Synthesis of 3-Aryl-3-(Furan-2-yl)Propanoic Acid Derivatives, and Study of Their Antimicrobial Activity

Reactions of 3-(furan-2-yl)propenoic acids and their esters with arenes in Brønsted superacid TfOH affords products of hydroarylation of the carbon–carbon double bond, 3-aryl-3-(furan-2-yl)propenoic acid derivatives. According to NMR and DFT studies, the corresponding O,C-diprotonated forms of the starting furan acids and esters should be reactive electrophilic species in these transformations. Starting compounds and their hydroarylation products, at a concentration of 64 µg/mL, demonstrate good antimicrobial activity against yeast-like fungi Candida albicans. Apart from that, these compounds suppress Escherichia coli and Staphylococcus aureus.

Based on our research on superelectrophilic activation in organic synthesis [13,14] and our investigation in furan chemistry [15,16], we undertook this study on the synthesis of First, reactions of acid 1a with benzene under the actions of various Brønsted or Lewis acids have been investigated. It was found that compound 2a as product of hydrophenylation of the carbon-carbon double bond has been formed ( Table 1). The highest yield of 2a (65%) has been achieved under the use of AlCl 3 at room temperature for 1 h (entry 4). Prolongation of the reaction time until 4 h leads to a decrease of the yield of 2a to 47% (entry 5). Reaction with AlBr 3 gives a comparable yield of the target compound (entry 7). Acidity of trifluoroacetic acid has been found to be not enough for electrophilic activation of starting substrate 1a (entry 1). On the other hand, H 2 SO 4 and FeBr 3 lead to the formation of mixtures of oligomeric materials (entries 2 and 3). Yields of 2a for reactions in TfOH under different conditions are moderate, 22-33%, at the complete conversion of starting acid 1a (entries 7-10).
Then, reactions of acid 1a with other arenes have been studied. Reactions with methylated arenes (o-, m-, p-xylenes, mesitylene, and durene) in TfOH at 0 • C for 2 h afford the corresponding products of hydroarylation 2b-f in good yields of 55-98% (Scheme 1). Contrary to the reaction with benzene (Table 1, entry 4), AlCl 3 -promoted transformations of 1a with these methylated arenes lead mainly to oligomeric compounds with the formation of small amounts of target compounds 2b-h. It should be noted that the reaction of 1a with electron-donating arenes, anisole (methoxybenzene), veratrole (1,2dimethoxybenzene), or electron-poor 1,2-dichlorobenzene under the action of both TfOH and AlCl 3 give oligomeric materials. Reaction of acids 1b-d, obtained from 5-HMF, with benzene under the action of TfOH or AlCl 3 furnishes compound 2g (Table 2). Apart from hydrophenylation of the carboncarbon double bond, the additional alkylation of benzene by the CH 2 OH or CH 2 oMe groups takes place for acids 1b,c. Yields of target compound 2g are comparable for a reaction with TfOH or AlCl 3 (compare pairs of entries: 2 and 6, 7 and 9, and 10 and 12). Hydroarylation of ester 1g by different arenes in TfOH at 0 • C for 2 h is shown in Scheme 2. These reactions lead to the formation of compounds 2h-q in good yields. In general, yield of esters 2 are higher with the use of TfOH rather than AlCl 3 (see yields for 2j in Scheme 2). Reactions with anisole and durene give mixtures of isomers 2n and 2o and 2p and 2q, respectively.
In the same transformation, diester 1h with benzene under the action of TfOH or AlCl 3 gives a product of double hydrophenylation 2r as an equimolar mixture of diastereomers in a moderate yield (Scheme 3).
Diacid 1e, and benzofuran derivatives 1f and 1i in reactions with benzene and other arenes under the action of both TfOH and AlCl 3 afford complex mixtures of oligomeric materials.
As it has been mentioned above, acids 1a (Table 1, entry 2) and 1b ( Table 2, entry 1) in reactions with benzene in H 2 SO 4 at room temperature for 1 h gave oligomeric compounds. According to the HPLC-HRMS analysis ( Figure S51), the latter were represented by a number (about 20-25 chromatographic peaks for each parent compound) of dimers-hexamers with molecular weights in the range of 200-700 Da, while the most intense signals belonged to trimeric and tetrameric compounds (Table 3).  A specific feature of these products is a surprisingly large number of oxygen atoms in their elemental compositions and RDB (ring and double bond equivalent or unsaturation degree) values lower than expected. Tandem mass spectra of the corresponding precursor ions presented in the Supplementary Materials demonstrate the loss of 1-4 (depending on compound) water molecules (−18.0106 Da), which is evidence of the presence of aliphatic hydroxyl groups in their structures. This makes it possible to assume that, under applied reaction conditions, the hydration of carbon-carbon double bonds in the side chain and furane ring occurs in addition to the hydrophenylation described above for other reaction systems and confirmed by the presence of the tropylium ion [C 7 H 7 ] + signal at m/z 91.0565. Moreover, in most cases, the same double bond simultaneously undergoes phenylation and hydroxylation. The further oligomerization proceeds through the addition of 1a or its hydrated derivatives and, thus, the formation of ether or ester bonds, in some cases along with the side processes of decarboxylation (the latter also can proceed during ESI in the ion source). In the case of starting compound 1b, the same patterns were observed; however, the structures of the oligomers typically included two phenyl moieties. The plausible structural formulas and tandem mass spectra for all products listed in Table 3 are presented in the Supplementary Materials ( Figures S52-S59). These oligomers are humin-like compounds similar to those obtained from furan derivatives in acidic media [17,18].
To investigate the reaction mechanism, we carried out a NMR study on the protonation of compounds 1 in TfOH. NMR monitoring of the solutions of compounds 1a-d,f,g,i, having only one conjugated enone system, has shown that these compounds are rapidly transformed into oligomeric materials in TfOH. That reveals a high electrophilic reactivity of intermediate cations. Contrary to that, diacid 1e and diester 1h, having two conjugated enone systems, give stable solutions of O,O-diprotonated species Ae and Ah, respectively (see 13 C NMR data in Table 4). Comparison of the chemical shifts of carbon atoms in starting compounds 1e and 1h and their protonated forms Ae and Ah show large down field shifts of the corresponding signals in cations. A positive charge is substantially delocalized from the carbonyl group into the carbon-carbon double bond and furan ring. Thus, differences in chemical shifts ∆δ for carbons C 3 and C 5 are around 7 and 27 ppm, correspondingly (Table 4). Table 4. 13 C NMR data of starting compounds 1e and 1h and their protonated forms Ae and Ah generated in TfOH.

