Design, Synthesis, and Biological Evaluation of Novel Fused Spiro-4H-Pyran Derivatives as Bacterial Biofilm Disruptor.

This study aims to synthesize novel fused spiro-4H-pyran derivatives under green conditions to develop agents having antimicrobial activity. The synthesized compounds were initially screened for in vitro antibacterial activity against two Gram-positive and three Gram-negative bacterial strains, and all the compounds exhibited moderate to potent antibacterial activity. However, compound 4l showed significant inhibition toward all the bacterial strains, particularly against Streptococcus pneumoniae and Escherichia coli with minimum inhibitory concentration values of 125 μg/mL for each. The toxicity studies of selected compounds (4c, 4e, 4l, and 4m) using human red blood cells as well as human embryonic kidney (HEK-293) cells showed nontoxic behavior at desired concentration. Growth kinetic and time-kill curve studies of 4l against S. pneumoniae and E. coli supported its bactericidal nature. Interestingly, compound 4l showed a synergistic effect when used in combination with ciprofloxacin against selected strains. Biofilm formation in the presence of a lead compound, as assessed by XTT assay, showed complete disruption of the bacterial biofilm visualized by scanning electron microscopy. Overall, the findings suggest 4l to be considered as a promising lead for further development as an antibacterial agent.

Introduction

Over the past decades, bacterial resistance toward commonly used antibiotics is an epidemic health plague worldwide, and it might become a serious threat to public health.[1] World Health Organization (WHO) has pointed out that the infections caused by resistant bacteria may increase severe complications for the health of the community in a near future or even death.[2] Additionally, biofilm formation also plays an important role in bacterial infection as well as drug resistance. Reports suggested that biofilm formation causes more than 80% of bacterial infections.[3] A number of bacterial infections such as periodontitis, endocarditis, chronic lung infections, skin infections, etc., occur due to biofilm formation on biotic surfaces.[4] Biofilm formation on abiotic surfaces such as catheters, surgical instruments, and other medical equipment lead to nosocomial infections.[5] Such problems have constantly emerged due to the limitation of nontoxic and effective antibacterial drugs and their overuse in medical treatments and animal farming.[6] Recently, different classes of compounds such as peptides, small-molecule inhibitors, and fatty acid conjugates are reported as antibacterial agents, which directly act on the biofilm.[7−9] The new discovery of appropriate antibiotics has been vital to human survival especially for immune-compromised patients and patients with AIDS because all microbes are resistant to almost all presented drugs.[10] In the present scenario, the development of newly discovered antibiotics speedily reduced after the great discovery of penicillin in 1950s and 1960s.[11] Currently, the Gram-positive bacterium Staphylococcus aureus responsible for health care-associated soft tissue infections is the most studied pathogens.[12] This clinically isolated S. aureus bacterium has developed resistant to the generally recommended antibiotics including β-lactams such as oxacillin, ampicillin as well as methicillin.[13] One the other hand, the Enterococcus faecium bacterial strain is also resistant to vancomycin drug, which is almost the last remedy to multidrug resistant bacterial strains.[14] Therefore, it is clear that the rapid development of effective antibacterial agents with an innovative and alternative mode of action to cure drug-resistant bacterial infections urgently solicited.

Spirocyclic core containing compounds has pronounced pharmacological activities and widely present in numerous natural and biologically active molecules.[15] It is a clear fact that the presence of a spiro carbon in a molecule leads to structural rigidity, which significantly influences the biological activities. Especially, the oxindole core containing a spirocyclic stereo center at the C-3 position embodies a privileged heterocyclic scaffold found in natural products as well as synthetic pharmaceuticals.[16] Spirooxindoles containing a pyran ring have drawn noteworthy attention to organic as well as medicinal chemists due to their varied range of pharmacological activities including antimicrobial,[17] antimycobacterial,[18] antifungal,[19] anticancer,[20] antioxidant,[21] and photochromic properties.[22] Similarly, 4H-pyran fused with dimedone/chromenes containing isatins showed a broad range of biological properties such as anticoagulant, spasmolytic, antioxidant, antibacterial, and diuretic activities.[23−25] Recently, Zarei et al. synthesized mono- and bis-spiro-4H-pyran and reported biological studies of the synthesized compounds.[26] They have also described that the spiro-4H-pyran derivatives bearing dimedone and isatin motifs have eminent antioxidant efficacies and exhibited strong antibacterial activities against the Escherichia coli bacterial strain compared to the positive control gentamicin. The naturally occurring alkaloids, for example, (+)-calanolide A and rhodomyrtone have also fused pyran and pyranocoumarin motifs (Figure ).[27] Similarly, the compounds pyranokunthone B and 4H-pyranonaphthoquinone (α-lapachones) exhibit antimalarial and antitumor activity, respectively, while zanamivir is used as a medication for the treatment of influenza caused by influenza A and B viruses (Figure ).[28,29]

Figure 1

Examples of naturally occurring pyran alkaloids and biologically active 1,4-naphthaquinone derivatives and comparison with target compounds.

Recently, (E)-N-methyl-1-(methylthio)-2-nitroethenamine (NMSM) has drawn significant attention as an important synergistic building block due to the presence of push–pull skeleton for the synthesis of various O/N-heterocyclic ring systems via multicomponent reactions (MCRs).[30] By using an MCR strategy and NMSM as a building block, we have synthesized 4H-chromen-5-ones and N-methyl-1,4-dihydropyridines under neat conditions.[31] Therefore, in continuation of our effort to develop green and sustainable methods for the synthesis of small heterocycles having biological activities,[32,33] herein, we investigate an expedient, environmentally benevolent, regioselective three-component reaction for the preparation of spiro-4H-pyrans using oxygen containing 1,3-dinucleophilic sources (cyclic-1,3-diketone, 4-hydroxycoumarin, and 2-hydroxy-1,4-naphthaquinone), isatins, and NMSM under catalyst-free conditions as an antibacterial agent (Scheme ).

