Heterocycles 38. Biocatalytic Synthesis of New Heterocyclic Mannich Bases and Derivatives

This paper describes the biocatalytic synthesis of new Mannich bases containing various heterocyclic rings (thiazole, furane, thiophene, pyridine) by applying the lipase catalyzed trimolecular condensation of the corresponding heterocyclic aldehydes with acetone and primary aromatic amines, in mild and eco-friendly reaction conditions. The obtained Mannich bases were acylated to their corresponding N-acetyl derivatives. All compounds were characterized by 1H-NMR, 13C-NMR and MS spectrometry.


Introduction
Mannich bases and derivatives are pharmacologically and chemically useful compounds due to their therapeutic potential and to the reactivity of their functional groups [1,2]. In addition, aromatic heterocyclic ring systems have a great value in the field of medicinal chemistry, being responsible for a variety of biological activities. In particular, the thiazole ring is a pharmacophore group intensely studied due to OPEN ACCESS its remarkable biological potential, including anti-inflammatory [3], antitumor [4], antibacterial [5], and antiviral [6] properties. The thiophene and furane moieties have attracted special interest in the development of new potent analgesic [7], anti-inflammatory [8], antimicrobial [9], and anticonvulsant [8] agents. The pyridine ring is often found in the structure of antimicrobial agents, the most relevant example being the antitubercular agent isonicotinic hydrazide.
The classical variant of the Mannich reaction involves the use of formaldehyde, a secondary amine hydrochloride and an enolisable carbonyl compound as CH-acidic substrate. The drawbacks of the classical procedure are more evident when primary amines or ketones with two reactive alpha positions are used in the reaction. In this case, the formation of secondary products cannot be avoided, because of the lack of selectivity. Modern variants of the Mannich reaction have been proposed, either starting from pre-formed enolates [10], immonium salts [11], or by organometallic/organocatalytic methods [12]. Despite the notable successes achieved, the use of these chemical methods is generally limited by economical reasons, by the low stability of the intermediates and because of the toxicity of the solvents, intermediates and catalysts used.
Biocatalytic processes have become a useful and green alternative in organic synthesis due to their multiple advantages. Moreover, the enzymes can be immobilized and this way they can be readily separated from the reaction mixture and reused in many cycles. Previous studies reported that, besides their natural role, lipases are able to catalyze different synthetic reactions, such as C-C and C-N bond forming [13]. The lipase-catalyzed direct Mannich reaction using as substrates some substituted benzaldehydes, primary amines and ketones was previously described and a possible mechanism was also proposed [14]. The method afforded Mannich bases that cannot be obtained by classical Mannich reaction. However, at present, there is a lack of information in this field and further studies are necessary to be made, in order to extend the applicability of this biocatalytic approache.
As a continuation of research devoted to the development of mild and eco-friendly biocatalytic synthetic routes and being aware of the pharmacological value of nitrogen, sulfur and oxygen containing heterocycles [3][4][5][6][7][8][9], we decided to exploit the potential of the lipase-catalyzed direct Mannich reaction by implementing for the first time this enzymatic reaction in the series of heterocyclic compounds. The novel obtained heterocyclic Mannich bases could have promising applications in medicinal chemistry, due to the therapeutic potential of the heterocyclic side chain and his multiple functionalization.

