Functionalization of 3-chloroformylcoumarin to coumarin Schiff bases using reusable catalyst: an approach to molecular docking and biological studies

Recently, heterogeneous catalysts have been explored eximiously in the synthesis of heterocyclic compounds. Therefore, here we used solid-supported heterogeneous silica sulfuric acid as a catalyst for the synthesis of Schiff's base of 3-chloroformylcoumarin in view of simplified procedure, reusability and acceptable efficiency, which are required in organic synthesis. An efficient and facile methodology is preferred for synthesis of a class of chromeno-3-substituted derivatives (1a–1l) with good yields. The molecular docking results showed excellent binding interactions with the Mycobacterium tuberculosis InhA-D148G mutant (PDB: 4DQU). The same biomolecules were screened for their in vitro anti-tubercular activity against the M.tb H37Rv strain and antimicrobial studies. Physico-chemistry, toxicity prediction with IC50 value and bioactivity score were also calculated for title compounds. Most active compounds were further tested for cytotoxicity studies and exhibited low-level cytotoxicity against Vero cells. The suggested conjugates are promising lead compounds for the subsequent investigation in search of new anti-tubercular agents. All the conjugates were obtained within the range and followed the Lipinski rule of 5, indicating more ‘drug-like’ nature.


Introduction
Owing to the growing concern of chemicals and their impact on the environment, cleaner reaction conditions in chemical synthetic procedures are needed to be incorporated. The intensive endorsement to maintain greenness requires us to avoid secondary substances (e.g. organic solvents, additional toxic reagents), to stop the overproduction of waste and minimize the consumption of energy [1]. Recently, the use of heteropolyacid catalysts, especially reusable solid catalysts, has gained a leading role in organic synthesis due to their environmental and economic considerations, and industrial utilization [2]. The high catalytic activity, moisture sensitivity, reusability and notably low cost makes solid-supported reagents attractive substitutes to conventional Lewis acids [3,4]. A number of synthetic schemes have been described for the synthesis of various heterocycles including the condensation of aldehydes with substituted aromatic amines [5][6][7][8]. However, most of these reports have some flaws such as lengthened reaction times, use of overpriced toxic solvents/chemicals, harsh reaction conditions, occurrence of several side products and/or lesser yields. Although the chemical utilization of solid-supported reagents for organic synthesis has been well explored, there are relatively few literature reports on the use of silica sulfuric acid (SSA) [9][10][11][12][13][14].
SSA is an acid catalyst, simply prepared by using chlorosulfonic acid with silica gel at ambient temperature (scheme 1) [15]. Easy handling, low price, efficiency, recoverability and reusability make SSA eco-friendly and a synthetically acceptable catalyst. We recognized that SSA would be an excellent proton entity compared to all the reported acidic reagents or acidic resins like polystyrene sulfonic acid and Nafion-H [16] under heterogeneous reaction conditions. SSA enhances reactivity and selectivity and has synthetic applicability in organic reactions such as oxidation [17], formation of carbon-carbon bonds [18,19], cycloaddition, [20] protection-deprotection steps [21,22], esterification [23] and the synthesis of heterocycles [24]. SSA is easier to handle than other acidic reagents and can be readily taken out of the reaction mixture by simple filtration. Additionally, it is recyclable and may be applicable on an industrial scale in pharmaceuticals. Therefore, we expanded the applications of SSA as a new versatile heterogeneous acid as a reusable catalyst for the title compound synthesis.
On the contrary, tuberculosis (TB) is a foremost communicable and infectious airborne, contagious, deadly disease caused by the pathogenic bacterium Mycobacterium tuberculosis (M. tb) of the 'tuberculosis complex' [25], which had evolved sometime around the seventh millennia BC, but has been making recent appearances and this malady has not yet been able to be completely eradicated. Robert Koch identified M. tb as the 'white plague' in 1882 [26]. It has attained epidemic proportions over wide geographical regions in the world. According to the World Health Organization, 10-12 million new TB cases are detected every year and has been estimated to be a major cause of death (2-3 million/year) [27,28]. A high rate of new cases of TB infections and deaths in HIV-positive patients gives a compelling view of AIDS in developing countries [29]. TB treatment lacks new medications, even after the development of potent drug therapy for M. tb treatment. The current drugs available in the market for standard TB treatment are limited by certain formidable challenges including lengthy treatment of 6-9 months, multiple drug regimens and chemical side effects [30]. The problem is becoming worse by increasing resistance to standard available drugs and synergy of this disease with HIV and infections in immunocompromised