Triethyl ammonium sulphate catalyst one pot , Solvent free synthesis of novel Coumarin derivatives as antimicrobial agents

The work reports synthesis of 15 novel 3-((dicyclohexylamino)(substituted phenyl/heteryl)methyl)-4-hydroxy-2H-chromen-2-one derivatives 4 (a-o) as potential antimicrobial agents in solvent-free condition using Triethyl ammonium sulphate [Et3NH][HSO4] as an efficient, eco-friendly and reusable catalyst. Compared to other methods, this new method consistently has advantages, including excellent yields, a short reaction time, mild reaction conditions and catalyst reusability. The heterocyclic compound Coumarin, is associated with diverse biological activities of immense importance. Due to the presence of coumarin moiety in various pharmaceutically active compounds, we planned the green synthesis of a series of 15 novel compounds containing coumarin moiety coupled with dicyclohexyl rings by an eco-friendly ionic-liquid mediated protocol at room temperature by stirring. The structures of the synthesized compounds were confirmed by spectral characterization such as IR, 1H NMR, 13CNMR and Mass spectral studies. All the synthesized compounds 4 (a-o) were evaluated for anti-fungal and antibacterial activities and have exhibited promising antimicrobial activity.


INTRODUCTION
Many drug-resistant human pathogenic microbes have been observed in the past few decades [1] and it is serious public health problem in a wide range of infectious disease [2,3].These resistant pathogenic microbes' strains cause failure in antimicrobial treatment and enhance the mortality risks, and sometimes contribute to complications.To overcome this problem the best way is the development of new bioactive compounds effective against resistant strains is highly needed.In spite of a large number of antibiotics and chemotherapeutics available for medical use, antimicrobial resistance created substantial medical need for new classes of antimicrobial agents.Design and synthesis of newer antimicrobials will always remain an area of immense significance [4][5].The novel and potent antimicrobial agents can be obtained by modifying the structure of a well known antimicrobial agent or the second strategy is to combine together two or more different antimicrobial pharmacophores in one molecule.
The Mannich reaction is one of the most important carbon-carbon bond forming reactions in organic synthesis because of its atom economy and potential application in the synthesis of biologically active molecules.In this reaction, an amine, two carbonyl compounds, and acid (or base) catalysts are used to produce β-amino carbonyl compounds, which constitute various pharmaceuticals, natural products and versatile synthetic intermediates [13,14].Conventional catalyst of the classic Mannich reaction involves inorganic and organic acids like HCl [15], proline [16], p-dodecybenzenesulfonic acid [17].Reaction using these catalysts, however, often suffers drawbacks including long reaction times, harsh reaction condition, and difficult product separation.
Considering the focus on green synthesis in recent years, ionic liquid have attracted attention many of researchers.Ionic liquid have been referred as "designer solvents/ green solvents" because their physical and chemical properties can be adjusted by varying the cation and anion.[21] and some other bronsted ionic liquids (22)(23).Although extensive work has been done in this area, the disadvantage of the above mentioned catalytic systems, are large amount of catalyst required, the necessity of an organic co-solvent, cost, the ionic liquids contain halogen, which in some ways, limits their "greenness".Thus synthesizing halogen free, water soluble, economic, reusable and easy to prepare ionic liquid was the main aim of our research team.Taking in consideration the above mentioned points we have carried out the synthesis of coumarin-dicyclohexyl coupled hybrid derivatives 4(a-o) using [Et 3 NH][HSO 4 ] as an solvent and easily recoverable green catalyst (Scheme 1).

Mannich reaction have been performed using various ionic liquid such as
All the synthesized compounds 4(a-o) were screened for in vitro antifungal and antibacterial activity.Minimum inhibitory concentration (MIC) values were determined using the standard agar method as per CLSI guidelines [24][25][26][27].

