Microwave-assisted preparation and antimicrobial activity of O-alkylamino benzofurancarboxylates

Abstract A series of derivatives of 2 and 3-benzofurancarboxylates were synthesized under microwave-assisted conditions. Their in-vitro antimicrobial properties were assessed. Inhibition by the compounds of the growth of antibiotic-susceptible standards and clinically isolated strains of Gram-positive and Gram-negative bacteria, yeasts, and a human fungal pathogen was moderate to significant. Methyl 5-bromo-7-[2-(N,N-diethylamino)ethoxy]-6-methoxy-2-benzofurancarboxylate hydrochloride was identified as the most active compound (MIC 3–12 × 10−3 μmol/cm3 against Gram-positive bacteria; MIC 9.4 × 10−2 μmol/cm3 against Candida and Aspergillus brasiliensis). The molecular and crystal structures of 2-(N,N-diethylamino)ethyl 6-acetyl-5-hydroxy-2-methyl-3-benzofurancarboxylate were established by single-crystal X-ray diffraction. Graphical Abstract .


Introduction
The benzofuran system, an important pharmacophore, is present in numerous compounds isolated from natural sources and in synthetic products. These heterocyclic compounds have a variety of pharmacological properties, and changes of their structure result in high diversity that has proved useful in the search for new therapeutic agents. It is widely known that numerous compounds containing the benzo[b]furan system, both synthetic and isolated from natural sources, have antimicrobial activity [1].
Eight flavaglines and six cyclopenta[b]benzofurans isolated from Aglaia odorata, Aglaia elaeagnoidea, and Aglaia edulis (Meliaceae) have been tested for antifungal properties against the three plant pathogens Pyricularia grisea, Fusarium auenaceum, and Alternaria citri. P. grisea, responsible for rice blast disease, was the fungus most susceptible to all the benzofurans, with rocaglaol the most active compound [2]. Thirteen compounds based on the benzofuran structure bearing aryl substituents at the C-3 position through a methanone linker have been synthesized and screened for antibacterial and antifungal activity against four bacteria: Escherichia coli, Staphylococcus aureus, Methicillin-resistant S. aureus, and Bacillus subtilis, and a fungus Candida albicans. Four hydrophobic benzofuran analogs were found to have favorable antibacterial activity better than that of control drugs [3].
As we have reported elsewhere, aminoalkylation the OH group of 7-hydroxycoumarin derivatives resulted in products with better antibacterial activity than the starting compounds [14]. Encouraged by this, and in continuation of our research, we designed the synthesis of a series of benzofurancarboxylates bearing O-aminoethyl substituents and assayed their antimicrobial activity. In this study we report their microwave-assisted preparation and discuss the advantages of this technique compared with synthesis under conventional conditions, described elsewhere [15].

