Ionic Liquid-Catalyzed Multicomponent Synthesis of Isoxazole-5(4H)-ones: in vitro Activities and Principal Component Analysis

Abstract

Heterocycles of the isoxazolone class stand out in the pharmaceutical field because they have numerous biological properties, such as antioxidant, antifungal and antibacterial properties. Thus, isoxazole derivatives have attracted the attention of organic researchers, and several procedures have been developed to obtain these compounds. The present study aimed to synthesize 17 derivatives of isoxazol-5(4H)-ones using a multicomponent reaction catalyzed by an acidic ionic liquid, which allowed yields from 20-96% of the products. To evaluate the bioactivity of the compounds, the synthesized derivatives were analyzed at various concentrations against bacteria and fungi; the derivatives, in general, showed a moderate activity of 125 μg mL^-1 against the fungus Candida tropicalis (ATCC 13803) and four derivatives showed an inhibition of < 15.62 μg mL^-1 for Gram-positive Staphylococcus epidermidis (ATCC 12228). Furthermore, the antioxidant potential of the derivatives was evaluated by measuring their ability to scavenge 2,2-diphenyl-1-picryl-hydrazyl (DPPH) free radicals, resulting in a remarkable reduction percentage of up to 81.06%. To gain further insights into the structure-activity relationships, a principal component analysis was conducted to classify the compounds into active and inactive classes, providing a comprehensive understanding of their bioactivity profiles.

Keywords:
isoxazolone derivatives; multicomponent reaction; bioactivity; antioxidant; quantum-chemical descriptors

Introduction

In the context of green chemistry, organic synthesis seeks the development of less toxic reactions and conditions that favor the environment and human health, as well as energy efficiency, product selectivity, operational simplicity, and economic synthesis.¹,² The great challenge of medicinal and pharmaceutical chemistry is to obtain fast and efficient compounds with bioactive properties.¹

Consequently, several procedures have been developed with the objective of obtaining these compounds.³ Multicomponent reaction (MCR) stands out as a methodology for obtaining heterocycles with favorable yields, atomic economy, procedural simplicity, high selectivity and easy productivity of molecules with active biological properties.³,⁴ In the literature, there are many studies on the use of basic catalysts in the synthesis of isoxazolone derivatives, such as 1,4-diazabicyclo[2,2,2]octane (DABCO),⁵ pyridine, aminated catalysts,⁶ in addition to ionic liquid acid with basic sites, such as synthetic enzyme (PEI-IL),⁷ among others. The application of ionic liquids (ILs) is a tool in the synthesis of organic compounds because they provide shorter reaction time, high yield and low toxicity.⁸

ILs exhibit properties such as low flammability, non-volatility, thermal stability, and recyclability, making them desirable for environmentally friendly processes in accordance with green chemistry.⁹,¹⁰ In this context, imidazolium ILs stand out and can be applied as catalysts and/or solvents in multicomponent reactions (MCRs), providing high yields and selectivities. The literature¹¹ reports the effect of this class of ILs on the formation, stabilization, and enhancement of reactivity of reagents and intermediates during a multicomponent transformation, attributed to a combination of cations and anions during the reaction.

MCRs encompass several types of reactions that produce different heterocyclic compounds, such as the reactions of Hantzsch, Mannich, Ugi, Passerini and Biginelli.¹² Isoxazole heterocyclics, which are compounds that have a nitrogen and an oxygen in the five-membered aromatic ring,¹³ can be obtained by linear divergent synthesis or multicomponent reaction. Several studies¹⁴ have shown drugs that have an isoxazole nucleus in their structure (Figure 1), for example, cycloserine, sulfisoxazole and zonisamide.

Figure 1
Chemical structures of drugs cycloserine, sulfisoxazole and zonisamide, which have an isoxazole core in their chemical structure.¹⁴

The isoxazole class presents some derivatives that alternate according to the type of substituent attached to the five-membered ring.¹⁵ Isoxazolone and 3,4-disubstituted derivatives (Figure 2) are used in the formation of fused heterocyclic compounds with biological activity.⁶,¹⁶ These compounds are obtained from the use of β-ketoesters, α-acetylenic aldehydes and α,β-unsaturated ketones.¹⁷

Figure 2
Chemical structure of isoxazolone and 3,4-disubstituted derivative.

In this context, the present research seeks to synthesize heterocyclic organic compounds derived from isoxazol-5(4H)-ones via multicomponent reaction, encompassing the principles of green chemistry, and biologically evaluate these organic products for pharmacological purposes, applying principal component analysis (PCA) to explain the experimental results.

