Hands-on synthesis of furanamides and evaluation of their antimicrobial activity

Abstract Diverse natural and synthetic furan derivatives have shown biological activity. Here, we describe the preparation of benzyl and arylethyl β-furanamides with OH or OMe aryl substituents by an adapted sustainable method from a furoic acid using methyl chloroformate. Symmetric and asymmetric β,β’-furanamides have instead been prepared using azabenzotriazole based catalyst (HATU). The products have been evaluated for their antimicrobial properties on Gram positive and Gram negative bacteria. Just a minimal not-significant activity has been observed in some derivatives.


Introduction
Furans have a prominent role among heterocycles. They are widespread in nature, often exhibiting important bioactivities, as well as in compounds of high industrial interest as pharmaceuticals or polymers (Keay et al. 2008). From a synthetic point of view, furans are useful building blocks for the elaboration of natural and synthetic organic materials (Lee et al. 2005;Merino et al. 2007), as well as synthons in multistep reactions, including total synthesis, and several furan-containing scaffolds serve as privileged structures in medicinal chemistry (Lo go glu et al. 2010). Therefore, great attention is constantly devoted to the search of more efficient methodologies for preparing functionalized furans or elaborating suitable derivatization reactions. The main difficulty in the development of synthetic approaches for furans is their sensitivity to acids, strong oxidants, and high temperatures (Nikbin et al. 2013). In this context, we have developed various mild methods to convert furans to derivatives of high synthetic and biological interest (Astarita et al. 2009;Iesce et al. 2015;Cermola et al. 2021) or to introduce useful functional groups. More recently, for example, to avoid acidic conditions, we have pointed out Tf 2 O-mediated Friedel-Crafts acylation and benzylation reactions on furoic acids to introduce aroyl and benzyl groups on furans. The research has led to the preparation of furolignan-type natural products, (Comegna et al. 2012;Iesce et al. 2016;Tufano et al. 2020;Tufano et al. 2020) and a few of them have shown antimicrobial properties (Vollaro et al. 2019;Tufano et al. 2020).
In our continuing studies on this topic, we decided to search a mild method to introduce the amide function. The importance of the amide functional group emerges from its presence in many crucial compounds, such as proteins, insecticides, plastics, drugs and so on. Anilines (Li et al. 2012), benzyl amines (Pavel et al. 2019) and arylethyl amines (Georgiev et al. 2013) are the most used nitrogen moieties. In many cases, when compared with the other derivatives, amides have shown much higher activity (Pavel et al. 2019). The presence and the position of the aromatic substituents have also a role (Li et al. 2012), and in many cases, an antimicrobial activity has been found and connected to the presence of OH substituents (Hollman 2001;Georgiev et al. 2013). Some examples of active furanamides have also been reported (Poojary et al. 2003).
In this work, we explored the possibility to prepare furanamides starting from 3furoic acids and benzyl and arylethyl amines with OH or OMe group as ring substituents using a recent sustainable procedure (DellaGreca and Longobardo 2020). The authors have prepared amides of hydroxycinnamic acids by a unique protection-activation system by ethyl chloroformate in the presence of a base in water at room temperature (DellaGreca and Longobardo 2020). This procedure appeared as the most promising one since it is compatible with both furan and phenol systems.
All the structures were confirmed by spectral data. The products were analyzed for their antimicrobial properties on Gram-positive and Gram-negative bacteria.

Results and discussion
The starting reagent was commercially available dimethyl furan-3,4-dicarboxylate (1) that was hydrolysed under mild basic conditions (Comegna et al. 2012) to give compound 2a (Scheme 1) together with small amount of 3,4-difuroic acid (2b) that was the main product by prolonged reaction time (see Scheme 3). Monoamides 4a-f were prepared starting from furoic acid 2a (Scheme 1) with amines 3a-f according to the reported procedure (DellaGreca and Longobardo 2020) but using acetone as solvent to improve solubility. In particular, acid 2a in acetone was treated with 2.2 equiv. of triethylamine (TEA) and methyl chloroformate, under vigorous stirring for 1 h; then, amine 3 was added and the mixture quenched after 3 h with aq. citric acid. Purification gave amide 4 in the yields reported in Scheme 1.
Attempts to apply this synthetic procedure to acid 5, obtained by methylester hydrolysis of furanamide 4b, (Scheme 2) or to diacid 2b failed and only tarry material was obtained together with small amount of the starting acid. Hence, to form amide bond we used, as coupling reagent, O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) that has been described as an excellent condensing reagent in peptide coupling chemistry (Carpino 1993). Under the described conditions (HATU in DMF as solvent and in the presence of H€ unig's base (N,N-diisopropylethylamine, DIPEA), the reaction, starting from acid 5, with amine 3e gave the benzyl phenethyl diamide 6 in good yield. (Scheme 2) Using HATU, as condensing reagent, diamide 7 was prepared starting from 3,4-furandicarboxylic acid (2b) and amine 3e.