Compound/Cation
13 C NMR, δ, ppm Then, we did DFT calculations of intermediate cations Aa-Ch derived under the protonation of 3-(furan-2-yl)propenoic acid derivatives 1a,g, diacid 1e, and diester 1h to estimate the electrophilic properties and reactivity of these species (Table 5) (Table 4), one may propose that these species may be reactive intermediates in reactions with aromatic nucleophiles. Electronic properties of dications Ae and Ah show that the reactivity of carbon C 3 is mainly explained as orbital factors, since this carbon possesses a negative charge (−0.19 e), but it gives an 11.4-11.6% contribution into LUMO (entries 5 and 8).
Based on the data obtained on the reactions of compounds 1 with arenes (Tables 1 and 2 and Schemes 1-3), NMR (Table 3) and DFT (Table 4) studies of intermediate cations, one may propose a plausible reaction mechanism of the reaction of compounds 1, except diester 1h (vide infra), with arenes leading to products of hydroarylation 2 (Scheme 4). The first protonation of substrates 1 in Brønsted superacid TfOH occurs onto carboxyl oxygen forming O-protonated species A. Then, the protonation of the carbon-carbon double bond may give O,C-diprotonated species B. In principle, both species A and B may take part in electrophilic aromatic substitution with arenes. However, taking into account a strong electron-donating character of furan substituent, the second protonation of the conjugated C=C bond may proceed, leading to dications B. Moreover, the formation of such O,C-diprotonated species from various conjugated enones, such as butenones [20], indenones [21], cinnamic acids, and their esters and amides [22][23][24][25], was proven by NMR in Brønsted superacids. These dications are key reactive intermediates in various processes of electrophilic aromatic substitution [20][21][22][23][24][25][26][27][28]. Thus, it is the most probable that dications B lie in the reaction pathway from compounds 1 to 2. Reactions under the action of AlCl 3 proceed in the same manner when the electrophilic activation of substrate 1 is achieved by coordination of this strong Lewis acid onto carbonyl oxygen of the carbon-carbon double bond, leading to reactive intermediate species. In the case of diester 1h, the reaction in TfOH proceeds through the intermediate formation of O,O-diprotonated species Ah, which reacts with benzene, affording bishydrophenylation product 2r (Scheme 5).

