Biological and Sensing Applications of a Few 1,3,4-Oxadiazole Based Donor-Acceptor Systems

1,3,4-Oxadiazole pharmacophore is still considered a viable biologically active scaffold for the synthesis of more effectual and broad-spectrum antimicrobial agents. Therefore, the present study is based on five 1,3,4-oxadiazole target structures, viz., CAROT, CAROP, CARON (D-A-D-A systems) and NOPON and BOPOB (D-A-D-A-D systems) bearing various bioactive heterocyclic moieties relevant to potential biological activities. Three of the compounds, CARON, NOPON and BOPOB were assessed in-vitro for their efficacy as antimicrobial agents against gram positive (Staphylococcus aureus and Bacillus cereus) and gram negative (Escherichia coli and Klebsiella pneumonia) bacteria; and two fungi, Aspergillus niger and Candida albicans; also, as an anti-tuberculosis agent against Mycobacterium tuberculosis. Most of the tested compounds displayed promising antimicrobial activity, especially CARON which was then analyzed for the minimum inhibitory concentration (MIC) studies. Similarly, NOPON portrayed the highest anti-TB activity among the studied compounds. Consequently, to justify the detected anti-TB activity of these compounds and to recognize the binding mode and important interactions between the compounds and the ligand binding site of the potential target, these compounds were docked into the active binding site of cytochrome P450 CYP121 enzyme of Mycobacterium tuberculosis, 3G5H. The docking results were in good agreement with the result of in-vitro studies. In addition, all the five compounds were tested for their cell viability and have been investigated for cell labeling applications. To conclude, one of the target compounds, CAROT was used for the selective recognition of cyanide ion by ‘turn-off’ fluorescent sensing technique. The entire sensing activity was examined by spectrofluorometric method and MALDI spectral studies. The limit of detection obtained was 0.14 µM.


Introduction
Extensive and indiscriminate application of antibacterial and antifungal agents have led to several pathogenic bacteria and fungi acquiring drug resistance.Under this scenario, discovery of new structural lead compounds that may be used for designing new antimicrobial agents that are more effective and less toxic compared to the already existing one's gains momentum.Tuberculosis (TB) is one among the common infectious disease known by mankind and the common causative agent is Mycobacterium tuberculosis.Presently, the treatment for TB offers the use of four-drug regimen including isoniazid, rifampin, ethambutol and pyrazinamide for the first two months and isoniazid and rifampin for further four months.But re-emergence of the TB infections along with the rise in MDR-TB (multidrug resistance tuberculosis), XDR-TB (extensively drug-resistant tuberculosis) and TDR-TB (totally drugresistant tuberculosis) has led to the resistance to two firstline TB drugs isoniazid and rifampin, additional resistance to second line TB drugs and resistance to broad range of first-line and second-line drugs respectively [1][2][3].
Another interesting candidate for the development of effective antimicrobial agent owing to their varied biological and chemical properties is phenothiazine, a nitrogen-, and sulfur-containing tricyclic compound, which has been known for over 100 years.Phenothiazines also possess similar biological properties as those described for 1,3,4-oxadiazole above [11].Moreover, they are commonly available, quite inexpensive, well-tolerated and nontoxic in nature.In view of these literature reports on the potential biological activities of 1,3,4-oxadiazole and phenothiazine, we surmised that molecules containing both phenothiazine and oxadiazole residues are likely to exhibit interesting, if not enhanced, bioactivity.Accordingly, we have investigated the antimicrobial and anti-tuberculosis activities of three donor-acceptor (D-A) compounds viz.CARON, NOPON and BOPOB containing phenothiazine as donor and oxadiazole as acceptor components [12,13].In continuation, in vitro anti-TB studies have been augmented with in silico molecular docking studies as well.
Fluorescent probes, selective towards various cell organelles serve as beneficial tool in drug designing for outlining the important functional and morphological changes that these organelles undergo in a diseased state [14,15].In particular, fluorescent dyes can be employed for the development of fluorescently tagged drug molecule for learning the cellular level interaction of these drug molecules with the corresponding targets [16] which is quiet useful in improving the efficiency of the drug.Hence fluorescent imaging is evolving as a promising method for drug discovery and development.Among these materials, those exhibiting emission in the 650-900 nm range (far red/near-infrared (FR/NIR) region) are of paramount importance in fluorescent bioimaging [17,18] owing to their low tissue absorption and autofluorescence in the FR/ NIR region leading to minimum background interference and improved image sensitivity.Taking advantage of this aspect, we have employed all the five fluorescent D-A systems of present interest in cell labeling applications in N2a cell line.
Anions find application in numerous areas such as biological, environmental, clinical and also in the field of catalysis.Hence, recognition of anions is of prime interest [19,20].Amongst various anions, cyanide ion is quiet useful in the field of textile manufacturing, metallurgy, herbicide preparation, silver production and also in gold extraction [21][22][23].Simultaneously, it is also a highly potent environmental pollutant due to its extreme toxicity towards mammals [24,25].Several analytical methods such as voltammetry, Induction of Coupled Plasma Mass Spectrometry (ICP-MS), selective ion electrodes, and atomic absorption/emission spectra [26][27][28] serve this purpose.But these methods suffer one or more disadvantages like complicated synthetic procedure, tedious purification methods etc.Here comes the need for the development of a convenient and robust detection method for CN − ion.In this context, the design and syntheses of small molecules exhibiting bright fluorescence as chemodosimeters for anions are the prime focus of research in supramolecular and anion coordination chemistry [29][30][31][32] since they are based on responses visible to naked eye.Moreover, these chemodosimeters work by drawing the benefit of the nucleophilic nature of cyanide ion.This led to the development of numerous chemosensors based on the color change as well as fluorescence response produced owing to the disturbance created by the nucleophilic addition of cyanide anion on olefinic double bond of the respective conjugated systems [33][34][35][36][37][38].
In this regard, we have unitized one of the D-A system of present focus with cyanoacrylic moiety as potential cyanide binding site, viz.CAROT as a representative candidate for fluorometric sensing of cyanide ion.To the best of our knowledge, this is the first cyanide chemosensor based on 1,3,4 oxadiazole/phenothiazine D-A system with cyanoacrylic moiety.Antimicrobial activity of these compounds was then determined in terms of mean diameter of the inhibition zone (in mm, known as the zone of inhibition).Zone of inhibition delivers a mean to classify the compounds according to the following classes [39] as shown in Table 1.