Experimental

General experimental procedures

All chemicals were obtained from Sigma-Aldrich Brazil (São Paulo, Brazil) and used without further purification. Reactions were monitored by thin layer chromatography (TLC) using chromatographic plates of silica gel impregnated on aluminum Merck 60 F-254 plates with ethyl acetate (Synth, São Paulo, Brazil) and n-hexane (Dinâmica, São Paulo, Brazil) (1:1). After elution, the TLC plates (Merck, São Paulo, Brazil, TLC silica gel 60 F254) were observed under ultraviolet light (λ = 254 nm) or developed in I₂ (Sigma-Aldrich, São Paulo, Brazil). ¹H and ¹³C nuclear magnetic resonance (NMR) spectra were acquired with a Bruker Avance III 500 MHz spectrometer for ¹H NMR, 11.7 T (Karlsruhe, Germany), with ATB (automation triple resonance broadband) and SW (switchable) probes, 5 mm internal diameter, at room temperature and pulse of 45 ºC for hydrogen and carbon, at the Institute of Chemistry at the Samambaia Campus of the Federal University of Goiás (IQ-UFG). Chemical shifts are reported relative to tetramethylsilane (TMS) in CDCl₃ as a reference. Multiplicities were defined in the usual way, s (singlet), d (doublet), dd (double doublet), t (triplet), q (quartet), qu (quintet), m (multiplet). Chemical shifts of ¹H and ¹³C{¹H} were acquired with dimethyl sulfoxide (DMSO-d₆) as deuterated solvent and with tetramethylsilane (TMS) as internal standard from Cambridge Isotope Laboratories Inc. (Massachusetts, USA). The NMR data are presented as follows: chemical shift (δ) in ppm, multiplicity, the number of hydrogens, and J values in hertz (Hz). Melting points were determined using a Microchemistry model MQAPF-301 (Palhoça, Brazil) apparatus and are uncorrected. Infrared (IR) spectra were recorded on the PerkinElmer Spectrum Frontier FT-IR MID-NIR PerkinElmer spectrometer (Waltham, USA) at the Center for Analyses, Innovation and Technology in Natural and Applied Sciences at Goiás State University (UEG)-CAITEC using the KBr purchased from Sigma-Aldrich (Cotia, São Paulo, Brazil) method in the 4000-400 cm^-1 region.

Optimization of reaction conditions for the synthesis of 4-arylidene-3-methylisoxazole-5(4H)-one derivatives (ILA)

In a round-bottom flask under heating benzaldehyde 1, ethyl acetoacetate 2 and hydroxylamine hydrochloride 3 in solvent (4 mL) and catalyst (Scheme 1) were added. The mixture was maintained under magnetic stirring and reflux, then left in the refrigerator for 24 h. The obtained crystals were vacuum filtered and washed with cold distilled water, later recrystallized in EtOH. The catalytic system, the influence of the solvent, temperature, reaction time and number of reagents in the reaction medium were evaluated. Finally, aromatic aldehydes with different substituents and different positions were employed to obtain various 4-arylidene-3-methylisoxazol-5(4H)-one derivatives.

Scheme 1
Synthesis of isoxazol-5(4H)-one derivatives via the multicomponent reaction.

Evaluation of biological activity

Minimum inhibitory concentration (MIC)

The method was adapted from the National Committee for Clinical and Laboratory Standards (CLSI)¹⁸ in an Elisa multiwell plate. To determine the minimum inhibitory concentration (MIC) of the microorganisms, the products were solubilized in 5% dimethylsulfoxide (DMSO) and diluted in the culture medium of each microorganism, obtaining solutions at different concentrations 2000, 1000, 500, 125, 62.5, 31.25, and 15.62 μg mL^-1, using chloramphenicol as a control antibiotic and miconazole nitrate for fungi. The biological assay was performed in triplicate. The standard ATCC strains chosen were Staphylococcus epidermidis (ATCC 12228) and Escherichia coli (ATCC 25312), Candida albicans (ATCC 10231) and Candida tropicalis (ATCC 13803). To determine the minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC), an aliquot of 100 μL was collected from wells with no growth of microorganisms and seeded on plates containing nutrient agar. They were incubated on these plates at 35 ºC for 24 h, noting the lowest concentration of compounds that provided total inhibition of bacterial and fungal growth, MBC and MFC were obtained.

Antioxidant

To evaluate the antioxidant activity, the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) free radical scavenging method was used. The samples were prepared from a stock solution with a concentration of 400 µM, and for dilutions, 100 µL of the stock solution were used (performed in triplicate). Then, 50 µL of the dilution performed on the samples were inserted into each well and 0.1 mM of the DPPH radical was added, kept in a dark environment for 30 min and the absorbances were later read in the UV-Vis spectrophotometer at 515 nm. A positive control was prepared (quercetin at a concentration of 400 µM) and 100 µL of the stock solution was inserted into the microplate to dilute the positive control. After reading the plates and obtaining the absorbance (Abs), the percentage of antioxidant activity (AAO) was calculated by equation 1.

(1) $\text{AAO}/\%=\left(\text{radical control Abs – sample Abs}\right)/\left(\text{radical}\text{control Abs}\right)$

Subsequently, the amount of sample needed to inhibit 50% of a given radical concentration by 50% (IC₅₀) was calculated using the straight-line equation for each analyzed sample, obtained from the graph of concentration versus percentage of inhibition. Finally, the efficient concentration (EC₅₀) of the derivatives synthesized were calculated with equation 2.

(2) ${\text{EC}}_{50}=\frac{{\text{IC}}_{50}}{{\left[\text{DPPH}\right]}_{\text{t}=0}}$

where [DPPH]_t=0 = DPPH concentration at time zero.

Computational procedures

In the absence of the crystallographic structure of the 4-arylidene-3methylisoxazol-5(4H)-ones (ILA) derivatives, it is necessary to perform a conformational analysis on the compounds to establish the most stable conformation of the molecules. In the conformational analysis, the geometry of the molecular structure is determined and the method of molecular mechanics with the molecular mechanics (MM+) force field was used. The 17 derivatives synthesized from ILA1 to ILA17 that showed lower energy in the conformational analyses had their geometries selected for a flatter optimization. The final optimization of the geometry of these derivatives was performed using density functional theory (DFT) at the Gaussian 16 program¹⁹ by the M06-2X/6-311++G**(d,p) level of theory. It is important to highlight that in all stages the global minimum geometries were confirmed by the absence of imaginary harmonic frequencies.