Scheme 5.
Plausible reaction mechanism of the reaction of diester 1h with benzene in TfOH, leading to compounds 2t.
At the final stage of this study, the antimicrobial activity of the starting furan derivatives 1 and products of their hydroarylation 2 were investigated relative to the bacteria Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 and to yeast-like fungi Candida albicans (ATCC 10231) (see details in SI). It was found that all the compounds 1 and 2 inhibited the growth of yeast-like fungi Candida albicans at a concentration of 64 µg/mL. Concerning to the S. aureus strain, a minimum inhibitory concentration (MIC) was 128 µg/mL for most of the tested objects. The best result demonstrated acid 2d, which suppressed the growth of microorganisms at a concentration of 64 µg/mL. However, the tested compounds 2i, 2m, and 2r did not show antimicrobial activity at the specified concentrations. For most of the tested compounds, the MIC against E. coli ranged between 64 and 128 µg/mL.

Conclusions
A novel method of synthesis of 3-aryl-3-(furan-2-yl)propenoic acid derivatives has been developed on the basis of hydroarylation of the carbon-carbon double bond of 3-(furan-2-yl)propenoic acids and their esters by arenes under superelectrophilic activation conditions in neat triflic acid TfOH. The obtained furans have demonstrated a high level of antimicrobial activity against yeast-like fungi Candida albicans, and they also can inhibit Escherichia coli and Staphylococcus aureus.

General Information
The NMR spectra of solutions of compounds in CDCl 3 were recorded on Bruker AM-500 spectrometer (Bruker Company, Germany) at 25 • C at 500 and 125 MHz for 1 H and 13 C NMR spectra, respectively. The residual proton-solvent peaks CDCl 3 (δ 7.26 ppm), DMSOd 6 (δ 2.50 ppm), CD 3 OD (δ 3.31 ppm), (CD 3 ) 2 CO (δ 2.05 ppm) for 1 H NMR spectra, and the carbon signals of CDCl 3 (δ 77.0 ppm), DMSO-d 6 (δ 39.52 ppm), CD 3 OD (δ 49.00 ppm), (CD 3 ) 2 CO (δ 29.84 ppm) for 13 C NMR spectra were used as references. NMR spectra in the superacids TfOH at room temperature were recorded on Bruker 400 spectrometer at 400 and 100 MHz for 1 H and 13 C NMR spectra, respectively. NMR spectra in TfOH were referenced to the signal of CH 2 Cl 2 added as the internal standard: δ 5.30 ppm for 1 H NMR spectra and δ 53.52 ppm for 13 C NMR spectra. HRMS was carried out with instruments Bruker maXis HRMS-ESI-QTOF and Varian 902-MS MALDI Mass Spectrometer. IR spectra of the compounds in KBr were taken with a FSM-1201 spectrometer. GC-MS spectra were taken with the Shimadzu GCMS QP-2010 SE machine. The preparative reactions were monitored by thin-layer chromatography carried out on silica gel plates (Alugram SIL G/UV-254), using UV light for detection.
The study of oligomeric products was carried out using a TripleTOF 5600+ highresolution quadrupole time-of-flight (QTOF) mass spectrometer (AB Sciex, Concord, ON, Canada) equipped with a Duospray ion source with ESI probe. A mass spectrometer was combined with an LC-30 Nexera HPLC system (Shimadzu, Kyoto, Japan) consisting of a DGU-5A vacuum degasser, two LC-30AD chromatographic pumps, an SIL-30AC autosampler, and an STO-20A column thermostat.
Chromatographic separation was achieved at 40 • C on a Nucleodur PFP column (Macherey-Nagel, Duren, Germany) with a pentafluorophenyl-propyl stationary phase, 150×2 mm, particle size 1.8 µm. A mixture of water (A) and acetonitrile (B) containing 0.1% formic acid was used as a mobile phase. The gradient elution was programmed as follows: 0-3 min: 10% B, 3-40 min: ramp to 100% B, and 40-45 min: 100% B. The mobile phase flow rate was 0.3 mL/min, and the injection volume was 5 µL. Nontargeted screening of reaction products was performed in a data-dependent acquisition mode using positive electrospray ionization (ESI+). The following ion source parameters were used: nebulizing, drying, and gas curtain pressure-40, 40, and 30 psi, respectively, capillary voltage-5500 V, and source temperature-400 • C. The parameters used for recording the mass spectra in a TOF MS mode were as follows: declusterization potential-80 V, m/z range-150-1200, and acquisition time-150 ms. Tandem (CID) mass spectra were recorded for precursor ions with signal intensities above a threshold of 100 cps. Nitrogen was used as the collision gas and collision energy-50 eV with a spread of 30 eV. The maximum number of simultaneously fragmented precursor ions-15, m/z range-20-1200. Data processing was performed using MasterView and Formula Finder (AB Sciex, Concord, ON, Canada) software packages. Elemental compositions of the detected compounds were determined based on the accurate masses of ions, their isotopic distributions, and product ions m/z. The following constraints were applied: maximal number of atoms: C-100, H-300, O-20, mass error < 5 ppm (MS) and <10 ppm (MS/MS), and signal-to-noise ratio (S/N) > 10.

