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. 2023 Jun 2;14(8):1549–1561. doi: 10.1039/d3md00115f

Design, synthesis and biological evaluation of phenanthridine amide and 1,2,3-triazole analogues against Mycobacterium tuberculosis†

Adinarayana Nandikolla a, Yogesh Mahadu Khetmalis a, Boddupalli Venkata Siva Kumar a, Ala Chandu b, Banoth Karan Kumar b, Gauri Shetye c, Rui Ma c, Sankaranarayanan Murugesan b, Scott G Franzblau c, Kondapalli Venkata Gowri Chandra Sekhar a,✉
PMCID: PMC10429663  PMID: 37593576

Abstract

The phenanthridine core exhibits antitubercular activity, according to reports from the literature. Several 1,2,3-triazole-based heterocyclic compounds are well-known antitubercular agents. A series of twenty-five phenanthridine amide and 1,2,3-triazole derivatives are synthesized and analyzed using ESI-MS, 1HNMR, and 13CNMR on the basis of our earlier findings that phenanthridine and 1,2,3-triazoles shown good antitubercular activity. The synthesized phenanthridine amide and 1,2,3-triazole analogues were tested in vitro against Mycobacterium tuberculosis H37Rv and minimum inhibitory concentration (MIC) values were determined utilizing non-replicating and replicating low-oxygen recovery assay (LORA) and microplate Alamar Blue assay (MABA) methodologies. The phenanthridine amide derivative PA-01 had an MIC of 61.31 μM in MABA and 62.09 μM in the LORA technique, showing intense anti-TB activity. Amongst the phenanthridine triazole derivatives, PT-09, with MICs of 41.47 and 78.75 μM against the tested strain of Mtb in both MABA and LORA was the most active one. The final analogues' drug-likeness is predicted using absorption, distribution, metabolism, excretion, and toxicity (ADMET) studies. The most active compounds PA-01 and PT-09 were further subjected to in silico docking studies. Using the Glide module of Schrodinger, molecular docking analysis was carried out to estimate the plausible binding pattern of PA-01 and PT-09 at the active site of Mycobacterial DNA topoisomerase II (PDB code: 5BS8). Further, molecular dynamics studies of PA-01 and PT-09 were also carried out.

Amongst the Phenanthridine amides and triazoles reported in this work, PA-01 and PT-09 emerged as the most active anti-tubercular agents against Mtb H37Rv strain in MABA and LORA assays.graphic file with name d3md00115f-ga.jpg

Introduction

Tuberculosis (TB), a chronic infection transmitted by the infectious bacterium Mycobacterium tuberculosis (Mtb), remains one of the world's top ten causes of death. In 2021, over 1.2 million and 0.25 million people died from TB infection and human immunodeficiency virus (HIV-TB) coinfection, respectively, according to the World Health Organization (WHO) statistics.1 This disease, caused chiefly by Mtb, is currently treated with conventional therapy of four front-line medications (isoniazid, rifampin, pyrazinamide, and ethambutol) for 6–9 months. Multidrug-resistant tuberculosis (MDR-TB), extensively drug-resistant tuberculosis (XDR-TB), and even entirely drug-resistant tuberculosis (TDR-TB) have recently emerged as crucial global health threats.2,3 Despite the US Food and Drug Administration's (FDA) approval of bedaquiline and delamanid for the treatment of MDR-TB in 2012 and 2014, respectively, following a 40-year hiatus, some side effects have been reported.4 The current medications prescribed for TB have several significant drawbacks, such as prolonged treatment time, inability to give essential drug regimens for the course of treatment, and poor drug quality.4 Drug discovery and development for TB have not evolved to the point where the disease can be eradicated entirely. As a result, it is critical to investigate innovative chemical entities for the treatment of drug-resistant types of this widely spread infection with minimal adverse effects.5

Phenanthridine is one of the most prospective fused bicyclic rings that is accepted as a “drug preconception” scaffold,6 due to its wide range of biological activities and applications in anti-cancer,6 anti-bacterial,7 anti-malarial,8 anti-HIV,9 anti-plasmodial,10 and antitubercular agents.11–16 Many phenanthridine derivatives are reported to have antitubercular activity. Recently, Cappoen and group reported novel tetrahydrobenzo-[j]-phenanthridine-7,12-diones against Mtb. Among all the compounds, A was the most potent with an MIC value of 6.33 μM against Mtb H37Ra.11 A series of 6-(4-substitutedpiperazin-1-yl) phenanthridine derivatives have also been reported as antitubercular agents by our group. Compound B showed the highest activity against the M. tuberculosis H37RV strain with an MIC value of 4.05 μM. In another work reported by our group, compound C was the most potent with an MIC of 4.23 μM against the Mtb H37Rv strain.12,13 In 2014, Cappoen et al. reported phenanthridine-7,12-diones as anti-TB agents. From this work, compound D displayed an MIC50 value of 0.21 μM against Mtb H37Rv (Fig. 1).14

Fig. 1. Structures of phenanthridine containing antitubercular compounds.

