Development of novel isatin–nicotinohydrazide hybrids with potent activity against susceptible/resistant Mycobacterium tuberculosis and bronchitis causing–bacteria

Abstract Joining the global fight against Tuberculosis, the world's most deadly infectious disease, herein we present the design and synthesis of novel isatin-nicotinohydrazide hybrids (5a–m and 9a–c) as promising anti-tubercular and antibacterial agents. The anti-tubercular activity of the target hybrids was evaluated against drug-susceptible M. tuberculosis strain (ATCC 27294) where hybrids 5d, 5g and 5h were found to be as potent as INH with MIC = 0.24 µg/mL, also the activity was evaluated against Isoniazid/Streptomycin resistant M. tuberculosis (ATCC 35823) where compounds 5g and 5h showed excellent activity (MIC = 3.9 µg/mL). Moreover, the target hybrids were examined against six bronchitis causing-bacteria. Most derivatives exhibited excellent antibacterial activity. K. pneumonia emerged as the most sensitive strain with MIC range: 0.49–7.81 µg/mL. Furthermore, a molecular docking study has proposed DprE1 as a probable enzymatic target for herein reported isatin-nicotinohydrazide hybrids, and explored the binding interactions within the vicinity of DprE1 active site.


Introduction
Tuberculosis (TB) is considered to be the world's top infectious killer 1 . It is a complicated devastating disease that accounts for the death of 1.5 million individuals in 2018 as declared by WHO 1 . About 23% of the world's population is expected to be latent TB carriers with a high risk to develop the disease 2 . The first-line drugs such as Isoniazid, Rifampicin, Ethambutol, and Streptomycin have shown significant activity against Mycobacterium tuberculosis however, the emergency of drug-resistant TB (DR-TB), especially multi-drug resistant TB (MDR-TB) is still a serious trouble 3 . The emerged drug resistance can be considered as a global health crisis, where only one out of three accessing proper remedy among the patients falling ill in 2018 1 .
Along with the emerged drug resistance, the development of secondary bacterial infection is considered to be a critical problem 4 . Different bacterial strains have been isolated from sputum samples including: Haemophilus influenzae, Klebsiella pneumoniae, Streptococcus pneumoniae, and Moraxella catarrhalis 4,5 . The tubercular and the bacterial co-infection can lead to inadequate treatment causing serious complications such as pneumoniae, bronchial anthracofibrosis, and chronic airflow obstruction [5][6][7][8] .
Unfortunately, many virulent bacterial strains have developed bacterial resistance against most of the currently used antibiotics. The death rate due to bronchitis has been increased up to an alarming level recording about 0.7 million cases of death every year. Thus, there is a crucial need to design and synthesise new candidates that may help in the battle of the developed bacterial resistance towards the currently available anti-tubercular and antibacterial drugs.
Isatin is a promising endogenous biologically active scaffold. It could be chemically modified to produce various heterocyclic compounds with different biological activities [9][10][11][12][13][14] . Isatin based derivatives have revealed promising antibacterial [15][16][17] and anti-TB effects 18,19 . Moreover, the nicotinohydrazide moiety has a large contribution to the field of medicinal chemistry. It has been incorporated in several active antimicrobial 20 and anti-tubercular agents 21 . In fact, the presence of a hydrazide group can be considered as a significant key for an optimum anti-tubercular activity 22 . Recently, several research teams adopted molecular hybridisation techniques between isatin and different moieties for the design of new anti-mycobacterial agents [23][24][25][26] , such as isatin-INH hybrids I (Figure 1) 25 and isatin-nalidixic acid hybrids II ( Figure 1) 26 . Later, our research team has identified the N-substituted isatin-thiazolidinone hybrid III ( Figure 1) as a promising antitubercular agent that produced an effective killing of M. aurum in infected macrophage model with broad-spectrum antimicrobial effect against sensitive and resistant bacterial strains 27 . SAR study revealed that N-benzylation or N-methylation of the 2-oxindole ring enhanced the activity by 2-4 folds as compared to the N-unsubstituted analogues.
In view of the findings stated above and in a continuation of our previous work for the discovery of potential anti-tubercular and anti-microbial agents 28 , we decided to extend our research through conjugating different N-substituted isatin moieties with a nicotinohydrazide moiety to furnish target hybrids 5a-I ( Figure 2). In order to explore the impact of C-5 substitution, as well as to ensure different lipophilic environments that may be suitable for the mycobacterial activity, both 5-Cl and 5-Br substituents were grafted at isatin scaffold. As it was suggested that N-substitution of isatin scaffold could enhance the mycobacterial activity 27,29 , N-alkylation, or N-benzylation of the herein reported hybrids was performed ( Figure 2). Thereafter, the impact of C-2 and C-6 substitution of the nicotinohydrazide moiety the anti-tubercular and antibacterial activities were investigated through developing the two sets 5j-m and 9a-c ( Figure 2). Finally, an isatin-3-pyridylamine hybrid 14 was synthesised to carry out further elaboration of the target scaffold ( Figure 2).
Herein seventeen new 2-oxindolin-3-ylidene-nicotinohydrazide derivatives (5a-m, 9a-c, and 14) were designed, synthesised, and evaluated for their anti-tubercular activity against drug-susceptible M. tuberculosis strain (ATCC 27294) and Isoniazid/Streptomycin resistant M. tuberculosis (ATCC 35823), together with the antibacterial effect against bronchitis causing-bacteria in an attempt to produce new candidates having potential activities with minimal bacterial resistance that may reduce the complications associated with the secondary bacterial infection.
Finally, molecular docking studies have been performed for the most active compounds into the active sites of two enzymes: flavoenzyme Decaprenylphosphoryl-b-D-Ribose 2 0 -Epimerase (DprE1) and enoyl-acyl carrier protein reductase (InhA) being critical enzymes in the metabolism and synthesis of M. tuberculosis cell wall 30,31 to explore the possible mechanism of action of the new hybrids.

