Synthesis and Evaluation of Novel 4-Hydroxycoumarin Derivatives as Potential Anti-Microbial Agents

Introduction

Inflammation is a complex phenomenon accompanied by interrelationships between humoral and cellular reactions induced by the diversity of inflammatory mediators such as toll like receptors (TLRs), interleukins (ILs), etc. The symptoms of the inflammation indicate many onsets within the body’s system. Although it is still a challenging endeavors to cop inflammation and its biological neutralization. It is critical need for better-tolerated and more efficient drugs which evade the suffering volunteers from such complications.¹ Moreover, the recent past years envisaged exploring the medicinal system concerning a better approach to treat many life-threatening infections caused by multi-drug resistant for gram-positive (+ve) and gram-negative (–ve) pathogenic bacteria.² Instead of antibiotic-resistant drugs, researchers are still engaged to generate the potential drug which not only resists the microorganism survivability but also obviates the infections which cause significant morbidity and mortality in a living organism. Although several clinical reports have been published concerning today’s emergence of antibiotics resistance.^{3, 4}

Coumarins or 2H-1-benzopyran-2-one are aromatic organic compounds that consist of a large class of phenolic compounds that occurred naturally or were derived by fusion of benzene and α-pyrone rings.⁵ As per the scientific reports, more than 1300 coumarins have been acknowledged as secondary metabolites from natural resources.⁶ Coumarins and their derived products are potentially acting as an anti-inflammatory, anticoagulant, antibacterial, antifungal, antiviral, anticancer, etc.⁷ The coumarin molecule has been accompanied by its unique anti-edema and anti-inflammatory activities. Thus, coumarin derivatives could be potentially active in the treatment of all high protein edemas.¹ In addition, it is suggested to have a critical need for the development of new and novel coumarins derivatives against the upstream infection of microorganisms especially for gram-positive and gram-negative bacterias as coumarins are usually associated with low toxicity and have raised considerable interest because of their potential benefits on human health.⁸ Furthermore, it has been scientifically validated that a series of novel Schiff’s base- accustomed coumarins compounds play a significant role against various diseases even acts as anti-inflammatory agents.

Taking all these facts into consideration, the present prospects of the study is associated to target base synthesis of several coumarin derivatives by using Schiff’s base. In addition, all the synthesized compounds were evaluated for their antibacterial and anti-inflammatory activity using in-silico, in-vitro, and in-vivo approaches. The target of our study is mainly associated to replace the drugs causing antibacterial resistance and development of newly antibacterial agent which may be facilitated as an effective and economic antibacterial drug.

Material and Method

Reagents, instruments and measurements

All reagents, solvents, and catalysts were of analytical grade and used directly. The melting point was determined in open glass capillary tubes and incorporated accordingly. The completion of reactions was monitored by TLC using silica gel-G coated glass plates and visualization was done by using iodine/UV lamp. IR spectra were recorded on Bruker alpha-T spectrophotometer using A.T.R. technique. 1H NMR spectra were recorded on a Bruker Advance II 400 NMR spectrometer in CDCl3 as the solvent and TMS as an internal standard. Microbial strains such as S. aureus (MTCC No. 3161), B. subtillis (MTCC No. 441), E. coli (MTCC NO. 1687) and P. aeruginosa (MTCC NO. 424) were collected from the institutional lab.

Chemistry

The synthesis process was completed in four steps as shown in figure 1. Compound 1a was prepared via amination of 4-hydroxycoumarin with ammonium acetate. Further, this compound was treated with ethylchloroacetate in presence of KOH/K₂CO₃ yielding N-alkylated product (2a). The reaction of this product with hydrazine hydrate yeilded desired hydrazide compound (3a). Schiff base derivatives (4a-4j) were obtained by reaction of 3a with different aromatic aldehydes in presence of glacial acetic acid in alcohol. The process for the synthesis of coumarin derivatives involves four steps as follows.

Synthesis of 4-aminocoumarin (1a)

During the process for the synthesis of 4-aminocoumarin (1a), a mixture of 4-hydroxycoumarin (0.01mol) and ammonium acetate (0.01mol) was stirred at 160⁰ for 3hrs in DMF. After completion of the reaction, the content was cooled at room temperature and then poured into water and stirred for 10 min. The completion of the reaction was checked by TLC. The solid was collected by filtration, washed, recrystallized from appropriate solvent and dried.