Fig. 1

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Imidazo-[1,2-a]-pyridin-3-carboxamide is one of the most promising heterocyclic scaffolds in medicinal chemistry because of its wide variety of pharmacological applications. Novel imidazo-[1,2-a]-pyridine-carboxamide derivatives have been described as strong anti-TB agents by Onajole et al. Compound E was found to be the most powerful in this series, with an MIC of 0.10 μM against the Mtb H37Rv strain.4 Imidazo-[1,2-a]-pyridine amide derivatives as anti-mycobacterial drugs were reported by Lv et al. in 2017. Compound F demonstrated considerable efficacy against the drug-resistant Mtb H37Rv strain, with an MIC of 0.041 μM.17 Moraski and colleagues synthesized effective anti-tuberculosis imidazo-[1,2-a]-pyridine compounds. Compound G was tested against seven non-tubercular strains and showed excellent efficacy against the drug-resistant Mtb H37Rv strain with an MIC90 of 1.07 μM.18 Reddyrajula et al. reported imidazo-[1,2-a]-pyridine-1,2,3-triazole derivatives as anti-TB agents. Compound H was the most active against the Mtb H37Rv strain, with an MIC of 1.56 μM mL−1.19 Ethyl imidazo-[1,2-a]-pyridine carboxamide derivatives were reported by Wang et al. as novel anti-TB agents. Compound I exhibited excellent inhibitory action against the H37Rv Mtb strain, with an MIC of 0.1196 μM.20 In another study, Li and colleagues designated imidazo-[1,2-a]-pyridine-3-carboxamide as an anti-tubercular agent. Compound J is the most active (MIC < 0.0354 μM) against the drug-resistant H37Rv Mtb strain in this series (Fig. 2).21

Fig. 2. Structures of imidazo-[1,2-a]-pyridin-3-carboxamide based anti-TB agents.

Fig. 2

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N-(Pyridinyl)-benzamide derivatives have a wide range of biological activities, and few researchers have developed N-(pyridin-2-yl)-amides as anti-TB agents. Compound K, reported by Nawrot and group, exhibited promising anti-TB activity with an MIC value of 108.81 μM against the Mtb H37Ra strain.22 Recently, our group reported (pyridine-3-yl)-benzamide derivatives that displayed antitubercular activity. Compound L, with an IC50 of 1.46 μM, showed the most potent activity against the Mtb H37Ra strain.23 Wang et al. reported nitrobenzamide derivatives as antitubercular agents. Compound M had considerable activity against the drug-sensitive Mtb H37Rv strain with an MIC value of 0.038 μM (Fig. 3).24

Fig. 3. Structures of N-(pyridinyl)-benzamide-based anti-TB compounds.

Fig. 3

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One of the most widely accepted methods for developing novel pharmacological scaffolds is molecular hybridization. Our group previously reported certain phenanthridine derivatives (B, C) as antitubercular agents.12,13 As an exploration of these phenanthridines as the primary scaffold and imidazo-[1,2-a]-pyridin-3-carboxamide/N-(pyridinyl)-benzamide as a subunit, we designed novel phenanthridine derivatives (PA-01 to PA-14) and (PT-01 to PT-11) as antitubercular agents in the current work (Fig. 4).

Fig. 4. Scaffold design strategy for new antitubercular agents.

Fig. 4

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Results and discussion

Results and discussion of in silico predicted ADMET parameters

Owing to the development of in silico methods, the US FDA approval of new molecule entities has increased rapidly in recent years. However, many drug candidates still fail to become drugs because of poor efficacy and safety. The parameters like absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties play a crucial role in all drug discovery and development stages. The in silico predicted physicochemical properties of the synthesized compounds reveal that all the compounds exhibited drug-likeness properties such as molecular weight, hydrogen bond donors (HBD), hydrogen bond acceptors (HBA), and partition co-efficient (log Po/w) within the recommended values. Except for aqueous solubility, all the other descriptors like the solvent-accessible surface area, Caco-2 permeability, brain/blood partition coefficient, number of rotatable bonds, and percentage of human oral absorption of the experimental compounds were in the prescribed range as that of marketed drugs. Further, nearly half of the title compounds showed low predicted values of log S when compared with recommended values (−6.5 to 0.5) which indicate poor aqueous solubility. Still, we included them for further studies by observing a significant percentage of oral absorption values of these compounds.