After optimizing the geometry of the derivatives (see the Supplementary Information (SI) section, Table S1-S17), the following molecular descriptors were obtained and catalogued: the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energies; the dipole moment; the GAP energy GAP = HOMO - LUMO; the ionization energy, which is given by the difference between the energy of HOMO and the energy of removing an electron; the electron affinity, which is given by the negative of the LUMO energy; the hardness; the electronic chemical potential; the electrophilicity index; the surface area; the volume; the refractivity; the polarizability; and the Log P (partition coefficient octanol-water), interatomic distances, angles, and torsions (dihedral angles) for the 17 derivatives. Finally, the natural bond order and the atomic charges derived from the electrostatic potential by the CHELPG method were catalogued. All these molecular descriptors were included in an electronic spreadsheet, which reached a total of 178 columns versus 17 lines of the structures in question. Subsequently, this spreadsheet was transposed to the R software²⁰ environment for statistical analysis and graphics, in which several statistical algorithms and data analysis tools are present. The data were used for PCA analysis in the R language using the prcomp function of the stats package in R.²⁰ The descriptors obtained in the theoretical calculations were related to the antibacterial, fungicidal and antioxidant activities through multivariate statistical methods, more precisely using PCA. With these selected descriptors and the chemometric methods used in this study, it was possible to qualitatively evaluate the biological activity of the molecules studied, separating them into two distinct groups, active and inactive molecules referring to antioxidant, bacterial and fungicidal activity. The procedures adopted in optimizing the geometry, obtaining quantum-chemical descriptors and statistical analysis are the same as those followed in recent publications.²¹,²²

Results and Discussion

Optimization of reaction conditions for the synthesis of 4-arylidene-3methylisoxazol-5(4H)-one (ILA) derivatives

Several catalysts were tested 1-methyl-3-carboxymethyl-imidazolium chloride (MAI.Cl),²³ 1,3-bis(carboxymethyl)-1H-imidazolium chloride (imidazole diacid), 1-methyl-3-(3-sulfopropyl)-1H-imidazolium-SiW₁₂O₄₀ ((MSI)₄SiW) and p-toluenesulfonic acid (p-TSOH) (see Table 1). Several reaction conditions were analyzed as shown in Table 1.

Table 1
Evaluation of reaction conditions in the synthesis of isoxazol-5(4H)-one

entry	Catalyst	Solvent	Temperature / °C	Yield / %
1	MAI.Cla	water	80	57
2	imidazole diacid	water	80	52
3	(MSi)4SiW	water	80	53
4	p-TSOH	water	80	29
5	MAI.Cl (10 mol%)	water	80	50
6	MAI.Cl (50 mol%)	water	80	46
7	MAI.Cl	tetrahydrofuran	80	30
8	MAI.Cl	dimethylformamide	80	–
9	MAI.Cl	ethanol	80	13
10	MAI.Cl	ethyl acetate	80	38
11	MAI.Cl	water	rt	3
12	MAI.Cl	water	40	14
13	MAI.Cl	water	60	44
14	MAI.Cl	water	90	49

• p-TSOH: p-toluenesulfonic acid; (MSi)₄SiW: 1-methyl-3-(3-sulfopropyl)-1H-imidazolium-SiW₁₂O₄₀; rt: room temperature. MAI.Cl: 1-methyl-3-carboxymethylimidazolium chloride;

The best catalyst in the reaction medium was MAI.Cl with a 57% yield of the 4-benzylidene-3-methylisoxazol-5(4H)-one product. The use of IL in organic reactions stands out for demonstrating benefits for the environment, such as its low toxicity.²⁴ The variation of solvents occurred between apolar, polar aprotic and protic used during the synthesis, in which it was observed that water was the best solvent in the reaction medium maintaining the yield of 57%. In the literature,²⁵ water is widely used, since it is a safe and non-toxic protic polar solvent, in line with the principles of green chemistry. In the temperature variation, it was determined that the temperature of 80 °C allowed the yield of 57%, and it is important to emphasize that reactions at higher temperatures favor the formation of the product. This occurs because the temperature provides energy to overcome the thermodynamic barrier (activation energy), which favors the reaction and the formation of the desired product.²⁶

In the time variation, it was possible to observe that the time of 2 h provided the best yield, observing lower yields in shorter times (30 min and longer times 4, 6 and 24 h). The last optimization performed was in the variation of the amount of reagent in the synthesis of the product isoxazol-5(4H)-one (ILA) with 76% yield, observing that the excess of hydroxylamine hydrochloride influences (Figure 3) the reaction yield.²⁷

Figure 3
Graph of evaluation of reagent amount in the synthesis of isoxazol-5(4H)-one ILA1.

After establishing a general protocol for the synthesis of 4-arylidene-3-methylisoxazol-5(4H)-ones (ILA) derivatives, different aromatic aldehydes with substituents in the ortho, meta and para positions were employed, as shown in Table 2.

Table 2
Synthesis of 4-arylidenyl-3-methylisoxazole-5(4H)-one derivatives

entry	Derivative	Yielda / %	Melting point / °C
1		76	137-139
2		72	176-177
3		75	188-190
4		85	232-233
5		88	202-203
6		96	131-133
7		78	247-248
8		76	216-219
9		90	232-234
10		74	219-221
11		20	181-182
12		90	137-139
13		92	200-203
14		48	141-143
15		85	170-172
16		34	142-143
17		76	225-227

• ^a
  Conditions: 2 mmol ethyl acetoacetate, 1 mmol aromatic aldehyde, 2 mmol hydroxylamine hydrochloride; 20 mol% MAI.Cl, 4 mL H₂O at 80 °C for 2 h.