Chemistry:
Herein we report the one-pot synthesis of 15  The re-usability of the ionic liquid [Et 3 NH][HSO 4 ] was also studied and the results obtained are as shown in Table 3.After the completion of the reaction, the reaction mixture was quenched with ice crystals and filtered recrystallized using ethanol.The residual ionic liquid was washed with diethyl ether, dried under vacuum at 60 0 C and reused for subsequent reactions.The recovered ionic liquid could be used for five times without obvious loss of catalytic activity.4 All the synthesized compounds were characterized by 1 H-NMR, 13 C-NMR, mass spectroscopy and IR. a Values are the average of three readings

In-vitro antibacterial activity
The newly synthesized compounds 4(a-o) were screened for in vitro antibacterial activity against different bacterial strains.All the compounds have shown good to moderate antibacterial activity as shown in Table 6 The compound 4b bearing 2,4 difluro was found to be the most active compound among the synthesized series having MIC values 48 µg/ml for E. coli, 50 µg/ml for B. subtilis and 52 µg/ml for S. aureus.Values are the average of three readings

General Information
All the reactions were performed in oven-dried glass-wares.All reagents and solvents were used as obtained from the supplier or recrystallized/redistilled unless otherwise noted.The purity of the synthesized compounds was monitored by ascending thin layer chromatography (TLC) on silica gel-G (Merck, Darmstadt, Germany) coated aluminum plates, visualized by iodine vapor and melting points were determined in open capillary tubes.The FTIR spectra were obtained using Jasco FTIR-4000 and peaks were expressed in terms of wave number (cm -1 ).The 1 H NMR and 13 C NMR spectra of synthesized compounds were recorded on Bruker Avance II 400 NMR Spectrometer at 400 MHz Frequency in CDCl 3 and using TMS as internal standard (chemical shift δ in ppm), Mass spectra were scanned on Water's Micromass Q-Tof system Elemental analyses (C, H, and N) were done with a FLASHEA 112 Shimadzu' analyzer (Mumbai, Maharashtra, India) and all analyses were consistent (within 0.4%) with theoretical values.

In-vitro antimicrobial activity
All the synthesized compounds were screened for in vitro antifungal and antibacterial  4).Minimum inhibitory concentration (MIC) values were determined using the standard agar method as per CLSI guidelines [24][25][26][27].
Use of green catalyst, i.e. triethyl ammonium sulphate as an ionic liquid helped us in the synthesis of expected derivatives in good yields and is advantageous being an eco-friendly method.The mild reaction conditions, excellent yields in shorter reaction time and evasion of cumbersome work-up procedures make this process economically lucrative for industrial application with the advantage of reusability of catalyst.In the present series the compound 4e with 2,4-di Fluro substituent on phenyl group found to be most potent antibacterial agent.The compound 4k with 4-hydroxy-3-methoxy on phenyl group found to be most potent antibacterial agent.
Scheme 1. One-Pot, three component synthesis of novel 3-((dicyclohexylamino)(substituted phenyl/heteryl)methyl)-4-hydroxy-2H-chromen-2-one derivatives 4 (a-o) In search of an efficient catalyst and the best experimental reaction conditions, the reaction of benzaldehyde (1a), dicyclohexylamine (2) and 4-hydroxy coumarin (3) at room temperature was considered as a standard model reaction to obtain 4a.Initially, the reaction was carried out in the absence of the catalyst; the product formed in a trace/negligible amount Table1, entry 1.To determine the appropriate concentration of the

of 3 -
((dicyclohexylamino)(substituted phenyl/heteryl)methyl)-4-hydroxy-2Hchromen-2-one derivatives 4 (a-o) A 25 mL a beaker was charged with a mixture of a suitable aldehyde (1.25mmol), dicyclohexyamine (1.25mmol), 4-hydroxy coumarin (1.25mmol), and 20 mol % of [Et 3 NH][HSO 4 ] as catalyst and the reaction mixture was stirred at room temperature.After completion of the reaction (monitored by TLC), the mixture was poured into ice cold water.The product obtained, was filtered and dried.The corresponding product was obtained in high purity after recrystallization of the crude product from ethanol.The authenticity of compounds was established by 1 H-NMR, 13 C-NMR, IR and Mass spectra.

Table 3
Reusability of [Et 3 NH][HSO 4 ] catalyst for model reaction 4a With these optimized reaction conditions for the model reaction 4a, i.e. 20 mol% [Et 3 NH][HSO 4 ] catalyst, room temperature and solvent-free conditions, we have synthesized 15

Table 4
Physical characterization of synthesized compounds 4 (a-o).

Table 5 .
In-vitro antifungal activity of synthesized compounds 4 (a-o)