Scheme 1

Use of NMSM for the One-Pot Synthesis of Novel Fused Spiro 4H-Pyrans

Results and Discussion

Chemistry

Initially, we have chosen dimedone 1a (1.0 mmol), isatin 2a (1.0 mmol), and NMSM 3a (1.0 mmol) as model substrates to optimize the reaction conditions, and for the same model substrates, a number of trial reactions were performed (Table ). In our initial endeavor using 10 mol % of I2, the reaction was carried out at room temperature for 24 h as well as on refluxing in ethanol for 8 h and provided product 4a in 28 and 84% yield, respectively (Table , entries 1 and 2). The proposed structure was confirmed by spectroscopic techniques such as IR, 1H NMR, and 13C NMR. The 1H NMR spectrum of 4a showed characteristic two signals of the N–H proton at δ = 10.45 and 10.48 ppm, and 13C NMR spectra revealed a signal at δ = 48.5 ppm for spiro carbon of the newly formed 4H-pyran ring. On the other hand, the IR vibrational stretching frequencies and HRMS data also supported our obtained product as 4a.

Table 1

Optimization Study for the Synthesis of Spiro 4H-Pyran Compound (4a)a

entry	solvent	catalyst (mol %)	temp.	time	yieldb (%)
1	EtOH	I2 (10)	rt	24 h	28
2	EtOH	I2 (10)	reflux	8 h	84
3	EtOH	piperidine (10)	reflux	12 h	40
4	EtOH	DBU (10)	reflux	10 h	25
5	EtOH	p-TSA (10)	reflux	8 h	79
6	EtOH	AcOH (10)	reflux	9 h	72
7	EtOH	l-proline (10)	reflux	10 h	66
8c	EtOH		reflux	5 h	89
9	H2O		100 °C	12 h	40
10d	H2O/EtOH		90 °C	10 h	43
11e	H2O/EtOH		90 °C	9 h	52
12	MeCN		reflux	10 h	58
13	MeOH		reflux	12 h	62
14	DMF		reflux	24 h	0

Reaction conditions: dimedone (1a, 1 mmol), isatin (2a, 1 mmol), and NMSM (3a, 1 mmol).

Isolated yields.

Optimized reaction conditions.

H2O/EtOH ratio, 1:1.

H2O/EtOH ratio, 1:3.

Encouraged by these above results, other acidic and basic catalysts were screened, using piperidine, DBU, p-TSA, AcOH, and l-proline (10 mol % of each) at refluxing ethanol, which provided the isolated yields of 4a as 40, 25, 79, 72, and 66%, respectively (Table , entries 3–7). Interestingly, the best yield of compound 4a as 89% was achieved when the reaction was performed without a catalyst in refluxing ethanol for 5 h (Table , entry 8). Next, to examine the solvent effect, the same model reaction was screened with different solvents or mixed solvents. We have observed that changing the solvent fails to improve the yield of the desired product (Table , entries 9–14). Thus, the catalyst-free conditions under refluxing ethanol emerge as optimal reaction conditions in terms of time and yield.

After getting the optimized reaction conditions in hand, we are delighted to extend our investigation toward assessing the generality of this protocol. For the same conditions, the different reactions were performed by using dimedone and NMSM with other isatin derivatives bearing functionalities such as 5-NO2, 5-Br, 5-Cl, and N-Me. All the reactions ensued smoothly to accomplish the desired products (4b–4e) with good yields (83–88%) (Table ).

Table 2

Substrate Scope of Functionalized Spiro 4H-Pyrans (4a–4m)a,b

Reaction conditions: 1,3-dinucleophilic sources (1.0 mmol), isatins (1.0 mmol), and NMSM (1.0 mmol) at refluxing ethanol.

Isolated yields.

Again, we carried out the reaction of cyclohexane-1,3-dione and 5-H/5-Cl/5-NO2 isatin with NMSM under optimized reaction conditions, which yielded the corresponding products 4f, 4g, and 4h with yields 88, 87, and 86%, respectively. Further, the feasibility of this reaction was also examined with other analogues of 1,3-dinucleophilic oxygen sources such as 4-hydroxycoumarin and 2-hydroxy-1,4-naphthaquinone with isatin derivatives and NMSM under optimal conditions. We were fruitful in isolating corresponding products 4i–4m in good yield (70–80%) as shown in Table .

The newly synthesized spiro-4H-pyran derivatives were fully characterized by standard spectroscopic techniques such as IR, 1H NMR, 13C NMR, HRMS, and elemental analysis. Finally, the structure of the compound 4e was unambiguously confirmed by X-ray crystallographic analysis (CCDC 1887180). A colorless single crystal was grown by slow evaporation of the CH3CN solvent, and the crystal was mounted for X-ray crystallography on a glass fiber. The ORTEP depiction of the representative compound 4e is shown in Figure .

Figure 2

ORTEP representation of compound 4e (CCDC 1887180).

The formation of compounds can be rationalized via a domino reaction pathway as illustrated with a plausible mechanism shown in Scheme . The first step is conceivable and simple condensation of dimedone (oxygen containing 1,3-dinucleophilic sources) with isatin to give a Knoevenagel condensate I, which underwent Michael-type addition with NMSM to an intermediate II. Then, the intermediate II undergoes imine–enamine tautomerism and leads to the formation of species III. Again, the intramolecular O-cyclization of species III furnished compound 4a via intermediate IV by the elimination of −MeSH. But during our investigation, we did not perceive 4a′ even in a trace amount, thereby rendering our protocol highly regioselective.

