A Facile Synthesis of Some Bioactive Isoxazoline Dicarboxylic Acids via Microwave-Assisted 1,3-Dipolar Cycloaddition Reaction

Abstract

The microwave-assisted 1,3-dipolar cycloaddition reaction of several aldoximes and dimethyl-2-methylene glutarate in the presence of diacetoxyiodobenzene as an oxidant produced four new isoxazoline-derived dimethyl carboxylates. Saponification followed by acidification of the latter yielded novel isoxazoline dicarboxylic acids in reasonable to high yields. The structures of these novel compounds were characterized by IR, ¹H-NMR, ¹³C-NMR, and HRMS spectroscopy. Their biological activities disclosed higher inhibition of the growth of E. coli organisms by the aromatic compounds than by the aliphatic derivatives, demonstrating their potential in antibiotics research.

1. Introduction

There is a drastic need to develop novel antibiotics due to the rise in resistance to the present arsenal of antimicrobial drugs used to combat bacterial-related infections. Such resistance is the bacteria’s natural response to develop new clones to confront the antibiotics, thus developing immunity to several classes of antibiotics [1]. The upsurge in antimicrobial resistance has increased morbidity and mortality rates in the United States [2]. Isoxazolines are an important class of heterocyclic compounds with known biological properties ranging from antiviral [3], antimicrobial [4], antifungal [5], pesticidal [6,7], and antineoplastic [8] properties.

Most notably, several antimicrobial and antituberculosis isoxazoles reported in the literature include compounds such as 1–7 [9] (Figure 1). Interestingly, of all these compounds, the pyridyl nitrofuranyl isoxazolines 5 had remarkable antibacterial activity against multiple drug-resistant (MDR) Staphylococcus strains [10]. Also, Aarjane and coworkers prepared the acridone-derived isoxazolines 6 and 7, which were highly potent against E. coli with MIC values of 26.95 and 35.85 μg/mL, respectively [11].

Several groups have reported on the synthesis of isoxazolines using microwave-assisted chemistry [12,13,14] and other green chemistry [15,16] methods. Compared to conventional heating, these methods dramatically reduce reaction time, improve product yield, and readily reduce undesired side products [16], especially when carried out under neat conditions or with water as a solvent [17,18].

Some well-documented reaction conditions for synthesizing isoxazolines via the 1,3-dipolar cycloaddition (1,3-DC) reactions include (i) the reaction of α,β-unsaturated carbonyl compounds with hydroxylamine hydrochloride in the presence of a base [3,4,19], (ii) 1,3-dipolar cycloaddition reaction of nitrones with alkynes as dipolarophiles [20,21], and most notably, (iii) the reaction of alkenes with nitrile oxides that are generated in situ from aldoximes [22,23,24,25].

In this paper, we report on a facile synthesis of several isoxazoline dicarboxylic acid compounds (12a–12d of Supplementary Materials) via (i) MW-assisted 1,3-dipolar cycloaddition reactions of dimethyl-2-methylene glutarate (10) and the oximes (9a–9d), and (ii) base hydrolysis of the resulting isoxazoline dimethyl esters (11a–11d of Supplementary Materials) followed by acidification on workup with 6 M HCl. The biological activity of compounds 12a–12d of Supplementary Materials against E. coli was also determined and reported in this article.

2. Materials and Methods

2.1. Materials and Instruments

All melting points (mp) were obtained on a Laboratory Device MEL-TEMP II melting point apparatus and are uncorrected. A Perkin-Elmer Spectrum 100 FTIR Spectrometer was used to obtain the IR spectra for each compound. A Bruker Avance 300 MHz FT-NMR spectrometer, using TMS or solvent peaks as a reference, was used to obtain ¹H and ¹³C NMR spectra in deuterated solvents. The high-resolution mass spectroscopy (HRMS) data were collected on an Agilent Technology 6200 series TOF/6500 Q-TOF LC/MS instrument. All novel synthesized compounds were characterized by IR, ¹H NMR, ¹³C NMR, and HRMS spectroscopy. Thin layer chromatography (TLC) was conducted on pre-coated silica gel plates that were visualized under UV light (254 nm) and developed with potassium permanganate stain solution or phosphomolybdic acid in ethanol solution. Microwave (MW) reactions were carried out with an Anton-Par Monowave 450 microwave reactor.

2.2. Synthesis of Dimethyl-2-Methylene Glutarate (10) [26]

Methyl acrylate (21.5 g, 250 mmol) was cooled to −10 °C, and tributylphosphine (5.1 g, 25 mmol) was added under an N₂ atmosphere in a 250 mL round-bottom flask. The reaction was allowed to warm to room temperature (rt) with stirring over 1.5 h. After 1.5 h, the reaction mixture was concentrated by rotary evaporation to give the crude product. Flash column chromatography of the crude product with 10% EtOAc/cyclohexane eluent gave 10 (11.8 g, 56%) as an oil.