Chemistry
The synthetic strategies adopted for the preparation of final compounds (5a-m, 9a-c and 14) were illustrated in Schemes 1-3. In the first scheme, different isatin derivatives 3a-c were alkylated with the appropriate alkyl halide in anhydrous DMF in the presence of K 2 CO 3 as an acid binder to furnish the N-substituted indoline-2,3-diones 4a-i. On the other hand, methyl nicotinate 1a and methyl 6-methoxynicotinate 1b was refluxed with hydrazine hydrate in methanol to produce the key intermediate nicotinohydrazides 2a-b which subsequently reacted with N-substituted indoline-2,3-dione derivatives 4a-i in absolute ethanol containing a catalytic amount of glacial acetic acid to get the final compounds 5a-m (Scheme 1).
In the last scheme, methyl 6-methoxynicotinate 1b was refluxed with NaOH in MeOH to get the free 6-methoxynicotinic acid 10, which then undergone to chlorination via SOCl 2 to furnish 6-methoxynicotinoyl chloride 11 where the chloride was subsequently substituted with an azide functionality to produce 6methoxynicotinoyl azide 12. The azid derivative 12 was subjected to Curtius Rearrangement to get 5-isocyanato-2-methoxypyridine 13, that refluxed with 5-bromoindoline-2,3-dione 3c in dry toluene to furnish the final compound 14.
1 H NMR spectra of the final compounds 5a-m and 9a-c were confirmed with the presence of a D 2 O exchangeable singlet signal attributable to the proton of the hydrazide linker at d (13.31-13.86) ppm. Moreover, 1 H NMR spectra for compounds 9a-c confirmed the presence of singlet signal in the aliphatic region for the CH 3 group around d 2.71 ppm, in addition to the extra aromatic protons for the 6-phenyl ring at a range of d 7.44-7.57 ppm, whereas, spectra for compounds 5j-m and 14 confirmed the appearance of singlet signal for OCH 3 groups at d (3.82-3.97) ppm.

