Pyridine appended 2-hydrazinylthiazole derivatives: design, synthesis, in vitro and in silico antimycobacterial studies

An array of pyridine appended 2-hydrazinylthiazole derivatives has been synthesized to discover novel chemotherapeutic agents for Mycobacterium tuberculosis (Mtb). The drug-likeness of pyridine appended 2-hydrazinylthiazole derivatives was validated using the Lipinski and Veber rules. The designed thiazole molecules have been synthesized through Hantzsch thiazole methodologies. The in vitro antimycobacterial studies have been conducted using Luciferase reporter phage (LRP) assay. Out of thirty pyridine appended 2-hydrazinylthiazole derivatives, the compounds 2b, 3b, 5b, and 8b have exhibited good antimycobacterial activity against Mtb, an H37Rv strain with the minimum inhibitory concentration in the range of 6.40–7.14 μM. In addition, in vitro cytotoxicity of active molecules has been observed against Human Embryonic Kidney Cell lines (HEK293t) using MTT assay. The compounds 3b and 8b are nontoxic and their cell viability is 87% and 96.71% respectively. The in silico analyses of the pyridine appended 2-hydrazinylthiazole derivatives have been studied to find the mode of binding of the active compounds with KasA protein of Mtb. The active compounds showed a strong binding score (−5.27 to −6.23 kcal mol−1).


