Lipase-Catalyzed Solvent-Free Amidation of Phenolic Acids

Abstract A series of N-alkyl-substituted amides, based on various phenolic acids, have been synthesized by the condensation of equimolar amounts of phenolic acids with different alkyl amines in the presence of Candida antarctica lipase at 60–90 °C in 16–20 h. The reactions were carried out in a solvent-free system without the use of any activating agents. All the products were obtained in appreciable amounts and the yields for different compounds varied between 75.6% and 83.5%. The synthesized compounds were characterized using spectroscopy techniques, namely infrared and NMR (1H and 13C). GRAPHICAL ABSTRACT


INTRODUCTION
Cinnamic acids and their synthetic esters, amides, and glycosides exhibit a wide range of activities, including the anti-oxidative effect on low-density lipoprotein (LDL), [1] the peroxyl radical scavenging effect, [2] and anti-inflammatory [3] and antimutagenic [4] effects. The cholesterol-lowering effect of several hydroxylated cinnamic acid derivatives of amino acids has been evaluated in mice fed with highcholesterol diets. [5] The presence of double bonds in hydroxylated cinnamide derivatives decreases the cholesterol-lowering activities and the number of free phenolic hydroxyl groups greatly affects the biological activity. Amides of phenolic acids having hydrophobic side chains exhibit potent cholesterol-lowering effects. [6] The amides of cinnamic and hydroxycinnamic acids with aliphatic monoamines have been synthesized and their radical scavenging activity against 2,2-diphenyl-1-picryl hydrazyl (DPPH) tests have been reported. [7,8] Substituted amides and amines are also important because of the presence of these functional groups in many compounds having both medicinal as well as pesticidal activities. They are reported to have antibacterial, antifungal, antiviral, insecticidal, nematicidal, and herbicidal properties. [9][10][11][12][13][14][15][16][17][18][19][20][21] Syntheses of these compounds have mainly been achieved by classical chemical reactions, which generally involve the generation of a reactive carboxy derivative, either an acid chloride or anhydride, followed by aminolysis with amine. Also, the conversion of esters to amides has some limitations as the commonly used reagents, that is, sodium methoxide, sodium hydride, sodium metal, or butyl lithium, often interfere with other functional groups present in the reacting species. Among the many significant advances in organic chemistry, attempts at environmentally benign processes in organic synthesis have highlighted the importance of enzyme-catalyzed reactions, particularly for large-scale industrial applications. [22] Enzyme-catalyzed organic reactions have provided a great impetus to organic synthesis during the past two decades. Enzymes, especially lipases, are known for their low cost and great tolerance toward substrates. Environmentally benign character of the enzymatic processes is desirable for large-scale industrial applications. Even though some enzyme-catalyzed reactions for the synthesis of amides were reported, most of them involved the use of activating agents or solvents and the bioactivity of these synthesized compounds was hardly studied. [23][24][25][26][27][28][29][30][31] Considering these facts, we got interested in the search for new phenolic acid analogs. We envisaged developing a one-pot biocatalytic amidation procedure using lipase as catalyst. In this report, we have carried out C. anatrctica lipase (CAL-B)catalyzed amidation of phenolic acids by different aliphatic amines in bulk (without solvent) at 60-90°C.

RESULTS AND DISCUSSION
The condensation of equimolar amounts of different phenolic acids, namely salicylic acid (1), 3-hydroxy cinnamic acid (2), p-coumaric acid (3), caffeic acid (4), ferulic acid (5), o-coumaric acid (6), and cinnamic acid (7), with amines [viz. propyl amine (8a), hexyl amine (8b), heptyl amine (8c), undecyl amine (8d), hexadecyl amine (8e), and octadecyl amine (8f)], catalyzed by Candida antarctica lipase (CAL-B) in a solvent-free system at 60-90°C in 16-20 h resulted in the formation of N-propyl- The feasibility of the solvent-free system was investigated. The reactions with equimolar amounts of amines 8a-8f and phenolic acids 1-7 were performed (Scheme 1). The reaction mixture was very viscous and the reaction temperature had to be raised up to 90°C in order to achieve efficient stirring in the system. There was a report in literature [32] where the reaction temperature was maintained at 55°C for the lipase-catalyzed amidation. In that case, the reaction did not go to completion as some unreacted acid and amine remained in the reaction mixture in salt form even after incubating the mixture for longer times. Such a situation did not arise in our study as the reaction temperature was kept between 60 and 90°C under reduced pressure. The conversion of acids to amides was found to be 100% in 75.6-83.5% yields. The synthesized amides were fully characterized from their spectral data.
In infrared (IR) spectra, the amide I band due to CO stretching appeared at approximately 1645 cm À1 , amide II band due to N-H bending appeared at Scheme 1. Synthesis of phenolic acid amides.