Anti-tubercular activity
All of the newly synthesised hybrids were evaluated for their antitubercular action towards M. tuberculosis (ATCC 27294) using the Microplate Alamar Blue Assay (MABA) 32 . INH was used as the reference drug. The obtained results for the anti-mycobacterial activity were summarised in (Table 1) and expressed as a minimum inhibitory concentration (MIC).
The results revealed that most tested compounds exerted potent to moderate anti-tubercular action with MIC range ¼ 0.24-7.81 mg/mL. Compounds 5d, 5g, and 5h were found to be as potent as INH with MIC ¼ 0.24 mg/mL. Compounds 5f, 5j, and 5k N-alkylation and Nbenzylation was reported to enhance the anti-TB activity (29) to explore the impact of  revealed potent activity with MIC range ¼ 0.48-0.98 mg/mL, whereas derivatives 5a-5c, 5e, 5i, 5l, 5m, and 14 exhibited a moderate activity with MIC range ¼ 1.95-7.81 mg/mL. Concerning the structure-activity relationship, it was observed that the anti-tubercular activity of compounds 5a-5i was affected by two main factors; N-substitution and incorporation of halogen at position 5 of the oxindole ring. Regarding N-substitution it was shown that, compounds bearing isobutyl group: 5b, 5f, and 5h exhibited remarkable activity with MIC values ¼ 1.95, 0.48, and 0.24 mg/mL, respectively. Similarly, compound 5d with N-benzyl moiety revealed a potent activity with MIC ¼ 0.24 mg/mL. In contrast, compounds 5c and 5i carrying the N-ethylcarboxylate group were found to be the least active derivatives (MIC ¼ 7.81 mg/mL). Moreover, the contribution of substitution at position 5 of oxindole ring on the activity was quite important. Incorporating 5-Br group in compounds 5g and 5h (MIC ¼ 0.24 mg/mL) showed better activity than 5-Cl substituted compounds 5e and 5f (MIC ¼ 0.48 and 3.9 mg/mL, respectively) and the unsubstituted congeners 5a-5c (MIC range ¼ 3.9-7.81 mg/mL).
Then we turned our attention to study the impact of substitution at position 6 on the nicotinohydrazide moiety of the new hybrids. Comparing the anti-tubercular activity of 6-methoxy nicotinohydrazide derivatives 5j-5m against the unsubstituted counterparts 5a-5d, it was found that the introduction of 6-methoxy group in compounds 5j-5l (MIC ¼ 0.98-3.9 mg/mL) enhanced the anti-tubercular activity by 2-4 fold compared to unsubstituted analogues 5a-5c (MIC range: 1.95-7.81 mg/mL) except for the benzyl derivatives 5d and 5m. Conversely, the introduction of 6phenyl moiety to the nicotinohydrazide moiety as in compounds 9a-c (MIC ¼ 15.63-31.25 mg/mL) led to a reduced anti-tubercular activity that may be due to steric factors.
In conclusion, increasing the lipophilicity of the isatin scaffold via C-5 substitution with halogens (Br > Cl) was more advantageous for activity. Also, the anti-mycobacterial action of the target hybrids was decreased in the following order; 6-methoxy nicotinohydrazide > unsubstituted nicotinohydrazide > 2-methyl-6-phenyl nicotinohydrazide (Figure 3).