Introduction
Tuberculosis (TB) is an infectious airborne disease caused by Mycobacterium tuberculosis (Mtb). TB remained the topmost infectious disease before the coronavirus (COVID- 19) pandemic. According to the Global tuberculosis report 2021, there were 9.9 million infections and 1.3 million deaths. The COVID-19 pandemic has halted years of global progress in reducing TB deaths. 1 The majority of existing TB drugs were discovered in the late 1950s, and due to the extensive usage of these drugs for the past seventy years, these drugs are less effective in controlling the disease as Mycobacterium has developed resistance to them. Bedaquiline and delamanid are used in conjunction with other TB medicines for curing MDR-TB. [2][3][4] Pretomanid has most recently entered into the market for treating XDR-TB, and it is used in combination with bedaquiline, and linezolid. 5 Developing new and effective TB drugs for the treatment of XDR-TB and MDR-TB with shorter and simpler regimens with negligible side effects is the need of the hour.
A broad range of established and new scaffolds were tested for their antitubercular activity over the last decade to identify novel anti-TB drugs. Thiazole derivatives are promising compounds to act as antitubercular agents as they are target-specic. Besides, thiazole compounds are known to act as the anticancer, 6 antitumor, 7,8 antimalarial, 9 antimicrobial, 10 anti-inammatory, 11 and anti-hypolipidemic 12 agents. Moreover, the thiazole moiety is structurally analogous to thiolactomycin, an antibiotic that exists naturally but with synthetic challenges. 13 During the biosynthesis of the Mtb cell wall, thiolactomycin inhibits b-ketoacyl-ACP synthase (KasA), leading to the death of Mtb. 14 Nitazoxanide (NTZ), a thiazole ring containing oral FDAapproved drug to treat protozoal infections, signicantly inhibits intracellular Mtb development. 15,16 Similarly, tizoxanide, a metabolite of NTZ has also been reported to inhibit non-replicative and replicative Mtb strains. 17 Interestingly, derivatives of 2-aminothiazole-4-carboxylate are known to be potent inhibitors of Mtb's KasA protein. 18 Due to the wide range of biological activity of 2-amino thiazole derivatives, in our research lab, we synthesized novel and effective 2-amino thiazole derivatives with antitubercular activity. 19,20 We found 2-(2-hydrazinyl)thiazole derivatives showed good antitubercular activity when the 2-pyridyl group was introduced at imine carbon of the thiazole ring. 21 To ne-tune and check the effect of substituents on the antitubercular activity of the derivatives of the pyridine group introduced at the 4th place of the thiazole ring, different functional groups on imine carbon have been incorporated and evaluated in the present investigation for their anti-TB activities. Interestingly, all the pyridine appended 2-hydrazinylthiazole derivatives discussed in the present report show better antitubercular activity than the previously reported derivatives of 2-hydrazinylthiazoles. The detailed results are presented in this paper. The thiazole derivatives discussed in the present investigation have been synthesized easily through classical Hantzsch thiazole methodologies. The advantage of this method is that there is much scope for generating a class of novel thiazole derivatives. 22 2. Results and discussion 2.1. Designing of pyridine appended 2-hydrazinylthiazole through molecular hybridization approach In the rst step of the present investigation, pyridine appended 2-hydrazinylthiazole derivatives have been designed, and molecular hybridization 23,24 approach was used for this purpose. As given in Fig. 1, the thiazole scaffold is linked to pyridine on one side and the hydrazine group on the other side for better pharmacological properties during the inhibition of TB.
Pyridine is one of the essential heterocyclic scaffolds found in natural substances, such as alkaloids (trigonelline), vitamins (vitamin B3 and B6), coenzymes (nicotinamide adenine dinucleotide), etc. Because of its versatile properties, such as water solubility, good chemical stability, and hydrogen bond-forming capability, pyridine moiety plays a vital role in medicinal chemistry. Pyridine derivatives show excellent antitubercular activity. For example, lansoprazole, a pyridine moietycontaining drug that inhibits gastric acid secretion by binding to the proton-pump receptor, has an intracellular effect against Mtb. Lansoprazole kills Mtb by attacking the cytochrome bc1complex, according to ex vivo pharmacokinetic studies. 25 Pyridomycin is another pyridine-containing and natural antibiotic drug that demonstrates intense activity against various Mycobacteria, including Mtb and M. smegmatis. 26 TBA-354 is another pyridine-containing drug that shows promising antitubercular activity with replicative and static action against Mtb. 27 The antitubercular activity of pyridine-containing drugs and pyridine-containing derivatives synthesized in our lab previously 21 prompted us to include a 2-pyridyl scaffold at the 4th place of the thiazole ring in the present investigation. Fig. 1(c) gives the structure of some of the pyridine-containing antitubercular drugs.
The hydrazone (R 1 R 2 C]NNH 2 ) is another essential and promising scaffold in medicinal chemistry. The lone pair electrons of amine nitrogen are conjugated with the imine group present in hydrazone compounds. The nitrogen atom of the hydrazone is nucleophilic, while the carbon is both electrophilic and nucleophilic. 28 These functional features make hydrazone a versatile chemical entity with antimalarial, antiviral, anti-HIV, anti-schistosomiasis, antimicrobial, anthelmintic, anticancer, antiplatelet, antidiabetic, antidepressant, anticonvulsant, anti-inammatory, analgesic, and antioxidant properties. 29,30 Moreover, the most promising antitubercular drugs such as rifampicin, isoniazid, and rifapentine contain hydrazone scaffold (see Fig. 1(a)) in their structures. With unique hydrogen bonding donor and receiving regions, hydrazones have gained much attention as potent anti-TB molecules. 31 The combination of 2-aminothiazole and hydrazone scaffolds in the derivatives of 2-(2-hydrazinyl)thiazole exhibited good inhibitory potentials against the strain of Mtb, H37Rv. 19,[32][33][34] The encouraging results of the in vitro studies and the higher possibility of synthesizing different types of substitutions motivated us to synthesize derivatives of pyridine appended 2-hydrazinylthiazole in nding out anti-TB agents with improved inhibitory potentials.
In the second step of this research, Lipinski and Veber rules were used to evaluate the drug-like molecule (DLM) nature of pyridine appended 2-hydrazinylthiazoles. 35,36 The DLM nature can be found out using molecular weight, log P value, number of hydrogen bond acceptors, and donors mentioned in the Lipinski rule. Besides, total polar surface area (TPSA) and the number of rotatable bonds (RBs) mentioned in the Veber rule can also be used to understand the DLM nature of the pyridine appended 2-hydrazinylthiazoles. Molinspiration server has been used to deduce the physicochemical properties of designed pyridine appended 2-hydrazinylthiazoles, and the deduced data are tabulated in Table 1. The results indicate that the molecular weights are in the range of 232.31-370.43, which is less than Lipinski's recommended value of 500. The log P values are between 1.64 and 4.10, and these values are less than 5 which is suggested by Lipinski rule. Likewise, the hydrogen bond acceptors of all compounds are between 4 to 7, and hydrogen bond donors are less than two that are far below the Lipinski rule's recommended values. The total number of RBs of all the compounds is in the range of 4-7, and the TPSA of the compounds is in the range of 50.17-96.00 A 2 , which is less than the recommended values. Therefore, all these compounds have not violated the Lipinski and Veber rules and have DLM characteristics. The boiled egg diagram of the pyridine appended 2hydrazinylthiazoles has been predicted through the Swiss ADME web-based tool, 37 and the graph is shown in Fig. 2. A compound should have high gastrointestinal absorption to act as an orally active drug (under white region). Most of the pyridine appended 2-hydrazinylthiazoles exhibited high gastrointestinal absorption except compounds 1b and 2b, and these compounds have exhibited blood-brain barrier permeation (under yellow region).