The predicted toxicity profile of the synthesized molecules suggests that the title compounds were non-toxic. Finally, all the expected ADMET profiles of the tested compounds were within the range of standard drugs, indicating that these compounds are unlikely to encounter any issues during the further development phase of drug discovery (Table 1).

In silico predicted ADMET parameters of the synthesized compounds.

S. no Comp. code Mol. wt. SASA Donor HB Acceptor HB log Po/w log S P Caco log BB Rotor % Human oral absorption
1 PA-01 194.24 406.45 2 1.5 2.42 −2.69 2099.72 −0.08 1 100
2 PA-02 292.38 613.82 1 3.5 4.39 −5.23 3329.29 −0.24 5 100
3 PA-03 320.43 673.37 1 3.5 5.08 −5.92 3973.74 −0.3 7 100
4 PA-04 300.32 554.92 1 6 2.69 −3.95 1393.54 −0.4 2 100
5 PA-05 274.32 563.37 1.5 3.5 3.76 −4.57 3346.48 −0.16 4 100
6 PA-06 313.36 577.45 2.5 4.5 3.16 −4.54 879.79 −0.67 3 100
7 PA-07 332.79 589.41 1 3.5 4.69 −5.73 3412.88 0.15 2 100
8 PA-08 424.24 593.57 1 3.5 4.81 −5.78 3867.85 0.19 2 100
9 PA-09 324.38 627.58 1 3.5 4.85 −5.78 2970.74 −0.24 4 100
10 PA-10 379.46 711.78 2 3.5 5.53 −6.95 1790.79 −0.58 5 100
11 PA-11 351.41 625.86 2 3.5 4.72 −5.74 2118.46 −0.3 3 100
12 PA-12 312.37 586.56 1 3.5 4.55 −5.21 3816.76 −0.03 3 100
13 PA-13 391.27 618.09 1 3.5 5.15 −6.11 3874.69 0.15 3 100
14 PA-14 313.36 581.14 2.5 4.5 3.04 −4.32 890.22 −0.67 3 100
15 PT-01 313.35 580.36 1.5 4 3.9 −4.87 3259.88 −0.1 3 100
16 PT-02 460.54 843.24 1 6 5.9 −8.71 780.37 −1.06 4 100
17 PT-03 500.48 795.47 1 6 6.1 −8.53 1402.34 −0.33 3 93.079
18 PT-04 466.93 760.53 1 6 5.59 −7.64 1593.67 −0.38 3 100
19 PT-05 477.48 776.34 1 7 4.37 −7.11 163.85 −1.74 4 92.144
20 PT-06 507.51 793.38 1 7.75 4.61 −6.96 308 −1.46 5 72.549
21 PT-07 500.48 791.89 1 6 6.08 −8.46 1395.23 −0.33 3 92.937
22 PT-08 460.54 804.37 1 6 5.73 −8.17 1489.04 −0.6 3 100
23 PT-09 460.54 809.71 1 6 5.86 −8.27 1156.28 −0.71 3 100
24 PT-10 556.38 791.04 1 7 4.78 −7.66 151.33 −1.62 4 80.988
25 PT-11 546.37 816.51 1 7 5.69 −8.34 391.25 −1.02 4 80.745
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Chemistry

Phenanthridin-6-amine (PA-01) is synthesized as per our earlier reported protocol.25 9H-Fluoren-9-one (1) on treatment with hydroxylamine hydrochloride and NaOAc in EtOH : H2O (1 : 1) at 25 °C yielded 9H-fluoren-9-one oxime (2). The oxime compound (2), when treated with polyphosphoric acid and P2O5 at 150 °C for 1 h, yielded phenanthridin-6(5H)-one (3). The phenanthridinone (3) was treated with POCl3 and N,N-dimethylaniline under reflux for 4 h and the intermediate compound 6-chlorophenanthridine (4) was obtained. Chloro substituted phenanthridine (4), when treated with 4-methoxybenzylamine and K2CO3 in DMF and heated at 150 °C overnight, resulted in N-(4-methoxybenzyl)-phenanthridin-6-amine (5). Treatment of N-benzyl phenanthridine (5) with trifluoro acetic acid at 25 °C overnight afforded phenanthridin-6-amine (PA-01). The phenanthridin-6-amine (PA-01) was coupled with different substituted alkyl/aryl acids using EDC·HCl, HOBt, and DIPEA in DMF at 25 °C yielding the formation of title compounds PA-02 to PA-14 as shown in Scheme 1.