Anti-tubercular activity towards Isoniazid and Streptomycin resistant M. tuberculosis (ATCC 35823)
The anti-tubercular activity against Isoniazid and Streptomycin resistant M. tuberculosis (ATCC 35823) was evaluated using MABA 32 . Investigating the results of the target compounds 5a-m, 9a-c, and 14 (Table 1), it was found that many of the tested derivatives exhibited potent to moderate activity with MIC range ¼ 3.9-62.5 mg/mL. Compounds 5g and 5h with the best anti-tubercular effect against M. tuberculosis (ATCC 27294), showed excellent activity (MIC ¼ 3.9 mg/mL) resistant M. tuberculosis strain (ATCC 35823), with more than 32 fold increase in the activity compared to INH and Streptomycin (MIC > 125 mg/mL). Moreover, compounds 5d, 5f and 5j elicited a good activity with MIC ¼ 15.63 mg/mL, whereas derivatives 5b, 5k, and 14 produced a moderate activity with MIC ¼ 62.5 mg/mL. Finally, compounds 5a and  5l were found to be least active showing MIC value ¼ 125 mg/mL, while the 6-phenyl pyridine derivatives 9a-c were inactive with MIC >125 mg/mL.
Interestingly, compounds 5g and 5h showing best anti-tubercular activity with the minimal bacterial resistance against resistant-TB, revealed a broad-spectrum antibacterial action against most of the tested bronchitis causing-bacteria. They were found to be more potent than the reference drug against H. influenzae, M. catarrhalis, and B. pertussis (MIC ¼ 0.24-3.9 mg/mL, Azithromycin MIC ¼ 3.9-7.81 mg/mL). Additionally, they were equipotent to Azithromycin against M. pneumoniae, S. pneumoniae and K. pneumoniae (MIC ¼ 1.95, 0.98, and 0.49 mg/mL, respectively).

Cytotoxicity against non-tumorigenic lung fibroblast WI-38 cells
In order to assess the cytotoxic impact of hybrids 5g and 5h on normal human cells, an MTT cytotoxicity assay was carried out against non-tumorigenic WI-38 cells. The obtained results ascribed to hybrids 5g and 5h non-significant cytotoxic action with IC 50 value ¼ 60.95 and 54.01, respectively (Table 3), thus providing favourable selectivity indices (SI) equal to 15.6 and 13.8, respectively.

Molecular docking
Over the past decade, several druggable targets have been utilised in TB-drug discovery to develop new therapies in order to fight the global epidemic of TB. Most of these enzyme targets have an essential role in energy production, cell wall biosynthesis, and DNA replication, repair, and transcription. Examples of these enzymes are; Alanine racemase (Alr), Maltosyltransferase (GlgE), N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU), Pantothenate synthetase (PS), Mtb ATP synthase subunit C (AtpE), Flavin-dependent thymidylate synthase (TS or ThyA), and flavoenzyme Decaprenylphosphoryl-b-D-Ribose 2 0 -Epimerase (DprE1) 30 .
On account of their superior anti-mycobacterial activity, compounds 5g and 5h were selected for further in silico investigation to explore their possible molecular target. Two promising drug enzymes in M. tuberculosis were suggested as potential targets for the target compounds in this study; the flavoenzyme Decaprenylphosphoryl-b-D-Ribose 2 0 -Epimerase (DprE1) and the enoyl-acyl carrier protein reductase (InhA) enzymes. Both enzymes play an essential role in the metabolism of the cell wall and the synthesis of mycolic acid, respectively 30,31 . The selection of these two enzymes is based on a pilot docking study that explored the plausible binding interactions and energy scores for the prepared compounds with different enzyme targets.
The crystal structures of the two enzymes were downloaded from the protein data bank PDB ID: 4NCR for DprE1 and 2B35 for InhA. Despite, the importance of docking in providing a predicated binding mode and an account for a ligand strength, it remains a theoretical technique that needs proper validation by comparison with an experimental reference. Therefore, both co-crystallised ligands were re-docked into their corresponding enzymes. The calculated RMSD values between the co-crystallised and docked pose were 0.52 and 0.71 for DprE1 and InhA, orderly which indicates a valid and reliable docking protocol. Compound 5h has achieved À10.1 and À7.9 kcal/mole energy score for DprE1 and InhA, respectively, whereas, compound 5g has achieved À9.7 and À7.3 kcal/mole for DprE1 and InhA, respectively. Interestingly, both compounds 5g and 5h achieved a higher energy score than the co-crystalized ligand (À9.3 kcal/mole) in DprE1. These findings suggested that DprE1 may be a plausible target for the herein reported compounds.
Inspection of the docking poses for the two examined compounds revealed their ability to fit perfectly in the active site of the DprE1 enzyme establishing different types of interactions (Figures 4 and 5). The oxindole ring in both compounds 5g and 5h was engaged in two H-bond interactions via its carbonyl oxygen (C ¼ O) functionality with His-132 and Tyr-415 amino acids. Furthermore, the NH of the hydrazide linker and the nitrogen atom of the pyridine ring were involved in H-bond interactions with His-132 and Asn-385, respectively. Moreover, the nitrogen atom of the pyridine ring in compound 5h achieved an extra hydrogen bond interaction with Cys-387 which may explain its higher energy score than 5g. Worthy of note, the N-isobutyl group of compound 5h established several hydrophobic interactions Table 2. MIC (mg/mL) for hybrids (5a-m, 9a-c, and 14) against bronchitis causing-bacteria as determined using XTT assay.  with non-polar residues lining a hydrophobic pocket, such as Pro-116, Val-121, Ile-131, and Ala-417. The 2 D diagrams for interactions of compounds 5g and 5h in InhA active site ( Figure S1), and the 3 D illustrations of the superimposition for the docking poses and the co-crystalized ligands in DprE1 and InhA active sites ( Figure S2), as well as the detailed interactions achieved by compounds 5g and 5h within the DprE1 binding site (Table S3) were provided in the Supporting Materials.

