Microwave-Assisted Synthesis of Aminophosphonic Derivatives and Their Antifungal Evaluation against Lomentospora prolificans

Lomentospora prolificans is a pathogenic and multidrug-resistant fungus that can infect both immunocompetent and immunocompromised patients, with mortality rates up to 87%. The World Health Organization (WHO) included this fungal species in its first list of 19 priority fungal pathogens, which focused on fungal pathogens that can cause invasive acute and subacute systemic fungal infections. Therefore, there is a growing interest in finding new therapeutic alternatives. In this work, the synthesis of twelve α-aminophosphonates by the microwave-assisted Kabachnik–Fields reaction and twelve α-aminophosphonic acids by a monohydrolysis reaction is reported. All compounds were evaluated by the agar diffusion method as a preliminary screening in comparison with voriconazole, showing inhibition halos for compounds 7, 11, 13, 22 and 27. The five active compounds in the preliminary tests were evaluated against five strains of L. prolificans following protocol M38-A2 from CLSI. The results showed that these compounds exhibit antifungal activity in the concentration range of 900->900 μg/mL. Cytotoxicity against healthy COS-7 cells was also evaluated by the MTT assay, and it was shown that compound 22 was the least cytotoxic, with a viability of 67.91%, comparable to the viability exhibited by voriconazole (68.55%). Docking studies showed that the possible mechanism of action of the active compounds could be through the inhibition of the enzyme lanosterol-14-alpha-demethylase in an allosteric hydrophobic cavity.


Introduction
Fungal infections affect more than one billion people each year, of which 150 million develop severe symptoms and approximately 1.6 million die [1]. Lomentospora prolificans is a highly virulent filamentous fungus that can cause a wide spectrum of clinical manifestations [2]. L. prolificans can infect both immunocompetent and immunocompromised patients, the immunocompromised patients being the most susceptible, causing disseminated infections with mortality rates up to 87% [3,4]. In fact, the World Health Organization (WHO) included this fungal species in its first list of 19 priority fungal pathogens, selected due to their perceived public health importance [5].

Chemistry
α-aminophosphonates 5-16 were synthetized by the microwave-assisted Kabachnik-Fields (KF) reaction without a catalyst (Scheme 1), in which the corresponding aldehydes 2a-e, an amine 3a-c and diphenyl phosphite 4 in ethanol were reacted at 80 °C for 10 min. The progress of the reaction was monitored by TLC. Once the reaction was completed, the reaction mixture reached room temperature and the product precipitated (Scheme 2) due to their low solubility in ethanol. Compounds 5-16 were isolated by vacuum filtration and washed with cold diethyl ether.