Synthesis of pyridine appended 2-hydrazinylthiazoles
In the third step of this research, as given in Scheme 1, all the designed pyridine appended 2-hydrazinylthiazoles with DLM nature have been synthesized. To synthesize pyridine appended 2-hydrazinylthiazole derivatives, rst, thiosemicarbazones were synthesized from thiosemicarbazide and the respective carbonyl compounds using the literature method. 19,38 The structure of thiosemicarbazones has been conrmed using different spectroscopic techniques. The characteristic imine proton present in thiosemicarbazones appears as a single peak between 8.5 ppm and 9.0 ppm in the 1 H nuclear magnetic resonance (NMR) spectra, and methyl protons at the imine group resonate between 2.28 ppm to 2.37 ppm. The proton present in -NH resonates between 10.05 ppm and 12.47 ppm, whereas protons present in aromatic groups resonate between 7.00 ppm and 8.4 ppm. In 13 C NMR spectra, the characteristic imine carbon resonates around 150 ppm, and in the infrared spectra, the stretching vibrations of the characteristic imine group appear in between 1542 and 1579 cm À1 . The spectral data of thiosemicarbazones presented here are similar to the already published data. 38 In the next step, as shown in Scheme 1, novel pyridine appended 2-hydrazinylthiazole derivatives have been synthesized from 2-(bromoacetyl)pyridine and corresponding thiosemicarbazones using the classical Hanzch-thiazole synthesis method. All the structures of the novel pyridine appended 2-hydrazinylthiazole derivatives have been conrmed using spectroscopic techniques. The characteristic thiazole proton resonates between 7.5-8.5 ppm, and the methyl protons attached to the imine group resonates around 2-2.5 ppm in 1 H NMR spectra. The -NH protons of thiosemicarbazones resonate at 9.0-9.5 ppm, whereas the same protons resonate downeld at 10.5-12.5 ppm in the derivatives of pyridine appended 2hydrazinylthiazoles, as a result of aromaticity present in the thiazole ring. The spectral data and spectra of all the novel compounds are given in ESI. † Structures of all the synthesized derivatives of pyridine appended 2-hydrazinylthiazole are given in Table 1.