Conclusion
In this work, different sets of isatin-nicotinohydrazide hybrids (5a-m, 9a-c) and isatin-3-pyridylamine hybrid 14 were designed and synthesised. The anti-mycobacterial activity towards M. tuberculosis (ATCC 27294), as well as towards the INH and Streptomycin resistant M. tuberculosis (ATCC 35823), were evaluated for the seventeen newly synthesised 2-oxindolin-3-ylidene-nicotinohydrazide derivatives. The obtained results identified compounds 5g and 5h as the most potent anti-tubercular agents in this study (MIC ¼ 0.24 mg/mL), with minimal mycobacterial resistance (MIC ¼ 3.9 mg/mL). Furthermore, all herein reported hybrids were examined for their antibacterial activity against six bronchitis causing-bacteria; M. pneumoniae, H. influenzae, K. pneumoniae, M. catarrhalis, B. pertussis, and S. pneumonia. Most hybrids exerted significant antibacterial activity in comparison to the reference drug Azithromycin against the tested bacterial strains. The most sensitive strain was K. pneumoniae with MIC range ¼ 0.49-7.81 mg/mL. Interestingly, compounds 5g and 5h displayed a broad-spectrum antibacterial activity against most of the tested bronchitis causing-bacteria. Moreover, an in silico molecular modelling study has proposed DprE1 as a potential enzyme target for herein reported isatin-nicotinohydrazide hybrids. Compounds 5g and 5h were able to fit perfectly in the active site of the DprE1 enzyme achieving a higher energy score (À9.7 and À10.1 kcal/mole, respectively) than the co-crystalized ligand (À9.3 kcal/mole). The oxindole ring was engaged in two H-bond interactions via its carbonyl oxygen (C ¼ O) functionality with His-132 and Tyr-415 amino acids, whereas, the NH of the hydrazide linker and the nitrogen atom of the pyridine ring were involved in H-bond interactions with His-132 and Asn-385, respectively. In addition, the N-isobutyl group of compound 5h established several hydrophobic interactions with non-polar residues lining a hydrophobic pocket.

General
I.R. spectra have been recorded on Schimadzu FT-IR 8400S spectrophotometer. NMR spectra were recorded by Bruker-Avance 400 spectrometer. 13 C NMR spectra were run at 100 MHz in deuterated  dimethylsulphoxide (DMSO-d 6 ). Chemical shifts (d H ) are reported relative to the solvent (DMSO-d 6 ). NMR analyses were carried out at the NMR Unit, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt, whereas IR analyses were carried out at the Faculty of Science, Mansoura University, Mansoura, Egypt. Elemental analyses were carried out at the Regional Centre for Microbiology and Biotechnology, Al-Azhar University, Cairo, Egypt. Compounds 2a,b 35 , 4a-i 36,37 , 8 38 , and 11 39 , were prepared as described previously.