patients [31]. Therefore, latent TB patients are more affected by HIV because HIV weakens their immune system and makes them much more prone to developing active TB. Patients who are co-infected with HIV have an up to 800 times more chance of becoming infectious with active TB [32]. The constant increase in multidrug-resistant strains of M. tb has additionally contributed to the demand for new anti-TB drugs. However, over a decade no new TB drugs have been introduced into clinical use. Drugs active against resistant forms of TB are less effective and more toxic, and need to be taken for an extended regimen of up to 18 months [33]. This has inspired us for new efforts to find potent anti-TB drug candidates, which comprises developing pipelines for drug discovery and enhancing the ease of synthesis, in particular trying to implement new treatments that can considerably shorten the duration of effective therapy, which would improve patient compliance and survivability with novel mechanisms of action [34][35][36].
Coumarin is an oxygenated heterocycle; structurally it is the least complex component and forms a huge group of conjugates belonging to the flavonoid class of plant secondary metabolites, which have a special role in nature with their wide spectrum of biological applications [37]. They have attracted increased attention in recent decades for their diverse pharmacological properties. Among all properties, their cytotoxic effects have been most extensively studied [38,39]. Naturally occurring calanolide A and B are conjugates of coumarins that have evoked appreciable interest for their dual activity against TB and HIV infections [40]. Some antibiotics viz. novobiocin, clorobiocin and coumermycin  It is evident from earlier reports that substitution of coumarin at all positions except in the one and two positions with various functionalities has led to potent anti-TB activity. The alkyl substituents at the third position may change to aryl and heterocyclic groups, and evinced excellent anti-TB activity. Especially, coumarin bearing substitutions at the third and fourth positions was found to be more active due to conjugation [41]. Cardoso et al. reported a series of N-benzylidene-2-oxo-2H-chromene-3carbohydrazides as substituents at the third position of coumarin, and examined their non-infected cell viabilities and anti-TB performance against M. tb, and the results are compared to pyrazinamide (PZA), which is a first-line anti-TB drug. Compound (5) in figure 1 exhibited an antimycobacterial activity at 50 mg ml −1 and was found more active than the reference drug PZA [42].
Imines with a hydroxyl moiety of aromatic heterocycles are of particular interest. It is known that bioactive compounds bearing an imine group, with variation in substitutions, are important structures because of their biological properties such as anticancer and anti-tubercular activities [43]. The reaction of heterocyclic aldehydes and substituted anilines resulted in substituted heterocyclic Schiff bases. This type of building block is able to coordinate with metal ions and hydrogen bonds, and accept protons at the cellular level. Along with enzymatic interactions and receptors, it also controls physico-chemical properties of desired molecules to exhibit a broad spectrum of bioactivity. Hence looking into the biological significance of coumarins, particularly in the field of TB, we anticipate that coumarins could be a good starting point for the development of new lead anti-tubercular drugs. Figure 2 presents the structures of some potent coumarin scaffolds exhibiting anti-tubercular properties.
Today, the Lipinski rule of 5 (RO5) is widely used by medicinal chemists worldwide to evaluate not only the absorption of compounds but also specific drug similarity [44]. Hence, considering the highlights of RO5, we designed coumarin compounds and analysed their physico-chemical properties set by RO5, drug-likeness, toxicity prediction with IC50 value and bioactivity scores. It was found that all the derived conjugates were obtained within the frame of RO5. Considering the diverse biological and physico-chemical properties of coumarin compounds, there has been a growing interest in the synthesis of coumarin Schiff bases. It was thought that these two active pharmacophores, i.e. coumarin and aromatic amines, linked together through an imine bond would generate novel molecular templates with the most potential to exhibit anti-tubercular properties. Hence, our present strategy is to prepare coumarin derivatives having all these subunits in one structural frame (figure 3) which might exhibit enhanced synergistic effect and activity. The broad spectrum of their biological activity makes them a promising subject for the synthesis of new derivatives to identify lead bioactive compounds. The ongoing work uses the (M. tb) H 37 Rv strain to screen the anti-tubercular property of synthesized coumarin drugs.   Thus, in the view of the current interest in environmental protection, we extended environmentally benign heterogeneous acid catalysts for simple synthesis of coumarinyl Schiff bases. These derivatives (1a-1l) are examined for in vitro anti-tubercular, antibacterial and antifungal activities.