Scheme 2

Plausible Mechanism for the Formation of Spiro 4H-Pyrans

Biology

In Vitro Antibacterial Activity

The inhibitory potential of synthesized compounds 4a–4m was evaluated against two Gram-positive, (Streptococcus pneumoniae MTCC 655 and Enterococcus faecalis MTCC 439) and three Gram-negative (E. coli ATCC 25922, Salmonella typhimurium MTCC 3224, and Pseudomonas auroginosa MTCC 2453) bacterial strains. Initially, in vitro screening was performed at 250 μg/mL as the single highest concentration to identify potent compounds and excluded those compounds that are ineffective at this concentration. At this concentration, compounds 4c and 4e were found to be selective inhibitors of P. auroginosa with complete growth inhibition. Similarly, compound 4m selectively inhibited S. typhimurium and S. pneumoniae with 100% growth inhibition. Among all, compound 4l emerged as the lead molecule, which showed complete inhibition of all the bacterial strains used in this study (Table ).

Table 3

In Vitro Screening of the Newly Synthesized Compounds (4a–4m)

	% inhibition at 250 μg/mL
compound	E. faecalis MTCC 439	S. typhimurium MTCC 3224	P. auroginosa MTCC 2453	S. pneumoniae MTCC 655	E. coli ATCC 25922
4a	41.482	57.71	37.322	29.72	43.18
4b	31.896	58.53	17.973	21.26	43.18
4c	38.062	61.4	100	21.48	40.66
4d	30.371	60.99	52.54	23.22	36.11
4e	38.563	66.12	100	28.2	23
4f	37.844	73.72	67.282	29.07	50.4
4g	30.284	65.7	65.899	23.65	36.9
4h	33.116	62.43	58.99	24.73	31.56
4i	7.5	31.01	6.58	1.96	15.39
4j	27.24	57.09	23.05	27.77	45.84
4k	18.083	37.58	4.61	19.74	32.03
4l	100	100	100	100	100
4m	67.19	100	58.07	100	84.31

Further, based on above observation, we selected compounds 4c, 4e, 4l, and 4m to calculate minimum inhibitory concentration (MIC) value (in μg/mL) against those bacterial strains, which are completely inhibited at 250 μg/mL. Compounds 4c and 4e showed moderate activity with MIC values of 250 μg/mL for each against P. auroginosa. Similarly, compound 4m showed moderate activity against S. typhimurium with an MIC value of 250 μg/mL and good activity against S. pneumoniae with an MIC value of 125 μg/mL as shown in Table .

Table 4

MIC ( in μg/mL) of Selected Compounds against Respective Bacterial Strainsa

compound	E. faecalis MTCC 439	S. typhimurium MTCC 3224	P. auroginosa MTCC 2453	S. pneumoniae MTCC 655	E. coli ATCC 25922
4c	n.d.	n.d.	250	n.d.	n.d.
4e	n.d.	n.d.	250	n.d.	n.d.
4l	250	250	125	125	125
4m	n.d.	250	n.d.	125	n.d.
CIP	<7.8	<7.8	<7.8	<7.8	<7.8

n.d. = not done; CIP = ciprofloxacin (standard).

Interestingly, compound 4l showed significantly good inhibition of all the bacterial strains, particularly against S. pneumoniae and E. coli with an MIC value of 125 μg/mL. Therefore, compound 4l with potent selective inhibition of bacterial strains was selected for further pharmacological investigations.

The minimum bactericidal concentration (MBC) was determined to explore the killing efficiency of compounds against selected strains. The bactericidal property of compounds was determined up to the three generations of bacteria. The results showed that P. auroginosa is a more sensitive bacterial strain to such spiro-4H-pyran-based compounds. The MBC values of compounds 4c and 4e were determined as ≤500 μg/mL, while for 4l, it was noted as ≤250 μg/mL. Compound 4m also showed a good killing effect with an MBC value of ≤500 μg/mL against S. typhimurium and S. pneumoniae bacterial strains (Table ).

Table 5

MBC (in μg/mL) of Selected Compounds against Respective Bacterial Strainsa

compound	E. faecalis MTCC 439	S. typhimurium MTCC 3224	P. auroginosa MTCC 2453	S. pneumoniae MTCC 655	E. coli ATCC 25922
4c	n.d.	n.d.	≤500	n.d.	n.d.
4e	n.d.	n.d.	≤500	n.d.	n.d.
4l	≤1000	≤1000	≤250	≤1000	≤500
4m	n.d.	≤500	n.d.	≤500	n.d.

n.d. = not done.

Structure–Activity Relationship

Based on aforementioned data of Table and Table , the following conventions could be presumed about the preliminary structural–activity relationship (SAR). It is quite clear from the above results that the fusion of the spiro-4H-pyran core ring with 1,3-dinucleophilic oxygen containing compounds had a pronounced influence on antibacterial activity. The compounds in which the 4H-pyran ring has fused by 2-hydroxy-1,4-naphthaquinone (4l and 4m) showed a good antibacterial activity as compare to the compounds having fusion of dimedone. While the fusion of the core 4H-pyran ring by 4-hydroxycoumarin (4i– 4k) displayed moderate activity. In general, it is perceived that the fusion of dimedone, 4-hydroxycoumarin, and 2-hydroxy-1,4-naphthaquinone with the 4H-pyran ring plays a dynamic role in the antibacterial activity.