Scheme 1. Preparation of final compounds PA-01 to PA-14. Reagents and conditions: (i) NH2OH·HCl (1.2 equiv.), NaOAc (1.2 equiv.), EtOH : H2O (2 : 1), 90 °C, 2 h (ii) PPA (8.0 equiv.), P2O5 (0.5 equiv.), heating at 150 °C, 1 h (iii) POCl3 (6.0 equiv.), N,N-dimethylaniline (0.5 equiv.), reflux 4 h (iv) 4-methoxy benzyl amine (3.0 equiv.), K2CO3 (5.0 equiv.), DMF, 150 °C, overnight (v) TFA, 25 °C, overnight (vi) substituted alkyl/aryl acid (1.1 equiv.), HOBT (1.5 equiv.), EDC·HCl (1.5 equiv.), DIPEA (3.0 equiv.), DMF, 25 °C, 5 h.

Scheme 1

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The mixture of PA-01 and ethyl 2-chloroacetoacetate dissolved in 1,4-dioxane at 100 °C overnight afforded ethyl-2-methylimidazo-[1,2-f]-phenanthridine-3-carboxylate (6). Hydrolysis of the ester (6) by LiOH at 70 °C afforded 2-methylimidazo-[1,2-f]-phenanthridine-3-carboxylic acid (7). The acid intermediate was dissolved in DMF, then later EDC·HCl, HOBt and DIPEA were added. Treatment of this mixture with propargyl amine at 25 °C yielded 2-methyl-N-(prop-2-yn-1-yl)-imidazo-[1,2-f]-phenanthridine-3-carboxamide (PT-01). The terminal acetylene on treatment with various substituted azides in the presence of CuSO4·5H2O, sodium ascorbate, and catalytic copper iodide in DMF : t-BuOH : water afforded PT-02 to PT-11 (Scheme 2).

Scheme 2. Synthesis of compounds PT-01 to PT-11. Reagents and conditions: (a) 1,4-dioxane, ethyl-2-chloroacetoacetate (3.0 equiv.), 100 °C, overnight (b) LiOH (5.0 equiv.), EtOH : water (1 : 1), reflux, 5 h (c) HOBt (1.5 equiv.), EDC·HCl (1.5 equiv.), DIPEA (3.0 equiv.), propargyl amine (1.10 equiv.), DMF, 25 °C, 5 h (d) substituted aryl azides, sodium ascorbate (1.5 equiv.), CuSO4·5H2O (5 mol%), CuI (5 mol%), DMF : t-BuOH : water (4 : 2 : 2), 25 °C, 6 h.

Scheme 2

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In vitro anti-mycobacterial evaluation of the final compounds

In the present work, the anti-mycobacterial activity of the title compounds (PA-01 to PA-14 and PT-01 to PT-12) was determined in vitro against non-replicating and replicating Mtb using the LORA and MABA methods, respectively. Table 2 shows the result of the anti-TB activities. Using the CLSI M24, 3rd edition,26 broth microdilution method, the research compounds were tested in vitro for tuberculostatic activity. The concentration of the studied new chemical entities ranged from 100 to 9.14 μg mL−1. A sterile control and a growth control were created on individual plates without antibiotics and inoculation, respectively. At 37 °C, the controls and plates were incubated for 7 days. After incubation, Alamar Blue solution was added to each plate well, and the plates were re-incubated for another 24 h. The colour change from blue to pink indicates growth, and the MIC is the lowest concentration of substance that prevents the colour change. For comparison, rifampicin, bedaquiline, and isoniazid were used as reference drugs.27,28

Antitubercular activity of compounds against Mtb H37Rv.