Material and methods
All the chemicals were purchased commercially. The open capillary method was used for detection of melting points, which are uncorrected. IR spectra were recorded using a Nicolet 5700 FT-IR (Nicolet, Madison, WI, USA) with KBr discs for all derivatives. 1 H and 13 C NMR spectra were recorded with a Bruker 400 MHz spectrometer using CDCl 3 /dimethylsulfoxide (DMSO) as solvents and are reported as δ values (ppm). Mass spectra were recorded with a Shimadzu GCMSQP2010S. The elemental analyses were carried out with a Hereaus CHN rapid analyser. Thin-layer chromatography (TLC) was used during the reaction for monitoring the progress of the reaction.

Chemistry
The conjugate of coumarinyl Schiff bases (1a-1l) was synthesized by using SSA. At first, 3formylchlorocoumarin (1) was obtained efficiently in good yields by the Vilsmeir-Hack formylation reaction of 3-acetylcoumarin. Further, 3-formylchlorocoumarin (1) (1 mmol) with substituted anilines (a-l) (1 mmol) in 10 ml ethanol and a catalytic amount of SSA at room temperature (RT) afforded 3-((1Z,14E)-1-chloro-3-(phenylimino)prop-1-enyl)-2H-chromen-2-one (1a-1l) under the conventional method, as is stated in scheme 2. It was observed that use of the SSA approach proved to be extremely fast, providing good to excellent yields (58-78%). The results are stated in table 1. The most evident advancement in the synthesis was the speed at which the reaction proceeded; the reactions were completed within 3 to 4 h. Optimization was also featured in different polar protic and aprotic solvents (table 2). The reaction carried out in acetonitrile gave the product in lesser yield at RT (entries 1 and 8). Use of solvent ethanol yielded the product with a shorter reaction time of 3 h at RT with an overall yield of 78% (entry 2). Further, dioxane in 6 h yielded the product with 38% yield (entry 4); in the case of tetrahydrofuran (THF) traces of the product were obtained, whereas no product was obtained in dimethylformamide (DMF), DMSO and acetone solvents even after a longer reaction time of 12 h. In the absence of SSA, even after longer time the reaction did not initiate at room temperature, resulting in incompletion of the reaction (entry 9). The reaction was carried out with silica alone, and a longer time for the completion of the reaction was noted. This may be due to the less acidic property of silica (entry 10). Here we generally saw evidence of imide formation in instances with excellent yields. This allowed us to state a mechanism for the formation of 1a using SSA as shown in scheme 3. Initially, the attack of electrons of aniline on the aldehydic carbon of coumarin takes    place. In the consecutive step, the protonation occurs from SSA, forming itself as a nucleophile in the pool of reaction mixture. The moment water is eliminated from the reaction mass, the nucleophilic SSA abstracts protons from nitrogen and gains stability by the formation of a double bond between C and N.
To the best of our knowledge, this represents the first time that SSA has been used to catalyse the direct Schiff's bases of 3-formylchlorocoumarins and substituted anilines under normal reaction conditions. However, the fact that there was an example where SSA proved to be a catalyst suitable for the cleavage of the carbon-nitrogen double bond of Schiff's bases in dioxane under conventional heating at reflux conditions raised a concern about our postulate that the overall conversion is merely a direct formation of double bond that happens when a catalytic amount is used at RT, rather than a cleavage of the double bond [45]. For example, the excess amounts of SSA at reflux is a condition which limits the applicability of such protocols. Therefore, the development of a new method which is free from such a problem is necessary. To understand the mechanistic insight further, several experiments were repeated with lesser SSA equivalents. Here we generally observed the formation of imide and in instances where a good yield was observed.