IR (neat, cm⁻¹): 2996, 2953, 2846, 1718, 1631, 1437. ¹H NMR (300 MHz, CDCl₃): δ 6.18 (s, 1H), 5.59 (s, 1H), 3.75 (s, 3H), 3.66 (s, 3H), 2.63 (t, J = 7.2 Hz, 2H), 2.51 (t, J = 7.2 Hz, 2H); ¹³C-NMR (75 MHz, CDCl₃): δ 173.2, 167.2, 138.8, 126.1, 52.0, 51.7, 33.0, 27.4.

2.3. General Procedure for the MW-Assisted 1,3-Cycloaddition Reaction

Under monomode microwave irradiation, alkene (1.2 eq), diacetoxyiodobenzene (DIB), and oxime (1.0 eq) were added to a 10 mL MW-reaction vial, then methanol (5.0 mL) was then added, and the vessel was placed in the cavity of the reactor. After closing the reactor cavity with the lid, the contents were irradiated (200 W, 180 °C) for 5–10 min with one minute hold time. After completion of the reaction, as was evidenced from the TLC analysis, the solvent was evaporated under vacuum. The crude product was then transferred to a silica gel column (120 mesh, column packed in hexane). The column was eluted using 20–40% ethyl acetate/hexane to afford the isoxazolines 11a–11d of Supplementary Materials as a yellow oil or white solid. The reported yields were based on isolated pure products.

Methyl-3-decyl-5-(3-methoxy-3-oxopropyl)-4,5-dihydroisoxazole-5-carboxylate, 11a of Supplementary Materials

The general procedure above yielded isoxazoline 11a of Supplementary Materials as a yellow oil.

Yield, 91%; IR (neat, cm⁻¹): 2960, 2914, 2848, 1733, 1438; ¹H NMR (300 MHz, CDCl₃): 0.78 (t, J = 5.5 Hz, 3H,), 1.09–1.20 (m, 14H), 1.35–1.51 (m, 2H), 1.96–2.56 (m, 6H), 2.79 (d, J = 17.3 Hz, 1H), 3.28 (d, J = 17.3 Hz, 1H), 3.57 (s, 3H), 3.68 (s, 3H); ¹³C-NMR (75 MHz, CDCl₃): δ 14.0, 22.6, 26.2, 27.4, 28.6, 29.06, 29.13, 29.20, 29.37, 29.45, 31.6, 31.8, 45.3, 51.6, 52.7, 86.5, 158.8, 172.0, 172.8.; HRMS (TOF): m/z calc. for C₁₉H₃₃NO₅Na [M + Na]⁺ 378.2251, found 378.2256.

Methyl 3-(tert-butyl)-5-(3-methoxy-3-oxopropyl)-4,5-dihydroisoxazole-5-carboxylate, 11b

Yellow oil, yield, 54%; IR (neat, cm⁻¹): 2961, 1733, 1619, 1438; ¹H NMR (300 MHz, CDCl₃): δ 1.18 (s, 9H), 2.06–2.49 (m, 4H), 2.90 (d, J = 17.3 Hz, 1H), 3.41 (d, J = 17.3 Hz, 1H), 3.66 (s, 3H), 3.77 (s, 3H); ¹³C-NMR (75 MHz, CDCl₃): δ 28.1, 28.8, 31.8, 33.3, 43.0,51.9, 53.0, 87.1, 165.9, 172.3, 173.

Methyl 3-([1,1′-biphenyl]-4-yl)-5-(3-methoxy-3-oxopropyl)-4,5-dihydroisoxazole-5-carboxylate, 11c

White solid, mp. 106–110 °C; Yield, 83%; IR (neat, cm⁻¹): 3010.8, 2957.0, 1728.4, 1600.3, 1579.4, 1489.7, 1436.1; ¹H NMR (300 MHz, CDCl₃): δ 300 MHz): δ 2.26–2.63 (m, 4H), 3.35 (d, J = 17.3 Hz, 1H), 3.69 (s, 3H), 3.83 (s, 3H), 3.87 (d, J = 17.3 Hz, 1H), 7.24–7.50 (m, 3H), 7.58–7.77 (m, 6H); ¹³C-NMR (75 MHz, CDCl₃): δ 28.9, 32.1, 43.6, 52.0, 53.2, 88.2, 127.3, 127.4, 127.6, 127.8, 128.1, 129.1, 140.2, 143.5, 156.1, 172.1, 173.0; HRMS (TOF): m/z calc. for C₁₉H₃₃NO₅Na [M + Na]⁺ 378.2251, found 390.1315.