Synthesis of target compounds 5a-m and 9a-c
In a round-bottom flask, the appropriate nicotinohydrazide derivative 2a-b or 8 (2.5 mmol) were added to an equivalent amount of N-substituted indoline-2,3-dione derivatives 4a-i in 15 ml of glacial acetic acid. The previous mixture was heated under reflux with TLC monitoring, after complete consummation of starting materials, the reaction flask was cooled to room temperature. The formed precipitate was collected under vacuum, washed with hexane, cold water and recrystallized from dioxane/methanol mixture to furnish final compounds 5a-m and 9a-c, respectively.

Synthesis of target compound 14
6-Methoxynicotinoyl azide 12 (0.3 g, 1.6 mmol) was dissolved in anhydrous toluene (20 ml) which then heated under reflux for 1 h. To the previous solution, an equivalent amount of 5-bromoisatin was added and the reaction mixture was refluxed for 5 h. The produced precipitate was filtrated while hot washed with hexane and recrystallized from acetonitrile to furnish compound 14.
Full characterisation details (NMR, IR, and elemental analysis) for the target hybrids (5a-m, 9a-c, and 14) have been provided in the Supporting Materials.

Anti-tubercular activity
Microplate Alamar blue assay (MABA) 32 has been utilised to determine MICs for all herein prepared hybrids (5a-m, 9a-c, and 14) against M. tuberculosis ATCC 27294 (Isoniazid-susceptible strain) and Mycobacterium tuberculosis ATCC 35823 (Isoniazid and Streptomycin resistant strain). The detailed procedures were presented in the supporting materials.

XTT Susceptibility Assay for the antibacterial activity
A colorimetric broth microdilution method was utilised to determine the MIC for the tested hybrids following the XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide]reduction assay 33,34 . MICs for the tested hybrids was assessed against bacteria causing bronchitis; Gram-negative bacteria: H. influenzae ATCC 10211, M. pneumoniae ATCC 15531, K. pneumoniae ATCC 43816, M. catarrhalis ATCC 25238, and Bordetella pertussis ATCC 9340, as well as S. pneumoniae ATCC 1659, representing Gram-positive bacterium, that obtained from the American type culture collection. The detailed experimental procedures have been added in the supporting materials.

Cytotoxicity assay
The MTT assay 40 was used to assess the cytotoxic impact of hybrids 5g and 5h towards the normal human lung WI-38 cells as reported earlier 41 .

Molecular docking
Aiming to define the molecular target for the best two lead compounds, the crystal structures of both DprE1 and InhA enzymes of TB were downloaded from the Protein data bank ID: 4NCR and 2B35, respectively 42,43 . Vina Autodock software was implemented in the current work to conduct the docking studies. The program demands both the ligand and the receptor in the pdbqt format, for that, M.G.L tools were used to prepare the two enzymes and the ligands into the right format 44 . Also, the active sites were determined by generating a grid box sized 24 Â 24 Â 24 Å surrounding the binding domain of each co-crystallised ligand with its corresponding enzyme (centre_x ¼ 17.3, centre_y ¼ À20 and centre_z ¼ 3.4 for 4ncr and centre_x ¼ 17, centre_y ¼ 12.5 and cen-tre_z ¼ 10.5 for 2b35). Docking validity and reliability were ensured by re-docking each co-crystallised ligands with their corresponding enzyme, then calculating the RMSD between the co-crystallised pose and the docked pose. Discovery studio 4.5 visualiser was employed in the analysis of docking results as well as in the evaluation of best candidates based on binding affinity score and interaction with receptor 45 .