Results and discussion
Chromeno-3-substituted Schiff's base hybrids were confirmed by spectroscopic analysis, as in the case The IR spectrum exhibited a band at 1723 cm −1 assignable to lactone carbonyl stretching, whereas the -C=N stretching appears at 1600 cm −1 .
Depiction of the product was further confirmed by the 1 H NMR spectrum, wherein one singlet corresponding to C4-H of coumarin appeared in the downfield region at δ 9.179 ppm. Two doublets corresponding to C13-H and C5-H of coumarin resonate at δ 8.670 and 8.055 ppm (J = 7.6 Hz); adjacent to it another sharp triplet was observed at δ 7.765 ppm (J = 8.4 Hz), which is assigned to the C7-H of the coumarin ring; next to it another doublet was observed at δ 7.553 ppm (J = 7.2 Hz), which corresponds to C8-H of coumarin. The C6-H of coumarin resonate as a triplet at δ 7.430 ppm (J = 7.6 Hz and 7.2 Hz), whereas the C12-H resonate as a doublet at δ 6.417 ppm. The remaining aromatic protons resonate in their expected aromatic region at δ 7.186-7.343 ppm. 13 C NMR provides additional support for the structure of 1a. The carbon of the lactone carbonyl The bond between the C11 and the Cl atom is polar, and significant fragmentations take place on these carbons, giving a peak at m/z 274. The mass peak at m/z 44 is due to elimination of CO 2 . All the remaining coumarin derivatives furnished satisfactory spectroscopic and analytical data. All data are in accordance with the assigned structures and are stated in the experimental section.

Biological evaluation
The coumarin derivatives were examined for the potential in vitro anti-tubercular properties against the M. tb H 37 Rv strain by the Microplate Alamar Blue Assay (MABA) [46]. The most active derivatives found were tested for their cytotoxicity against Vero cells by the MTT [47] assay. Further, title compounds were tested for their antifungal and antibacterial properties by the disc diffusion method. The molecular docking study was used to find out the interactions of small coumarin-derived molecules and receptors in proteins. The crystal structure of the M. tb InhA-D148G mutant (PDB ID: 4DQU) in complex with NADH (2.45 Å X-ray resolutions) was used for this study. Physico-chemical, in silico toxicity prediction with IC50 value and bioactivity score were also calculated for the title compounds.

Anti-tubercular screening
The title (1a-1l) compounds were initially examined for in vitro anti-TB activity at a concentration of 6.25 µg ml −1 against the M. tb H 37 Rv strain in BACTEC 12B medium using the MABA. Compounds exhibiting inhibition ≥ 90% in the initial evaluation were tested at below 6.25 µg ml −1 using twofold dilution in the range of 3.12-0.2 µg ml −1 to find out the actual minimum inhibitory concentration (MIC). The anti-tubercular results are presented in table 3. In the primary screening, most of the compounds (1b, 1c, 1e, 1h, 1i and 1k) displayed 90-100% inhibition. At the second level, two derivatives (1e and 1k) showed inhibition with MIC < 0.2 µg ml −1 and four compounds 1b, 1c, 1h and 1i with MIC < 2 µg ml −1 , when checked with standard isoniazid (MIC 0.02 µg ml −1 ). From table 3, it is observed that the electronreleasing -OCH 3 -substituted compounds (1e) and (1k) have shown more significant inhibitory activity with an MIC of 0.05 and 0.19 µg ml −1 , respectively. The activity increased with the change in the position of -OCH 3 group in the following sequence p-OCH 3 > m-OCH 3 . The halogens -Cl and -Br, substituents at the para and meta position of the phenyl ring (1b, 1c, 1h and 1i), exhibited MIC in the range of 1.21-3.12 µg ml −1 , while the -CH 3 -substituted compounds were found to be unreactive.
From the results, it is concluded that most of the screened coumarin derivatives have been found to exhibit a good safety profile. Among the tested compounds, -OCH 3 substituents (1e and 1k) showed the          A 5% DMSO solution is used to dissolve compounds, and the disc diffusion method is used to determine the antimicrobial activity in terms of the zone of inhibition [49]. All incubations were performed in triplicate. Among the tested compounds, the compounds 1b, 1c, 1e, 1h, 1i and 1k 1b, 1c, 1e, 1h, 1i and 1k turned out to be active against all tested bacterial strains and exhibited an excellent zone of inhibition bearing -Cl, -Br and -OMe groups varied at the para and meta positions, and 1f and 1l showed good activity, whereas 1a, 1d, 1 g and 1j were found to be less active against some bacterial strains or inactive. The results are tabulated in table 5 and are expressed in terms of the diameter of growth of the inhibition zone (in mm).
The enhanced antifungal and antibacterial activities of the compounds 1b, 1c, 1e, 1h, 1i and 1k could be attributed by the presence of -OCH 3 , and halogen (-Cl and -Br) groups varied at the phenyl ring. However, based on this promising observation, it is premature to arrive at the conclusion on the structure-activity aspect of these molecules, and further evaluation is needed to use them for clinical use.   Molecular docking studies revealed that the compact skeleton of coumarin is the basic reason of how it holds strong contacts with the important amino acid (hydrophobic and hydrophilic) side chains inside the active site as well as the adjoining sites of the enzyme ( figure 8a,b), thus preventing its protubercular active role. It has been concluded that hydroxyl and methoxy substituted Schiff bases were accommodated more in the predicted allosteric active site than in the metal-binding site. The docking results are presented in table 6.