S. no. Entry R/R′ MABA MIC (μM) LORA MIC (μM) HepG2 CC50 (μM)
1 PA-01 — 61.31 62.09 30.53 ± 1.64
2 PA-02 graphic file with name d3md00115f-u1.jpg >342.02 79.83 79.52 ± 9.17
3 PA-03 graphic file with name d3md00115f-u2.jpg >312.07 300.36 >50
4 PA-04 graphic file with name d3md00115f-u3.jpg >332.98 >332.98 52.99 ± 2.49
5 PA-05 graphic file with name d3md00115f-u4.jpg >364.54 >364.54 22.62 ± 1.63
6 PA-06 graphic file with name d3md00115f-u5.jpg 78.28 85.78 32.09 ± 1.64
7 PA-07 graphic file with name d3md00115f-u6.jpg >300.49 >300.49 NTa
8 PA-08 graphic file with name d3md00115f-u7.jpg >235.71 >235.71 NT
9 PA-09 graphic file with name d3md00115f-u8.jpg >308.28 >308.28 NT
10 PA-10 graphic file with name d3md00115f-u9.jpg 70.57 218.31 29.96
11 PA-11 graphic file with name d3md00115f-u10.jpg 125.39 68.97 32.14 ± 3.64
12 PA-12 graphic file with name d3md00115f-u11.jpg 294.01 267.31 >50
13 PA-13 graphic file with name d3md00115f-u12.jpg >255.58 >255.58 >50
14 PA-14 graphic file with name d3md00115f-u13.jpg 149.51 150.53 56.90 ± 6.14
15 PT-01 — >319.12 >319.12 NT
16 PT-02 graphic file with name d3md00115f-u14.jpg >217.14 >217.14 NT
17 PT-03 graphic file with name d3md00115f-u15.jpg >199.80 92.97 84.42 ± 7.33
18 PT-04 graphic file with name d3md00115f-u16.jpg >214.16 >214.16 NT
19 PT-05 graphic file with name d3md00115f-u17.jpg 84.29 87.37 71.93 ± 9.05
20 PT-06 graphic file with name d3md00115f-u18.jpg 94.01 105.68 NT
21 PT-07 graphic file with name d3md00115f-u19.jpg 183.79 89.45 82.32 ± 7.51
22 PT-08 graphic file with name d3md00115f-u20.jpg >217.14 208.30 90.73 ± 9.33
23 PT-09 graphic file with name d3md00115f-u21.jpg 41.47 78.75 NT
24 PT-10 graphic file with name d3md00115f-u22.jpg 179.73 179.73 NT
25 PT-11 graphic file with name d3md00115f-u23.jpg >183.02 >183.02 NT
26 Rifampicin — 0.073 0.145 —
27 Bedaquiline — 0.054 0.216 —
28 Isoniazid — 2.625 560.015 —
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a

NT – not tested.

Cytotoxicity evaluation

The Alamar Blue assay was used to test the cytotoxicity of all final compounds in the HepG2 cell line at 100 μM. The results showed that most of these analogs were nontoxic to HepG2 at the tested concentration. The cytotoxicity concentration (CC50) was determined by further investigating compounds that are cytotoxic to HepG2 cells.25 The most active compound, PA-01, showed a CC50 value of 30.53 ± 1.64 μM (Table 2).

Structure–activity relationship (SAR) studies

Compounds with an amino group linked to the phenanthridine carbon showed exceptional antitubercular efficacy among the synthesized PA derivatives. PA-01 showed significant antitubercular activity with an MIC of 61.31 μM in the MABA method, and 62.09 μM in the LORA method. Amongst the aromatic substituted phenanthridine analogues, PA-06 with (4-aminophenyl) substitution was the best, with MICs of 78.28 and 85.78 μM in MABA and LORA methods, respectively. Heterocyclic amides (PA-10, PA-11) showed decent antitubercular potency. PA-10 exhibited an MIC of 70.57 μM in MABA and 218.31 μM in the LORA method. Meanwhile PA-11 displayed MICs of 125.39 μM and 68.97 μM in MABA and LORA methods, respectively. In the case of the PA series of compounds, PA-14 emerged to be the next best compound with MIC values of 149.51 and 150.53 μM in MABA and LORA, respectively. In both methods, aromatic amide compounds containing halogens (Cl, Br, I) displayed much lower activity (PA-07, PA-08, PA-13) with MIC > 100 μg mL−1.