Methyl 5-(3-Methoxy-3-oxopropyl)-3-phenyl-4,5-dihydro-isoxazole-5-carboxylate, 11d [27]

The general procedure above yielded isoxazoline 11d as a yellow oil.

Yield, 70%; IR (neat, cm⁻¹): 3002, 2955, 1732, 1601, 1258, 905, 759; ¹H NMR (300 MHz, CDCl₃): δ 2.23–2.60 (m, 4H), 3.33 (d, J = 17.3 Hz, 1H), 3.67 (s, 3H), 3.81 (s, 3H), 3.83 (d, J = 17.3 Hz, 1H), 7.36–7.45 (m, 3H), 7.62–7.65 (m, 2H); ¹³C-NMR (75 MHz, CDCl₃): δ 28.9, 32.1, 43.6, 52.0, 53.3, 88.2, 127.3, 127.4, 127.6, 127.7, 128.1, 129.1, 140.3, 143.5, 156.2, 172.1, 173.0; HRMS (TOF): m/z calc. for C₁₅H₁₇NO₅Na [M + Na]⁺ 314.1005, found 314.0999.

2.4. General Synthesis of 2,5-Disubstituted Isoxazoline Dicarboxylic Acids

Next, 6 M (KOH solution (3 eq) was added to a stirred solution of each isoxazoline-dimethyl carboxylate compound (1 eq) in 20 mL of EtOH/H₂O (2:1 ratio). The reactions were monitored using TLC until all the esters were hydrolyzed. The reactions were acidified to pH 2–3 using 6 M HCl aq. All solid products (12c and 12d) were collected via vacuum filtration using a Buchner funnel. Compound 12d was recrystallized from CH₂Cl₂/toluene. The liquid (12a of Supplementary Materials) and wax (12b of Supplementary Materials) products were extracted from the aqueous reaction mixture with ethyl acetate (3 × 20 mL). The organic layers were washed with brine, dried using anhydrous magnesium sulfate, and concentrated under a vacuum. Flash column chromatography of the resulting crude products using 5–10% MeOH/CH₂Cl₂ produced the diacid products as racemic mixtures.

5-(2-carboxyethyl)-3-decyl-4,5-dihydroisoxazole-5-carboxylic acid, 12a of Supplementary Materials

Yellow oil, yield, 93%; IR (neat, cm⁻¹): 3391, 3010, 2922, 1727, 1652, 1437.; ¹H NMR (300 MHz, CDCl₃): δ 0.89 (t, J = 6.8 Hz, 3H), 1.20–1.40 (m, 14H), 1.46–1.70 (m, 2H), 2.15–2.70 (m, 6H), 2.93 (d, J = 17.6 Hz, 1H), 3.43 (d, J = 17.6 Hz, 1H), 8.01 (brd s, 2H); ¹³C-NMR (75 MHz, CDCl₃): δ 14.2, 22.7, 26.4, 27.7, 29.0, 29.3, 29.4, 29.6, 29.6, 31.9, 32.0, 41.0, 45.6, 86.7, 159.1, 173.9, 174.9; HRMS (TOF): m/z calc. for C₁₇H₃₀NO₅ [M + H]⁺ 328.2118, found 328,2124.

3-(tert-butyl)-5-(2-carboxyethyl)-4,5-dihydroisoxazole-5-carboxylic acid, 12b

White wax, yield, 99%; IR (neat, cm⁻¹): 3010–2636 (brd signal), 1734, 1702, 1618, 1367;¹H NMR (300 MHz, acetone-d₆): δ 0.86 (s, 9H), 2.95–3.42 (m, 4H), 3.98 (d, J = 17.4 Hz, 1H), 4.33 (d, J = 17.4 Hz, 1H); ¹³C-NMR (75 MHz, acetone-d₆): δ 28.3, 32.2, 33.6, 42.8, 87.7, 166.2, 172.2, 173.7; HRMS (TOF): m/z calc. for C₁₁H₁₇NO₅Na [M + Na]⁺ 266.0999, found 266.1004.