Physico-chemical properties
The theoretical calculation of absorption, distribution, metabolism, excretion and toxicity properties for synthesized compounds are calculated and compared with RO5. This is expressed as the octanol/water partition coefficient called logP; other theoretical calculations such as topological polar surface area, number of hydrogen bond acceptors and number of hydrogen bond donors were also performed. All these physico-chemical properties of synthesized compounds are stated in table 7. All the coumarin compounds showed good agreement and followed RO5, indicating more 'drug-like' nature, and none of the compounds violate RO5.

In silico toxicity prediction
The best way to calculate the toxic effect of drug molecules is developing animal models, which is ideal. Based on compounds of known drug candidates and their toxicity by toxic fragments or chemical structure, the Web server ProTox estimates rodent oral toxicity [50]. This compares the similarity of synthesized compounds which will be loaded in a server with a database of compounds having known toxicity previously, recognizing the toxic fragments of coumarin compounds and possible toxicity targets. The server requires only the two-dimensional structure for which prediction is to be carried out.  Table 7. Drug-likeness property (RO5) of compounds (1a-1j). HBA, number of hydrogen bond acceptors (n-ON); HBD, number of hydrogen bond donors (n-OHNH); LogP, logarithm of partition coefficient between n-octanol and water (miLogP); TPSA, topological polar surface area; GPCR, G-protein-coupled receptors.
Lipinski  promising role in influencing the activity of the ortho-substituted derivatives. The attribution from the preliminary SAR analysis has led to the determination of some key structural requirements for the 3-((1Z,14E)-1-chloro-3-(substituted phenylimino)prop-1-enyl)-2H-chromen-2-one hybrids to exert their anti-TB property, which provides insights into further structural modifications.

Conclusion
In summary, a simple and efficient protocol for the synthesis of coumarin Schiff base derivatives using SSA was accomplished. In the primary screening for anti-TB, most of the compounds (1b, 1c, 1e, 1h,  1i and 1k) displayed about 90-100% inhibition. In the secondary level, two compounds (1e and 1k) inhibited M. tb with MIC < 0.2 µg ml −1 , which is in good agreement with molecular docking results. Four compounds (1b, 1c, 1h and 1i) were found with MIC < 2 µg ml −1 , when compared with isoniazid. Further, among the tested compounds, -OCH 3 substituents (1e and 1k) exhibited an excellent safety profile with over 90% survival rate of Vero cells, indicating good selectivity. Compounds with -Cl, -Br and -OMe groups varied at the para and meta positions showed high potency, in that methyl substituents exhibited good results towards both antifungal and antibacterial activity. Molecular docking studies provided the binding insights consistent with the acceptor and the donor of the title compounds. These studies have been used to correlate and support our experimental results. Also we found that compounds (1j and 1k) with -OH and -OCH 3 -substituted coumarin molecules make hydrogen bonding interactions, which were explored in hydrophobic binding residues, with the electron-donating group in hydrophilic residues probably beneficial for enhancing the binding interactions. The toxicity prediction study reveals that coumarin compounds can act as the lead compounds for further investigations and potent applications of pharmacological interest. From the overall findings, this study suggests that all the potentiality is because of the oxygenated coumarin heterocycle, which later was enhanced by condensing substituted anilines and could be used as a drug to inhibit the occurrence of TB. However, further detailed investigation of coumarin is needed for the exploration of its potency, which can provide lead candidates for drug development in the treatment of such diseases.