In general, it is observed that the reaction conditions provided yields between 20 and 96% of the synthesized derivatives. For the synthesis of derivatives of isoxazol-5(4H)-ones, the nature of the substituent was not crucial for the formation of the product.²⁸ Product ILA11 (entry 11) resulted in a 20% yield. In contrast, aromatic aldehydes with electron-donating substituents ILA1 (entry 1) and ILA6 (entry 6) provided a high yield of 76 and 96%. Aromatic aldehydes with electron-withdrawing substituents (NO₂, Cl, N, OH) of the aromatic ring provided good yields for the products ILA2, ILA3, ILA4, ILA5, ILA7, ILA8, ILA9, ILA10, ILA12, ILA13 and ILA17, with the respective results: 72, 75, 85, 88, 78, 76, 90, 74, 90, 92 and 76%. However, low yields are observed for derivatives with the NO₂ group in the ortho positions (entry 14, ILA14) and for (entry 16, ILA16), respectively 48 and 34%.

Thus, the reaction mechanism (Scheme 2) is determined by the presence of the type of acid catalyst and protic solvent used in the reaction medium. The first step is related to the reaction of ethyl acetoacetate with hydroxylamine hydrochloride and then the condensation of Knoevenagel with aromatic aldehyde.²⁴

Scheme 2
Mechanistic proposal for the formation of isoxazolones via MCR.

First, ethyl acetoacetate 1 undergoes activation through proton donation by the catalyst, followed by the nucleophilic attack of hydroxylamine hydrochloride 2, resulting in the formation of the intermediate oxime 4.⁵,²⁷ Subsequently, cyclization takes place, yielding the isoxazole 5. This formed heterocycle undergoes Knoevenagel condensation 6, ultimately leading to the desired product. Given that hydroxylamine hydrochloride was in excess, it reenters the cycle, and the catalytic mechanism initiates another iteration.⁷

Evaluation of biological activity

Evaluation of biological activity against bacteria

The antibacterial action of the compounds was lower for Escherichia coli bacteria than for Staphylococcus epidermidis . This occurs due to the complex cell wall, and the presence of lipopolysaccharide, porin, lipoprotein, peptidoglycan and phospholipid in Gram-negative bacteria. Because of this complex cell wall Gram-negative bacteria are known to be more resistant to the action of antibiotics.²⁵ The isoxazole derivatives that best inhibited the growth of E. coli bacteria showed a MIC of 250 and 500 µg mL^-1, OH (ILA7), 2-Cl (ILA13), 3-NO₂ (ILA15) and 2,4-OH (ILA17). These electron-withdrawing substituents are responsible for increasing the lipophilic nature of the compounds, which accentuates the interaction with the cell wall of Gramnegative E. coli.²⁵

In a study conducted by Ferouani et al.,²⁹ derivatives of isoxazol-5(4H)-ones with the substituents 4-F, 4-CH₃ and 2-OH exhibited significant antibacterial activity. However, the derivative with the 4-N(CH₃)₂ substituent showed no bacteriostatic and bactericidal activity, which aligns with the findings in our present research (entry 8, ILA8).

Gram-positive bacteria have a cell wall made up of peptidoglycan (approximately 90%) and are less complex than the previous bacteria.¹⁸ The compounds showed better inhibitory action < 15.62 to 250 µg mL^-1 against Gram-positive S. epidermidis, which occurs due to the more effective interaction between compounds and the cell wall of this bacterium. This high bacteriostatic action was also observed by Oraby et al.²⁶ with an MIC of 50 mg mL^-1. In the works of Banpurkar et al.,²⁸ derivatives of 4-(substituted-phenyl)-3-methyl-4H-isoxazol-5ones obtained antibacterial results against the bacteria Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus pyogenes. In general, the synthesized derivatives showed moderate bacteriostatic action, noting that ILA4, ILA5, ILA6 and ILA16 provided inhibitory action at concentrations lower than 15.62 µg mL^-1 for S. epidermidis. In contrast, the bactericidal action of these derivatives was 1000 µg mL^-1 or greater than 1000 µg mL^-1, which demonstrates that they are more prone to bacteriostatic action for both tested bacteria.

Table 3 presents the results obtained in the evaluation of the activity of the synthesized derivatives against the bacteria Staphylococcus epidermidis (ATCC 12228) and Escherichia coli (ATCC 25312).

Table 3
Evaluation of bacteriostatic and anti-bactericidal activity of 4-arylidene-3-methylisoxazol-5(4H)-one derivatives

Compound	Staphylococcus epidermidis (ATCC 12228)		Escherichia coli (ATCC 25312)
	MIC / (µg mL-1)	MBC / (µg mL-1)	MIC / (µg mL-1)	MBC / (µg mL-1)
ILA1	> 1000	> 1000	> 1000	> 1000
ILA2	1000	> 1000	> 1000	> 1000
ILA3	> 1000	> 1000	> 1000	> 1000
ILA4	<15.62	> 1000	> 1000	> 1000
ILA5	<15.62	1000	> 1000	> 1000
ILA6	<15.62	> 1000	500	> 1000
ILA7	250	> 1000	500	> 1000
ILA8	> 100	> 1000	> 1000	> 1000
ILA9	125	> 1000	1000	> 1000
ILA10	> 1000	> 1000	> 1000	> 1000
ILA11	> 1000	> 1000	> 1000	> 1000
ILA12	1000	> 1000	1000	> 1000
ILA13	> 1000	> 1000	250	> 1000
ILA14	125	> 1000	1000	> 1000
ILA15	500	> 1000	500	> 1000
ILA16	<15.62	> 1000	1000	> 1000
ILA17	250	> 1000	500	> 1000

• MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration.