Results and discussion
Our strategy was based on preparation of a series of derivatives of 2 and 3-benzofurancarboxylic acids (Fig. 1). Acids 1-6 were prepared as described elsewhere [15] and converted to their ammonium salts to improve solubility in polar solvents. Acids 1-4 and 6 were esterified with methanol to protect the carboxyl group against O-alkylation.
As the first step of our research we obtained O-alkylamino derivatives of methyl benzofurancarboxylates 1b-4b and 6b by microwave-assisted O-alkylation of the appropriate esters (compounds 1a-4a, 6a, Scheme 1, routes i and ii, Fig. 2), using 2-chloroethyl-N,N-diethylamine hydrochloride as alkylating agent.
The syntheses were performed in acetone under phasetransfer conditions, using anhydrous potassium carbonate as a base and Aliquat 336 (N-methyl-N,N-dioctyloctan-1ammonium chloride) as phase-transfer catalyst (PTC). Preparation of hydrochloride salts of the resulting bases was necessary to prevent decomposition and improve their solubility in polar solvents. These compounds were previously synthesized conventionally [15]. Microwave assistance resulted in reduced reaction time (from 16 to 20 h to 24 min); however, we did not notice any meaningful increase in product yield.  Benzofurancarboxylic acids 1-4, 6, and 7 reacted with 2-chloroethyl-N,N-diethylamine under similar conditions. Microwave-assisted alkylation of these compounds resulted in a mixture of two products. An example of this synthetic route (for compound 1) is presented in Scheme 1, route ii. Separation by column chromatography on silica gel yielded the product of esterification 1c and the product of O-alkylation and esterification 1d. The isolated compounds 1c, 1d, 3c, 4c, 6c, and 7c ( Fig. 2) were converted to their hydrochloride salts. Spectroscopic data (IR, 1 H and 13 C NMR, and MS) confirmed the structures of all the products.
In this investigation eighteen derivatives of 2 and 3-benzofurancarboxylic acids were assayed for in-vitro antimicrobial activity. The ammonium salts of benzofurancarboxylic acids 1-7 ( Fig. 1) were also tested. They did not inhibit the growth of any of the microorganisms (MIC [ 30 lmol/cm 3 ). Methyl esters 1a-7a of the acids [15] were not tested for antimicrobial activity.
The results show that the pattern of substitution of the benzofuran moiety is important to the activity. The most potent compound is 6bÁHCl; at concentrations in the range 3-12 9 10 -3 lmol/cm 3 it inhibits growth of Gram-positive bacteria strains. Given its structure, we may speculate that the 2-(N,N-diethylamino)ethoxy function at C-7, the bromine substituent at C-5, and the methoxy group at C-6 are responsible for the high activity. The isomeric compound 6cÁHCl is, however, less active; exchanging the positions of the 2-(N,N-diethylamino)ethoxy and methoxy functions results in reduction of both antibacterial and antifungal activity.
It is worth noting that the derivative of the substituted 3-benzofurancarboxylic acid 1bÁHCl is more active against Gram-positive bacteria strains than compounds 2bÁHCl, 3bÁHCl, and 4bÁHCl, obtained from the substituted 2-benzofurancarboxylic acids. Introducing the lipophilic methoxy group at the C-5 position resulted in increased antimicrobial activity (compound 3bÁHCl is more active then 2bÁHCl). Similarly, the 7-(p-methoxycinnamoyl) group increases the activity of 4bÁHCl compared with 2bÁHCl against Gram-positive bacteria ( 2-(N,N-diethylamino)ethyl esters 1cÁHCl, 3cÁHCl, and 4cÁHCl, with unsubstituted phenolic groups, are more active against Gram-positive bacteria but less active against Gram-negative bacteria than 1bÁHCl, 3bÁHCl, and 4bÁHCl (Table 2). It is worth noticing that compound 4cÁHCl is the most active against yeast strains. Compound 7cÁHCl was inactive in our assay.

X-ray structure analysis
The molecular and crystal structure of 1c in the solid state were analyzed by single-crystal X-ray diffraction. The molecular structure with the atomic numbering scheme is illustrated in Fig. 3 (the drawings were performed with Mercury software [16]). The results indicate that the compound crystallizes in the monoclinic space group P 2 1 /n with one molecule in the asymmetric unit. Selected bond lengths, bond angles, and torsion angles are listed in Table 3. The benzofuran moiety is nearly planar with a maximum deviation of 0.020(1) Å for C3a. The C8, C9, C10, O16, O17, and O18 atoms are almost coplanar with the two-ring framework (the appropriate torsion angles are given in Table 3). The orientation of the substituent at C3 relative to the benzofuran ring can be described by the torsion angle C2-C3-C9-O19 of -0.2(3)°. For the (N,Ndiethylamino)ethyl fragment we observed structural disorder as a result of conformational freedom and from X-ray data we found alternative positions of the C12 and C13 atoms. Strong intramolecular hydrogen bonding is present between O16 and O17 atoms ( Fig. 3; Table 4). The angle  The packing of the molecules viewed down the a axis ( Fig. 4) shows that the molecules are stacked in blocks with partly overlapping benzofuran systems and an interlayer spacing of ca. 3.5 Å . The molecules are linked by C7-H7AÁÁÁO18, C11-H11A(D)ÁÁÁO1 hydrogen bonds forming infinite chains along the a axis. These chains interact via C13D-H13GÁÁÁO17, C15-H15CÁÁÁC9, C8-H8BÁÁÁC10 contacts and pÁÁÁp stacking forces to create the blocks mentioned above. The bulky aminoethyl substituents are oriented outside these blocks and connect them via C13C-H13FÁÁÁO16 hydrogen bonds. Geometric data for all intra and intermolecular interactions are given in Table 4.