Chemistry
α-aminophosphonates 5-16 were synthetized by the microwave-assisted Kabachnik-Fields (KF) reaction without a catalyst (Scheme 1), in which the corresponding aldehydes 2a-e, an amine 3a-c and diphenyl phosphite 4 in ethanol were reacted at 80 °C for 10 min. The progress of the reaction was monitored by TLC. Once the reaction was completed, the reaction mixture reached room temperature and the product precipitated (Scheme 2) due to their low solubility in ethanol. Compounds 5-16 were isolated by vacuum filtration and washed with cold diethyl ether. Once the reaction was completed, the reaction mixture reached room temperature and the product precipitated (Scheme 2) due to their low solubility in ethanol. Compounds 5-16 were isolated by vacuum filtration and washed with cold diethyl ether.
Products 5, 7-13 and 15-16 were isolated by filtration without any further purification, with good yields (42-85%). However, compounds 6 and 14 were obtained in low yields when the proposed reaction conditions were followed; the reason for this may be because 4-dimethylaminocinnamaldehyde 2d is a very unstable compound [37], which favored the formation of undesirable by-products. For this reason, it was necessary to synthesize the aminophosphonates 6 and 14 through another methodology. Therefore, the KF reaction was carried out between 4-dimethylaminocinnamaldehyde 2d, diphenyl phosphite 4 and the corresponding amines 3b,c using vortex mixing (Scheme 3) [35]. Remarkably, there was an increase in the yields of compounds 6 and 14 from 11% and 29%, to 87% and 81%, respectively. All the spectra of synthesized aminophosphonates are supplied in the Supplementary Materials. Once the reaction was completed, the reaction mixture reached room temperatur and the product precipitated (Scheme 2) due to their low solubility in ethanol. Com pounds 5-16 were isolated by vacuum filtration and washed with cold diethyl ether.
Products 5, 7-13 and 15-16 were isolated by filtration without any further purification, with good yields (42-85%). However, compounds 6 and 14 were obtained in low yields when the proposed reaction conditions were followed; the reason for this may be because 4-dimethylaminocinnamaldehyde 2d is a very unstable compound [37], which favored the formation of undesirable by-products. For this reason, it was necessary to synthesize the aminophosphonates 6 and 14 through another methodology. Therefore, the KF reaction was carried out between 4-dimethylaminocinnamaldehyde 2d, diphenyl phosphite 4 and the corresponding amines 3b,c using vortex mixing (Scheme 3) [35]. Remarkably, there was an increase in the yields of compounds 6 and 14 from 11% and 29%, to 87% and 81%, respectively. All the spectra of synthesized aminophosphonates are supplied in the Supplementary Materials. It is important to note that, in this work, it was not possible to carry out the KF reaction without a solvent due to the heterogeneity of the components. Some authors have suggested using water as a green alternative; however, it is common that the organic components are not water-soluble, which may limit its use [25,[38][39][40]. Furthermore, Mu et al. reported a study in which ethanol was used as a solvent under conventional heating conditions; however, the reaction did not proceed, and without the solvent, the reaction time was 2 h [24]. Furthermore, Keglevich et al. studied the microwave-assisted KF reaction with different solvents and concluded that, among the investigated solvents, ethanol proved to be the most efficient, with good yields, but in comparison with the results observed in this work, the reaction time was 25 min, and it was necessary to use an excess of the phosphorous compound [33], while in this work, the reaction time under microwave irradiation was only 10 min, it was not necessary to use a large excess of the phosphorate compound and, as mentioned above, the aminophosphonates had the advantage of crystallizing in ethanol; therefore, it was not necessary to use column chromatography to pu- It is important to note that, in this work, it was not possible to carry out the KF reaction without a solvent due to the heterogeneity of the components. Some authors have suggested using water as a green alternative; however, it is common that the organic components are not water-soluble, which may limit its use [25,[38][39][40]. Furthermore, Mu et al. reported a study in which ethanol was used as a solvent under conventional heating conditions; however, the reaction did not proceed, and without the solvent, the reaction time was 2 h [24]. Furthermore, Keglevich et al. studied the microwave-assisted KF reaction with different solvents and concluded that, among the investigated solvents, ethanol proved to be the most efficient, with good yields, but in comparison with the results observed in this work, the reaction time was 25 min, and it was necessary to use an excess of the phosphorous compound [33], while in this work, the reaction time under microwave irradiation was only 10 min, it was not necessary to use a large excess of the phosphorate compound and, as mentioned above, the aminophosphonates had the advantage of crystallizing in ethanol; therefore, it was not necessary to use column chromatography to purify them. The next step in the synthesis involved basic monohydrolysis of the α-aminophosphonates 5-16 using potassium carbonate by microwave irradiation, in an ethanol/water mixture (3:1) as the reaction medium (Scheme 4). The reaction mixture was heated at 140 • C for 20 min. The progress of the reaction was monitored by TLC, and the solvents were evaporated under reduced pressure. The reaction crude product was purified by column chromatography in a gradient system, starting with ethyl acetate to remove the less polar impurities and ending with methanol/ethyl acetate (1:1) to obtain the monoacids as pure solids. It is noteworthy that the hydrolysis reaction proceeded by selectively hydrolyzing one of the -O-phenyl substituents on the phosphonate group, which was confirmed by 1 H NMR and 13 C NMR, where the observed signals agreed with the expected ones for com pounds 17-28 (Table 1). Potassium carbonate and water reacted to form a catalytic numbe of hydroxide ions, which are the species responsible for promoting the specific monohy drolysis reaction [35]. All the spectra of synthesized aminophosphonic acids are supplied in the Supplementary Materials. Monoacids 18 and 25 were not obtained at 140 °C for 20 min because the α-amino phosphonates 6 and 14 are unstable at high temperatures. Therefore, to obtain these mon oacids, the microwave reaction time was decreased from 20 min to 10 min, favoring the formation of 18 and 25 with yields of 37% and 67%, respectively.
The structures of the compounds 5-28 were confirmed by 1 H and 13 C NMR, as wel as high-resolution mass spectroscopy. It is important to point out some specific coupling It is noteworthy that the hydrolysis reaction proceeded by selectively hydrolyzing one of the -O-phenyl substituents on the phosphonate group, which was confirmed by 5 25 Monoacids 18 and 25 were not obtained at 140 • C for 20 min because the α-aminophosphonates 6 and 14 are unstable at high temperatures. Therefore, to obtain these monoacids, the microwave reaction time was decreased from 20 min to 10 min, favoring the formation of 18 and 25 with yields of 37% and 67%, respectively. The structures of the compounds 5-28 were confirmed by 1 H and 13 C NMR, as well as high-resolution mass spectroscopy. It is important to point out some specific coupling constants (J H-P and J C-P ), which were some of the key points in corroborating the union of the three components of the KF reaction (Table 1). In 1 H NMR, the characteristic hydrogenphosphorus coupling constants were observed for both the α-aminophosphonates 5-16 and the corresponding monoacids 17-28, ranging between 20 and 26 Hz. On the other hand, characteristic C-P coupling constants corresponding to the C-P bond (between 138 and 164 Hz) were observed in 13 C NMR for each of the α-aminophosphonates and αaminophosphonic acids.