Similar results were previously reported by Oliveira et al.,⁷ who tested these compounds against the bacteria E. coli. The compounds synthesized in our study, akin to those in the mentioned research, yielded results consistent with our findings. This underscores the intriguing bactericidal potential exhibited by certain products synthesized in this study.

Evaluation of biological activity against fungi

For fungi, moderate growth inhibition action was observed and no fungicidal action for the synthesized derivatives of isoxazol-5(4H)-ones. The derivatives that most inhibited fungal growth were those that showed the highest lipophilicity. Their internalization is more effective due to the composition of the cell wall and plasmatic membrane of these microorganisms.³⁰ This was observed with the derivatives ILA1, ILA6, ILA13, ILA14, ILA15, which demonstrated MIC of 125 and 500 µg mL^-1 against the fungus C. tropicalis. This antifungal action was also observed by Banpurkar et al.,²⁸ who obtained antifungal results for derivatives of 4-(substituted-phenyl)-3-methyl-4H-isoxazol-5-ones against Candida albicans, Aspergillus niger, Aspergillus clavatus. For the fungus C. albicans, a better MIC result is observed when the substituents are electron withdrawing, such as 2,4-OH and 4-NO₂. In this situation, only the derivatives ILA1, ILA14 and ILA17 presented a MIC of 500 µg mL^-1. The same could be observed in the results of work by Srinivas et al.,³¹ in which methylene-bis-tetrahydro-[1,3]-thiazole-[4,5-c]-isoxazole derivatives with electron-withdrawing groups, such as 4-Cl and 4-F on the aromatic ring, presented MIC of 3.75 µM against C. albicans (ATCC 10231). A study by Padmaja et al.³² synthesized derivatives of iminium isoxazolones and found a high inhibitory activity against the fungi Fusarium solani (NCIM No.1330), Curvularia lunata (NCIM No. 716) and Aspergillus niger (NCIM No. 596). Table 4 presents the results obtained in the evaluation of the activity of the synthesized derivatives against Candida albicans (ATCC 10231) and Candida tropicalis (ATCC 13803).

Table 4
Evaluation of the antifungal activity of 4-arylidene-3methylisoxazol-5(4H)-one derivatives

Compound	Candida albicans (ATCC 10231)		Candida tropicalis (ATCC 13803)
	MIC / (µg mL-1)	MBC / (µg mL-1)	MIC / (µg mL-1)	MBC / (µg mL-1)
ILA1	500	>1000	125	>1000
ILA2	>1000	>1000	1000	>1000
ILA3	>1000	>1000	>1000	>1000
ILA4	>1000	>1000	1000	>1000
ILA5	>1000	>1000	1000	>1000
ILA6	1000	>1000	500	>1000
ILA7	>1000	>1000	1000	>1000
ILA8	>1000	>1000	>1000	>1000
ILA9	1000	>1000	>1000	>1000
ILA10	>1000	>1000	>1000	>1000
ILA11	>1000	>1000	>1000	>1000
ILA12	1000	>1000	1000	>1000
ILA13	1000	>1000	500	>1000
ILA14	500	>1000	500	>1000
ILA15	1000	>1000	500	>1000
ILA16	1000	>1000	1000	>1000
ILA17	500	>1000	1000	>1000

• MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration.

Evaluation of antioxidant activity

The compounds showed good to moderate antioxidant activity (Table 5). The standard used, quercetin, demonstrated a percentage inhibition value of 68.5%. The compounds showed an antioxidant activity between 81.06 and 9.31%, demonstrating antioxidant activity. From this, a low antioxidant activity was determined for a percentage < 60%, moderate for 61-80% and high for 81-100%.

Table 5
Antioxidant activity (AAO), IC₅₀ and EC₅₀ results of 4arylidene-3-methylisoxazol-5(4H)-one derivatives using the DPPH free radical scavenging method

Compound	DPPH free radical
	Percentage of free radical scavenginga	IC50 / µM	EC50 / (mg mL-1 DPPH)
ILA1	79.5	0.609	0.018
ILA2	68.0	0.726	0.027
ILA3	15.1	2.199	0.220
ILA4	53.5	1.006	0.051
ILA5	48.6	1.547	0.118
ILA6	77.7	0.660	0.022
ILA7	78.9	0.660	0.022
ILA8	9.3	5.244	1.462
ILA9	68.5	0.715	0.026
ILA10	61.5	0.764	0.030
ILA11	76.9	0.524	0.018
ILA12	78.4	0.621	0.019
ILA13	80.1	0.612	0.019
ILA14	76.9	0.642	0.020
ILA15	76.0	0.634	0.020
ILA16	72.5	0.659	0.022
ILA17	71.1	0.700	0.025
Quercetin	68.5	0.700	0.025

• ^a
  Percentage of antioxidant activity (AAO / %) calculated for the concentration of 400 µM. IC₅₀: amount of sample needed to inhibit 50% of a given radical concentration by 50%; EC₅₀: efficient concentration defined by equation 2; DPPH: 2,2-diphenyl-1-picryl-hydrazyl.

Derivatives of isoxazol-5(4H)-ones with the hydroxy group in position 2 and 4 (ILA17, 2,4-OH) provided an inhibition percentage of 71.1%, while the methoxy group (ILA10) in the respective positions showed 61.5% of moderate antioxidant action. Sherin and Rajashekharan³³ observed better antioxidant activity of the compounds 3,5-bis-(styryl)-isoxazoles with methoxy and hydroxy groups in the aromatic ring, as reported in the present research.