Single crystal X-ray studies
In the next step, the single crystals of some of the pyridine appended 2-hydrazinylthiazole derivatives have been grown to Rifampicin understand the crystal packing and spatial orientation. As a representative structure, the crystal packing of a single crystal of 29b is given in Fig. 3, and the crystal parameters of 29b are tabulated in  against Mtb, H37Rv are given in Table S2 (see ESI †  In the third category, pyridine appended 2-hydrazinylthiazole derivatives substituted with methyl, methoxy, hydroxy, nitro, and diamine groups on the phenyl ring are considered.  Fig. 4.
Similarly, the correlation between log P values and their antitubercular activity of pyridine appended 2-hydrazinylthiazole derivatives is shown in Fig. 5. The log P value is gradually increased by introducing lipophilic groups at imine carbon of pyridine appended 2-hydrazinylthiazole. In the rst category, the log P values of pyridine appended 2-hydrazinylthiazoles are in the range of 1.64 to 3.97, and their MIC values are in the range of 6-431 mM. In the case of pyridyl substituted pyridine appended 2-hydrazinylthiazole derivatives, as in the case of compounds 29b and 30b, least log P values of 1.64 and 2.02, respectively was observed, and antitubercular activity of these compounds was also poor. When the log P value was increased to 2.04, the antitubercular activity decreased for the compound 30b with the MIC value 430.46 mM. Further, an increase in log P value to 3.26 leads to the increased antitubercular activity of the pyridine appended 2-hydrazinylthiazole derivatives. The compounds 2b and 3b with the log P values 2.81 and 3.26 show good antitubercular activity with MIC values 7.13 mM and 6.79 mM, respectively. When the log P value is increased to 3.97, as in compound 4b, it has shown moderate antitubercular activity with a MIC value of 75.66 mM.
In the second category, the halogen-substituted pyridine appended 2-hydrazinylthiazole derivatives have shown the log P value in the range of 2.93-4.1. Compound 5b has the log P value of 2.93 and showed excellent antitubercular activity with a MIC value of 6.7 mM. When the log P value is increased to 2.98, the compounds 6b and 7b have shown moderate antitubercular activity with MIC value 83.79 mM. Interestingly, a further increase in log P to 3.42, compound 8b has shown excellent antitubercular activity with MIC value 6.4 mM. When the log P value is increased to 3.47, the antitubercular activity is decreased for 11b and 12b with MIC values 79.42 mM and 317.66 mM, respectively. In the case of compound 13b, which has the log P value of 3.49, it showed moderate antitubercular activity with a MIC value of 79.42 mM. When the log P value is increased to 3.62, the compounds 15b and 16b have shown similar and inferior antitubercular activity with MIC value 278.36 mM. The compounds 9b and 10b have the log P values 3.62 and 3.68 and showed similar and moderate antitubercular activity with MIC value 71.77 mM. Further increase in the log P value to 4.1, the compound 14b has shown poor antitubercular activity with MIC value 286.33 mM. In the third category, the pyridine appended 2-hydrazinylthiazole derivatives have log P values in the range of 2.15-3.26 and showed moderate to poor antitubercular activity. The compounds 24b and 19b have log P values 2.15 and 2.31 and showed poor antitubercular activity with MIC values 306.39 mM and 337.43 mM, respectively.
Further increasing the log P value to 2.33, the compound 20b has shown antitubercular activity with MIC value 84.36 mM. When the log P value increased to 2.46, the compounds 23b and 22b have shown moderate antitubercular activity with MIC values 269.96 mM and 293.76 mM, respectively. Compounds 25b and 18b with log P values of 2.53 and 2.75 respectively showed antitubercular activity 73.44 mM and 337.43 mM, respectively. When the log P value is increased to 2.77, the compounds 26b and 27b have shown similar antitubercular activity with MIC value 307.36 mM. Further increasing the log P value to 2.87, the compound 21b has shown moderate antitubercular activity with a MIC value of 80.55 mM. When the log P value increased further to 3.26, the antitubercular activity decreased for the compounds 28b and 17b with MIC values 309.19 mM and 339.7 mM, respectively. Overall, most of the active pyridine appended 2-hydrazinylthiazole derivatives have shown good antitubercular activity when the log P value in the range of 2.81-3.42.

In vitro cytotoxicity studies of pyridine appended 2hydrazinylthiazole derivatives
Aer in vitro antitubercular evaluation, the active pyridine appended 2-hydrazinylthiazoles have been tested against Human Embryonic Kidney Cell lines (HEK 293t) to understand the cytotoxicity of the target thiazole derivatives. The active compounds 2b, 3b, 5b, and 8b were treated with HEK 293t cells at 6.5 mM concentrations, and MTT assay was used to analyse the cell growth. The cell viability of the compounds is in the range of 38-97%, and the results are presented in Fig. 6. Pyridine appended 2-hydrazinylthiazoles have shown more than 50% cell viability considered as nontoxic. The compounds 3b and 8b have shown cell viability at 87.52% and 96.71%, and hence, these compounds are termed to be nontoxic.