Synthesis of ethyl [(2-oxo-2H-1-benzopyran-4-yl) amino] acetate (2a)

4-aminocoumarin,1a (0.01mol) was treated with ethylchloroacetate (0.01mol) in the presence of KOH/K₂CO₃ and solvent (70 mL). The solution was refluxed for about 3hrs. After that the solution was cooled and poured to cold water, the precipitate was filtered off, washed with water, and dried to yield N-substituted compound 2a.

Synthesis of 2-[(2-oxo-2H-1-benzopyran-4-yl) amino] acetohydrazide (3a)

Compound 2a (0.01mol) was reacted with hydrazine hydrate (0.01mol) in presence of ethanol and refluxed for 3-4 hrs. The reaction mixture was cooled and poured into cold water under vigorous stirring. The crude products obtained were filtered off, dried and recrystallized by using an appropriate solvent to yield desired compound 3a.

General procedure for the synthesis of Schiff bases (4a-4j)

Compound 3a was reacted with a different aromatic aldehyde in the presence of glacial acetic acid in alcohol and refluxed for 3-4 hrs. After completion of the reaction (checked by TLC), the reaction mixture was cooled and poured into cold water with continuous stirring. The solid thus obtained was filtered, dried and recrystallized from appropriate solvent to yield corresponding Schiff bases (4a-4j). Further, the percentage yield and melting point of the entire synthesized compound were determined. The purity of the compounds was checked by TLC using appropriate solvent systems and the structures of all the derivatives were validated by mass spectroscopic IR, and ¹HNMR data.^9-12

The schematic representation of the process in chemistry involved in the synthesis of coumarin derivatives is summarised in figure 1.

Figure 1: Schematic representation of reaction process for the synthesis of coumarin derivatives Click here to View figure

Figure 2: Antibacterial activity of coumarins derivatives (1, 2, 7, 8, 10), figure (A’) showed control plate and treated plates against S. aureus.  Click here to View figure

Figure 3: MS spectra of compound 4a-4j. Click here to View figure

2-[(2-oxo-2H-1-benzopyran-4-yl)amino]-N‘-[(E)-phenylmethylidene] acetohydrazide (4a)

MS (ESI) m/z: 322.14 (M+1); (FTIR, υmax, cm−1); 3413.71, 3381.52 (=N, -NH), 1758.31 (C=C/-C=N), 1698.03 (C=O), 1437.62, 1228.44 (C-O-C and C-H), 1158.11, 1037.32, 815 (aliphatic amines aromatic C-H vibrations). ¹H-NMR (CD3OD, 500MHz): δ 10.164, 8.753, 8.121 (3H, 3s, 2NH, CH), 7.934 (1H, m, Ar, CH-5), 7.703, 7.389 (3H, m, Ar, CH-2, 6 & 7), 7.162 (3H, m, Ar, CH-3, 4, 5, benzylidene), 6.936 (2H, m, Ar, CH-6, 7), 4.472, 3.813 (3H, 2s, Ar, CH-3, CH₂).

N‘-[(E)-(4-chlorophenyl)methylidene]-2-[(2-oxo-2H-1-benzopyran-4-yl)amino]acetohydrazide (4b)

MS (ESI) m/z: 356.08 (M+1); (FTIR, υmax, cm⁻¹); 3531.05, 3419.72, 2912.95 cm^-1 (=N, -NH, CH₂)_, 1697.37 (C=O) 1438.28, 1228.44 (C-O-C and C-H), 1158.12, 1037.32 and 702.37 (aliphatic amines, aromatic C-H and C-Cl vibrations). ¹H-NMR (CD3OD, 500MHz): δ 10.182, 8.953 and 8.321 (3H, 3s, 2NH, CH), 7.9742 (1H, m, Ar, CH-5), 7.751 and 7.412 (3H m, Ar, CH-2, 6 & 7), 7.184 and 6.927 (4H, m, Ar, CH-6, 8 & 3, 5), 4.292 and 3.733 (3H, 2s, Ar, CH-3, CH₂).