Anwar et al.³⁰ obtained the best antioxidant activity for isoxazolones with NO₂ and Cl substituents on the aromatic ring at the position for, respectively, 33 and 34% of antioxidant activity. Derivatives in the present research, with these groups (NO₂ and Cl) presented a moderate to high antioxidant action, namely using derivatives ILA12 (4-Cl) and ILA13 (2-Cl) with 78.38 and 80.15%, respectively. As for the nitro substituent, moderate antioxidant action was seen, with a percentage of 76.87% for ILA14 (2-NO₂), 75.98% for ILA15 (3-NO₂) and 72.46% for ILA16 (4-NO₂). It was observed that derivatives ILA3, ILA4, ILA5, ILA8, ILA10 and ILA11 provided lower percentage values between 9.31 and 53.52%, with low antioxidant activity. IC₅₀ values were established as optimal (IC₅₀ ≤ 0.700 μM), medium (between 0.701-0.800 μM) and poor (IC₅₀ ≥ 1.000 μM). The IC₅₀ and EC₅₀ values obtained for the quercetin standard were 0.700 μM and 0.025 mg of quercetin per mg of DPPH, respectively, that is, the standard presented an excellent concentration of inhibition against the free radical DPPH. Therefore, compounds ILA1, ILA2, ILA6, ILA7, ILA9, ILA11, ILA12, ILA13, ILA14, ILA15, ILA16 and ILA17 showed optimal inhibition concentration. The other derivatives, ILA3, ILA4, ILA5 and ILA8, showed a weak concentration of DPPH free radical inhibition. In a similar way, for the efficient concentration (EC₅₀), compounds ILA1, ILA2, ILA4, ILA6, ILA7, ILA9, ILA10, ILA11, ILA12, ILA13, ILA14, ILA15, ILA16, ILA17 were those that showed efficient action from great to average. Compounds ILA3, ILA5 and ILA8 showed weak efficient action in the free radical scavenging method by DPPH.

Principal component analysis

The theoretical study is based on the different types of substituents of the aromatic aldehyde, as well as the position in which these substituents are found. This characteristic is related to the biological activity of the derivatives, thereby enhancing our comprehension of the experimental findings. The compound illustrated in Figure 4 can assume several different geometric conformations, since it presents different degrees of rotation in the connections of its structural composition. From the combination of four substituents, R¹, R², R³ and R⁴ in Figure 4, 17 compounds were formed, as shown in Table 1.

Figure 4
Representation of the numbered molecular structure of 4-benzylidene-3-methylisoxazol-5(4H)-ones (ILA1).

After applying principal components analysis, it was possible to differentiate the active and inactive compounds based on the molecular descriptors obtained. The PCA method was applied to all tested biological activities.

Conformational analysis and geometry optimization of Isoxazole-5(4H)-one against S. epidermidis

In the R environment, principal component analysis was used to test various combinations of molecular descriptors. The combination that resulted in a clear distinction between active and inactive compounds included the R₁ distances between elements 13 and 21, the T₁ torsion angle involving atoms 6, 8, 9, and 12, the A₁ angle formed by atoms 10, 11, and 19, the T₂ torsion of atoms 20, 12, 13, and 21, and the BO₂₁ bond order between elements 12 and 20. These specific numbers are illustrated in Figure 4.

It is valid to state that the combined descriptors represented up to 68.15% of the main components PC1 and PC2, so it is possible to characterize the compounds as active and inactive in relation to the bacterium Staphylococcus epidermidis. Figure 5a shows the first two principal components, PC1 and PC2, and a vertical continuous line is highlighted, which separates the blue (active) compounds from the red (inactive) compounds. For a better graphical representation, only the compound numbers shown in Table 2 are highlighted.

Figure 5
(a) Graph displaying the scores for active and inactive molecules. (b) Weighted graph depicting the descriptors influencing the antimicrobial activity of derivatives against S. epidermidis bacteria.

By analyzing only PC1 in Figure 5a, it is possible to separate the compounds into active and inactive. In the first PC (47.18%), the active compounds showed more positive values, contrasting with the inactive compounds that showed negative values in the same PC1. It was observed that the derivatives ILA4, ILA5, ILA6, ILA14, ILA15, ILA16 and ILA17, which are considered active, showed moderate to good antibacterial activity against S. epidermidis. Another analysis of interest is in the graph of Figure 5b, the weights of the descriptors, which allowed a characterization of trends between the molecular descriptors selected for the activity of the compound. Table 3 displays the values of the geometric properties calculated with the DFT method M062X/6-311++G**(d,p) selected by the PCA against S. epidermidis bacteria.

The variables responsible for the separation of compounds capable of distinguishing active and inactive molecules for Staphylococcus epidermidis were solely geometric descriptors. By combining them with their weights, we can obtain equation 3.

(3) $\text{PC}1=0.514{\text{R}}_1+0.336{\text{T}}_1+0.456{\text{A}}_1+0.535{\text{T}}_2-0.358{\text{BO}}_{21}$

When analyzing equation 3, which lists the variables and coefficients of the main component, PC1, it is possible to predict how the biological activity for Staphylococcus epidermidis can be justified. Variable R₁, for distance, has a value in the score projected in PC1 on the positive side, very similar to the other variables of T₁ torsion angle, A₁ bond angle and T₂ torsion angle. Therefore, with this positive score value (0.514) and positive torsion angles T₁ (0.336), T₂ (0.535) and A₁ (0.456) (Table 6), this variable must have higher values for the compounds to be part of the group of active molecules.