LIPASE-CATALYZED AMIDATION OF PHENOLIC ACIDS
approximately 1560 cm À1 , and amide III band due to C-N stretching appeared at approximately 1250 cm À1 , in all the products that confirmed the condensation of the carbonyl group with the amino group. In the 1 HNMR spectra, a quartet at δ 3.15-3.3 for CONH-CH 2 and a singlet at δ 7.97-8.31 for the N-H proton were characteristic of all the synthesized amides. In 13 C NMR, the peaks at δ 41.4-43.1 for CONHCH 2 and at δ 168-172.4 for CONH were prominent for all the compounds.
In addition to these characteristics, bands at approximately 3425 cm À1 (broad multiplet due to N-H stretching), 2932 cm À1 (strong), and 783 cm À1 (strong) were also present for all the test compounds in IR spectra. In 1 H NMR spectra, the signals in δ range 6.6-7.4 are for aromatic protons (Ar-H), a singlet at δ 4.80-5.29 for Ar-OH, a singlet at δ 3.7-3.8 for Ar-OCH 3 , and doublets at δ 6.2-7.7 for vinylic protons were observed for the synthesized phenolic acid amides.
In 1 H NMR spectral analysis, the H-2′ protons of all the propyl derivatives of phenolic acid amides appear as a multiplet in δ range 1.19-1.68, whereas the aliphatic protons of the side chain of other amides appear in δ range 1.16-1.84. The 1 H and 13 C NMR of one representative amide from salicylic acid and phenolic acids are discussed here.
To the best of our knowledge, the results presented in this study demonstrate for the first time the phenolic acid amide synthesis in solvent-free system by the direct lipase-catalyzed reaction of amines with phenolic acids. The present enzymatic procedure offers some important advantages over the chemical one for application in industrial processes.

EXPERIMENTAL
Laboratory-grade reagents and solvents were procured from Qualigens. Different amines were procured from M/S Sigma Aldrich and used as received. Candida antarctica lipase, an immobilized enzyme, was a gift from Novozymes, Denmark. Thinlayer chromatography (TLC) was performed on 200-µm-thick aluminium sheets with silica gel as adsorbent. The solvent system used for developing the thin-layer chromatography (TLC) plate was ethyl acetate / hexane, 2:8. Spots were visualized in an ultraviolet (UV) / iodine chamber. The IR spectra were recorded on a Perkin-Elmer 2000 Fourier transform IR (FT-IR) spectrometer. The 1 H NMR and 13 C NMR spectra were recorded on a Bruker AC-300 Avance spectrophotometer at 400 and 100 MHz, respectively using tetramethylsilane (TMS) as an internal standard. The chemical shift (δ) values are expressed in parts per million (ppm) and the coupling constants (J) values are in hertz (Hz). The elemental analysis was done on a Eurovector Elemental analyzer 3000 using sulfanilamide as standard with linear calibration.

CONCLUSION
A novel, efficient, and environmentally benign method for the synthesis of phenolic acid amides has been developed, which can be utilized for the synthesis of analogous compounds as this method avoids the use of activating agents for the conversion of acids to amides, which is otherwise not possible chemically.

FUNDING
This work was supported by the Department of Science and Technology, New Delhi, and Indian Council of Agricultural Research, New Delhi.

SUPPORTING INFORMATION
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