Exploration of novel TOSMIC tethered imidazo[1,2-a]pyridine compounds for the development of potential antifungal drug candidate

New candidates of imidazo[1,2-a]pyridine were designed by combining 2-amino pyridine, TOSMIC and various assorted aldehydes to explore their antioxidant and antifungal potential. The design of these derivatives was based on utilizing the antifungal potential of azoles and TOSMIC moiety. These derivatives were synthesized by adopting multicomponent reaction methodology, as it serves as a rapid and efficient tool to target structurally diverse heterocyclic compounds in quantitative yield. The resulting imidazo [1,2-a]pyridine derivatives were structurally verified by 1 HNMR, 13 CNMR, HRMS, and HPLC. The compounds were analyzed for their antioxidant and fluorescent properties and it was observed that compound 15 depicted highest potential. The compounds were evaluated for their antifungal potential to highlight their medical application in the area of Invasive Fungal Infections (IFI). Compound 12 gave the highest antifungal inhibition against Aspergillus fumigatus 3007 and Candida albicans 3018. To elucidate the antifungal mechanism, confocal images of treated fungi were analyzed, which depicted porous nature of fungal membrane. Estimation of fungal membrane sterols by UPLC indicated decrease in ergosterol component of fungal membrane. In silico studies further corrobo-rated with the in vitro results as docking studies depicted interaction of synthesized heterocyclic compounds with amino acids present in the active site of target enzyme (lanosterol 14 alpha demethylase). Absorption, distribution, metabolism, and excretion (ADME) analysis was indicative of drug-likeliness of the synthesized compounds.

The synthesis of imidazo [1,2-a]pyridine structure remains a subject of intense research and efforts have been made to develop new synthetic approaches (Devi et al., 2016) which includes multicomponent reaction, condensation reaction, oxidative coupling, tandem reaction etc. From this point of view, multicomponent reactions (MCR's) in combination with post cyclizations is a powerful tool to access complexity and diversity in one step (Ahmadi et al., 2017;Neochoritis et al., 2019;Rotstein et al., 2014;Kumar et al., 2010) to form imidazo [1,2-a]pyridine. Among these synthetic strategies, imidazole synthesis merged with TOSMICs has been recognized as one of the most significant building blocks in N-heterocyclic synthesis. It has also been acknowledged that the combination of any two privileged scaffolds potentially creates more active entities with enhanced biological properties (Ji et al., 2008), such types of fused heterocyclic compounds have been reported as important antifungal agents (Fuentefria et al., 2018). An estimated 1.5-2.0 million people die of fungal infections each year and approx 1.2 billion individuals are affected by fungal disease each year (Fuentefria et al., 2018). These emerging threats of fungal infections, direct the synthesis of novel chemical entities with antifungal potential.
Literature witnessed many reported imidazo [1,2-a]pyridine as antifungals, yet in many instances either the potential of the reported compound is minimal or the complex methodology limit its use. The importance of imidazo [1,2-a]pyridine core as prominent antifungal is also evident from the fact that this nucleus is prevalent in various reported derivatives as shown in Figure 1 (Al-Tel et al., 2011;Jafari et al., 2017). Encouraged by these observations and in continuation of our research work to discover novel compounds (Azad & Saxena, 2015;Azad et al., 2016) and by the lead from our previous publication (Shukla et al., 2019), the authors aimed to synthesize TOSMIC fused imidazo [1,2-a]pyridine compounds by using Groebke-Blackburn-Bienayme (GBB) multicomponent reaction. GBB reaction is a three-component-reaction (3CR) to afford the highly complexed structures by using 2-aminopyridine, aldehyde and an isocyanide and proceeds through subsequent formal [4 + 1] cycloaddition to form highly substituted heterocyclic compounds in one pot. Therefore, in the present work the author's attempt to design an ecofriendly methodology for the synthesis of TOSMIC fused imidazo [1,2-a]pyridine compounds. All the synthesized analogs were characterized by 1 HNMR and 13 CNMR. The compounds were additionally evaluated for their antioxidant and fluorescence potential. All the synthesized compounds were tested against both filamentous (Aspergillus fumigatus 3007) and unicellular (Candida albicans 3018) fungi for their antifungal effect. The potential antifungal compounds were analyzed by HPLC for their purity. Fungal membrane permeabilization was examined by observing the stained mycelia under confocal laser scanning microscope. Additionally, to strengthen our understanding of the antifungal effect of the synthesized compounds sterols were extracted from compound treated fungus and estimated using Ultra High Performance liquid chromatography (UPLC). To further establish the interaction of the compounds with fungal system, in silico studies were conducted which depicted interaction of the compounds with amino acid present in the fungal enzyme system. Computational methods predicting drug-likeliness of the compounds were used along with determination of absorption, distribution, metabolism, and excretion (ADME) properties.