In Vitro Assays
Compounds 5-28 underwent biological testing to determine whether any of the compounds could inhibit the growth of different multidrug-resistant strains of L. prolificans.
The in vitro susceptibility of the 24 synthesized compounds described above was preliminarily evaluated by the agar diffusion method against Lomentospora strains 11-2242, 10-1167 and 09-1125; this method is based on the visual evaluation of the diameter of the growth inhibition zones. For this purpose, agar plates were inoculated with an inoculum of strains ,  and (09-1125). Then, filter paper discs (5 mm in diameter) containing the test compounds at a concentration of 1 mg/mL were placed on the agar surface. Subsequently, the Petri dishes were incubated for 72 h. For this experiment, voriconazole (VRC) was used as a positive control, and it was observed that α-aminophosphonates 5, 6, 8-10, 12, 14 and 15 did not inhibit the growth of any of the L. prolificans strains tested, and only compounds 7, 11 and 13 exhibited inhibition halos. On the other hand, within the series of α-aminophosphonic acids, compounds 22 and 27 also showed inhibition halos.

Antifungal Activity Evaluation (M38-A2 Protocol)
Compounds that exhibited inhibition halos in the preliminary assay (7, 11, 13, 22 and 27) were evaluated against five strains of L. prolificans (11-2242, 05-21909, 10-1167, 13-196 and 09-1125) at different concentrations using the M38-A2 protocol, which challenges different concentrations of the drugs against a standard inoculum, visually registering changes in the fungal growth at 48 and 72 h of incubation. The drug concentration range was 600 µg/mL to 1000 µg/mL. The MIC 100 are shown in Table 2, and these were determined for compounds 7, 11, 13, 22 and 27 and antifungal VRC. It was observed that the compounds inhibited the growth of the five strains tested at concentrations of 900 µg/mL, while voriconazole inhibited growth at concentrations of 16 µg/mL.

Molecular Docking
Voriconazole acts by inhibiting lanosterol-14-alpha-demethylase (PDB ID: 5HS1), a protein involved in ergosterol biosynthesis, the main target of azole antifungal drugs [41]. Based on this, and given that VRC has been used as a control in the present study, it is proposed that active compounds 7, 11, 13, 22 and 27 could exert their antifungal action through a similar mechanism of action; therefore, molecular docking studies were carried out to support this hypothesis.
The methodology for the validation of the active site of the lanosterol-14α-demethylase protein (PDB ID: 5HS1) was performed and described in a previous work by Cordero-Díaz et al. [35] where validation was performed with the native co-crystallized ligand Voriconazole, with an RMSD of less than 2.5 Å. The Autodock-Vina program (https://vina. scripps.edu/; accessed on 15 January 2023) was used to predict molecular docking [42,43], using 1000 modes, 1000 exhaustiveness and 10 replicates for each one, selecting the lowest energy value [35,44]. All figures were visualized with Pymol (Schrödinger, San Diego, CA, USA; http://www.pymol.org/; accessed on 15 December 2022) and Discovery Studio Visualizer 4.5 software.
Molecular docking studies showed that compounds 7, 11, 13, 22 and 27 interact with the lanosterol-14-alpha-demethylase protein in an allosteric hydrophobic cavity. In analyzing the docking results, it should be noticed that the active compounds 7, 11 and 27 share the substituent at R 1 , which consists of an aromatic ring 4-aminoalkyl substituted. Both 4-dimethylamino and 4-diethylamino substituents are electro-donating groups that could concentrate the electron density in the aromatic ring, favoring different types of interactions. In this context, compound 7 displayed π-anion and π-alkyl interactions with Asp233 and Pro201, respectively (Figure 2a). Compound 27 presented π-sigma and π-alkyl interactions with Leu96 and Ala69, respectively (Figure 2e), and a π-alkyl interaction with Ile205 was observed for compound 11 (Figure 2b).
In the case of compounds 13 and 22, even though the chain length supporting the phenyl group at R 1 was increased, the in vitro inhibitory activity was preserved. The additional double bond containing a π bond did not favor additional interactions with the therapeutic target in this part of the molecule, but the aromatic ring interacted with a greater number of amino acids in this position. For compound 13, interactions with Ala320, His317, Pro201 and Leu512 were observed (Figure 2c), while compound 22 showed interactions with Leu95, Phe241, Phe384 and Met509 (Figure 2d).
On the other hand, it was observed that electro-withdrawing groups at the R 2 position favor a higher number of hydrophobic interactions with the aromatic ring. As shown in  (Figure 2b,d).
Finally, it was observed that the less sterically hindered aminophosphonic acids 22 and 27 interact with the protein in a different region from the proposed active site, while compounds that are more sterically hindered, such as aminophosphonates 7, 11 and 13, remained at the proposed active site. Interestingly, as the size of the substitution in the phosphonate group decreases, the ligand interacts in a region of the protein that is not the proposed active site.
Based on the previous analysis, it is recommended to consider some structural features in the design of future aminophosphonates and aminophosphonic acid derivatives. In the case of R 1 , it is important to include para-substituted phenyl rings with electro-donating groups. In addition, the R 1 with higher steric hindrance, at least in the molecular docking, is relevant to remain in the proposed active site. On the other hand, electron-withdrawing substituents at R 2 could favor a greater number of interactions with the biological target, and it is important to preserve the phosphonate group (Scheme 5).