Hemolytic and Cytotoxicity Assays

The cytotoxicity of selected compounds, that is, 4c, 4e, 4l, and 4m, was determined by hemolytic assay on human red blood cells (RBCs). Ciprofloxacin was used as the reference drug (Figure A). Approximately 30 to 40% cell lysis was observed at 400 μg/mL concentration of compounds. Although at higher concentration, that is, at 400 μg/mL, the hemolytic activity of compound 4l showed slightly high toxicity with 32% cell lysis than ciprofloxacin, but at lower concentration, it showed comparability to less or almost equal hemolysis. Compound 4l at 200 and 100 μg/mL (conc. near its MIC values) showed 20 and 13% cell lysis, respectively. Thus, results indicated negligible toxicity of the tested compounds, especially 4l, which showed potent antibacterial activity. Further, to extend the cytotoxicity evaluation on the human normal cell line, the synthesized compounds were also evaluated on HEK-293 cells using MTT assay. The selected compounds were tested in a concentration range of 0–400 μg/mL for 48 h, and interestingly, it was found that the treatment of these compounds does not affect the viability of the HEK-293 cell even at more than 200 μg/mL concentration. However, in the case of compounds 4e and 4m, minor cytotoxicity was observed at higher concentration (400 μg/mL). The cell cytotoxicity results suggested that the selected compounds are noncytotoxic to normal human cells in the studied concentration range (Figure B).

Figure 3

Cytotoxicity studies. (A) Hemolytic activity of compounds 4c, 4e, 4l, and 4m. (B) Cell viability of HEK-293 cells incubated with increasing concentrations of compounds 4c, 4e, 4l, and 4m measured by MTT assay.

Disk Diffusion Assay

The antibacterial potential of compound 4l was also determined by disk diffusion assay on a solid nutrient agar medium at the concentration corresponding to 0.5MIC (62.5 μg/mL), MIC (125 μg/mL), and 2MIC (250 μg/mL).

The dose-dependent zones of clearance were observed in the presence of various concentrations of test compound 4l. Moreover, 18, 17, and 14 mm clear zones of inhibition with an additional 3 ± 1 mm opaque zone of inhibition with a standard deviation of 0.47 were measured around the disk of 2MIC, MIC, and 0.5MIC, respectively, on the lawn culture of E. coli. Similarly, 17, 15, and 14 mm clear zones of inhibition with an additional 3 ± 1 mm opaque zone of inhibition with a standard deviation of 0.47 were measured around the disk of 2MIC, MIC, and 0.5MIC, respectively, on the lawn culture of S. pneumoniae (Figure ). The results indicated the inhibitory potential of 4l at various tested concentrations.

Figure 4

Disk diffusion assay for (a) E. coli and (b) S. pneumoniae showing disk (i) DMSO, (ii) 0.5MIC, (iii) MIC, (iv) 2MIC of compound 4l, and (v) blank.

Spot Assay

The viability of bacterial cells in the presence of various concentrations of test compound 4l was also determined by the spot assay. The bacterial cells were exposed for 6 h to the test compound 4l and cultured on a nutrient agar medium.

The results shown in Figure clearly indicated the bactericidal nature of compound, which significantly affects the viability of E. coli and S. pneumoniae even in 6 h. A confluent growth of E. coli and S. pneumoniae at 0.5MIC showed no visible effect on the cell, but at higher concentration, that is, MIC and 2MIC, significant inhibition occurred. Moreover, no S. pneumoniae growth was observed after treated with 2MIC of 4l. The experiment was performed in triplicate at three different times.

Figure 5

Spot assay showing growth in terms of (+) or (−) sign as (i) untreated and treated with (ii) 0.5 MIC, (iii) MIC, and (iv) 2MIC of compound 4l for (a) E. coli and (b) S. pneumoniae.

Growth Kinetic Studies

To investigate the effect of the potent compound 4l on the growth of S. pneumoniae and E. coli bacterial cells, growth kinetic studies were accomplished. The bacterial cells were exposed to different concentrations of the test compound (2MIC, MIC, and 0.5MIC). Ciprofloxacin-treated cells were used as a positive control, and untreated cells were used as a negative control. The growth curve of untreated bacterial cells showed slightly diauxic growth with clear lag, exponential or log, brief stationary, and decline phases of the cell cycle. The bacterial cells did not display any substantial growth when exposed to the 2MIC and MIC of 4l with a continuous lag phase of 24 h.

However, at sub-MIC of 4l, growth was observed between 16 and 18 h in E. coli, which increased with a very slow growth rate. Similarly, at the same concentration, that is, 0.5MIC, the growth in S. pneumoniae was observed between 18 and 20 h. The results clearly showed the bactericidal nature of compound 4l against both Gram-negative as well as Gram-positive bacterial strains as no significant growth detected even after 24 h at higher concentrations. The study suggested that compound 4l is an effective inhibitor of pathogenic with tested bacterial strains (Figure ).

Figure 6

Growth kinetic assay showing no growth of E. coli and S. pneumoniae at MIC and 2MIC of 4l favors its bactericidal nature.

Time–Kill Curve Assay

To determine the bacteriostatic or bactericidal nature of lead compound 4l, time–kill kinetics was performed against S. pneumoniae and E. coli bacterial strains at MIC and 2MIC.

The killing activity was observed up to 24 h. Compound 4l showed bactericidal activity at 2MIC.

Although no complete eradication of bacterial population was observed, significant decrease in log10 CFU/mL with respect to time was observed at MIC. At higher concentration, which equals to 2MIC, complete eradication of E. coli was observed after 24 h. Similarly, in the case of S. pneumoniae, 2MIC of compound 4l almost completely killed bacterial cells (Figure ). Moreover, results clearly indicated the bactericidal nature of compound 4l at higher concentration.

Figure 7

Time–kill curve of compound 4l against (a) E. coli and (b) S. pneumoniae.

Synergistic Study

In vitro synergistic antibacterial activity of compound 4l was performed with standard drug ciprofloxacin (CIP) against all the five strains: P. aeruginosa, S. typhimurium, E. faecalis, S. pneumoniae, and E. coli. Many-fold decrease in MIC values of compound 4l was observed when used in combination with CIP.