Among the aromatic substituted triazole-(N-((1-methyl-1H-1,2,3-triazol-4-yl)-methyl)-imidazo-[1,2-f]-phenanthridine-3-carboxamide) analogues, PT-09 showed excellent anti-Tb activity in the MABA technique with an MIC of 41.47 μM and 78.75 μM in the LORA method. In the MABA and LORA methods, PT-05 with electron-withdrawing NO2 on the meta position of the phenyl ring showed significant antitubercular activity, with MIC values of 84.29 and 87.37 μM. Compound PT-06 with a nitro group on the ortho position and methoxy on the para position of the phenyl ring with MICs of 94.01 and 105.68 μM in MABA and LORA methods emerged the next best. The rest of the phenanthridine-3-carboxamide triazole derivatives displayed lower activity. In summary, amongst the PA and PT derivatives, we observe that PA derivatives are superior in activity compared to PT derivatives.

In silico molecular docking studies

Docking studies were performed to explore the binding interaction of the potential compound within the active site of the receptor using the Schrodinger Glide module (Version 2019-1). The Mycobacterial DNA topoisomerase II (PDB code: 5BS8) crystal structure was retrieved from the protein data bank with a resolution of 2.4 Å.29,30 The protocol was validated by checking the RMSD of the docked pose of the co-crystal ligand with the X-ray pose of the co-crystal ligand. The RMSD was found to be 1.76 Å (Fig. 5a). The native ligand and significantly active compounds PA-01 and PT-09 were docked on topoisomerase II, and the results are reported in Table 3. The binding scores of the co-crystal, PA-01, and PT-09 were found to be −7.9 kcal mol−1, −6.68 kcal mol−1, and −3.5 kcal mol−1, respectively. From the docked orientation of the co-crystal ligand, the amino acid residue Arg128 forms a hydrogen bond contact with the acid group present in the ligand. Also, the native ligand containing a quinolone moiety bound to the receptor through π–π stacking interaction with DA15 (4.15 Å) and DG11 (3.65 Å) at the minor groove of DNA (Fig. 5b). Based on the results of RMSD, the docking study was initiated for the test compound. Apart from the Mycobacterial DNA topoisomerase II, another suitable target namely GyrB ATPase (PDB code: 4BAE) was also used for docking,31,32 and the results and discussion are included as Annexure-I in the ESI.†

Fig. 5. (a) Super-imposed view of the co-crystal ligand (green) and its re-docked pose (cyan) at the active site of target protein 5BS8 (RMSD 1.76 Å). (b) Surface view of the docked complex along with 2D and 3D contacts of the co-crystal ligand (MFX) at the active site of topoisomerase II (5BS8). (c) Surface view of the docking complex and 2D and 3D contacts of the potent compound PA-01 at the active site of topoisomerase II (PDB: 5BS8). (d) Surface view of the docked complex along with 2D and 3D contacts of the significantly active compound PT-09 at the active site of topoisomerase II (5BS8).

Fig. 5

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Molecular docking results of the co-crystal ligand and compounds PA-01 and PT-09 at the active site of the target protein (PDB: 5BS8).

Comp. code Binding residues Bond distances (Å) Type of interaction Glide score (kcal mol−1)
Co-crystal ligand (MFX) DA: 15(G) 4.15 Pi–pi stacking
DG: 11(H) 3.65 Pi–pi stacking −7.9
3.74 Pi–pi stacking
3.83 Pi–pi stacking
ARG128 2.49 Hydrogen bond
4.8 Salt bridge
PA-01 DG: 11(H) 4.2 Pi–pi stacking −6.68
3.92, 4.49 Pi–cation
DA: 15(G) 3.98, 3.64, 4.38 Pi–pi stacking
DT: 10(H) 4.25 Pi–pi stacking
PT-09 DA: 15(G) 4 Pi–pi stacking −3.5
3.86 Pi–pi stacking
DG: 11(H) 3.66 Pi–pi stacking
4.36 Pi–pi stacking
ARG482 3.27 Pi–cation
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DNA topoisomerases are ubiquitous enzymes that alter the architecture of DNA by separating and rejoining the strands and also play a significant role in the physiological control of the genome. The biologically active compound (PA-01) from the phenanthridine amide series was docked against the crystal structure of topoisomerase II (PDB 5BS8, resolution: 2.4 Å) obtained from the protein data bank. The glide score of compound PA-01 was found to be −6.68 kcal mol−1 and binding residues involved in the interaction are tabulated in Table 3. The docked pose of compound PA-01 mostly exhibited the pi–pi stacking linkage with DNA bases DG11, DA15 and DT10 at the active site. For instance, the A and C rings of the inhibitor form stacking interactions with DA15 and DT10 at a distance of 3.64 Å, 4.38 Å and 4.25 Å, respectively. Further, the B ring of the phenanthridine also displayed two pi–pi stacking contacts with DG11 (4.2 Å) and DA15 (3.98 Å) and a pi–cation contact with DG11 (3.92 Å and 4.49 Å) at the binding pocket of topoisomerase II (Fig. 5c). The most active compound PA-01 had a comparatively low binding affinity with the target protein, which might be due to the absence of hydrogen bond interaction with the receptor that was observed with the native ligand (MFX).

