5-metoxyindazole-3-carbaldehyde (2.0 mmol) 4 in abs. ethanol was added. The solution was refluxed for 2–3 h. The solid product formed was collected by filtration and recrystallized with ethanol.
Synthesis of indol and indazole containing aroylhydrazones
N’-[(E)-(5-methoxy-1H-indol-3-yl)methylidene]furan-2- carbohydrazide, 3a Yield: 81%; m.p. 222–224 °C. FTIR(ATR) νmax: 3266 NH, 3211 NH, 1641(cis), 1630(trans) C = O, 1607 C = N, 1572 C = C cm−1 ; 1 H NMR (DMSO-d6, 600 MHz): 1:0.23 mixture of conformers; signals for major synperiplanar conformer around the amide bond: δ 3.787 (s, 3H, OCH3), 6.685 (dd, J = 1.6, 3.3 Hz, 1H, H-4’), 6.853 (dd, J = 2.5,
8.8 Hz, 3H, H-6), 7.232 (d, J = 3.3 Hz, 1H, H-5’), 7.335 (d, J = 8.7 Hz, 1H, H-7), 7.762 (d, J = 2.7 Hz, 1H, H-2), 7.819 (d, J = 2.3 Hz, 1H, H-4), 7.913 (brs, 1H, H-3’), 8.592 (s, 1H, H-8), 11.469 (brs, 1H, CONH), 11.513 (brs, 1H, NH) ppm. 13C NMR (DMSO-d6, 150 MHz): 55.75 (OCH3), 104.67 (C-4), 111.88 (C-3), 112.41 (C-4’), 112.66 (C-6), 112.89 (C-7), 114.44 (C-5’), 125.42 (C-3a), 131.14 (C-2), 132.46 (C-7a), 145.62 (C-8), 145.77 (C-3’), 147.72 (C-1’), 154.20 (C=O), 154.87 (C-5) ppm. High-resolution mass spectrometry (HRMS) (electrospray ionization (ESI)) m/z: calcd: [M + H]+ 284.10244. Found: [M + H]+ 284.102968.
N’-[(E)-(5-methoxy-1H-indol-3-yl)methylidene]pyridine-3-carbohydrazide, 3b
Yield: 76%; m.p. 232–233 °C FTIR(ATR) νmax: 3350 NH, 3270 NH, 1637 C = O, 1610 C = N, 1602 C = C; 1 H NMR (DMSO-d6, 600 MHz): 1:0.19 mixture of conformers; signals for major synperiplanar conformer around the amide bond: δ 3.797 (s, 3H, OCH3), 6.860 (dd, J = 2.6, 8.8 Hz, 1H, H-6), 7.346 (d, J = 8.8 Hz, 1H, H-7), 7.565 (ddd, J = 0.8, 4.8, 7.9 Hz, 1H, H-5’), 7.799 (d, J = 2.8 Hz, 1H, H-2), 7.845 (d, J = 2.5 Hz, 1H, H-4), 8.258 (ddd, J = 1.8, 2.2, 7.9 Hz, 1H, H-6’), 8.584 (s, 1H, H-8), 8.745 (dd, J = 1.6, 4.8 Hz, 1H, H-4’), 9.070 (1H, dd, J = 0.8, 2.3 Hz, 1H, H-2’), 11.500 (brd, J = 1.9 Hz, 1H, CONH), 11.692 (brs,
1H, NH) ppm. 13C NMR (DMSO-d6, 150 MHz): δ 55.38 (OCH3), 104.19 (C-4), 111.36 (C-3), 112.40 (C-6), 112.57 (C-7), 123.66 (C-5’), 125.02 (C-3a), 129.82 (C-1’), 131.12 (C-2), 132.09 (C-7a), 135.35 (C-6’), 145.80 (C-8), 148.54 (C-2’), 151.97 (C-4’), 154.56 (C-5), 161.00 (C=O) ppm. HRMS (ESI) m/z: calcd: [M + H]+ 312.114281. Found: [M + H]+ 312.11343.