3-([1,1′-biphenyl]-4-yl)-5-(2-carboxyethyl)-4,5-dihydroisoxazole-5-carboxylic acid, 12c

Off-white solid decomposes. 182–185 °C, yield = 54%; IR (neat, cm⁻¹): 338.57, 3076.6, 3028.8, 2920, 1702, 1650.5, 1611.8, 1594.6, 1486.0; ¹H NMR (300 MHz, CD₃OD): δ 2.26–2.63 (m, 4H), 3.54 (d, J = 17.5 Hz, 1H), 3.90 (d, J = 17.5 Hz, 1H), 7.37–7.54 (m, 3H), 7.64–7.84 (m, 6H); ¹³C-NMR (75 MHz, CD₃OD): δ 30.0, 33.2, 44.4, 89.6, 128.3, 128.6, 128.7, 129.3, 129.4, 130.3, 141.6, 144.8, 158.3, 175.0, 176.5; HRMS (TOF): m/z calc. for C₁₃H₁₄NO₅ [M + H]⁺ 340.1179, found 340.1182.

5-(2-carboxyethyl)-3-phenyl-4,5-dihydroisoxazole-5-carboxylic acid, 12d

White solid. Mp. 137–138 °C; Recrystallized yield, 57%; IR (neat, cm⁻¹): 3551, 3029, 26,711,708, 1686, 1607, 1449; ¹H NMR (300 MHz, CD₃OD): δ 2.26–2.59 (m, 4H), 3.53 (d, J = 17.6 Hz, 1H), 3.87 (d, J = 17.6 Hz, 1H), 7.44–7.54 (m, 3H), 7.70–7.80 (m, 2H); ¹³C-NMR (75 MHz, CD₃OD): δ 30.0, 33.2, 44.4, 89.5, 128.2, 130.2, 130.6, 131.9, 158.6, 175.0, 176.5; HRMS (TOF): m/z calc. for C₁₃H₁₄NO₅ [M + H]⁺ 264.0866, found 264.0871.

2.5. Biological Activity of Compounds (12a–12d of Supplementary Materials) Against E. coli

The isoxazoline dicarboxylic acids (12a–12d of Supplementary Materials) were each dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions of 1 mg/mL for each compound. These compounds were separately tested against E. coli using the microdilution method described previously. Then, 640 μg/mL working solution of each compound was diluted 1:10 into Mueller Hinton Broth (MHB) and then serially diluted into a 96-well plate containing 50 μL of MHB in each well. Next, 50 μL microbial inoculum, adjusted to 0.5 McFarland scale, was then added to each well and incubated at 37 °C for 24 h [28]. The absorbance of the 96-well plate’ content was measured using a plate reader at 625 nm for the pure compound, pure culture, and pure MHB broth. Each compound in MHB was used as a control.

3. Results and Discussion

3.1. Chemistry

The aldoximes (9a–9c) used in this synthesis were prepared from hydroxylamine hydrogen chloride and the readily available carbaldehydes 8a–8c in high enough yield, varying from 75 to 99% (Scheme 1).

In our research efforts to create novel isoxazolines with potential antibacterial properties, we began by synthesizing the benzylic isoxazoline dicarboxylic acid compound 12d. This compound was chosen based on its aromatic characteristics and its potential to exhibit antibacterial activities [10]. The latter was derived from the dimethyl ester 11d in quantitative yield; it is a key intermediate in the synthesis of the biologically active 2-(2-amino-2-phenyl ethyl)-5-oxotetrahydro-furan-2-carboxylic acid [27]. Compound 11d was first attempted at room temperature using benzaldoxime (9d) and dimethyl-2-methylene glutarate (10) at room temperature with diacetoxyiodobenzene (DIB) as oxidant. This reaction gave 34% of compound 11d after 110 min. The main byproduct was presumably the dimer of the nitrile oxide from oxime 9d. Upon the introduction of microwave heating, the reaction’s yield improved drastically to 70% in 10 min when the reaction was performed at 180 °C [27]. Following the successful synthesis of 11d, three other isoxazoline dimethyl dicarboxylate esters (11a, 11b, and 11c of Supplementary Materials) were prepared using MW-assisted 1,3-DC reactions and hydrolyzed to their respective isoxazoline dicarboxylic acids 12a, 12b, 12c of Supplementary Materials. The complete syntheses are outlined in Scheme 2 below.

The scope of the MW-assisted 1,3-dipolar cycloaddition reaction was extended to prepare the aromatic dimethyl isoxazoline-esters 11c and the aliphatic isoxazolines 11a and 11b of Supplementary Materials by reacting compound 10 and the aldoximes 9a–9d with DIB also at 180 °C in MeOH in a microwave reactor. An acceptable mechanism for this reaction involves the in situ generation of the nitrile oxides 13a–13d from the oxidation of the aldoximes 9a–9d with DIB followed by an immediate 1,3-dipolar addition reaction with the alkene 10 (Scheme 3) [29].