Molecular docking studies
Docking studies for pyridine appended 2-hydrazinylthiazole derivatives against Mtb, KasA protein was carried out in order to determine the mode of action because the 2-aminothiazole pharmacopore is structurally similar to the thiolactomycin (TLM) which is naturally occurring antibiotic. 13,19 TLM inhibits bketoacyl-ACP synthase (KasA) in mtFabH fatty acid synthesis, which in turn inhibits cell wall biosynthesis, resulting in Mtb death. 14 In the mycolic acid biosynthesis, 42 the long-chain fatty acid, KasA protein, plays an important role, thus, KasA protein is selected as a target for docking studies in the present investigation. All the active antitubercular agents have shown good interaction with KasA protein by forming a strong hydrogen bonding with the pyridine appended 2-hydrazinylthiazole molecules. The binding score of the complex was found to be in the range of À4.517 kcal mol À1 to À6.233 kcal mol À1 . Some molecules showed a better binding score than standard drug isoniazid, i.e., À5.830 kcal mol À1 . The in silico results and interaction diagram with Mtb, KasA protein are given in Table 1 and Fig. 7

Conclusion
The derivatives of pyridine appended 2-hydrazinylthiazole have been designed based on Lipinski and Veber rules. The designed thiazole derivatives were synthesized from 2-(bromoacetyl) pyridine and a variety of thiosemicarbazones. The resulting compounds have been evaluated against the H37Rv strain of Mtb to understand their antitubercular activity. Pyridine appended 2-hydrazinylthiazole derivatives with phenyl (2b and 3b), phenyl substitution with 2-and 4-uoro (5b and 8b) groups at imine carbon have shown excellent inhibitory potential with MIC values of 6-8 mM. Human embryonic kidney cell lines were used to determine the in vitro cytotoxicity properties of active pyridine appended 2-hydrazinylthiazoles at 6.5 mM. The active compounds 3b and 8b show cell viability of more than 87.52% and 96.71%, respectively, and are nontoxic. The docking studies of pyridine appended 2-hydrazinylthiazoles have been done against Mtb, KasA protein, and the molecules showed a binding score in the range of À5.27 to À6.23 kcal mol À1 .

Materials and methods
Bruker Avance-II NMR spectrometer (400 MHz for 1 H nucleus and 101 MHz for 13 C nucleus) was used to get NMR spectra. Thermo Nicolet 6700 Fourier transform infrared (FT-IR) spectrometer was used to get FT-IR spectra. Thermo Scientic High-Resolution Magnetic Sector MS DFS mass spectrometer was used to record mass spectra (MS). Rigaku-Oxford Xcalibur Eos single-crystal X-ray diffractometer with Mo-Ka radiation (l ¼ 0.71073 A) was used to obtain single crystal X-ray data. For empirical absorption correction, the implemented SCALE3 ABSPACK scaling algorithm was used. The XL in the Olex 2-1.2 package was used for the renement and SHELXS-97 was used for the structural solution.

Synthesis of thiosemicarbazone derivatives
Carbonyl compounds (1.1 mmol) and appropriate thiosemicarbazide (1 mmol) were dissolved in ethanol. To this solution, acetic acid (glacial) (a catalytic amount) was added. The resulting solution was reuxed for 3-6 hours and then cooled to room temperature. The resulting precipitate was ltered to obtain the corresponding thiosemicarbazone.

4.3.
General procedure for the synthesis of pyridine appended 2-hydrazinylthiazole derivatives 2-(Bromoacetyl)pyridine (1.1 mmol) and appropriate thiosemicarbazone (1.0 mmol) were dissolved in ethyl alcohol. The resulting solution was reuxed for 30 min-6 h and aer cooling to room temperature, the reaction mixture was neutralized with an aqueous NaHCO 3 solution. Finally, the solid obtained was ltered and recrystallized from ethyl alcohol to get the nal products.