4-(Dimethylamino)-N’-[(E)-(5-methoxy-1H-indol-3-yl) methylidene]benzohydrazide, 3c
Yield: 77%; m.p. 160–161 °C. FTIR(ATR) νmax: 3314 NH, 3215 NH, 1623 C=O, 1599 C=N; 1583 C=C cm−1; 1 H NMR (DMSO-d6, 600 MHz): 1:0.19 mixture of conformers; signals for major synperiplanar conformer around the amide bond: δ 2.991 (s, 6H, NCH3), 3.794 (s, 3H, OCH3), 6.756 (d, J = 8.8 Hz, 2H, H-3’ and H-5’), 6.844 (dd, J =2.3, 8.7 Hz, 1H, H-6), 7.326 (d, J = 8.7 Hz, 1H, H-7), 7.723 (d, J = 2.6 Hz, 1H, H-2), 7.820 (d, J = 8.8 Hz, 2H, H-2’ and H-6’), 7.856 (d, J = 1.9 Hz, 1H, H-4), 8.574 (s, 1H, H-8), 11.218 (s, 1H, CONH), 11.401 (s, 1H, NH) ppm. 13C NMR (DMSO-d6, 150 MHz): δ 39.77 (NCH3), 55.34 (OCH3), 104.21 (C-4), 110.92 (C-3’ and C-5’), 111.77 (C-3), 112.22 (C-6), 112.37 (C-7), 120.32 (C-1’), 125.01 (C-3a), 128.83 (C-2’ and C-6’), 130.06 (C-2), 132.02 (C-7a), 143.68 (C-8), 152.24 (C-4’), 154.34 (C-5), 162.30 (C=O) ppm. HRMS (ESI) m/z: calcd: [M+H]+ 337.165902. Found: [M+H]+337.16517.
4-Methoxy-N’-[(E)-(5-methoxy-1H-indol-3-yl)methylidene] benzohydrazide, 3d
Yield: 86%; m.p. 259.1 °C. FTIR(ATR) νmax: 3370 NH, 3256 NH, 1635 C=O, 1608 C=N, 1574 C=C cm−1; 1 H NMR (DMSO-d6, 600 MHz): 1:0.15 mixture of conformers; signals for major synperiplanar conformer around the amide bond: δ 3.796 (s, 3H, OCH3), 3.833 (s, 3H, PhOCH3), 6.852 (dd, J = 2.5, 8.8 Hz, 1H, H-6), 7.057 (d, J = 8.8 Hz, 2H, H-3’ and H-5’), 7.334 (d, J = 8.7 Hz, 1H, H-7), 7.757 (d, J = 2.3 Hz, 1H, H-2), 7.854 (d, J = 2.3 Hz, 1H, H-4), 7.918 (d, J = 8.7 Hz, 2H, H-2’ and H-6’), 8.587 (s, 1H, H-8), 11.394 (brs, 1H, CONH), 11.442 (brs, 1H, NH) ppm. 13C NMR (DMSO-d6, 150 MHz): δ 55.33 (OCH3), 55.41 (PhOCH3), 104.20 (C-4), 111.58 (C-3), 112.24 (C-6), 112.41 (C-7), 113.65 (C-3’ and C-5’), 124.99 (C-3a), 126.14 (C-1’), 129.29 (C-2’ and C-6’), 130.45 (C-2), 132.03 (C-7a), 144.55 (C-8), 154.39 (C-5), 161.68 (C-4’), 161.89 (C=O). HRMS (ESI) m/z: calcd: [M+H]+ 324.134268. Found: [M+H]+: 324.13366.
N’-[(E)-(5-methoxy-1H-indol-3-yl)methylidene]-4-methyl- 1,2,3-thiadiazole-5-carbohydrazide, 3e
Yield: 92%; m.p. 260.2 °C. FTIR(ATR) νmax: 3234 NH, 3137 NH, 1658, 1627 C=O, 1603 C=N 1585C=C cm−1; 1 H NMR (DMSO-d6, 600 MHz): 1:0.07 mixture of conformers; signals for major synperiplanar conformer around the amide bond: δ 2.970 (s, 3H, CH3), 3.828 (s, 3H, OCH3), 6.876 (dd, J = 2.5, 8.8 Hz, 1H, H-6), 7.381 (d, J = 8.8 Hz, 1H, H-7), 7.697 (d, J = 2.3 Hz, 1H, H-4), 7.907 (d, J = 2.9 Hz,1H, H-2), 8.382 (s, 1H, H-8), 11.706 (brs, 1H, NH), 12.022 (brs, 1H, CONH) ppm. 13C NMR (DMSO-d6, 150 MHz): 15.26 (CH3), 55.83 (OCH3), 102.67 (C-4), 110.92 (C-3), 113.53 (C-6 and C-7), 124.44 (C-3a), 132.44 (C-7a), 133.30 (C-2), 136.91 (C-1’), 144.16 (C-8), 155.20 (C-5), 159.42 (C=O), 163.05 (C-5’) ppm. HRMS (ESI) m/z: calcd: [M+H]+ 316.08565. Found: [M+H]+ 316.086271.