N‘-[(E)-(3-chlorophenyl)methylidene]-2-[(2-oxo-2H-1-benzopyran-4-yl)amino]acetohydrazide (4c)

MS (ESI) m/z: 356.29 (M+1); (FTIR, υmax, cm⁻¹); 3447.96, 3385.34 and 1731.06 (=N, -NH and C=C/C=N), 1665.03 (C=O)_, 1445.23 and 1392.58 (C-O-C and mono-substituted alkynes), 1107.23 and 725.03 (aliphatic amines and C-Cl vibrations). ¹H-NMR (CD3OD, 500MHz): δ 10.173, 8.413 and 8.281 (3H, 3s, 2NH, CH), 8.142 (1H, m, Ar, CH-5), 6.891 and 6.732 represents for seven protons (7H, 2m, Ar, CH-6, 7, 8 of coumarin ring and CH-2, 3, 4, 6 of benzylidene ring), 4.912 and 3.973 (3H, 2s, Ar, CH-3, CH₂).

N‘-[(E)-(3-nitrophenyl)methylidene]-2-[(2-oxo-2H-1-benzopyran-4-yl)amino]acetohydrazide (4d)

MS (ESI) m/z: 337.01 (M+1); (FTIR, υmax, cm⁻¹); 3521.88, 3458.01 and 1701.43 (=N, -NH and C=C/C=N) 1659.72 (C=O)_, 1479.05 (C-O-C), 1112.95, 1069.28 and 768.25 (aromatic and aliphatic amines and C-H scissoring vibrations). ¹H-NMR (CD3OD, 500MHz): δ 10.113, 8.793 and 8.423 (3H, 3s, 2NH, CH), 8.232 (1H, m, Ar, CH-5), 7.613 (1H, m, Ar, CH-7), 7.191 and 6.692 (6H, 2m, Ar, CH-6, 8 of coumarin ring and CH-2, 3, 4, 6 of benzylidene ring), 5.284, 4.752 and 3.973 (5H, 3s, NH₂, CH-3, CH₂).

N‘-[(E)-(4-hydroxyphenyl)methylidene]-2-[(2-oxo-2H-1-benzopyran-4-yl)amino]acetohydrazide (4e)

MS (ESI) m/z: 337.98 (M+1); (FTIR, υmax, cm⁻¹); 3587.15, 3421.49, 3188.25 and 1754.59 (=N/-NH, -OH, –CH₂ C=C/C=N), 1665.21 (C=O)_, 1601.83, 1495.24, 1443.47, 1339.62 (diketones, C-O-C, aromatic C-H/mono-substituted alkynes), 1062.91, and 735.38 (aromatic and aliphatic amines and scissoring vibrations).¹H-NMR (CD3OD, 500MHz): δ 10.113, 9.431, 8.769 and 8.459 (4H, 4s, 2NH, OH, CH), 8.213 (1H, m, Ar, CH-5), 7.729 (3H, 2m, J=7.12 Hz, Ar, CH-2, 6 and 7), δ 7.213 (2H, 1m, Ar, CH-6, 8), 6.711 (2H, d, J=1.84, Ar, CH-3, 5), 4.892 and 4.073 (3H, 2s, CH-3, CH₂).

N‘-[(E)-(3,4-dihydroxyphenyl)methylidene]-2-[(2-oxo-2H-1-benzopyran-4-yl)amino]acetohydrazide (4f)

MS (ESI) m/z: 353.24 (M+1); (FTIR, υmax, cm⁻¹); 3585.17, 3412.94, 3179.27 and 1745.95 (=N/-NH, -OH, –CH₂ C=C/C=N), 1661.25 (C=O) 1603.81, 1495.42, 1443.47, 1339.67 (diketones, C-O-C, aromatic C-H and mono-substituted alkynes), 1062.19, 1012.78 and 735.38 (aromatic and aliphatic amines and scissoring vibrations).¹H-NMR (CD3OD, 500MHz): δ 10.113, 9.431, 8.769 and 8.459 (5H, 3s, 2NH, 2OH, CH), 8.231, (1H, m, Ar, CH-5), 7.782 (1H, m, Ar, CH-7), 7.313 (2H, m, Ar, CH-6, 8), 6.811 and 6.412 (3H, 2d, J=1.98, 6.97, Ar, CH-2, 3, 6 of dihydroxybenzylidene ring), δ 4.892 and 4.062 (3H, 2s, CH-3, CH₂).