In-vitro Antimicrobial Study
The results of preliminary antimicrobial activities of the studied compounds are summarized in Fig. 1 and their zone of inhibition values are collated in Table 2.
The target compounds presented varying zone of inhibition (Fig. 2) against before mentioned bacteria.CARON displayed the highest zone of inhibition among the tested compounds against all the studied bacterial strains.The compound was strongly inhibitory against the bacterial strain Klebsiella pneumonia and moderately inhibitory against the other bacterial strains.Moreover, it was the only compound among the three which displayed an inhibitory action against antifungal strain, Aspergillus niger.Similarly, NOPON CARON that was identified as the most active among the tested compounds was subjected to the determination of minimum inhibitory concentration (MIC) against all the studied microbial strains and the results are collated in Table 3.

In-vitro Antituberculosis Study
Positive results on antimicrobial activity of the target compounds and th fact that the compounds incorporating 1,3,4-oxadiazole and phenothiazine core display excellent anti-tuberculosis activity [40], encouraged us to investigate the anti-tuberculosis activity of these compounds.Thus, all the three target compounds were screened for their inhibitory activity against the growth of Mycobacterium tuberculosis (MTCC No:6) strain by a method described in literature [41,42] with LB broth as negative control.All experiments were carried out in triplicate and the activity of the compounds are expressed in terms of their Minimum Inhibitory Concentration (MIC) values as shown in Table 4. MIC values obtained were in the range of 800-1200 µg/mL, with NOPON exhibiting the highest activity.