1-Benzyl-N’-[(E)-(5-methoxy-1H-indol-3-yl)methylidene] pyrrolidine-3-carbohydrazide, 3f
Yield: 78%.; m.p. 136–137 °C. FTIR(ATR) νmax: 3482 NH, 3184 NH, 1647 C=O, 1622 C=N, 1582C=C; 1 H NMR (600 MHz, DMSO-d6) 1:0.40 mixture of conformers; signals for major synperiplanar conformer around the amide bond: δ 2.090–2.145 (m, 1H, b-CH2-4-H-pyrrolidine), 2.412–2.462 (m, 1H, a-CH2-5-H-pyrrolidine), 2.525 (dd, J = 7.5, 8.9 Hz, 1H, a-CH2-2-H-pyrrolidine), 2.613–2.656 (m, 1H, b-CH2-5-H-pyrrolidine), 3.037 (t, J = 8.8 Hz, 1H, b-CH2-2-H-pyrrolidine), 3.546 (d, J = 13.1 Hz, 1H a-CH2-benzyl), 3.602 (d, J = 13.1 Hz, 1H, b-CH2-benzyl), 3.764 (s, 3H, OCH3), 3.775–3.829 (m, 1H, CH-3-pyrrolidine),
6.818 (dd, J = 2.5, 8.8 Hz, 1H, H-6-indole), 7.206 (m, 1H, p-phenyl), 7.282–7.322 (m, 4H, m,o-phenyl), 7.311 (d, J =8.8 Hz, 1H, H-7-indole), 7.625 (d, J = 2.5 Hz, 1H, H-4-indole), 7.677 (d, J = 2.7 Hz, 1H, H-2-indole), 8.122 (s, 1H, CH=N), 10.946 (brs, 1H, NHCO), 11.372 (brs, 1H, NH); 13C NMR (151 MHz, DMSO-d6) signals for major synperiplanar conformer around the amide bond: δ 26.61 (C-4-pyrrolidine), 39.52 (C-3-pyrrolidine), 53.77 (C-5-pyrrolidine), 54.93 (CH3O), 56.65 (C-2-pyrrolidine), 59.43 (CH2-benzyl), 102.76 (C-4-indole), 111.32 (C-3-indole), 112.58 (C-6-indole), 112.59 (C-7-indole), 124.51 (C-3a-indole), 126.78 (o-phenyl), 128.15 (p-phenyl), 128.40 (m-phenyl), 130.34 (C-2-indole), 131.87 (C-7a-indole), 139.14 (ipsophenyl), 140.14 (CH=N), 154.33 (C-5-indole), 174.35 (C=O). HRMS (ESI) m/z: calcd: [M+H]+ 377.197202; Found [M+H]+: 377.19643.
Antimycobacterial activity
Resazurin microtiter assay (REMA) (Martin et al. 2003; Nateche et al. 2006; Ramírez and Marquina 2017) is a colorimetric method (the results are a color reaction) to determine the minimum inhibitory concentration (MIC). A 96-well microplate was used and in each well Middlebrook 7H9 broth at different concentrations of the tested compound as well as a suspension of the reference strain Mycobacterium tuberculosis H37Rv (103–105 cells/ml) were added. A parallel control contained culture medium and suspension of the reference strain, without compound.
After 7 days of incubation at 37 °C, bacterial growth was measured by adding to each well the redox indicator—0.1% of resazurin. The microplate was re-incubated overnight at 37 °C; a change in color of the indicator resulting from a chemical transformation of the reagent indicated bacterial growth in the presence of the tested compound at different concentrations. The MIC was calculated and defined as the lowest concentration resulting in a complete inhibition of bacterial growth and reproduction. The MIC values are given as µM.