The results indicated that the compound 4l showed synergy with CIP against Gram-positive strains E. faecalis and S. pneumoniae with fractional inhibitory concentration index (FICI) values of 0.27 and 0.29, respectively. Further, no synergistic relation was found against P. aeruginosa and S. typhimurium with high FICI values, that is, 1.06 and 1.01, respectively. Moreover, partial synergistic activity was found against E. coli with an FICI value of 0.53 (Table ).

Table 6

In Vitro Synergistic Antibacterial Activity of Compound 4l with Ciprofloxacin

	MIC alone (μg/mL)		MIC in combination (μg/mL)
bacterial strain	4l	CIP	4l	CIP	FICIa	mode of interaction
P. aeruginosa	250	0.5	15.63	0.5	1.06	indifferent
S. typhimurium	250	0.25	3.90	0.25	1.01	indifferent
E. faecalis	250	0.25	3.90	0.0625	0.27	synergistic
S. pneumoniae	125	0.25	3.90	0.0625	0.29	synergistic
E. coli	125	0.25	3.90	0.125	0.53	partial synergistic

Synergy and antagonism were defined by FIC indices of <0.5 and >4, respectively. An FIC index result of >0.5 but <4 was considered indifferent.

Confocal Laser Microscopy

The cellular uptake of compound 4l by bacterial cells was determined by performing confocal laser microscopy on the E. coli strain. A nucleic acid binding dye, DAPI (4′,6-diamidino-2-phenylindole), was used to stain bacterial cells. DAPI is a fluorescent dye, which strongly binds to the A-T rich region of DNA and, upon excitation at 358 nm, emits blue fluorescence as emission spectra at 461 nm. Untreated as well as treated cells (exposed MIC of compound 4l for 3 h) were stained and observed under a confocal laser microscopy. Figure demonstrates that untreated cells are live and do not emit blue fluorescence, while in the treated sample, reduction in the number of living cells occurs. A significant number of cells emit fluorescence, which clearly indicates cell lysis due to the presence of compound 4l. Thus, compound 4l has potent antibacterial property, which may further be explored to develop better pharmacophore.

Figure 8

Confocal laser microscopic images of E. coli cells (a) untreated and (b) treated with 4l at MIC.

Biofilm Assessment by XTT Assay

Biofilm formation is an important virulence attribute of pathogenic microorganisms. Gram-negative E. coli and Gram-positive S. pneumoniae were used to determine the effect of compound 4l on biofilm formation. The concentrations equal to 4MIC, 2MIC, and MIC were used to explore its biofilm inhibition activity. The results indicated that compound 4l almost completely disrupted biofilm formation in the Gram-positive strain S. pneumoniae even at lower concentration, which equals to its MIC value. Similarly, 97, 80, and 77% reduction in absorbance was observed, which clearly indicates the reduction of biofilm formation in the E. coli strain. Thus, the compound 4l was found to be a potent disruptor of biofilm formation. Moreover, at sub-MIC, that is, MIC/2, MIC/4, and MIC/8, significant reduction in biofilm formation was observed (Figure ).

Figure 9

Biofilm inhibition by XTT assay and % of biofilm inhibition in E. coli and S. pneumoniae in the presence of 4MIC, 2MIC, MIC, MIC/2, MIC/4, and MIC/8 concentrations of compound 4l.

SEM Analysis of Biofilm

The quantitative assessment of biofilm formation by XTT assay was further examined under scanning electron microscopy (SEM). Biofilm formation on the surface of materials occurs due to secretion of the extracellular matrix, which primarily consists of polysaccharide. Two concentrations equal to 2MIC and MIC values of compound 4l were taken to compare the efficacy against biofilm formation with untreated cells.

The cells in untreated samples appeared in clusters and embedded in the extracellular matrix (Figure a). The sample treated with 2MIC showed very few scattered bacterial cells. Similarly, significant damage in biofilm formation showed at MIC (Figure ). The results clearly indicated the inhibition of biofilm disruption after exposed to compound 4l and strongly supported the data of the previous experiment.

Figure 10

SEM image of biofilm formation by E. coli in (a) untreated sample, (b) sample exposed to MIC, and (c) 2MIC for 24 h.

Docking Studies

Methionine aminopeptidases (MetAPs) are first-row transition metalloenzymes with five conserved metal ion-binding residues in the active sites, which involve in the cleavage of N-terminal methionine during protein synthesis. This process is highly crucial for the survival of bacterial cells, which enable MetAP as an emerging drug target for bacterial infections. Although more information is needed to prove MetAP as a selective drug target of compound 4l, our previous studies on heterocyclic compounds as inhibitors of MetAP and its crucial role in post-translational modifications of bacterial proteins prompt us to determine the binding pattern of 4l using an in silico approach.[34] Thus, docking studies were performed with MetAPs of E. coli (PDB ID: 1c21) and S. pneumoniae (PDB ID: 4km3) to support in vitro antibacterial results. Docking results of ligand 4l with EcMetAP and SpMetAP showed good interaction with binding energies of −7.6 and −7.0 kcal/mol, respectively.

It interacted with MET112, GLN130, TYR234, and ASP253 residues within the binding site of EcMetAP (Figure b). Moreover, MET112 is a common residue, which also interacts with the natural substrate methionine of MetAP. Similarly, ligand 4l interacted with ASP195, HIS199, and HIS252 amino acid residues within the binding pocket of SpMetAP (Figure d). HIS199 is a metal-binding residue of SpMetAP and present in the active binding site of protein. Thus, on the basis of docking score and interacting residues, the results clearly indicate ligand 4l as a possible inhibitor of MetAPs and also support in vitro antibacterial potential.