Scheme 1. Preparation of furanamides 4a-f
In order to obtain information on their antimicrobial properties, compounds 4a-f, 6 and 7 were tested on three Gram-positive strains Staphylococcus aureus ATCC 29213, S. aureus ATCC 43300 (MRSA) and S. epidermidis ATCC 35984 (biofilm producer), and two Gram-negative strains, Pseudomonas aeruginosa ATCC 27853 and Klebsiella pneumoniae ATCC BAA1705. The antibiotics vancomycin, oxacillin, tobramycin, and imipenem were used as controls against S. epidermidis, S. aureus, P. aeruginosa, and K. pneumoniae, respectively, according to European Committee on Antimicrobial Susceptibility Testing (EUCAST version 9.0, 2019). As reported in Supporting file (Table  S1) none of the compounds tested was able to significantly affect the growth of the microorganisms studied. However, a poor activity was observed for compounds 4c, 4d and 4e that at 128 mg/mL reduced the growth of both S. aureus strains by 15, 25 and 20%, respectively. This could be due to the presence of the aryl OH according to the well-known role of the phenolic and cathecolic function in anti-oxidative processes (Foti 2007). The minimal activity was annulled by steric factors either on ethyl chain and on furans as observed for amide 4f and b,b'-furan diamides 6 and 7, respectively. However, a wider range of similar compounds could be performed to establish structure activity relationship.
Considering that amide 4d showed the less poor bioactivity we decided to synthetize the corresponding acid 8 to verify the influence of the presence of a carboxylic function (Scheme 4). In order to avoid oxidation of the cathecolic function, amide 4d was hydrolysed using sodium tert-butoxide under nitrogen. As shown in Table S1, the conversion of 4d to 8 resets even the minimal bioactivity.
Furan derivative prepared showed bioactivity neither on S. epidermidis nor on gram-negative strains.

Synthesis of compounds
3.1.1. Preparation of 4-(methoxycarbonyl)furan-3-carboxylic acid (2a) and furan-3,4-dicarboxylic acid (2b) Dimethyl furan-3,4-dicarboxylate (1, 184 mg; 1 mmol) was dissolved in 3 mL of methanol and the flask was immersed in a water bath at r.t. Under stirring, 0.75 mL of a solution of 2 M KOH were added. After 30 min, methanol was removed in vacuum. The mixture was dissolved in H 2 O (5 mL) and treated with diethyl ether (5 mL; 1:1 v/v). The aqueous layer was treated with a solution of 2 M HCl up to pH $1 observing the formation of a white precipitate. The precipitate was filtered and washed with H 2 O until neutrality. The filtrate was extracted with EtOAc (3 Â 5 mL) and the combined organic layers were washed with water until neutral pH and dried (Na 2 SO 4 ). Evaporation of the solvents under reduced pressure afforded a crude residue, which was purified by column chromatography using a gradient of CH 2 Cl 2 -EtOAc (100/0 to 80/20 v/v) as eluent to afford 2a (yield 79%).
In another experiment, the diester 1 was treated with 2 M KOH as above and the resulting mixture allowed to stand under stirring for 20 h. Methanol was then removed in vacuum and the mixture was dissolved in H 2 O (5 mL) and washed with diethyl ether (5 mL; 1:1 v/v). The aqueous layer was treated with a solution of 2 M HCl up to pH $1 observing the formation of a white precipitate. The solid was filtered under vacuum, washed with H 2 O until neutrality and dried to afford the crude difuroic acid 2b (80%) that was used without purification.

General procedure for the synthesis of furanamides (4a-4f)
To a magnetically stirred solution of furan-3-carboxylic acid 2a (85 mg; 0.5 mmol) in acetone (2.5 mL), triethylamine (2.2 equiv.; 1.1 mmol; 150 mL) was added and the flask was immersed in a water bath. Methyl chloroformate (2.2 equiv.; 1.1 mmol; 140 mL) was added dropwise. A white precipitate rapidly appeared and stirring was continued for 1 h at r. t. until a TLC control (hexane-EtOAc, 7/3 v/v) showed no more starting material presence. The mixture was filtered, and the filtrate was washed with further 1-3 mL of acetone. The collected filtrate was then added dropwise to 1.5 equiv. of amine 3, dissolved or suspended in 2 mL of acetone. The mixture was kept under stirring for 3 h. Then, most of acetone was removed in vacuum and to the residue 3 mL of 5% aq. citric acid were carefully added under stirring. When a white precipitate appeared, it was filtered and washed with H 2 O until neutrality and dried. In other Scheme 4. Synthesis of amide 8. cases the residue was extracted with EtOAc (3 Â 5 mL) and the combined organic layers were washed with water until neutral and dried (Na 2 SO 4 ).
Evaporation of the solvent under reduced pressure afforded a crude residue, which was purified by column chromatography using a gradient of CH 2 Cl 2 -EtOAc (100/0 to 70/30 v/v) as eluent to afford the desired furanamide.
3.1.3. Preparation of 4-((4-methoxybenzyl)carbamoyl)furan-3-carboxylic acid (5) The furanamide (4b) (117 mg; 0.4 mmol) in 1.2 mL of methanol was treated with 0.3 mL of a solution of 2 M KOH and the resulting mixture allowed to stand under stirring for 30 min. Then, the mixture was dissolved in H 2 O (3 mL) and worked as for acid 2a.
The column chromatography of the residue using a gradient of CH 2 Cl 2 -EtOAc (100/ 0 to 80/20 v/v) as eluent afforded acid 5 in 70% yield.

Antimicrobial susceptibility testing
Minimal inhibitory concentrations (MICs) of all the compounds were determined in Mueller-Hinton medium (MH) by the broth microdilution assay, following the procedure already described (Buommino et al. 2021). The compounds were added to bacterial suspension in each well yielding a final cell concentration of 1 Â 106 CFU/mLand a final compound concentration ranging from 2 to 128 lg/mL. Negative control wells were set to contain bacteria in Mueller-Hinton broth plus the amount of vehicle (DMSO) used to dilute each compound. Positive controls included vancomycin (2 lg/ mL), tobramycin (2 lg/mL), oxacillin (2 and 10 lg/mL) and imipenem (4 and 8 lg/mL). The MIC was defined as the lowest concentration of drug that caused a total inhibition of microbial growth after 24 h incubation time at 37 C. Medium turbidity was measured by a microtiter plate reader (Bio-Rad mod 680, Milan, Italy) at 595 nm.