Under MW conditions, the rate of the 1,3-DP reaction is faster than the dimerization of the nitrile oxide; hence, the relative yields (54–91%) obtained of the 2,5,5-trisubstituted isoxazolines (11a–11d of Supplementary Material) products obtained revealed the effectiveness of the MW method in synthesizing these isoxazolines. One drawback in preparing these compounds was the low yield for compound 11b of Supplementary Material (54%). This may be due to the steric hindrance encountered during the 3 + 2 cyclization reaction of dipolarophile 10 with the bulkier tert-butyl nitrile oxide (13b).

Compounds 11a–11d of Supplementary Materials were characterized using ¹H-NMR, ¹³C NMR, and/or HRMS spectroscopy. All the isoxazolines possessed distinct resonances for the methylene protons (-N=C-CH₂-) as doublets between 2.90 and 3.83 ppm with coupling constants of 17.3 Hz. The signals for the sp²-hybridized carbons (-N=C-) of the isoxazoline ring resonated at 158.8, 165.9, 156.1, and 156.4 ppm, respectively, in the ¹³C NMR spectra of these compounds. The other quaternary carbons at C-5 within the isoxazoline core resonated at the expected frequency between 86.5 and 88.0 ppm [19].

Base hydrolysis of the dimethyl ester isoxazolines 11a–11d of Supplementary Materials was readily achieved using a 6 M KOH solution. The dipotassium carboxylate salt formed was then reacidified with HCl solution to pH 2–3 to give the dicarboxylic acids (12a of Supplementary Materials) as a white wax, 12b as a yellow oil, and both 12c and 12d as white solids. The yields for the dicarboxylic acids isolated ranged between 54 and 99%. Generally, this saponification reaction produced very high yields. However, the low yields for compounds 12c and 12d resulted from the recrystallization of these acids after isolation. Notably, the isoxazoline rings are quite stable to base hydrolysis, and only saponification of the ester moieties was observed in these reactions. Compounds 12a–12d of Supplementary Materials were eventually characterized using ¹H-NMR, ¹³C-NMR, and HRMS spectroscopy. Of concern was the methylene protons in the isoxazoline rings remained as an AB quartet between 3.4 and 4.0 ppm, with coupling constant J = 17.4 Hz. Also, these compounds’ quaternary carbon (C-5) signals resonated between 84 and 95 ppm. In the IR spectra for these diacids, the vibrational modes for the carbonyl (C=O) (1730–1700 cm⁻¹) and hydroxyl (3500–2630 cm⁻¹) functionalities indicated the carboxylic acid groups within compounds 12c–12d. Once fully characterized, all four isoxazoline derivatives were tested for their activities against E. coli bacteria.

3.2. Bioassay

Our biological assays of compounds 12a–12d of Supplementary Materials against E. coli were conducted using a broth microdilution procedure [29]. The compound 12d showed an inhibition of 87.3% at 15.1 μM. Compound 12c controlled bacterial growth relative to DMSO by 80.4% at a concentration of 47.1 μM. These aromatic isoxazoline derivatives were more potent than the aliphatic derivatives, with compounds 12b and 12a of Supplementary Materials showing much lower potency of 40.7% at 32.9 μM and 30.7% at 97.7 μM, respectively (

Table 1. The % inhibition of E.coli by compounds 12a–12d of Supplementary Materials.

Compound	Concentration/(μM)	% Inhibition of E. coli
12a	97.7	30.7
12b	32.9	40.7
12c	47.1	80.4
12d	15.1	87.3

). We are encouraged by these preliminary results and plan to pursue this avenue further, particularly because of its applicability to developing new antibiotics.

4. Conclusions

The isoxazoline dicarboxylic acids (12a–12d of Supplementary Materials) were rapidly synthesized using a MW-assisted 1,3-dipolar cycloaddition reaction in three steps. This involved the known aldoximes (11a–11d of Supplementary Materials) and dimethyl-2-methylene glutarate with DIB as the oxidant. Saponification of these new isoxazolines followed by acidification yielded these isoxazoline dicarboxylic acids in reasonable to high yields (53% and 85% over three steps). The structures of these compounds were confirmed using spectroscopic data from their NMR and HRMS spectra. The biological activity of all isoxazoline dicarboxylic acids (12a–12d of Supplementary Materials) was determined against E. coli organisms. Compound 12d of Supplementary Materials was the most effective antibiotic of the synthesized compounds, with an activity of 15.1 μM at 87.3% inhibition. This study will be expanded to determine the antibacterial effect of additional derivatives of compound 12d towards E. coli and other Gram-negative bacteria. This study is essential for developing new antimicrobial agents against drug-resistant bacteria.