Experimental
Reagents of the highest grade available were purchased from Aldrich and used without further purification. Solvents were used as received from commercial suppliers, and no further attempts were made to purify or dry them. Melting points were determined with an ElectroThermal 9001 digital melting point apparatus (ElectroThermal, Essex, UK). A Plazmatronika 1,000-W microwave oven equipped with a single mode cavity suitable for microscale synthesis and microwave choked outlet connected to an external condenser set to 30 % power was used (http:// www.plazmatronika.com.pl). High-resolution mass spectra were recorded on a Quattro LCT (TOF). 1 H NMR, 13 C NMR, HSQC, and HMBC spectra in solution were recorded at 25°C with Varian NMRS-300 or a Varian Unity  Preparation of compounds 1b-6b has been described elsewhere [15].     were in agreement with the data reported in our paper [15]. One or two basic products were isolated. The bases were converted into their hydrochlorides as described above.     The cylinder-plate method was used in the preliminary antimicrobial activity tests [18]. A suspension of the tested compound (20 mg/cm 3 , 0.05 cm 3 , in 0.08 M phosphate buffer, pH 7.0, containing 10 % DMSO) was placed in the cylinder. The cylinders were placed on a Muller-Hinton 2 or Sabouraud agar plate inoculated with one of the tested strains. The bacterial strains were incubated at 37°C for 24 h and the fungal strains at 30°C for 48 h. Minimal inhibitory concentration (MIC) was obtained by mixing with 19 cm 3 Mueller-Hinton 2 agar and cooling to 56°C with 1 cm 3 of the appropriate dilution of the tested compound. Then, 2 9 10 -3 cm 3 of a particular cell suspension of optical density 0.5 unit on the McFarland scale was applied to the surface of the agar. The lowest concentration of tested compound which totally inhibited growth of the examined strain was evaluated as MIC value [19]. For control samples, MIC values of ciprofloxacin ranged between 0.14 and 0.37 9 10 -3 lmol/cm 3 for bacterial strains and MIC values of fluconazole ranged between 3.9 9 10 -4 and 8.4 9 10 -1 lmol/cm 3 for yeast strains.

Crystallography
Crystals of 1c suitable for X-ray analysis were grown by slow evaporation of a solution in toluene-isopropanol (1:1). Diffraction data were collected on an Oxford Diffraction SuperNova diffractometer using CuK a radiation at room temperature. Data reduction was performed with SuperNova software [20]. The unit cell parameters were determined by least-squares treatment of setting angles of the highest-intensity reflections chosen from the whole experiment. The structure was solved by direct methods, by use of SHELXS-97 software, and refined on F 2 by the fullmatrix least-squares method, again by use of SHELXL97 software [21]. Two reflections were excluded from the reflection file because of their large ð F o j j 2 À F c j j 2 Þ differences. The function Rw F o j j 2 À F c j j 2 2 was minimized with w À1 ¼ ½r 2 . Non-hydrogen atoms were refined with anisotropic thermal data and the atoms of O-aminoethyl substituent were found to be disordered. So, the C12, C13A, and C13B atoms were located in two alternative positions and their occupancies were refined to 0.487(5) for C12A/C13A/C13B and 0.513(5) for C12B/C13C/C13D. The coordinates of the hydrogen atoms were generated geometrically and refined ''riding'' on their parent atoms with U iso set at 1.2 (1.5 for methyl group) times U eq of the appropriate carrier atom. All details concerning data collection, crystal data, and structure refinement are given in Table 5. The supplementary information in the CIF form is available from Cambridge Crystallographic Database Centre, no. CCDC-949328.