The glide score of compound PA-09 was found to be −6.68 kcal mol−1 and binding residues involved in the interaction are tabulated in Table 3. Similarly, compound PT-09 forms hydrophobic interactions with DA15, DG11 and Arg482, as seen in Fig. 5d. The triazole and dimethyl benzene rings of the significantly active compound form π–π stacking interaction with the receptor which confirmed the stability of the ligand at the minor groove of the DNA–topoisomerase complex. Further, there was a π–cation interaction (3.27 Å) between Arg482 and the imidazole ring present in PT-09 (Fig. 5d). The low docking score of the most active molecule could be due to the lack of hydrogen bond interaction with the receptor, which was seen with the native ligand (moxifloxacin-MFX).

Results and discussion of molecular dynamics (MD) studies

Molecular dynamics (MD) simulation analysis

The stability and conformational variations have been assessed for 5BS8 apoprotein, complexes of 5BS8 with the co-crystal ligand (MFX), compound PA-01 and compound PT-09 throughout the MD simulation period. The steadiness of protein–ligand complexes was compared with the corresponding apoenzyme to monitor the deviations of backbone atoms during the entire simulation. The root mean square deviation (RMSD) of the apoenzyme was found to be less than 7.5 Å. The ligand RMSD of compounds PA-01 and PT-09 was found to be lower than the co-crystal ligand RMSD which is about 15 Å, indicating that the ligands are stable and bound to the active site.

Additionally, the root mean square fluctuation (RMSF) plot depicts the flexibility of each residue during the simulation. When compared to the apoenzyme and enzyme with the co-crystal ligand, PA-01 and PT-09 complexes exhibited minimal fluctuation with RMSF values less than 3 Å for most residues of the binding pocket, as shown in Fig. 6. This implies no significant fluctuation of active site residues at the binding site of the target protein (5BS8).

Fig. 6. Root mean square fluctuation (RMSF) trends of the apoenzyme, co-crystal ligand, compound PA-01 and compound PT-09 complexes during 100 ns MD simulation.

Fig. 6

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For complex PT-09, the RMSD was less than 5 Å for the first 14 ns and following a rise of 10 Å up to 25 ns later, the complex attained steadiness throughout the MD simulation of 100 ns. For complex PA-01, the ligand RMSD was in the range of 2.8–10 Å, initially reaching a maximum of nearly 10 Å and then gradually declining in RMSD with minimal fluctuations throughout the MD simulation (Fig. 7).

Fig. 7. Root mean square deviation (RMSD) graphs of the apoenzyme, co-crystal ligand, compound PA-01 and compound PT-09 complexes during 100 ns MD simulation.

Fig. 7

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The binding pattern of the co-crystal ligand, compound PA-01 and compound PT-09 at the active site of the target protein is depicted in Fig. 8–10, respectively. The MD results of the protein–ligand complex (PLC) interactions are comparable with the molecular docking predictions. The co-crystal ligand majorly forms water-mediated hydrogen bonds with most of the residues present at the binding pocket. The residues like Glu501 and Arg482 form hydrogen bond interaction with the native ligand and contribute to 52% and 10% of simulation time, respectively. The complex is also strengthened by water-bridged interactions with the other amino acid residues like Lys484, Asn499, Gly483, Arg128, Lys441 and Asp461 at the binding site during the simulation (approx. 15%) (Fig. 8). Similarly, compound PA-01 primarily revealed water-mediated and hydrogen bond interactions with the target active site residues. Glu501 (17%) and Arg482 (17%) residues in close proximity to the amino group of PA-01 in the active site stabilize the complex to a larger extent. Additionally, Thr500, Asp461 and Lys484 residues were also engaged in coupling with the ligand during the simulation of 100 ns (Fig. 9).

Fig. 8. Protein–ligand contacts of 5BS8 with the co-crystal ligand complex during 100 ns MD simulation.