Table 6
Values of geometric and electronic properties calculated with the DFT method M062X/6-311++G**(d,p) selected by PCA against S. epidermidis bacteria

Compound		R1 distance / Å	T1 torsion / degree	A1 angle / degree	T2 torsion / degree	BO21
ILA1	inactive	1.0830	–179.9955	119.3322	0.0003	0.9248
ILA2	inactive	1.0825	–179.9992	118.9135	0.0000	0.9241
ILA3	inactive	1.0853	–179.9881	119.7905	0.0003	1.0198
ILA4	active	1.3628	178.7372	119.7181	2.0436	0.9173
ILA5	active	1.3625	179,4987	125,3298	2,0032	0,9172
ILA6	active	1.0844	179.9628	119.1046	0.0100	0.9253
ILA7	active	1.3601	–179.9904	119.4663	0.0004	0.9184
ILA8	inactive	1.0807	179.9993	118.3653	0.0000	0.9247
ILA9	active	1.3670	179.6508	121.5356	1.9401	0.9160
ILA10	inactive	1.0803	179.9984	119.3893	0.0001	1.0112
ILA11	inactive	1.0823	–179.4724	118.2417	0.6538	1.0185
ILA12	inactive	1.0820	–179.9964	120.4848	0.0002	0.9228
ILA13	inactive	1.0820	180.0019	119.4949	0.0000	1.0597
ILA14	active	1.0813	138.7439	119.3973	1.1051	0.9085
ILA15	active	1.4801	179.9978	119.4747	–0.0006	0.9027
ILA16	active	1.0812	179.9944	121.3229	–0.0003	0.9212
ILA17	active	1.0864	180.0001	121.5325	0.0000	1.0263

• R₁: distances between elements 13 and 21; T₁: torsion angle involving atoms 6, 8, 9, and 12; A₁: angle formed by atoms 10, 11, and 19; T₂: torsion of atoms 20, 12, 13, and 21; BO₂₁: bond order between elements 12 and 20.

On the other hand, variable BO₂₁, which is bond order, has a negative score, as noted in equation 3, but positive values as shown in Table 6. Therefore, for the compounds in question to be part of the group of active molecules, these values must be reduced.

The correlation matrix of the selected descriptors is presented in Table 7, in which their values help to measure the relationship between the variables. This relationship can be linearly positive, when values are close to +1, and linearly negative or inverse, when values tend to –1.

Table 7
Pearson correlation coefficient calculated between the descriptors selected by PCA using R software environment²⁰

	R1 distance	T1 torsion	A1 angle	T2 torsion	BO21
R1 distance	1.00	0.23	0.36	0.53	–0.46
T1 torsion	0.23	1.00	0.31	0.32	–0.04
A1 angle	0.36	0.31	1.00	0.51	–0.14
T2 torsion	0.53	0.32	0.51	1.00	–0.30
BO21	–0.46	–0.04	–0.14	–0.30	1.00

• R₁: distances between elements 13 and 21; T₁: torsion angle involving atoms 6, 8, 9, and 12; A₁: angle formed by atoms 10, 11, and 19; T₂: torsion of atoms 20, 12, 13, and 21; BO₂₁: bond order between elements 12 and 20.

The correlation between the descriptors shown in Figure 5b indicates that variables R₁, T₁, A₁ and T₂ have a slight and direct correlation; thus, the increase of one of the variables leads to the reduction of the other. There is the same inverse negative association of geometric descriptors with the BO₂₁ bond order.

Conformational analysis and geometry optimization of isoxazole-5(4H)-one derivatives against E. coli

Figure 6a presents the percentages related to the main components and their ability to explain the biological activity of the compounds for E. coli as a function of the percentage of the components used to separate active from inactive molecules. The values present on the axes of Figure 6a, the first component, PC1, with 89.45% of the total variance of the data, the second component, PC2, with 9.51%, and the third with the remaining 1.03%.

Figure 6
(a) Graph displaying the scores of active and inactive molecules. (b) Weighted graph depicting the descriptors influencing the antimicrobial activity of derivatives against Escherichia coli bacteria.

As shown in Figures 6a and 6b, it is observed that the variables responsible for separating the samples capable of distinguishing active and inactive molecules for E. coli were the variables GAP, A₁, A₂, T₁ angles (see numbering in Figure 4) and BO₂₄ bond order. It was observed that derivatives ILA7, ILA13, ILA15 and ILA17 were considered active, and these showed moderate antibacterial activity against E. coli. Table 8 exposes all the values of the geometric and electronic properties calculated with the DFT method M062X/6-311++G**(d,p) selected by the PCA against E. coli bacteria.