Validation of Antituberculosis Study by Molecular Docking Study
Molecular docking serves as an enormously valuable method for structure-based drug designing methods to find out targets for different ligands as well as the associated thermodynamic interactions with the target enzyme, particularly during the non-availability of resources to perform the experimental studies.Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis (Mtb).Mycocyclosin is a secondary metabolite that has a critical role in the survival of Mycobacterium tuberculosis [43].Biochemical pathways studies suggested that mycocyclosin synthase (CYP121) is the enzyme responsible for producing mycocyclosin by catalyzing C-C bond formation between the carbons ortho to the phenolic hydroxyl of cyclo (L-tyr-L-tyr) (cYY).This enzyme is encoded by the Cytochrome P450 121 gene.The involvement of CYP121 in essential pathways of Mtb marks it as a potential drug target to inhibit Mtb infections [44][45][46][47][48].Moreover, it could be considered as a probable target for the development of new drugs for TB since it is established to be essential for viability of the bacterium.Hence docking studies were performed with the cytochrome P450 cyp 121 enzyme of Mycobacterium tuberculosis, 3G5H to identify the binding mode of the target compounds into the active site of this enzyme.
Various binding interactions of CARON with the amino acid residues present in the active site of the enzyme are depicted in Fig. 3a.CARON showed van der Waals interaction with almost 20 amino acid residues present in the binding cavity of the studied enzyme.A couple of conventional hydrogen bonding interaction was obtained between the N atom of cyanide moiety and hydrogen atom of the carboxylic acid side chain of the cyanoacryllic group present in the compound with the donor atoms of the amino acid residues, ARG A 286 and PHE A 338 respectively.The π electron cloud of the aromatic rings of the phenothiazine moiety and 1,3,4 oxadiazole interacts with the PHE A 168 amino acid residue of the receptor molecule via π-π stacking interaction.
Similarly, the π electron cloud of the naphthyl moiety exhibits a π-σ interaction with the amino acid residues, TRP A 182 and ALA A 167. Also, the π electrons of the 1,3,4 oxadiazole moiety and the phenyl group of the naphthyl side chain is also involved in π alkyl contact with the VAL A 78 and VAL A 228 amino acid residues respectively.The octyl side chain of the compound is associated with the MET A 62 and VAL A 83 amino acid residues via an alkyl interaction.The binding score obtained for CARON was − 11.3 kcal mol − 1 (Table 5).
Likewise, Fig. 3b portrays the different binding interactions of NOPON with the amino acid residues present in the active site of the enzyme.To begin with, the compound shows van der Waals interaction with about 23 amino acid residues of the receptor.The π electron cloud of the one of the naphthyl moiety is involved in a π donor hydrogen bonding interaction with the donor atom of CYS A 35 amino acid residue.Whereas the π electrons of the other naphthyl moiety and the phenothiazine ring are engaged in π-σ interaction with the TRP A 182 and ALA A 167 as well as ALA A 233 amino acid residues respectively.Additionally, the π electron cloud of the naphthyl moiety is also involved in a π sulfur interaction with the S atom of the CYS A 345 amino acid residue.Similar to CARON, the octyl side chain of the NOPON is also associated with the ASN A 85 and CYS A 345 amino acid residues via alkyl interactions.Finally, to end with, the π electron cloud of the phenothiazine moiety is involved in π alkyl interaction with ALA A 233 and CYS A 345 amino acid residues.Hence the presence of naphthyl moiety in NOPON is responsible for most of the interaction of the same with enzyme.This may be attributed to the highest docking score of NOPON (-14.4 kcal mol − 1 ) among all the three target compounds.
BOPOB portrays the following binding interactions with the amino acid residues present in the active site of the enzyme as displayed in Fig. 3c.The compound shows van der Waals interaction with various amino acid residues of the receptor.The π electron cloud of the phenyl moieties is involved in a π donor hydrogen bonding interaction with the donor atom of CYS A 37 and ALA A 218 amino acid residues respectively.Additionally, the π electron cloud of the naphthyl moiety is also involved in a π sulfur interaction with the S atom of the CYS A 343 amino acid residue.Similar to CARON and NOPON the octyl side chain of the BOPOB is also associated with the ASN A 85 and CYS A 345 amino acid residues via alkyl interactions.Finally, the π electron cloud of the phenothiazine moiety is involved in π alkyl interaction with ALA A 213 and CYS A 343 amino acid residues.The docking scores of all the three target compounds are tabulated in Table 5.All the three compounds exhibited almost similar activity (docking score) and interactions in comparison to other reported compounds [49][50][51][52].