In vitro cytotoxicity
The cytotoxicity of selected promising agents was evaluated in the human embryonic kidney cell line HEK-293 cells (Mosmann 1983; Konstantinov et al. 1999). They were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ GmbH, Braunschweig, Germany). The cells were grown in a controlled environment—cell culture flasks at 37 °C in an incubator ”BB 16-Function
Line” Heraeus (Kendro, Hanau, Germany) with humidified atmosphere and 5% CO2. The cells were reset by tripsinization and supplementation with a fresh medium two times a week. The cell lines were maintained in 90% RPMI-1640 + 10% fetal bovine serum. The cell viability was assessed using the standard MTT-dye reduction assay. In brief, exponentially growing cells were seeded in 96-well flatbottomed microplates (100 μl/well) at a density of 2 × 104 cells per ml. After 24 h of incubation at 37 °C, they were treated with the tested compounds for 72 h. For each concentration, a set of at least 8 wells were used. After the exposure period, 10 μl MTT solution (10 mg/ml in phosphate-buffered saline (PBS)) aliquots were added to each well. Thereafter, the microplates were incubated for 4 h at 37 °C and the MTT-formazan crystals formed were dissolved through addition of 100 μl/well 5% formic acid (in 2-propanol). The absorption was measured using a Beckman Coulter DTX-800 multimode microplate reader at 580 nm. Cell survival fractions were calculated as percentage of the untreated control. In addition, half-maximal inhibitory concentration (IC50) values were derived from the concentration response curves, using non-linear regression analysis (Curve-fit, GraphPad Prism Software package).
Selectivity index (SI)
For calculation of the selectivity index, dividing the IC50 value by the MIC value (SI ratio = IC50/MIC) for each compound, IC50 values from Table 1 were used.
Animals
Adult mice were kept under controlled temperature (ambient temperature 20 ± 2 °C in a 12/12 light/dark cycle). The animals were purchased from the National Breeding Centre, Slivnitsa, Bulgaria. The animals were given free access to the food diet and water. A minimum of 7-day acclimatization was allowed before the commencement of the study and their health was monitored regularly by a veterinary physician. The Vivarium (certificate of registration of farm No. 0072/01.08.2007) was inspected by the Bulgarian Drug Agency in order to check the husbandry conditions (No. A-11-1081/03.11.2011). All performed procedures were approved by the Institutional Animal Care Committee, Bulgarian Food Safety Agency, and made according to Ordinance No. 15/2006 for humaneness behavior to
experimental animals.
Acute toxicity
The compound was suspended in distilled water, using 1–2 drops of Tween 80. The solution was administered via intraperitoneal (i.p.) or per oral (p.o.) route in 0.1 ml of 10 g animal body weight (b.w.). In order to decrease the number of experimental animals the acute toxicity tests after i.p. and p.o. administration of the tested compound 3e was assessed using the simple alternative method of Schlede et al. (2005).
The experiment was performed on 12 female mice: 6 of them were used for intraperitoneal administration of the tested compound and six for oral gavage. The test was started with a dose of 2000 mg/kg b.w. given i.p. to three female mice. Because of lack of toxicity or death, the same dose of 2000 mg/kg b.w. was administered to another three female mice. The animals that survived the acute
administration were observed for 14 days. The same procedure was conducted for oral administration of the tested substance.
Docking evaluation
Molecular docking studies were carried out using Molecular Operating Environment (MOE, version 2016.08) of Chemical Computing Group (https://www.chemcomp.com/MOE Molecular_Operating_Environment.htm). Docking simulations were performed on the crystal structure of Mycobacterium tuberculosis enoyl reductase (InhA) complexed with 1-cyclohexyl-N-(3,5 dichlorophenyl)-5-oxopyrrolidine-3-carboxamide, extracted from Protein Data Bank (http://www.rcsb.org/, PDB ID 4TZK). During the docking process, water molecules were removed while the
co-factor NAD was kept. “Protonate 3D” tool of MOE was applied to pose the missing hydrogen atoms in order of the correct ionization states to be assigned to the protein structure. “Docking” module in MOE was run to perform the molecular docking. Docking procedure has been implemented with default settings. The top 30 poses as ranked by London dG were kept and minimized using MMFF94x within a rigid receptor. The GBVI/WSA dG (Generalized-Born Volume Integral/Weighted Surface area) scoring function was then applied to score the resulting poses and 5 best poses were recorded.