N‘-{(E)-[4-(dimethylamino)phenyl]methylidene}-2-[(2-oxo-2H-1-benzopyran-4-yl)amino]acetohydrazide (4g)

MS (ESI) m/z: 364.27 (M+1); (FTIR, υmax, cm⁻¹); 3432.89, 3349.72, 3250.02, 2991.10 and 1724.11cm^-1 (=N/-NH, –CH₃/CH₂, C=C/C=N), 1663.92 (C=O), 1619.05, 1467.72 and 1112.02 (diketones, C-O-C, aromatic and C-H stretching), 1053.29, 912.32 and 629.18 (aromatic and aliphatic amines and acetylenic vibrations). ¹H-NMR (CD3OD, 500MHz): δ 10.113, 8.769 and 8.459 (3H, 3s, 2NH, CH), 8.225 (1H, m, Ar, CH-5), 7.631 (3H, m, Ar, CH-6, 7, 8; coumarin), 6.983, 6.427 (4H, 2m, Ar, CH-2, 3, 5, 6; benzylidene), 4.892, 3.762 and 3.056 (9H, 3s, Ar, CH-3, CH₂, 2CH₃).

N‘-[(E)-(3,4,5-trimethoxyphenyl)methylidene]-2-[(2-oxo-2H-1-benzopyran-4-yl)amino]acetohydrazide (4h)

MS (ESI) m/z: 412.36 (M+1); (FTIR, υmax, cm⁻¹); 3476.58, 3409.95, 3276.82, 3052.47, and 1901.93 (=N/-NH,–CH₃ C=N), 1681.46 (C=O), 1583.47, 1457.17 and 1349.83 (diketones, C-O-C, aromatic and mono-substitution), 1247.26 (carbonyls), 1093.44 and 897.14 (aliphatic amines and scissoring vibrations).¹H-NMR (CD3OD, 500MHz): δ 10.102, 8.769 and 8.459 (3H, 3s, 2NH, CH), 8.236 (1H, 2d, J=7.32 Hz, Ar, CH-5), 7.632 (1H, m, Ar, CH-7), 7.023 (2H, m, Ar, CH-6, 7) 6.318 (2H, s, Ar, CH-2, 6), 4.892, 3.762 3.502, and 3.253 (12H, 4s, Ar, CH-3, CH₂, 3CH₃).

N‘-[(E)-(3,4-dimethoxyphenyl)methylidene]-2-[(2-oxo-2H-1-benzopyran-4-yl)amino]acetohydrazide (4i)

MS (ESI) m/z: 382.78 (M+1); (FTIR, υmax, cm⁻¹); 3479.02, 3388.74, 3239.98, 3057.12 and 1903.49 (=N/-NH,–CH₃/CH₂, C=N), 1735.15 (C=O)_, 1607.7, 1547.83, 1512.42, 1482.97 and 1269.23 (C-O-C, aromatic C-H and mono-substitution/carbonyls), 1079.32, 981.07 and 907.35 (aromatic, aliphatic amines and scissoring vibrations).¹H-NMR (CD3OD, 500MHz): δ 10.102, 8.769 and 8.459 (3H, 3s, 2NH, CH), 8.236 (1H, m, Ar, CH-5), 7.622 (1H, m, Ar, CH-7), 7.323 (1H, d, J= 1.12, Ar, CH-2), 7.023 (2H, m, Ar, CH-6, 7), 6.632 and 6.286 (2H, 2d, J=1.87, 1.73, Ar, CH-5, 6), 4.892 and 3.573 (3H, 2s, Ar, CH-3, CH₂), 3.253 (6H, d, J=7.01, 2CH₃).

N‘-[(E)-(4-bromophenyl)methylidene]-2-[(2-oxo-2H-1-benzopyran-4-yl)amino]acetohydrazide (4j)

MS (ESI) m/z: 401.11 (M+1); (FTIR, υmax, cm⁻¹); 3401.82, 3357.48, 3049.95 and 1752.29 (=N/-NH, CH₂, C=C/C=N), 1653.54 (C=O), 1607.82, 1501.29, 1459.48, 1385.47 and 1209.25 (diketones, C-O-C, aromatic C-H and mono-substitution/carbonyls group), 1098.39, 1025.07, 907.59 and 785.48 (aromatic, aliphatic amines, C-Br and scissoring vibrations).¹H-NMR (CD3OD, 500MHz): δ 10.102, 8.769 and 8.459 (3H, 3s, 2NH, CH), 8.236 (1H, 2d, J=7.32 Hz, Ar, CH-5), 7.613 (5H, m, Ar, CH-7, 2, 3, 5, 6), 7.192 (2H, m, Ar, CH-6, 8), 4.892 and 4.073 (3H, 2s, CH-3, CH₂). The structures of synthesized compounds with the melting point, percentage yield, and TLC retardation factor are summarized in table 1.