DFT Calculations
All computations were carried out at the DFT/HF hybrid level of theory using hybrid exchange functional B3LYP by using GAUSSIAN 2009 program packages [32]. The geometries optimization was performed using the 6-311+G(2d,2p) basis set (standard 6-311G basis set added with polarization (d,p) and diffuse functions). Optimizations were performed on all degrees of freedom, and solvent phase optimized structures were verified as true minima with no imaginary frequencies. The Hessian matrix was calculated analytically for the optimized structures in order to prove the location of correct minima and to estimate the thermodynamic parameters. Solvent-phase calculations used the Polarizable Continuum Model (PCM, solvent = water).

Study of Biological Activity
MICs of furan compounds against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, and Candida albicans ATCC 10231 were determined using broth microdilution as described in ISO 20776-1:2019 and ISO 16256:2021. Stock solutions of furan compounds in neat (pure) DMSO were prepared in sterile tubes and used on the same day. Two-fold dilutions of the furan compounds in the appropriate culture medium were added to the wells of a 96-well plate. The final concentrations of the test substances (after inoculation) were 256, 128, 64, 32, 16, 8, 4, 2, and 1 µg/mL. Solutions of furan compounds were added to the wells of the plates, 50 µL per well for S. aureus and E. coli and 100 µL for C. albicans.
RPMI-1640 medium, buffered with MOPS (3-(N-morpholino)propanesulfonic acid) containing l-glutamine and lacking sodium bicarbonate was used for C. albicans. The medium for E. coli and S. aureus was Mueller-Hinton broth. The fungus inoculum was prepared in the test medium and adjusted to match the turbidity of a 0.5 McFarland standard. A 1:100 dilution followed by a 1:20 dilution was performed for the yeast strain to obtain a final inoculum ranging from 0.5 to 2.5 × 10 3 CFU/mL. Then, 100 µL of the fungal inoculum was added to each well containing furan compounds.
Bacterial inoculums were prepared in sterile sodium chloride solution and adjusted to the 0.5 McFarland standard. A volume of 50 µL of this suspension was diluted in 10 mL of Mueller-Hinton broth until a concentration of approximately 5 × 10 5 CFU/mL was reached. Of this suspension, 50 µLwas inoculated into each furan compounds-containing wells.
To ensure that the inoculum contained the required number of cells, the viability of the inoculum suspensions was counted. One hundred microliters of the inoculum was taken from the growth control tube immediately after inoculation and diluted in 9.9 mL of sodium chloride solution. One hundred microliters of this dilution were applied to the surface of a suitable agar plate (Sabouraud dextrose agar plate for C. albicans and Trypticase soy agar plate for S. aureus and E. coli), which were then incubated overnight.
After inoculation, the plates were incubated at 37 • C for 18 h for bacterial strains, 22 h for C. albicans. The susceptibility to furans was assessed on the basis of visual observation of growth the strains in the culture media. The minimal inhibitory concentration (MIC) is the lowest concentration of an antimicrobial that inhibits visible growth of a bacterial culture under a defined set of experimental conditions. Malonic acid (0.91 g, 8.9 mmol) and substituted furan-2-carbaldehyde (8.9 mmol) were added to pyridine (10 mL). Then, piperidine (0.23 g, 2.7 mmol) was added dropwise for 5 min, and the mixture was stirred 4 h at 115 • C. The mixture was poured into water (50 mL), and aqueous HCl was added to a slightly acidic medium (pH 5-6), while orange precipitate was observed. A precipitate was filtered off and washed with water. The solution of NaOH (0.29 g, 7.2 mmol) in MeOH (3 mL) was added to a stirring mixture of acids 1 (7.2 mmol) in MeOH (5 mL). Dimethyl sulfate (1.21 g, 8.6 mmol) was added dropwise for 5 min, and the mixture was stirred for 1h at 60 • C. The mixture was poured into water (50 mL) and extracted with diethyl ether (3 × 50 mL). The extracts were combined, washed with water, and dried with Na 2 SO 4 ; the solvent was distilled under reduced pressure.