“Ligand Interactions”
MOE tool was further used to analyze the molecular docking results by a visualization of the protein–ligand interactions in the active site of the complex. The tool presents in a diagram form an identification and visualization of the interactions between the ligand and the receptorinteracting entities, solvent molecules, and ions in the active site of the protein. Among the main interactions included are hydrogen bonds, salt bridges, hydrophobic and cation–π interactions, and solvent exposure.
Results and discussion
The synthesis of the novel series of hydrazide–hydrazones 3a–k and 5l, m was performed as outlined in Scheme 1 using an established procedure (Angelova et al. 2017a). The progress of the reaction was monitored using TLC. The novel aroylhydrazones 3a–k, and 5l–m were confirmed by 1 H NMR, 13C NMR, and HRMS spectral data and their melting points. The spectral analyses were in accordance
with the assigned structures. Stereochemistry was unambiguously confirmed with the help of cross-peak intensities observed in 2D NOESY (nuclear Overhauser effect spectroscopy) spectrum.
Although the four isomers were considered (Fig. S1)
(Martins et al. 2014; Oliveira et al. 2017) for the aroylhydrazones with indole scaffold, E/Z isomerization was generally not observed and the Z geometric isomers were absent. Only the 1 H NMR spectrum of compound 3i taken in DMSO-d6 at 20 °C shows a 1:1 mixture of conformers. According to the confirmed nuclear Overhauser effect (NOE) between the methylidene proton and the NH proton, the most stable were E isomers around C=N double bond and the synperiplanar conformer around the amide O=C–N-N bond. Therefore, we concluded that a single E geometrical isomer was observed and the duplication pattern of novel hydrazone derivatives to be due to the presence of syn/anti amide conformers in DMSO-d6. Additionally, for the purposes of structure elucidation the NMR spectra of compound 3i were measured at 373 K to achieve a fast exchange. After cooling down to 293 K the 1 H NMR spectrum remained unchanged (Fig. S2). Further, the stability of compounds 3a–k and 5l–5m was tested by 1 H NMR spectra taken in DMSO-d6 at 20 °C. All studied compounds remained unchanged for more than a month (typically stacked spectrum in Fig. S3).
The designed aroylhydrazone framework (Fig. 1) was made into two parts: indole or indazole as a mainstay and 4-phenyl-substituted aromatic rings or various heterocycles connected to hydrazide moiety to intensify the desired pharmacophoric behavior with drug-like properties and aliphatic or aromatic groups adjoined to another side (5th position) of the indole or indazole scaffold.
As can be seen, distinctions in the proposed scaffold lead to accomplished of different values of log P (Table 1). The in vitro evaluation of the antimycobacterial activity of the synthesized indole and indazole compounds against reference strain Mycobacterium tuberculosis H37Rv (Table 1) revealed that the compounds were bactericidal in nature and some of them were found to be more potent than first-line
antimycobacterial drugs (isoniazid, ethambutol). Structure–activity relationship of compounds from indole and indazole series with respect to their antitubercular activity revealed that compounds with 5-methoxy substituted indole scaffold, 3a, 3c, 3d, 3e, 3f, and with 5- benzyloxindole scaffold 3k, were found to be the most potent molecules with MIC values in the 0.39–0.77 µM range. Thus, the compound 3e with 4-methyl-1,2,3-thiadiazolyl moiety and 5-metoxindole scaffold is an excellent antimycobacterial agent, the activity of which is twofold more potent than that of the isoniazid and fourfold higher than that of ethambutol. The other synthesized derivatives with the indole scaffold and a methoxy group at 5th position 3c, 3d, 3f, and 3k displayed high antimycobacterial activity equal to that of isoniazid.