Cell Staining Studies
The excellent fluorescent emission properties exhibited by all five studied compounds [12,13] under investigation prompted us to investigate the potential capability of these compounds as new fluorescent cell staining agents for livecell imaging.Optical merit of the compounds alone is not sufficient for effective cell imaging, they should possess good cell compatibility as well.Therefore, before moving on to the imaging interrogations, the cytotoxicity of these compounds was evaluated based on two cell lines, U87 (Uppsala 87 Malignant Glioma), a human primary glioblastoma cell line, and N2a cell line, a fast-growing mouse neuroblastoma cell line by MTT assay.As shown in Fig. 4, on incubation of both the cell lines with different doses (10-500 ng) of the target compounds for 72 h, almost 90% of the cells are alive and this shows that the compounds are non-toxic, hold good biocompatibility and have the potential for intracellular bio-imaging.Now, to realize the probable capacity of the target compounds as visible bio-imaging probes, the cell imaging experiments was executed using the cancer N2a cell line by fluorescence microscopy.The cells were brightly illuminated retaining good morphologies after 6 h of incubation with 50 ng/mL of each target compound (Fig. 5).This may be attributed to the accumulation of these compounds inside the cell.CAROT, CAROP, CARON and BOPOB was found to selectively target the cell Cytoplasm, whereas NOPON seems to be targeting Endoplasmic Reticulum.Thus, the imaging study reveals that the target compounds are permeable to the cell.
In addition to this, the cells emit multicolor fluorescence (Fig. 5), i.e., blue, red and green color under lasers of different wavelength range of 430-490, 602-655 and 512-555 nm respectively.This multicolor emission widens the opportunities of these target compounds over other tag reagents since the former can provide a considerable space to choose the wavelength of observation.
Consequently, the cell viability and imaging studies sum up the possibility of these biocompatible target compounds as promising candidate for cell staining or bio-imaging applications in near future.

CAROT as "Turn Off" Fluorometric Sensor for Cyanide Ion Selectivity and Sensitivity of CAROT for Cyanide Ion
Exceptional selectivity and sensitivity are essential criterion for the practical application of any sensor.To perceive these matters, the fluorescence changes of the probe (3.3 µM) on addition of different anions (4.8 µM) was investigated.It was observed, the probe exhibited very high selectivity for cyanide ion compared to other anions.The cyanide ion portrayed a very noticeable quenching effect (Fig. 6a) on the fluorescence spectra of the probe, while the other anions resulted in negligible response to the fluorescence intensity of the probe.This signifies the interaction of the probe with the cyanide ion.Moreover, the cyanide ion induced fluorescence change stated above was naked eye detectable (Fig. 6b) under UV lamp (at 354 nm), which designates that CAROT can readily differentiate cyanide ion by remarkable fluorescence "turn-off" response.
Further insight into the sensitivity of the probe (CAROT) towards the analyte (CN) was deduced by the fluorescent titration experiment of the former (3.3 µM) with the latter  7a) and showed a very good linear relationship with CN − concentration with a correlation coefficient of R 2 = 0.989 as portrayed in Fig. 7b.The high affinity of the sensor towards cyanide ion is confirmed from these observations.

Binding Ratio, Binding Constant and Limit of Detection (LOD)
Assuming a favorable coordination between CAROT and cyanide ion for the reason behind fluorescence quenching, MALDI studies were performed to determine the stoichiometry of the binding ratio between the sensor and the analyte.An ESI-MS peak of the sensor-analyte complex was observed at m/z = 583 (Fig. 8b) which specified the probability of a 1:1 stoichiometry of CAROT (m/z = 557) to cyanide ion.The binding or association constant for the interaction of chemosensor CAROT with cyanide ion was estimated to be 2.75 × 10 6 M −1 from the data obtained from fluorescence titration studies described above and using the Benesi-Hildebrand (B-H) plot (Figure).The free energy change, (ΔG) was obtained as -45.98 kJ mol −1 .Similarly, the fluorescence limit of detection (LOD) and limit of quantification (LOQ) of CAROT for the selective detection of cyanide ion was obtained as 0.14 × 10 − 6 and 0.17 × 10 −6 M respectively from the Stern-Volmer plot.Fortunately, the limit of detection so obtained was much lower than the permissible limit of cyanide ion in drinking water (1.9 µM) prescribed by WHO guideline and at the same order of magnitude as reported for most of the "turn off" fluorescence cyanide ion chemosensor   [53].The obtained limit of detection value is compared with some of the previousy reported results (Table 6) [54−56].