Table 8
Values of geometric and electronic properties calculated using the DFT method M062X/6-311++G(d,p) selected by PCA against E. coli bacteria

Compound		GAP / Hartree	A1 angle / degree	A2 angle / degree	T1 torsion / degree	BO24
ILA1	inactive	0.25000	120.0379	119.9977	0.0003	0.9227
ILA2	inactive	0.23305	121.8233	115.7892	0.0000	1.0262
ILA3	inactive	0.24303	119.6284	119.3736	0.0003	0.9225
ILA4	inactive	0.23350	119.7986	117.3639	2.0436	1.0336
ILA5	inactive	0.22229	119.5846	120.2868	2.0032	1.0489
ILA6	inactive	0.24303	119.7970	121.0464	0.0100	1.0352
ILA7	active	0.24438	117.2698	119.9932	0.0004	0.9228
ILA8	inactive	0.20875	119.0877	121.2177	0.0000	1.1683
ILA9	inactive	0.22482	129.0770	109.2898	1.9401	0.9982
ILA10	inactive	0.22915	122.5838	115.0118	0.0001	1.0266
ILA11	inactive	0.21056	118.0288	121.3365	0.6538	1.1711
ILA12	inactive	0.24437	121.1991	119.2631	0.0002	1.0726
ILA13	active	0.26305	119.3560	119.4544	0.0000	0.9207
ILA14	inactive	0.25225	119.1077	119.7524	1.1051	0.9183
ILA15	active	0.24427	118.5232	119.7667	–0.0006	0.9014
ILA16	inactive	0.25047	122.0835	118.5278	–0.0003	0.8992
ILA17	active	0.26352	120.0380	122.1570	0.0000	1.0432

• A₁: angle formed by atoms 12, 13, and 21; A₂: angle formed by atoms 13, 14 and 22; T₁: torsion of atoms 20, 12, 13, and 21; BO₂₄: the bond order between elements 14 and 22.

Equation 4 lists the descriptors and their coefficients of the principal component of PC1.

(4) $\text{PC}1=-0.400\text{GAP}+0.534{\text{A}}_1-0.554{\text{A}}_2+0.447{\text{T}}_1+0.216{\text{BO}}_{24}$

The analysis of equation 4 describes PC1 and is sufficient to separate the compounds. It can be noted that all variables have positive values, as shown in Table 8. However, only the GAP and the A₂ angle have negative coefficients, according to equation 4. Therefore, in order for the compounds to be classified as part of the active molecule group, it is necessary to increase the value of the variables in the positive direction: A₁ angle, T₁ torsion and BO₂₄ bond order in the principal component analysis. The other variables that contribute to the separation are the GAP and the A₂ angle, which have negative weights and positive values (Table 8). Hence, for the compounds under consideration that belong to the active group, these values must be reduced.

The Pearson correlation matrix of the descriptors is presented in Table 9. The correlation between the descriptors, selected in Figure 6a, says that the A₁ angle has a strong inverse correlation with the A₂ angle. The same can be observed between the variables GAP and BO₂₄ bond order.

Table 9
Pearson correlation coefficient calculated between the descriptors selected by PCA using R software environment²⁰

	GAP	A1 angle	A2 angle	T1 torsion	BO24
GAP	1	–0.13	0.22	–0.36	–0.71
A1 angle	–0.13	1	–0.88	0.32	–0.01
A2 angle	0.22	–0.88	1	–0.40	0.12
T1 torsion	–0.36	0.32	–0.40	1	0.15
BO24	–0.71	–0.01	0.12	0.15	1

• A₁: angle formed by atoms 12, 13, and 21; A₂: angle formed by atoms 13, 14 and 22; T₁: torsion of atoms 20, 12, 13, and 21; BO₂₄: the bond order between elements 14 and 22.

Conclusions

Based on the obtained results, it can be concluded that the methodology applied for synthesizing isoxazolone derivatives proved to be an efficient alternative. It is chemically clean since the IL catalyst acid used is non-toxic, has a low reaction time and requires only a low temperature. It was possible to synthesize 17 different derivatives of 4arylidene-3-methylisoxazol-5(4H)-ones with the proposed methodology, with yields ranging from 20 to 96%. The results of antimicrobial screening revealed the potential of the compounds obtained as new antibacterial agent candidates, showing inhibitory action of less than 15.62 µg mL^-1 for Gram-positive S. epidermidis (ATCC 12228) and 250 µg mL^-1 for Gram-negative E. coli (ATCC 25312). For fungi, a moderate inhibitory action of 125 µg mL^-1 against C. tropicalis (ATCC 13803) and 500 µg m L ^-1 against C. albicans (ATCC 10231) was observed. The synthesized isoxazole derivatives showed optimal to average antioxidant activity, with a percentage between 81.1 and 9.31%. The theoretical structure-activity study, performed using molecular quantum mechanics calculations based on density functional theory with the M062X hybrid exchange and correlation functional and the 6-311++G**(d,p) basis sets, effectively described the geometric and electronic descriptors of the compounds. Through the application of multivariate statistical methods, specifically PCA, it was possible to distinguish the structures into two groups based on the selected quantum descriptors for each bacterium: the first group exhibited activity, while the second group showed inactivity.

Acknowledgments

The authors are grateful for the financial support from the Universidade Estadual de Goiás: Pró-Projetos No. 05/2021 (No. 000024942033 process SEI No. 000024942033), Pró-laboratórios No. 28/2022 (No. 000036339469, process SEI No. 202200020022734) Pesquisa and Pró-Programas No. 21/2022 (No. 45362693, process SEI No. 202200020020883) and CAPES. This research is also supported by the High-Performance Computing Center at the Subsecretaria de Tecnologia da Informação (STI), in the Secretaria de Desenvolvimento e Inovação (SEDI), Goiás, Brazil and by the High-Performance Computing Center at the Universidade Estadual de Goiás (UEG).

Supplementary Information

Supplementary information (IR, NMR ¹H and ¹³C spectroscopy) is available free of charge at http://jbcs.sbq.org.br as a PDF file.

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Edited by

Editor handled this article: Brenno A. D. Neto

Publication Dates

• Publication in this collection
  26 Feb 2024
• Date of issue
  2024

History

• Received
  31 Aug 2023
• Accepted
  28 Nov 2023