The replacement of phenyloxindol scaffold with methyloxindol induced a decrease in activit (compare 3a and 3k). On the other hand, introducing electron donating groups in aroilhydrazone moiety as
p-methoxyphenyl 3d or p-dimetilaminophenyl 3c (as an Ar-group) does not cause a significant change in the bioactivity. Unfortunately, the activity of the derivatives with a 5-bromoindole scaffold 3g, 3h, 3i, and 3j irrespective of substitution in hydrazone moiety was weaker than that of ethambutol (compare 3b and 3h). Furthermore, conversion of 3-pyridyl functional group in compound 3h into 3- indolyl in compound 3i or 3-indolylaceto group in 3k proved to be not effective. The compounds with indole scaffold 3b and with indazole scaffold 5l and 5m showed activity, comparable to the one of the reference substance ethambutol (MIC 1.69, MIC 1.32 and 1.66 µM, respectively). However, replacing the 5-metoxindazole scaffold in a compound 5m with 5-methoxindole scaffold in 3f was attributed to enhanced activity, whereas the presence of a thienyl group in 5l showed better activity in contrast to 5m.
In order to examine the selectivity (SI) of the antiproliferative effects, the cytotoxicity of the compounds was assessed against the human embryonal kidney cell line 293T, after 72 h of exposure (Konstantinov et al. 1999; Mosmann 1983). The compounds which exhibited the highest activity 3a and 3e (MIC 0.44 μM and MIC 0.39 μM, respectively) were also not cytotoxic against the human
embryonic kidney cells and displayed a very good selectivity index (SI = 633.49 and SI > 1978.83, respectively).
The other synthesized derivatives exhibited moderate toxicity in the human embryonic kidney cell line HEK-293T. A comparison of the antimycobacterial activity of derivatives with 5-methoxindole scaffold 3a–3f with 5-methoxindazole containing analogs 5l–5m allows the conclusion that the compounds with indole scaffold have a better bioactivity against the M. tuberculosis H37Rv strain than the compounds with an 5-methoxindazole scaffold (3f is more active than 3m) and showed a moderate cytotoxicity (ranging from 184.1 to 275.5 and n.d. for 3e) as well as a very low selectivity index.
Based on the activity of 3e (log P = 2.51), varying the lipophilicity (expressed in terms of log P) would have a significant effect on activity. We found that rendering the molecule as more lipophilic did not necessarily increase the antimycobacterial activity. Thus, introducing moieties which increase lipophilicity as in compounds 3k (log P = 4.39) did not improve their activity which was lower than 3a. It appears that another property of the compounds (probably their stability) surpassed the contribution of the lipophilicity to display better antimycobacterial properties against M. tuberculosis H37Rv. Obviously, the more pronounced activity of nontoxic indole derivatives 3е and 3a can be explained by the presence of the hydrazone linker with 4-methyl-1,2,3-thiadiazolyl or furyl moieties connected to 5-metoxindole scaffold and with lower values of log P, commensurate with that of ethambutol.
Furthermore, acute toxicity for the most active compound 3e was determined. According to the results, 3e did not cause any mortality at the tested dose of 2000 mg/kg given i.p. No toxic reactions were noticed until day 14. No adverse effects or deaths were also noticed for oral administration of the tested substance. For comparison, the acute toxicity of drug isoniazid for a mouse is as follows: median lethal dose (LD50) (p.o.) = 176 mg/kg; LD50 (subcutaneous) = 160 mg/kg; LD50 (intramuscular) = 140 mg/ kg; LD50 (intravenous) = 149 mg/kg (Saarstickstoff-Fatol).
According to the Globally Harmonized Classification System (GSH) (http://www.chemsafetypro.com/Topics/GHS/GHS_classification_criteria_acute_toxicity_category.html) the tested compound 3e (LD50 is higher than 2000 mg/kg b. w.) might be classified in class 5 (minimal hazard, between 2000 and 5000 mg/kg b.w.) or as slightly toxic, according to Hodge and Sterner scale.
To explain the activity order of 3a–k and 5l–m against M. tuberculosis H37Rv, the compounds were docked into the binding site of mycobacterial enoyl reductase (InhA). When performing docking, there are two major points to be considered when analyzing docking results: (1) the top score: the pose with the highest rank; and (2) the best pose: the pose with the lowest root mean square deviation (RMSD) to the reference ligand from the experimentally solved structure. The docking scores (Table S1 lists the essential of docking results for the synthesized compounds) of compounds 3a–k and 5l–m were found satisfactory in the range of −7.99 to −6.65 (Column S-score in Table S1).