Plausible Mechanism of Sensing
In view of the above results, Scheme 1 shows the conceivable mode of interaction between the probe (CAROT) and the analyte (CN-) inducing the fluorescence quenching.The sensor consists of a cyanoacrylic group as the side chain that is susceptible to a nucleophilic attack by cyanide ion.This binding could presumably suppress the Intramolecular charge transfer (ICT) that could occur from the phenothiazine-1,3,4-oxadiazole part of the sensor to the cyanoacrylic group contained in the same leading to the fluorescence turn off.The proposed mechanism is consistent with results obtained by the MALDI studies.

Interference Studies of Competitive Anions
Selectivity of the sensor, CAROT to cyanide ion was revealed from the preliminary fluorescent response studies of the same against different anions (Fig. 9a).Nevertheless, the furthermost important criteria for a selective anion chemosensor lies in its capacity to detect a specific anion in the vicinity of other competing ions.With a view to understand this, the sensor CAROT was subjected to competition studies in presence of other interfering anions.For this, the fluorescence intensity of mixed solution of sensor in presence of other interfering anions in acetonitrile solution was measured which displayed no change fluorescence intensity.But when the above solution was treated with 10 µM cyanide ion, the fluorescence intensity was significantly quenched.Thus, it is established that the fluorescence intensity remains unchanged even in presence of 100 equivalents of the competing anions which makes CAROT a selective fluorescent "turn-off" chemosensor for the detection of cyanide ion in the presence of competing ions.

Recognition time on Cyanide Sensing
Rapid response and short response time is an indispensable criterion for a designed fluorescent chemosensor to be employed in practical applications.Henceforth, time dependent fluorescent intensity response of CAROT towards cyanide ion was inspected in acetonitrile solution and the result is portrayed in Fig. 10.The study revealed that the addition of cyanide ion to the sensor solution quenched its fluorescence instantly and attained stability.This observation broadens the range of applicability of the present chemosensor regarding the rapid detection of cyanide ion in real samples.

Sensor CAROT-based test Strips
When it comes to the case of practical applications, the strip platform provides numerous advantages over the solutionbased platform.Hence, test strips were fabricated as mentioned in the experimental section for the detection of cyanide ion employing the sensor, CAROT.The test strip produced a visible change under the UV light only in presence of cyanide ion among the tested anions (Fig. 9b) even though the concentration of the other anions were 10 times the concentration of cyanide ion.Moreover, the test strips are easy to read and the result is stable and consistent.Thus, CAROT can be used as a solid indicator to detect cyanide ion in aqueous solution.

Conclusion
In conclusion, a series of D-A systems based on 1,3,4-oxadiazole-phenothiazine backbone has been evaluated for their various in-vitro biological activities.Antimicrobial and antituberculosis studies of the selected systems identified CARON and NOPON as respective compounds with highest activity.The antituberculosis study supplemented with molecular docking study revealed that these compounds can better interact with various groups present in the binding pocket of the cytochrome P450 CYP121 enzyme of Mycobacterium tuberculosis, 3G5H.Results of MTT assay and cellular imaging study revealed the non-toxic nature and permeability of these compounds towards cells and potential use of these compounds in cell staining applications.Finally, the anion sensing studies of CAROT with potential cyanide binding site revealed that it can serve as an excellent fluorescent chemosensor for cyanide ion in aqueous solution.On the whole, the present donor-acceptor systems serve as potential multifaceted bioactive compounds.

Synthesis of the 1,3,4-oxadiazole-based Donor-acceptor Systems
The synthesis and characterization of the target compounds, CAROT, CAROP, CARON, NOPON and BOPOB have been already published by our group [12,13] and Scheme 2 shows the structures of the studied compounds.