Results and Discussion
The aim of this study was to exploit the biocatalytic promiscuity of lipases to promote the trimolecular Mannich type condensation, in order to obtain new compounds with biological potential. We oriented our research to the extension of this methodology in the series of heterocyclic compounds. The first goal was to find the optimal reaction conditions for the enzymatic condensation between acetone, primary aromatic amines and various heterocyclic aldehydes. In order to investigate the substrate scope of this method, different types of heterocyclic aldehydes were used as substrates: 2-arylthiazole-4-carbaldehyde, furane-2-carbaldehyde, thiophene-2-carbaldehyde and pyridine-3-carbaldehyde. Thiazolic aldehydes 1a-d were synthesized as previously described in the literature, by Hantzsch condensation of various thiobenzamides with 1,3-dichloroacetone, followed by Sommelet reaction [15].
First, various lipases have been evaluated as biocatalysts for the direct Mannich condensation between thiazolic aldehydes, aniline and acetone, in aqueous media (Table 1). Kun Li et al. previously demonstrated that the presence of water in the reaction media strongly influence the activity of lipases in the Mannich type condensation. In their experiments, the optimal water concentration was found to be between 40% and 50% [14]. Based on these reported data and taking into consideration the low solubility of heterocyclic aldehydes in water, we choose an acetone/water 1:1 (v/v) system as the reaction media, in order to increase the solubility of substrates.  [3][4][5]. In order to confirm the catalytic activities of lipases in the Mannich reaction, control experiments were also performed. In the absence of the enzyme or in the presence of bovine serum albumin, the formation of Mannich bases was not detected. Lipase B from Candida antarctica (CaL-B, Novozym 435) was chosen for further studies.
Encouraged by these results, we decided to investigate also the ability of lipases to accept thiazole-derived methylketones as CH-acidic substrates, instead of acetone. Consequently, the condensation of benzaldehyde with aniline and 4-methyl-2-phenylthiazol-5-yl ethanone was investigated, but in this case the lipases were not active.
Further experiments were oriented on the condensation of heterocyclic aldehydes with acetone and other primary aromatic amines (p-toluidine, p-Cl-aniline, p-nitroaniline), using CaL-B as biocatalyst. Similar good results were obtained when p-toluidine was used as substrate. CaL-B was inactive in the enzymatic Mannich reactions involving p-Cl-aniline and p-nitroaniline (Table 2, entries 9 and 10).
Because amines are generally susceptible to oxidative degradation and their purification is sometimes laborious, we opted for the use of the corresponding amine salt, because of its better stability and its easier manipulation and storage. Similar good results were obtained when aniline sulfate was used in the enzymatic reactions, in the presence of an equimolar quantity of sodium acetate (Scheme 2).
The preparative scale enzymatic synthesis of Mannich bases 4a-k was performed, in the presence of CaL-B as biocatalyst. The enzymatic reactions were conducted at room temperature, under continuous shaking at 1200 rpm. The aromatic amine was added in a small excess (1.1 eq.), thus ensuring the total conversion of the heterocyclic aldehyde. The Mannich bases 4a-k were obtained in 66%-80% yield, starting from the corresponding aldehydes (0.075 mol/L), aromatic amine (0.0825 mol/L), in the presence of CaL-B (Novozym 435), in an acetone/water 1/1 (v/v) mixture (Table 2). Compound 4a was easily isolated, because it precipitated in the reaction media, and no purification was necessary. The other enzymatic reaction products 4b-k were isolated by extraction and purified on column chromatography. By measuring the optical rotations of the obtained Mannich bases, it was found that the enzymatic reactions occurred without enantioselectivity. In order to increase the stability of the obtained Mannich bases, further acylation of these compounds was performed, using acetic anhydride and a catalytic amount of pyridine, affording compounds 5a-k in good yields (Scheme 3). All synthesised compounds were purified, physically and chemically characterized by 1 H-NMR, 13 C-NMR and MS spectroscopy.

Scheme 3. Synthesis of N-acetylated
In the 1 H-NMR spectra of Mannich bases 4a-k, all signals corresponding to the protons located on the benzene rings are present in the aromatic region. The proton located in the 5th position of the thiazole ring appears as a singlet at 7.08-7.15 ppm. The CH proton located in the β position of the butanone chain appears as a triplet at 4.89-5.18 ppm, while the CH2 protons from the α position are appearing as a doublet, at chemical shifts of 2.96-3.17 ppm. In the 1 H-NMR spectra of the corresponding N-acylated derivatives 5a-k, the β CH proton gives a triplet at 6.41-6.47 ppm, while the α CH2 protons are appearing as a doublet of doublet of doublets, at chemical shifts between 3.05 and 3.11 ppm.
In the 13 C-NMR spectra of compounds 4a-k, a characteristic signal between 206 and 208 ppm indicates the presence of the carbonyl group. In the case of N-acetylated compounds 5a-k, two characteristic carbonyl signals are present, indicating the presence of the ketone group (205-207 ppm for C=O) and, respectively, the presence of the amide group (170-170.9 ppm for CO-NH). All aromatic and aliphatic signals are also present, thus confirming the chemical structures of the products.
The MS spectra of the Mannich bases and their N-acylated derivatives are revealing similar fragmentation pathways. The chemical structures are confirmed by the presence of the molecular peak, which in most cases is accompanied by the peak [M + H] + .