| Chemistry
In this article, the reaction conditions were optimized using 2-amino pyridine (1, 1 mmol), anisaldehyde (2, 1 mmol), and TOSMIC F I G U R E 1 Examples of imidazo[1,2-a]pyridine derivatives as antifungals (3, 10 mmol) as the model substrate for the reaction. The reactants were dissolved in ethanol and the reaction was allowed to remain on stirring at room temperature in the absence of any catalyst for 10 h.
The reaction was monitored by TLC and it failed to generate the desired product even after 10 h (Table 1, entry 1). Initial effort was to screen the optimal catalyst for the reaction, therefore the same set of experiments was then performed with Sc(OTf) 3. Reaction was catalyzed with 10 mol% of Sc (OTf) 3 , it afforded the desired product in 67% of yield (Table 1, entry 2). With the satisfactory result, the reaction was further screened for different catalyst to optimize the reaction conditions, the experimental results are summarized in Table 1 (entries 3- 10). Among the screened catalyst L-proline was found to be the best for the reaction in terms of yield and reaction time (Table 1, entry 10), this encouraged the authors to use L-proline as a catalyst for the present reaction. Screening of mol% of catalyst loading is mandatory for the reaction as it affects the yield considerably. When 20 mol% of Lproline was loaded the yield of the reaction increased significantly ( ting the desired compound in 90% yield by complete conversion of the reactants into products it was established that the reaction medium influenced the process, where L-proline was used as a catalyst and EtOH as a solvent. Interestingly, the use of L-proline not only catalyzed the formation of iminium ion, but also helps in the transformational diversity . With the optimized conditions in hand, the aldehydic substrate scope on the reaction system was studied ( Based on literature reports (Dömling et al., 2012) and experimental results, the authors proposed a plausible mechanism for the formation of N-(2-(4-methoxyphenyl)imidazo [1,2-a]pyridin-3-yl)- Figure 2.

4-methylbenzenesulfonamide (4) in
Initially, the mechanism involves the reaction between anisaldehyde [2] and L-proline leading to the formation of an iminium electrophile which when reacts with the nucleophile (2-amino pyridine [1]

| Antioxidant activity
Among the synthesized compounds, compounds 7, 11, 9, 15, and 13 were believed to be responsible for good antioxidant activity as compared to other synthesized compounds. DPPH inhibitory test was conducted at six different concentrations. The inhibitory effects of synthesized compounds on DPPH are shown by IC 50 values, calculated and shown in Table 3. Significant change in activity was observed with different functionalities of imidazole cascade. The compound (15) demonstrated the potential antioxidant activity when compared to the reference ascorbic acid because of the presence of fivemember ring substitution (pyrrole) adjacent to imidazole ring, which stabilizes the unpaired electron and enhances the antioxidant potential of the molecule. Compound (7) having single methoxy on the 4-position of phenyl ring also showed comparable activity with that of reference due to the presence of electron donating methoxy group as reported in literature (Naik, 2012). The lower activity of compound 11 and 9 made us infer that the presence of bulkier naphthalene ring decreases the antioxidant potential of the compounds (Brullo et al., 2012). The authors found that although compound (13) possesses 2-methoxy group with naphthalene, does not favors for the enhanced activity as this method is selective for the small molecule, and bulkier group shows slow or inert reaction toward antioxidant (Lloyd et al., 1966). The other derivatives of aldehyde eventually hamper the antioxidant activity regardless of the substituent present.

| Analysis of fungal membrane permeabilization
Cell membrane disruption by inhibition of fungal ergosterol biosynthesis pathway has been documented as the mode of action of azole antifungal agents (Geißel et al., 2018). The cell membrane integrity and porous nature can be determined on the basis of cellular ability to exclude or penetrate dyes by live cells and damaged/necrotic cells (Dananjaya et al., 2017). In the present study, PI dye was used to examine the fungal membrane integrity and thus to generate an insight for a probable antifungal mechanism by the novel synthesized compounds. PI is a plasma membrane impermeable and DNA staining fluorescent probe, which when enters into the cell, intercalates between the DNA bases giving fluorescence (Fosso et al., 2015). In  7 and 8). A. fumigatus 3007 cells were grown in the presence of compound 12 for 96 h and eventually exposed to PI dye. The prepared slides when observed under CLSM depicted fluorescence ( Figure 7). The same section under bright field showed fungal mycelia and when the two images (bright field and fluorescent) were superimposed it was apparent that the fluorescence was emitted from within the cells. The untreated fungal mycelium did not display ant detectable red colored hyphae, thus indicating an uncompromised and intact fungal membrane. Similarly, when C. albicans 3018 cells were exposed to compound 12 for 24 h, it was observed that the cells gave fluorescence upon staining with PI ( Figure 8) F I G U R E 1 0 (a) Ribbon representation of CYP51 from Aspergillus fumigatus (yellow) and (b) Candida albicans (green) docked to compound 12 (tan ball and stick model) highlighting the protein-ligand interactions within 5 Å (shown in pink) alongwith hydrogen bonds in red fungal growth inhibition and the probable mechanism of the compounds.