Materials and Methods
All the reagents and solvents were purchased either from Sigma-Aldrich or from Alpha Aesar and were used as received.
Infrared spectral studies were performed on a Jasco FTIR spectrophotometer.The absorption and emission spectra were recorded using Evolution 201 UV-Vis spectrophotometer and Horiba Fluorolog3 Spectroflurophotometer, respectively.MALDI-TOF mass spectrum was recorded using Bruker Autoflex max LRF.

Molecular Docking Studies
Molecular modelling studies were performed using the AUTODOCK 4.0 software as implemented through the graphical user interface AUTODOCK TOOLS (ADT 1.4.6).The crystal structure of the cytochrome P450 cyp 121 enzyme receptor of Mycobacterium tuberculosis was obtained from the PDB (http:// www.pdb.org/ pdb/ home/ home.do, PDB ID 3G5H at a resolution of 1.4 Å).In all docking, a grid box size of 60 × 60 × 60 Å pointing in X, Y and Z directions was build.The three-dimensional structure of the target compounds was generated from their optimized structures as FCHK file.The further conversion of the FCHK file to the PDB format was achieved with the help of OPEN BABEL-2.4.1 software.The AUTO-DOCK TOOLS program was used to generate the docking input files in pdbqt format and the molecular images was produced with the aid of Discovery Studio Visualizer 4.5 package.Muller Hinton Agar medium (HIMEDIA-M173) was used for determination of susceptibility of microorganisms to the studied antimicrobial agents.38 g of the medium was suspended in 1000 ml distilled water and heated to boil to dissolve the medium completely.Further, it was sterilized by autoclaving at 15 lbs pressure (121 °C) for 15 min.Finally, cooled to 45-50 °C, mixed well and poured into sterile petri plates.

Antimicrobial Studies of the Compounds
Potato Dextrose Agar MH096 Himedia used for determination of susceptibility of fungal strains to antifungal agents.31.55 g of it was suspend in 1000 ml distilled water and heated to boil to dissolve the medium completely.Later, sterilized by autoclaving at 15 lbs pressure (121 °C) for 15 min.Cooled to 45-50 °C, mixed well and poured into sterile Petri plates.
(A) Antibacterial assay by Agar well diffusion method.
Agar well diffusion method is widely used to evaluate the antibacterial activity of the test samples.Mueller-Hinton agar (15-20 mL) was poured on glass petri plates of same size and allowed to solidify.Standardized inoculum of the test organism was uniformly spread on the surface of the plates using sterile cotton swab.Four wells with a diameter of 7 mm (20 mm apart from one another) were punched aseptically with a sterile cork borer in each plate.The test sample (40 and 80 µL) was added into the wells T1 and T2 from 10 mg/ml stock.Gentamycin (40 µl from 4 mg/ml stock) and the solvent used for sample dilution were added as positive and negative control respectively.The plates were incubated for 24 h at 36 °C ± 1 °C, under aerobic conditions.After incubation, the plates were observed and the zone of bacterial growth inhibition around the wells was measured in mm [57].(B) Antifungal assay by Agar well diffusion method.
Agar well diffusion method is also widely used to evaluate the antifungal activity of the test sample.Mueller-Hinton agar (15-20 mL) was poured on glass petri plates of same size and allowed to solidify.Standardized inoculum of the test organism was uniformly spread on the surface of the plates using sterile cotton swab.Four wells with a diameter of 8 mm (20 mm apart from one another) were punched aseptically with a sterile cork borer in each plate.The test sample (40 and 80 µL) was added into the wells T1 and T2 from 10 mg/ml stock.Clotrimazole (40 µl from 300 mcg/ml stock) and the solvent used for sample dilution were added as positive and negative control respectively.The plates were then incubated for 24 h at 27 °C ± 1 °C, under aerobic conditions.After incubation, the plates were observed and the zone of bacterial growth inhibition around the wells was measured in mm [58].

(C) Determination of Minimum Inhibitory Concentration (MIC).
The MIC of CARON was determined through microdilution method as described by the Clinical and Laboratory Standards Institute (CLSI) [59].Briefly, 1% of test pathogens (0.4 OD at 600 nm) were added to Luria-Bertani (LB) broth (Hi-Media, Mumbai, India) containing serially diluted CARON at concentrations ranging from 1600 to 12.5 µg/ml and incubated at 37 °C for 24 h.The MIC was recorded as the lowest concentration of CARON that showed complete inhibition of bacterial growth as like medium control.