Figure 11

Docking of compound 4l with MetAPs of (a) E. coli (PDB ID: 1c21) and (c) S. pneumoniae (PDB ID: 4km3). Compound 4l showing interaction with residues of (b) EcMetAP and (d) SpMetAP.

Conclusions

In summary, we have developed an environmentally benevolent protocol for the synthesis of fused spiro-4H-pyran derivatives in good to excellent yields from the reaction of oxygen containing cyclic-1,3-dinucleophiles, isatins, and NMSM in the absence of the catalyst. The promising features of the presented protocol include clean reaction profile, easy workup, avoidance of toxic catalysts, etc. The newly synthesized compounds were tested for their in vitro antibacterial activity, and compounds 4a–4l exhibited moderate to potent antibacterial activity against S. pneumoniae, E. faecalis, E. coli, S. typhimurium, and P. auroginosa. The compound 4l showed significant inhibition of all the bacterial strains with potent activity against S. pneumoniae and E. coli with MIC values of 125 μg/mL for each. The hemolytic and MTT assays showed the nontoxic nature of compound on human red blood cells (hRBCs) as well as HEK-293 cells. The study suggests that compound 4l has good potential to disrupt the biofilm formed by bacterial strains and thus may be a possible antibacterial agent against biofilm-associated bacterial infections. Moreover, docking results also supported candidature of compound 4l as a possible inhibitor of bacterial MetAP drug target for bacterial protein synthesis. Thus, the potential of compound 4l to inhibit a broad range of pathogenic bacterial strains prompts us to select it as a lead compound for further optimization and SAR studies to explore its mechanistic and other biological properties.

Experimental Section

Typical Procedure for the Preparation of Spiro 4H-Pyrans (4a–4m)

In an oven-dried 10 mL round-bottom flask was charged with a mixture of isatins (1.0 mmol) and 1,3-dinucleophilic oxygen sources (cyclic-1,3-diketone, 4-hydroxycoumarin, and 2-hydroxy-1,4-naphthaquinone) (1.0 mmol) with NMSM (1.0 mmol) at refluxing ethanol (5 mL). The resulting mixture was left for stirred, and reaction progress or consumption of starting materials was monitored by TLC. After completion of the reaction as indicated by TLC, the resulting precipitate was cooled. Then, the precipitate was filtered by using a Buchner funnel and washed with a cold ethanol solvent. The crude products were recrystallized from hot acetonitrile, which provide the pure products.

7,7-Dimethyl-2-(methylamino)-3-nitro-7,8-dihydrospiro[chromene-4,3′-indoline]-2′,5(6 H)-dione (4a)

Isolated as a white solid; yield: 89%; mp: 290–292 °C. IR (KBr, cm–1): 3194, 3111, 3058, 2927, 2887, 1729, 1561, 1496, 1356, 1246, 1172, 1070, 721. 1H NMR (400 MHz, DMSO-d6): δ 0.95 (s, 3H), 1.02 (s, 3H), 2.06 (d, J = 15.6 Hz, 1H), 2.19 (d, J = 15.6 Hz, 1H), 2.59 (d, J = 17.7 Hz, 1H), 2.70 (d, J = 17.7 Hz, 1H), 3.10 (s, 3H), 6.73 (d, J = 7.6 Hz, 1H), 6.80 (t, J = 7.8 Hz, 1H), 7.00 (d, J = 7.2 Hz, 1H), 7.99 (t, J = 7.6 Hz, 1H), 10.45 (s, 1H), 10.47 (s, 1H). 13C NMR (100 MHz, DMSO-d6): δ 26.6, 27.6, 28.4, 31.6, 48.5, 50.4, 107.7, 108.4, 113.2, 120.8, 122.3, 127.9, 130.9, 144.5, 156.8, 161.6, 176.6, 194.2. Anal. calcd (%) for C19H19N3O5: C, 61.78; H, 5.18; N, 11.38; found: C, 61.70; H, 5.17; N, 11.40; EI-HRMS: Anal. calcd for [C19H19N3O5 + H+]: 370.1397; found, 370.1388.

Biological Evaluation

In Vitro Screening of Synthesized Compounds

All the synthesized compounds 4a–4m were screened for their antibacterial properties against Gram-negative (E. coli ATCC 25922, S. typhimurium MTCC 3224, and P. auroginosa MTCC 2453) and Gram-positive (S. pneumoniae MTCC 655 and E. faecalis MTCC 439) bacterial strains. The sensitivity of these tested organisms toward synthesized compounds was initially determined at 250 μg/mL concentration. DMSO (dimethyl sulfoxide) was used to dissolve synthesized compounds and appropriately diluted in a test medium. All the five bacterial strains were grown up to a mid-log phase and diluted with sterilized nutrient broth to achieve suspension of cells according to McFarland standards. Inoculums (200 μL) were poured in 96-well plates, and 250 μg/mL conc. of test compounds was added. The plates were incubated at 37 °C and 160 rpm for 24 h. After an incubation period, the growth was measured in terms of optical density (O.D.) at 580 nm using a Thermo Scientific MultiskanGo plate reader. The % inhibition of growth was measured using the following formulawhere A stands for absorbance. The experiment was repeated twice in two different times.