Table 1: Chemical structures/IUPAC name and physicochemical characters of synthesized compounds

Antibacterial activity

In-vitro antibacterial activity of the screened coumarin derivatives compounds was performed through zone inhibition assay/well-diffusion assay against S. aureus, B. subtilis (gram-positive) and E. coli, P. aeureginosa (gram-negative) bacterial strain using the standard protocol with some modification.¹³ In brief, the bacterial inoculum was evenly spread over the surface of the agar Petri dishes plate using a sterile cotton swab. Thereafter, four holes of 5 mm diameter were made using a sterile tip. 10 µL of drug solution (1mg/ml) was added for two wells and the next two wells were treated with 10 µL of autoclaved double-distilled water as a control treatment. Plates were incubated for 72 h under aerobic conditions and at 37ºC temperature. The antimicrobial effect was determined by the clear zone in the agar, which was measured after the completion of treatment.

Results and discussion

Schiff bases are the ketone or aldehyde organic compounds which are widely used in synthesis of many organic compounds including coumarins. Although the classical synthesis mainly involves condensation of carbonyl compounds and used for highly electrophilic carbonyl and nucleophilic amine compounds.¹⁴ In our study, several coumarin derivatives were synthesized through Schiff base reaction. During the synthesis process, the reaction was characteristically monitored by thin-layer chromatography (TLC) to determine purity of compounds. The percentage yield and melting points of each synthesized compound were determined successfully. The resulted outcomes of the study have been summarized in table 1. 

Table 1: Chemical structures/IUPAC name and physicochemical characters of synthesized compounds. Click here to View table

Figure 4: FT-IR spectra of compound 4a Click here to view figure

Figure 5: FT-IR spectra of compound 4b Click here to View figure

Figure 6: FT-IR spectra of compound 4c Click here to View figure

Figure 7: FT-IR spectra of compound 4d Click here to View figure

Figure 8: FT-IR spectra of compound 4e Click here to View figure

Figure 9: FT-IR spectra of compound 4f Click here to View figure

Figure 10: FT-IR spectra of compound 4g Click here to View figure

Figure 11: FT-IR spectra of compound 4h Click here to View figure

Figure 12: FT-IR spectra of compound 4i Click here to View figure

Figure 13: FT-IR spectra of compound 4j Click here to View figure

Antibacterial activity of compound

Zone inhibition assay or agar disk-diffusion method is one of the most widely and economic methods used for many clinical microbiology laboratories for routine evaluation of antimicrobial testing. In this method, generally agar plates are inoculated with the testing bacterial strain and potency of the tested compound is evaluated by diffusing the test sample into the prepared disk. Furthermore, after a period of incubation, zone of inhibition against the bacterial growth is evaluated, that reveals the potency of tested compound.¹⁵ In our study, in-vitro antibacterial activity of the screened coumarin derivatives compounds was performed through zone inhibition assay/well-diffusion assay against S. aureus, B. subtilis (gram-positive) and E. coli, P. aeureginosa (gram-negative) bacterial strain. The inhibitory action to bacterial strain was considered with the direct proportion of obtained zone observed in the agar plate. The resulted outcomes suggest that compounds 4a, 4b, 4h, and 4j showed potential inhibitory effects against S. aureus while compounds 4a, 4b, 4h, and 4i showed potential inhibitory effects against B. subtilis. Moreover, 4a, 4b, 4g, 4h, 4i and 4j showed good potential inhibitory effects against the survivability of E. coli while compounds 4a, 4b, 4h, and 4j showed potential inhibitory effects against P. aeureginosa survivability. Ceftriaxone and erythromycin were used as a positive control to determine the correlative effect of synthesized compounds. Out of 10 compounds, compounds 4a, 4b, 4h, and 4j showed significant inhibitory potential even against all microbial strains. The average inhibitory effect by all the ten compounds and the standard drug has been summarized in table 2.