Antituberculosis Studies of the Compounds
Inoculums and Culture Media Details for Antimicrobial Studies Mycobacterium tuberculosis (MTCC No. 6, incubation 37 °C for 24 h) for antituberculosis studies of the target compounds were procured from the Microbial Type Culture Collection (MTCC) Chandigarh.

Preparation of Sample Stock Solution and Dilution
Range 10 mg/ml of the sample stock solution was prepared.The test sample was then diluted in LB broth to get a final concentration as 50, 100, 200, 400, 800, 1000 and 1200 mcg.Untreated LB broth was kept as negative control (NC).

Procedure
A single colony of organism, picked from a LB streak plate was dissolved in 3 ml LB broth and incubated overnight at 37 °C, 220 rpm and optical density at 600 nm, OD 600 was checked (1 OD 600 = 10 9 CFU/ml) with a spectrophotometer.The bacterial solution was then diluted with LB broth to get 0.1 OD 600 suspensions and incubated at 37 °C, 220 rpm till mid-log phase (~ 2 h).1ml of mid-log phase bacterial solution was put into in 1.5 ml Eppendorf tube and centrifuged at 6,000 rpm for five minutes and was washed with 1 ml PBS (Phosphate Buffer Solution).The washing procedure was repeated twice, and the bacterial pellet was dissolved with 1 ml LB broth.20 µl of the above bacterial solution was then mixed with 180 µl LB broth, OD 600 was checked with a spectrophotometer.The bacterial concentration was then adjusted to 1 × 10 7 CFU/ml with LB broth (1 OD 600 ~10 9 CFU/ml).Later 50 µl log phase culture of Mycobacterium smegmatis (~10 8 cfu) was added to all experimental wells and the bacterial cultures were inoculated uniformly to each well of 96 well plate containing different concentrations of the test samples.An uninoculated broth was kept as Blank and finally, all the tubes were incubated at 37 °C for 20-24 h.To end with, OD 600 was checked with a spectrophotometer and the MIC endpoint was read as the lowest concentration of test compound at which there is no visible growth of bacteria (no solution turbidity on naked eyes), and the absorbance measured keeping uninoculated broth as blank [41].
Details of cell lines used for the in vitro biocompatibility studies.

Details of Cell Lines Used for the In-vitro Biocompatibility Studies
U87 and N2a cell lines for the in-vitro biocompatibility studies were acquired from ATCC.

General Procedure for Cell Culture and MTT Assay
The cells were grown in High glucose DMEM (Himedia-AL007A) supplemented with 10% FBS (Sigma) and antibiotics at 37 degree C, 5% CO 2 .The cells were seeded in 96 well plates with the density of 10,000 cells per well and treated with the compound dissolved in ethanol to concentrations 10, 25, 50, 100, 250, 500 ng/mL.After 72 h of incubation with the molecules, MTT (Sigma) at a concentration of 0.5 mg/ml was added to the wells and further incubated for 4 h.The cells were lysed with DMSO and absorbance was measured at 590 nm.The percentage of cell viability was calculated using the following equation.

Cell Sample Preparation for Bioimaging Studies
N2a cells were treated with the dye at a concentration of 50 ng/mL and incubated for 2 h at 5% CO 2 at 37 ˚C.Plates were washed with PBS and cells were fixed with a 1:1 mixture of methanol and acetone at -20 ˚C for 20 min.The cells were washed once with PBS and stored in 10% glycerol and were visualized under fluorescent microscopy with various emission and excitation range.