The results presented in Table S1 clearly indicated that compounds exhibited significant binding affinities towards the M. tuberculosis InhA protein. The lowest RMSD (between the pose before and after refinement) was obtained for compound 3g, followed by 5l, 3h, and 3a, while the highest rank was recorded for compound 3d, followed by 3k, 5m, and 3f. The docking score of the most active compound of the series, 3e, was found to be −7.26. The differences in the docking scores of the compound justify the experimentally observed antimycobacterial potency of those compounds. The discrepancy between the molecular docking results and experimentally observed antimycobacterial potency of synthesized compounds might be ascribed to pharmacokinetic and biopharmaceutical properties, which ensure to a given compound better accessibility to the receptor site even when the binding interaction is a less prominent one.
Additionally, ”Ligand Interactions” tool of MOE was used to obtain the protein–ligand interactions diagrams of the co-crystallized ligand of M. tuberculosis InhA in the ligand-binding domain (Fig. 2), as well as for the predicted binding pose in the binding site of M. tuberculosis InhA protein of the most active compound 3e (Fig. 3a) and of the top score compound 3d (Fig. 4a). Protein–ligand interactions illustrated in Figs. 2, 3a and 4a were at the maximum distance of 4.5 Å between heavy atoms of the ligand and receptor. Figures 3b and 4b demonstrate the corresponding docking conformations of compounds 3e and 3d with their Connolly surface.
As seen from Fig. 2, only Tyr158 is involved in protein–ligand interactions. Residues Gly96, Phe97, Met103, Phe149, Met199, Ile215, and Leu218 are at receptor exposure, very close to the ligand, but still not at a binding distance. As seen from Fig. 3a (with the legend equal to that presented in Fig. 2), the most active compound 3e demonstrated one newly appeared interaction with Phe149 (arene-H interaction) that was at receptor exposure in Fig. 2, but not exhibiting any strong interaction. Most of the residues mentioned as important were still very close. The top score pose of the compound 3d (Fig. 4a, with the legend equal to presented in Fig. 2) repeated the interaction from Fig. 2 with Tyr158, although it is arene-H interaction instead of HB. As in the case of compound 3e, most of the residues mentioned as important were still very close.
All synthesized compounds 3a–k and 5l–m, as docked in the ligand-binding domain of M. tuberculosis enoyl reductase complexed with 1-cyclohexyl-N-(3,5-dichlorophenyl)-5-oxopyrrolidine-3-carboxamide, are presented in Fig. 5 with Connolly surface. As seen from the figure, all synthesized compounds occupied the same binding site as that of the ligand 1-cyclohexyl-N-(3,5-dichlorophenyl)-5-oxopyrrolidine-3-carboxamide (641), formed a cluster, and fitted well in the ligand-binding domain of M. tuberculosis InhA. Within the frame of this study, the docking data suggest interactions within the binding site of mycobacterial enoyl reductase that may induce the activity of the tested compounds.
Conclusions
In summary, in search of different scaffolds for antitubercular agents, we have designed and synthesized two new series of substituted indole and indazol-linked hydrazide–hydrazones 3a–k and 5l–m in excellent yields. All compounds demonstrated significant MICs ranging from 0.39 to 2.91 μM against a referent strain M. tuberculosis H37Rv. The cytotoxicity against the human embryonic kidney cell line HEK-293 was also evaluated and the selectivity (SI) of the antiproliferative effects was thus assessed. All compounds displayed good SI values ranging from >1978.83 to 12.04. In general, derivatives 3a–f and 3k possess potent antimycobacterial activity combined with low cytotoxicity that result in SI values higher than that of their 5-bromo substituted analogs 3g–j and of the derivatives with indazole scaffold 5l, m. In addition, the probability of the most promising antimicrobial compounds to inhibit the binding cavity of M. tuberculosis Enoyl-ACP reductase was studied theoretically via molecular docking.
The results revealed the importance of the 1,2,3-thiadiazole moiety in the connecting side chain and will help for future research in the development of novel antitubercular agents. Among the tested compounds with impressive antimycobacterial potency and selectivity, as well as very low toxicity, the hybrid of 5-metoxyindole-3-aroylhydrazone scaffold with thiadiazole moiety 3e identified is a potentially promising candidate for developing novel selective and slightly toxic antitubercular agents with bactericidal activity.
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