In vitro antimycobacterial assay
The antimycobacterial activity of pyridine appended 2-hydrazinylthiazole derivatives was evaluated using Luciferase Reporter Phage (LRP) assay method with modication. 43 The brief procedure as follows. About 400 mL of sterile Middlebrook 7H9 broth containing pyridine appended 2-hydrazinylthiazole derivatives at concentrations of 100 mg mL À1 were aliquoted into test cryovials (T) and 400 mL of Middlebrook 7H9 broth containing rifampicin (2 mg mL À1 ) was transferred to drug control cryovial (D). Next, the control cryovial (C) was aliquoted with 400 mL of sterile below-mentioned 7H9 broth (Himedia). Mtb H37Rv cell suspension (100 mL) (McFarland no. 2) were used to inoculate all cryovials and incubated at 37 C for 72 h. Then, 40 mL of 0.1 M CaCl 2 and 50 mL of mycobacteriophage (phAE202) were added into all the cryovials (cell-phage mixture) and incubated for 4 h at 37 C. Then, 100 mL of the cell-phage mixture was transferred to a luminometer cuvette and added with 100 mL of D-luciferin substrate (Cayman chemicals, USA). The relative light unit (RLU) was measured at 10 s integration time in a luminometer (Lumat 9508, Berthold, Germany). The compounds considered as an antitubercular agent if test compounds showed a 50% reduction in RLU when compared to the control RLU. The % of RLU reduction was calculated by using the below-mentioned formula.

RLU reduction ð%Þ ¼
control RLU À test RLU control RLU Â 100 The active compounds were further tested for their antitubercular activities at 25 mg mL À1 and 2 mg mL À1 concentrations by LRP assay, as mentioned above.

4.5.
In vitro cytotoxicity assay 4.5.1. Cell culture. The human embryonic kidney cells (HEK 293t) were maintained at 37 C under 5% CO 2 and 100% humidity in Dulbecco's modied eagle media and supplemented with 10% fetal bovine serum and antibiotics (200 mL mL À1 penicillin G, 200 mg mL À1 streptomycin). When the cells reached conuence, they were trypsinized and plated in 96 well culture plates at a concentration of 1 Â 10 4 cells per well, and incubated for 24 h at 37 C under 5% CO 2 to obtain a monolayer culture. The cells were treated with four pyridine appended 2hydrazinylthiazole derivatives at 6.5 mM concentration. Cells with no drug treatment and only with DMSO were maintained as control. The experiments were carried out in triplicate. Following a 24 h incubation period at 37 C under 5% CO 2 , the cell viability was assessed.
4.5.2. MTT assay. The media was aspirated from each well of the culture plate. First, 20 mL of MTT reagent (5 mg mL À1 ) was added and incubated for 4 h at 37 C in the CO 2 incubator, and then added 180 mL of dimethyl sulphoxide. Next, the formation of purple crystal formazan was solubilized by placing the plates on a shaker. Then, at a wavelength of 570 nm, the absorbance was measured using a microplate reader. By calculating (absorbance of treated cells/absorbance of untreated cells) Â 100, the percentage of cell viability was calculated.

Molecular docking
To perform the molecular docking study, the protein was retrieved from the RCSB protein data bank (https:// www.rcsb.org/pdb/home/home.do). Before the docking study, the target protein was prepared using the protein preparation wizard, in which the protein was pre-processed, optimized, and minimized. Similarly, to prepare the ligands for docking in maestro format, Schrodinger v11.5 (LigPrep module) was used. A grid box was generated around the protein in which the ligands bind to the protein's active site. The prepared protein and ligands were docked using Schrodinger v11.5. The glide score was calculated within the Schrodinger v11.5 soware. The active site prediction was made using the COACH server, 44 which can be accessed freely. The COACH server is generally used to predict protein-ligand binding sites. The LIGPLOT (version 4.5.3) program was used to generate schematic representations of protein-ligand complexes. UCSF Chimera (version 1.5.3) has been used for 3-D visualization and structural analysis of protein-ligand complex.

Conflicts of interest
There are no conicts to declare.