Anion Sensing Studies
Motivated by the importance of anion sensing and also based on molecular structure and photophysical properties [12,13] of the discussed organic compounds, one of the compounds, CAROT was selected as a suitable candidate for anion sensing application.
The stock solution of CAROT was prepared in acetonitrile with the concentration of 1 × 10 − 5 M and further diluted according to the requirement of each experiment.The aqueous solutions of different anions like F − , Cl − , Br − , NO 2 − , OAc − , CN − , N 3 − , C 2 O 4 2− , CO 3 2− , SO 4 2− and SCN − with a concentration of 1 × 10 − 3 M were prepared using their sodium salts.Fluorescence spectra of CAROT in presence of anions was used to investigate the selective response of the sensor towards various anions.For this, 150 µL of the aqueous solution of each anionic species was added to the 1 mL acetonitrile solution of CAROT and made up to 2 mL as final volume using acetonitrile solvent.After mixing them for 10 s, the fluorescence spectra were recorded at room temperature.Similarly, the sensitivity of CAROT for cyanide ion was studied by the fluorometric titration experiment of CAROT (3.3 µM in CH 3 CN) with aqueous solution of cyanide ion of 0.0-7.4µM concentration.
The binding ratio of cyanide ion with the sensor was determined by recording the MALDI-TOF spectrum of the sensor CAROT in presence and absence of cyanide ion.CAROT-CN solution was prepared by taking 1:1 stoichiometry using sinapinic acid as the matrix.Binding constant and Gibb's free energy was determined from the fluorometric titration studies and using Benesi-Hildebrand equation [60] as follows: Here the terms I and I 0 are the fluorescence intensity of the sensor, CAROT with a specific targeted anion concentration and in the absence of the anion respectively.Where, Ks, [CAROT] and [CN − ] are the association constant, concentration of CAROT and concentration of cyanide ion respectively.1/[CN − ] Vs 1/(I 0 -I) plots is then constructed which is identified as the B-H plot.The ratio of the intercept and the slope of the B-H plot give the binding constant.Then the free energy change, ΔG at experimental temperature (298 K) can be obtained from the relation, The limit of detection (LOD) and limit of quantification was calculated by the equation; LOD = 3σ/S and LOQ = 10σ/S where σ is the standard deviation of the blank measurements (10 runs) and ρ, is the slope between intensity versus sample concentration.

Test Strip Preparation
Whatman 40 filter papers were tailored to suitable size (5 cm x 2 cm) and immersed in acetonitrile solution of the sensor (1 × 10 − 5 M) for 2 h and thoroughly dried in air at room temperature.

Fig. 3
Fig. 3 Docked images of the a CARON, b NOPON and c BOPOB* (shown inside circles) in the active pocket of the cytochrome P450 cyp 121 enzyme of Mycobacterium tuberculosis, 3G5H and the various binding interactions of the respective compounds with the amino

Fig. 4 Fig. 5
Fig. 4 Percentage of cell viability of the five target compounds against U87 and N2a cells

Fig. 6 aFig. 7 a
Fig. 6 a Fluorescence emission spectra of CAROT (1 × 10 − 5 M) in acetonitrile upon the addition of 150 µL of various anions and b Fluorimetric responses of CAROT in (1 × 10 − 5 M) in acetonitrile under UV lamp, upon the addition of various anions

Fig. 8
Fig. 8 MALDI spectra of CAROT in the a absence and b presence of cyanide ion and c Benesi-Hildebrand linear analysis plot of CAROT (3.3 µM) with varying concentration of cyanide in acetonitrile

Scheme 1 Fig. 9 a
Scheme 1 Proposed binding mechanism of CAROT with cyanide ion

Table 1
Classification of compounds based on the value of their zone of inhibition

Zone of inhibition (in mm) Activity of the compound
Fig. 1 Zone of inhibition of the standard drug, Gentamycin, CARON, NOPON and BOPOB against the bacterial strains Staphylococcus aureus, Bacillus cereus, E. Coli, Klebsiella pneumonia, and Aspergil-lus niger and zone of inhibition of standard drug, Clotrimazole and CARON against fungal strain Candida albicans

*Gentamycin: Standard drug for antibacterial studies and Clotrimazole: Standard drug for antifun- gal studies, Sa: Staphylococcus aureus, Bc: Bacillus cereus, Ec: E. Coli, Kp: Klebsiella pneumonia
, An: Aspergillus Niger, Ca: Candida albicans; NA: Not Active Compounds Microorganism and Inhibition zone (mm) Fig. 2 The petri plate images of CARON, NOPON and BOPOB with different bacterial and fungal strains

Table 4
Minimum inhibitory concentration (MIC) values of CARON, NOPON and BOPOB against Mycobacterium tuberculosis

Table 5
The docking score of the target compounds with the active site of the 3GSH enzyme