Analytical Methods
The 1 H-NMR and 13 C-NMR spectra were recorded on a Brucker Avance DPX-300 spectrometer operating at 600 and 150 MHz, respectively. Chemical shifts on the δ scale are expressed in ppm values from TMS as internal standard. GC-MS measurements were performed with a Shimadzu QP 2010 Plus machine (Shimadzu Europa GmbH, Duisburg, Germany). EI-MS spectra were recorded with the spectrometer operating at 70 eV.
Melting points were determined on open glass capillaries using an Electrothermal IA 9000 digital apparatus (Bibby Scientific Limited, Staffordshire, UK).

Reagents and Solvents
The commercial chemicals and solvents were products of Sigma Aldrich or Fluka (Sigma Aldrich Chemie GmbH, Steinheim, Germany; Fluka production GmbH, Buchs, Switzerland). All solvents were purified and dried by standard methods as required. Lipase B from Candida antarctica (CaL-B, Novozym 435) was purchased from Novozymes (Bagsvaerd, Denmark). Lipase from Candida rugosa (CrL) was purchased from Fluka. Lipases from Pseudomonas fluorescens and Burkholderia cepacia (BcL) were from Amano, England.

Lipase Screening for the Enzymatic Mannich Reaction
To a solution of 2-phenylthiazol-4-carbaldehyde 1a (14.16 mg, 75 μmol) dissolved in acetone (600 μL), aniline (7.5 μL, 82.5 μmol), deionized water (600 μL) and different lipases (15 mg) were added in this order. The reaction mixtures were shaken at 500 rpm for 20 h and then analyzed by TLC using a mixture of petroleum ether: ethyl acetate 4:1 (v/v) as eluent. The enzyme was removed by centrifugation and filtration and then it was washed three times with acetone in order to recuperate the product. The filtrate was brought to dryness by rotatory evaporation under reduced pressure. The crude product was purified by column chromatography, using a mixture of petroleum ether: ethyl acetate 4:1 (v/v). The purified product was dried and weighed.

Analytical Scale CaL-B Catalyzed Direct Mannich Reaction Using Different Aldehydes and Aromatic Amines as Substrates
To a solution of 2-arylthiazol-4-carbaldehyde 1a-d (25 μmol) dissolved in acetone (200 μL), aromatic primary amine (27.5 μmol of aniline sulfate/p-toluidine/4-chloroaniline/4-nitroaniline), deionized water (200 μL) and CaL-B (4 mg) were added in this order. In the case when aniline sulfate was used as substrate, sodium acetate (2.26 mg, 27.5 μmol) was added to the reaction mixture. The reaction mixtures were shaken at 500 rpm for 20 h and then analyzed by TLC using a mixture of petroleum ether: ethyl acetate 4:1 (v/v) as eluent.

Synthesis of N-Acetylated Mannich Bases 5a-k
To a solution of Mannich base 4a-k (0.6 mmol) in anhydrous dichloromethane (4 mL), acetic anhydride (3 mmol) and 1 drop of pyridine were added. The reaction mixture was stirred overnight. After completion of the reaction (checked by TLC using dichloromethane as eluent), the reaction mixture was quenched with water. The organic layer was dried over anhydrous Na2SO4 and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography on silicagel, in a gradient separation procedure, using a mixture of dichloromethane: acetone from 25:1 to 9:1 (v/v) for compounds 5a-j and respectively a mixture of dichloromethane:acetone 9:2 v/v for compound 5k.