Based on screening, we determined MIC values of selected compounds for respective bacterial strains using the broth microdilution technique according to the standard protocol for antimicrobial assessment by CLSI.[35] Ciprofloxacin (CIP) was used as a positive control. Concentration gradient (250 to 0.122 μg/mL) of test compounds was maintained in 100 μL of nutrient broth medium into 96-well plates. Further, each well was inoculated with 100 μL of freshly prepared bacterial cell suspension containing approximately 5 × 106 cells and incubated at 37 °C with constant stirring at 160 rpm. Further, the bactericidal efficiency of test compounds was determined by minimum bactericidal concentration (MBC). Briefly, the cells treated with a concentration gradient from 2000 to 62.5 μg/mL selected compounds in 96-well plates were incubated 37 °C for 24 h. After an incubation period, the 10 μL of medium from the wells in which no visible growth appeared was transferred to 100 μL of fresh medium and incubated at 37 °C for 24 h. The MBC was determined as the concentration range at which no growth occurred even after three generations. The experiment was performed in triplicate.[36]

Cytotoxicity Studies by Hemolytic and Cell Viability Assays

The hemolytic activity of compounds 4c, 4e, 4l, and 4m on human RBCs was determined according to the previously reported method.[36] Briefly, human blood from a healthy individual was collected in EDTA-containing tubes and immediately centrifuged at 2000 rpm and room temperature for 10 min to harvest erythrocytes. The erythrocytes were then washed with phosphate buffer saline (PBS) solution and diluted to obtained 10% (v/v) erythrocytes/PBS suspension. The suspension was further diluted a 1:10 ratio in the same buffer. A dilution series of 400 to 3.125 μg/mL test compound was maintained in 100 μL of PBS solution in microcentrifuge tubes and introduced to the same value of diluted erythrocytes/PBS suspension. Ciprofloxacin was used as the reference drug, while 100% cell lysis was obtained using 1% Triton X-100. After incubation for 1 h at 37 °C, the microcentrifuge tubes were centrifuged at 2000 rpm for 10 min. To obtain the absorbance at 450 nm, 150 μL of supernatant fluid was transferred to a 96-well plate, and values were measured using a Thermo Scientific MultiskanGo plate reader. The hemolysis percentage was calculated by the following equation

For human cell-based studies, HEK-293 cells were procured from National Centre for Cell Sciences, Pune, India and maintained in DMEM media augmented with 10% heat-inactivated FBS and 1% antibiotic cocktail solution in a 5% CO2incubator at 37 °C. To observe the cytotoxicity of selected synthesized compounds, MTT assay was used as per the published protocols.[37] In short, the cells were plated at a density of 9000–10,000 cells/well of a 96-well cell culture plate and grown overnight. After 24 h, the cells were treated with increasing concentration of each compound (0–400 μg/mL) for 48 h at 37 °C in a CO2 incubator. The stock solutions of each compound were made in DMSO, and the working solutions (1000μg/ml) were made by diluting the stock in phosphate buffer saline (PBS), pH 7.4. The mixture of compounds and culture medium were aspirated after a stipulated time period, and cells were washed three times with PBS (pH 7.4). Subsequently, from a 5 mg/mL stock solution of MTT, 20 μL of MTT and 100 μL of cell culture medium were added to each well, and cell plates were incubated for 4–5 h (37 °C in the CO2 incubator). After that, 4–5 h incubation, the supernatant was pipetted out, and the purple formazan crystals were then dissolved by adding 150 μL of DMSO. The absorbance was measured at 570 nm using multiplate ELISA reader (BioRad). The % of cell viability was calculated and plotted as a function of concentration of compound.

Based on antibacterial potential and hemolytic activity, we select compound 4l for further exploration of their biological properties. We also determined the antibacterial properties of 4l using disk diffusion assay. The nutrient agar medium was autoclaved, allowed cooling up to approximately 45 °C, and poured into a sterilized Petri dish. On the basis of susceptibility, Gram-negative E. coli ATCC 25922 and Gram-positive S. pneumoniae MTCC 655 were used as test organisms. After solidification, freshly prepared inoculums of E. coli and S. pneumoniae were spread over agar plates, and 6 mm sterile disk of Whatman no. 1 filter paper was put at appropriate distance for various concentrations of test compounds. DMSO (14 μL) was used as a negative control, while one disk was left blank. Three disks were impregnated by 2MIC, MIC, and 0.5 MIC of 4l followed by the incubation of agar plates at 37 °C for 24 h. After incubation, the zone of inhibition was measured using HiAntibiotic Zone Scale-c. The experiment was performed in triplicates at three different times.

Spot assay was performed to check the killing effect of compound 4l. Mid-log phase cells of Gram-negative E. coli ATCC 25922 and Gram-positive S. pneumoniae MTCC 655 were harvested and diluted with sterile PBS to obtained cell suspension according to McFarland standards. Suspension (2 mL) of each bacterial strain was exposed to 2MIC, MIC, and 0.5MIC of test compound and further incubated at 37 °C and 160 rpm for 6 h. In the meantime, nutrient agar plates were prepared as mentioned previously. After incubation of 6 h, a spot (2 μL) from each sample was put at equidistance on the nutrient agar plate. The plates were again incubated at 37 °C for 24 h. The growth of bacterial cells was observed after an incubation period. The experiment was performed in triplicate in three different times.

Growth Curve Studies

Growth curve study was performed against E. coli (Gram-negative) and S. pneumoniae (Gram-positive) bacterial strains. Concentrations, equivalent to 2MIC, MIC, and 0.5MIC, of compound 4l were dispensed into the separate conical flasks containing 50 mL of freshly prepared sterile nutrient broth medium. The medium was inoculated with approximately 5 × 106 cells/mL obtained from the log phase of E. coli and S. pneumoniae and incubated at 37 °C and 160 rpm. Aliquot (1 mL) from each sample was removed at a regular time interval (i.e., 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h), and growth was measured turbidometrically at 580 nm using a Thermo Multiskan spectrophotometer. A graph was plotted between time duration (in h) and O.D. to determine the effect of compound 4l on the growth cycle of test organisms.[38]