| Fungal sterol composition
To further strengthen the understanding of the effect of synthesized compounds on the structure of fungal membrane, estimation of fungal sterols in the presence and absence of compounds was carried out.
Taking into consideration the multistep sequential synthesis of ergosterol, it is apparent that inhibition of enzyme lanosterol 14α demethylase would lead to the accumulation of lanosterol and thus reduction of ergosterol in fungal system. The sterol profile graphs ( Figure 9) obtained after the UPLC analysis were consistent with the given hypothesis. In the control sample (without the compound)-64.91% and 30.78% peak area was reported for lanosterol and ergosterol, respectively. However, when the fungus was grown in the presence of compound 12, there was marked reduction in the peak of ergosterol (7.92%) with an increase in the peak of lanosterol (90.79%), thus highlighting the inhibition of target enzyme (lanosterol 14 αdemethylase) by the synthesized compound. The depletion of ergosterol from fungal membrane leads to structural deformities resulting in fungal death.  12 (Figure 10a). Similarly, a good binding energy value of À51.28 kcal/mol was seen for CYP51-compound 12 dock complex from Candida. Analysis of the binding site showed the presence of both hydrophobic and polar amino acids with His 377 and Tyr 505 forming hydrogen bonds with the compound (Figure 10b). Further, the interaction of compounds with human counterpart of CYP51 enzyme showed many compounds with comparable binding to the target protein. However, as anticipated compound 12 displayed a lower binding to human CYP51 and is, thus, expected to preferably bind to fungal enzymes over its human counterpart.

| In silico analysis
The physicochemical properties of a compound are key determinants of its pharmacokinetic behavior (ADME: absorption, distribution, metabolism and excretion). In silico prediction of such properties is a critical step in drug discovery process and aids in screening the most promising compounds among many. Our in-house compound library was evaluated for the physicochemical properties using  (Table 6).

| Analysis of antioxidant activity
Determination of DPPH radical-scavenging activity: The radical inhibition action of the synthesized compounds was accessed by DPPH assay method. The DPPH method was selective for the antioxidant activity because it is the most effective method for calculating radical scavenging capacity by a chain-breaking mechanism (Abdel-Wahab et al., 2011). As indicated by the methodology, 3 ml of 0.004% DPPH was mixed with 1 ml of (1-500 μg/ml) standard or synthesized compounds. The standard used for activity is ascorbic acid.
The solutions were well mixed and the reaction was incubated in dark for 30 min at RT. Furthermore, the absorption activity was analyzed spectrophotometrically at 517 nm. The absorbance value of DPPH in methanol alone was presented as absorbance of blank. The absorbance value of compounds and standard when mixed with DPPH in methanol was presented as absorbance of sample and standard, respectively.
The percentage radical scavenging activity was computed by the formula: %Radical scavenging activity where 'D b ' is colony diameter in control and 'D a ' is colony diameter in the test plates. and incubated overnight with shaking at 35 C. One hundred microliters of actively growing yeast culture was then transferred to 900 μl of double distilled water and readjusted to achieve OD 600 of 0.12 ($1 Â 10 6 cfu/ml). The cell suspension was further diluted to achieve 1 Â 10 3 to 5 Â 10 3 cfu/ml in RPMI1640 medium [RPMI medium was made as per the CLSI guidelines and was buffered to pH 7.0 with and their physicochemical properties were calculated using QikProp. Prior to molecular docking, ligand structures were prepared using LigPrep module (Sastry et al., 2013). The structures of target proteins were prepared using protein preparation wizard (Schrodinger, LLC, New York, NY, 2019) which involved addition of hydrogen atoms, assigning of bond orders, formation of disulfide bonds and removal of heteroatoms while retaining heme cofactor followed by energy minimization and refinement.
Subsequently, molecular docking was done using extra-precision (XP) mode of Glide (Friesner et al., 2006) where ligand molecules were docked into the binding site defined by the cocrystallized (inhibitor) molecule. A binding free energy (ΔG bind) for each docked pose was further estimated using Prime MM-GBSA method ENREF_26 (Rastelli et al., 2010).