Table 2: Antibacterial activity of synthesized compounds against S. aureus, B. subtilis and E. coli,P. aeureginosa using well diffusion assay or zone inhibition assay

Sr. No.	Compound name	Staphylococcus aureus	Bacillus subtilis	Escherichia coli	Pseudomonas aeureginosa
1.	4a	6.36 ± 0.162	5.60 ± 0.049	3.61 ± 0.176	5.64 ± 0.021
2.	4b	7.29 ± 0.339	5.53 ± 0.459	3.35 ± 0.226	5.55 ± 0.042
3.	4c	5.37 ± 0.368	4.25 ± 0.228	2.09 ± 0.266	4.28 ± 0.258
4.	4d	4.28 ± 0.279	3.27 ± 0.238	3.01 ± 0.237	3.96 ± 0.245
5.	4e	3.57 ± 0.115	3.01 ± 0.023	2.58 ± 0.138	2.99 ± 0.247
6.	4f	3.98 ± 0.019	2.98 ± 0. 038	2.95 ± 0.438	3.01 ± 0.543
7.	4g	5.62 ± 0.862	3.55 ± 1.944	3.82 ± 0.014	5.92 ± 0.650
8.	4h	7.10 ± 0.544	5.11 ± 0.183	3.95 ± 0.226	4.94 ± 0.494
9.	4i	5.89 ± 0.098	4.85± 0.993	3.99 ± 0.286	4.27 ± 0.118
10.	4j	6.46 ± 1.725	4.53 ± 0.261	3.83 ± 0.791	5.40 ± 0.049
11.	Ceftriaxone	6.35 ± 0.216	5.37 ± 0.227	6.81 ± 0.286	6.02 ± 0.176
12.	Erythromycin	6.29 ± 0.123	6.37 ± 0.851	6.69 ± 0.248	6.31 ± 0.284

Figure 14: 1HNMR spectra of compound 4a Click here to View figure

Figure 15: 1HNMR spectra of compound 4b Click here to View figure

Figure 16: 1HNMR spectra of compound 4c Click here to View figure

Figure 17: 1HNMR spectra of compound 4d Click here to View figure

Figure 18: 1HNMR spectra of compound 4e Click here to View figure

Figure 19: 1HNMR spectra of compound 4f Click here to View figure

Figure 20: 1HNMR spectra of compound 4g Click here to View figure

Figure 21: 1HNMR spectra of compound 4h Click here to View figure

Figure 22: 1HNMR spectra of compound 4i Click here to View figure

Figure 23: 1HNMR spectra of compound 4j Click here to View figure

Previous studies have been reported that coumarin and its derivatives are the potential candidates which show strong antibacterial activity. A study conducted by Singh et al., 2015 reported that coumarin derivatives showed potent antimicrobial activities against seven of the nine microbial strains examined in the study. The inhibitory potential was expressed in form of MIC values ranging between 1.09 and 25 µg/mL and was the most active compound of the series.¹⁶ Furthermore, many reported studies strongly emphasized to have the strong antibacterial potential of coumarins.^{17, 18} In a study conducted by Chattha et al., evaluated antibacterial activity of coumarins -3-acetic acid derivatives and the reported that some of coumarins -3-acetic acid derivatives were found to have potential inhibitory effect against several gram positive and negative bacterial strains.¹⁹ Our study clearly demonstrate the potential antimicrobial compounds which would work against the drugs seems to have antibacterial resistant. Moreover, the study demonstrate an silent answer to the continual demand for new antibiotics for the unceasing emergence of antibiotic-resistant strains and the growing interest in the substitution of synthetic antibiotic.

Conclusion

In this study, ten coumarins 4-aminocoumarin derivatives were synthesized, characterized and evaluated for their antibacterial activity, successfully. The coumarins 4-aminocoumarin derivatives showed significant inhibitory potential against the gram positive and negative bacterial strains. Furthermore, compound 4a, 4b, 4h, and 4j showed significant inhibitory potential even against all microbial strains. These analogues are chemically tractable and hence provide ample prospects for further adaptation to obtain potent antimicrobial agents the antibacterial resistant drugs.

Acknowledgment

The authors thank Professor Vijay Bhalla, Principal, SGT College of Pharmacy, SGT University, Gurugram-122505, India, for providing necessary facilities.

Conflict of interest

The authors declare no conflict of interest.

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