Fig. 8

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Fig. 9. Protein–ligand contacts of target protein 5BS8 with the compound PA-01 complex during 100 ns MD simulation.

Fig. 9

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Fig. 10. Protein–ligand contacts of 5BS8 with the compound PT-09 complex during 100 ns MD simulation.

Fig. 10

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Compound PT-09 also exhibited primarily water-mediated interactions, followed by hydrogen bond and hydrophobic interactions. The residues Arg495 (4%), Glu501 (26%), Arg482 (4%), Thr500 (6%) and Lys484 (12%) were actively involved in water-assisted interactions with the ligand for most of the simulation period. The complex is also stabilized by van der Waals interactions with Pro123 and Lys441 at the binding pocket of target protein 5BS8 (Fig. 10).

Fig. 11 depicts different features of the ligand, including RMSD, radius of gyration (RG), intramolecular hydrogen bonds (intraHB), molecular surface area (MolSA), solvent accessible surface area (SASA) and polar surface area (PSA). The RMSD for compounds PA-01, PT-09 and the co-crystal ligand remained stable and was found to be less than 3 Å throughout the simulation study period. Subsequently, the RG trend remains stable with slight fluctuations for the native ligand and compound PA-01, whereas compound PT-09 initially fluctuated up to 5.5 Å and then exhibited steadiness over a period of 100 ns. Additionally, no intramolecular hydrogen bonds were identified for all three ligands during analysis. The trends of MolSA, SASA and PSA graphs showed minor fluctuations throughout the simulation period for the native ligand. Compound PA-01 displayed an equilibrium with the trends of MolSA and PSA graphs, while mild instabilities were observed with the SASA graph during the simulation period. Similarly, compound PT-09 revealed instability at the beginning (30 ns) of the study for the same parameters, and beyond it, no substantial departure in the properties of compound PT-09 was observed.

Fig. 11. Ligand properties of compounds PA-01 (a), PT-09 (b) and the co-crystal ligand (C) with respect to the active site of target protein 5BS8 over 100 ns simulation period.

Fig. 11

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Conclusion

In conclusion, we have designed and synthesized twenty-five phenanthridine core containing amides and 1,2,3-triazoles and screened them for in vitro anti-mycobacterial activity against the Mtb H37Rv strain in replicating and non-replicating forms of the bacteria. 1HNMR, 13CNMR, LCMS, and elemental analysis were used to characterize the synthetic analogues developed. The compounds demonstrated significant to mild antitubercular efficacy. According to the findings, among the phenanthridine amide derivatives, PA-01, which has an electron-donating amino (NH2) group on phenanthridine, showed excellent anti-TB activity with an MIC value of 61.31 μM in MABA and 62.09 μM in the LORA technique. With MIC values of 41.47 and 78.75 μM respectively, against the tested strain of Mtb, compound PT-09, a phenanthridine triazole derivative, was the most active in MABA and LORA methods. The ADMET properties of the compounds were also predicted in silico. Finally, a molecular docking analysis of the most active compound, PA-01, was performed using the Glide module of Schrodinger software to estimate the plausible binding pattern of the compound at the active site of Mycobacterial GyrB ATPase from Mycobacterium TB (PDB 4BAE) as PDB code. Molecular dynamics studies of PA-01 revealed hydrophobic interactions to be predominant, followed by H-bonds and water bridges with the active site.

Experimental section

In the ESI† section, the general procedure for synthesized intermediates and final products is described in detail. Procedures related to biology experiments viz., anti-TB MABA assay, cytotoxicity studies, and docking studies are outlined in the ESI.†

Conflicts of interest

There is no conflict of interest to declare.

Supplementary Material

MD-014-D3MD00115F-s001

Acknowledgments

KVGCS gratefully acknowledges support from the Council of Scientific and Industrial Research, New Delhi (CSIR) (F. No. 02(392)/21/EMR II), and DST-FIST (F. No. SR/FST/CSI-240/2012), New Delhi, India. He also acknowledges the Central Analytical Laboratory facilities of BITS Pilani Hyderabad Campus. All the authors thankful to BITS-Pilani, Pilani campus for providing adequate facilities to do this research. Additionally, Banoth Karan Kumar is thankful to the Ministry of Tribal Affairs, Government of India for providing financial assistance (Award No.: 201920-NFST-TEL-01497).

†

Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3md00115f

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Supplementary Materials

MD-014-D3MD00115F-s001
MD-014-D3MD00115F-s001.pdf (6.7MB, pdf)

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