Time–kill curve assay was performed to determine the bactericidal or bacteriostatic nature of the lead inhibitor 4l against Gram-negative E. coli and Gram-positive S. pneumoniae strains. The freshly prepared sterile nutrient broth medium was inoculated by fresh culture (approximately 2 × 106 cells) of test organisms in different conical flasks. The cells were further two-fold diluted and exposed to the test compounds at the concentrations corresponding to its 2MIC and MIC values, separately. Untreated cells were used as a negative control. All the conical flasks were incubated at 37 °C and 160 rpm. At predetermined time points (0, 2, 4, 8, and 24 h after incubation with agitation at 37 °C), a 100 μL aliquot was removed from each solution and properly diluted in sterile saline water. A 50 μL aliquot from each dilution was spread over the nutrient agar plate. Then, the colony counts were determined after incubation at 37 °C for 24 h. All the time–kill curve experiments were conducted in duplicate, and mean colony count data (log10 CFU/mL) were plotted as a function of time for each strain.[39]

Synergistic Assay

The fractional inhibitory concentration index (FICI) or synergistic activity of compound 4l with antibacterial drug ciprofloxacin was determined by the previously reported microdilution checkerboard method in 96-well plates.[40] Briefly, compound 4l was serially diluted from 250 to 0.122 μg/mL conc. horizontally, while CIP was diluted from 2 to 0.016 μg/mL conc. vertically in a sterile nutrient broth medium. The plates were inoculated with approximately 2 × 106 cells of freshly prepared P. aeruginosa, S. typhimurium, E. faecalis, S. pneumoniae, and E. coli strains. The plates were incubated at 37 °C for 24 h. The concentrations at which no visible growth appeared were determined as the MIC values of compound 4l and CIP in combination. The FICI was calculated using the following formula

Confocal Laser Microscopy

Confocal laser microscopy was performed to determine the bacterial cell lysis caused by lead inhibitor 4l. The log-phase bacterial cells of E. coli were harvested by centrifugation at 3000 rpm for 15 min and washed twice with phosphate buffer saline. The cell suspension was adjusted according to McFarland standards, and then cells were exposed to MIC of 4l for 3 h at 37 °C. After exposure to test compound, the cells were again washed with PBS and counterstained with DAPI for 30 min in the dark. The cells were further washed to remove extra dye using PBS buffer solution. The slides for treated and untreated samples were prepared, fixed using poly(l-lysine), and observed under a confocal laser scanning microscope Leica DMRE equipped with a confocal head TCS SPE (Leica, Wetzlar, Germany) and a 60× water immersion objective with a laser of 532 nm wavelength.

Biofilm Assessment by XTT Assay

Biofilm formation in E. coli and S. pneumoniae was determined by the semiquantitative method using XTT (2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium-hydroxide) reduction assay with slight modification in the previously known method.[29] Briefly, aliquots of 100 μL of standardized cell suspension of S. pneumoniae and E. coli prepared in a sterile nutrient broth medium containing 0.5% additional glucose and poured into the wells of the microtiter plate. The plate was incubated at 37 °C for 24 h in static conditions to establish the biofilm. After an incubation period, the medium was discarded and gently washed two times with PBS to remove nonadherent cells. Further, 100 μL of freshly prepared medium containing 4MIC, 2MIC, and MIC of compound 4l was added to the wells. The plate was again incubated at 37 °C for 24 h under static conditions to determine the metabolic activity of the biofilm. The medium was discarded and washed with PBS to remove the nonadherent cells followed by the addition of 100 μL of prepared XTT salt solution (HiMedia, India). The plate was incubated at 37 °C in dark for 90 min. The bacterial dehydrogenase activity reduces XTT tetrazolium salt to XTT formazan, resulting in a colorimetric change (turns to orange) that was correlated with cell viability. The colorimetric changes were measured spectrophotometrically at 490 nm. The % inhibition data were interpreted from dose–response curves.

Biofilm Formation Assessment by SEM

Biofilm inhibition property of compound 4l was also determined by scanning electron microscopy (SEM) in E. coli. Sterile nutrient broth (3 mL) containing 0.5% additional glucose was poured into the six-well plate (Tarson) and incubated with freshly prepared primary culture of E. coli. Glass pieces (10 mm2) were sterilized and dispensed into the medium for biofilm formation on the surface. The plate was incubated at 37 °C for 6 h followed by addition of test compound. The plate was then incubated at 37 °C for the next 24 h. After incubation, the glass pieces were removed and gently washed with PBS and kept in fixative (2.5% glutaraldehyde and 2% paraformaldehyde (PF) in 0.1 M phosphate buffer, pH 7.4) overnight. Samples were again washed with PBS and dried to examine under a scanning electron microscope.

The interaction between ligand and targeted protein was determined by molecular docking using the computational method. Docking studies were performed with methionine aminopeptidases of E. coli (PDB ID: 1c21) and S. pneumoniae (PDB ID: 4km3). The 3D coordinates of both proteins retrieved from RCSB protein data bank (www.rcsb.org/pdb) in PDB format. The heteroatoms and water molecules were removed from the crystal structure of MetAPs, which followed by energy minimization using a Swiss PDB Viewer tool. The structure of compound 4l was drawn using ChemDraw, and its 3D coordinate was generated using online SMILES translator. The energy minimization of ligand was done using a Swiss PDB Viewer tool. The desired format (PDBQT) for docking was obtained using an ADT tool. Autodock Vina 4.2 software was used for the docking process.[41] Grid dimensions of X, Y, and Z covering completely EcMetAP protein molecule were 44, 46, and 46, respectively, with 1 Å spacing, and those covering the SpMetAP protein molecule were 46, 50, and 42, respectively with 1 Å spacing. Dimensions of the center grid box were 14.24, −13.11, and 11.173 in the case of whole EcMetAP and 14.08, −55.61, and 5.29 for SpMetAP molecule blind docking. Complex with the minimum binding energy and involving interactions with important residues of binding pocket was chosen as the basis for further interface analysis. The visualization of docked outfile and interacted residues was done in PyMOL.[42]