Cytotoxicity
In order to compare the cytotoxicity at high concentrations of the active compounds 7, 11, 13, 22 and 27 compared to VRC, cytotoxicity studies were performed using the MTT test in COS-7 cells at a concentration of 1000 µM (Table 3). In this study, compound 22 was found to be the least cytotoxic, with a viability of 67.91%, comparable to the viability exhibited by VRC: 68.55%. Both 4-dimethylamino and 4-diethylamino substituents are electro-donating groups that could concentrate the electron density in the aromatic ring, favoring different types of interactions. In this context, compound 7 displayed π-anion and π-alkyl interactions with Asp233 and Pro201, respectively (Figure 2a). Compound 27 presented π-sigma and πalkyl interactions with Leu96 and Ala69, respectively (Figure 2e), and a π-alkyl interaction with Ile205 was observed for compound 11 (Figure 2b).  In the case of R1, it is important to include para-substituted phenyl rings with electrodonating groups. In addition, the R1 with higher steric hindrance, at least in the molecular docking, is relevant to remain in the proposed active site. On the other hand, electronwithdrawing substituents at R2 could favor a greater number of interactions with the biological target, and it is important to preserve the phosphonate group (Scheme 5).

Cytotoxicity
In order to compare the cytotoxicity at high concentrations of the active compounds 7, 11, 13, 22 and 27 compared to VRC, cytotoxicity studies were performed using the MTT test in COS-7 cells at a concentration of 1000 µM (Table 3). In this study, compound 22 was found to be the least cytotoxic, with a viability of 67.91%, comparable to the viability exhibited by VRC: 68.55%.

Synthesis of α-Aminophosphonates 5-16
In a 10 mL microwave (Anton Paar Monowave 300) vial with a magnetic stirrer, 1 eq. of the corresponding aldehyde 2a-e, 1 eq. of the corresponding amine 3a-c, 1.1 eq. of diphenyl phosphite 4 and 3 mL of absolute ethanol were added. The vial was placed in the microwave cavity, and the mixture reacted at 80 • C for 10 min. The reaction mixture was allowed to cool at room temperature to induce the precipitation of the product, which was isolated by vacuum filtration and washed with cold diethyl ether. This method was used for the synthesis of all the following molecules.

In Vitro Antifungal Activity
A total of five isolates of the L. prolificans were tested: 11-2242, 05-2190, 10-1167,  and 09-1125. All isolates were obtained from the Microbiology Department of the School of Medicine of Universidad Autónoma de Nuevo León and were previously identified by Elizondo-Zertuche et al. [45]. The strains were incubated on potato dextrose agar (PDA) at 35 • C for their metabolic reactivation.
For the in vitro susceptibility studies, a preliminary test was carried out by the agar diffusion method, and all synthesized compounds (α-aminophosphonates 5-16 and monohydrolyzed α-aminophosphonic acids 17-28) were evaluated against three strains of the Lomentospora (11-2242, 10-1167, 09-1125); thus, the most active compounds were selected based on the diameter of the growth inhibition zones. For this, conidia were inoculated as a lawn on Müller-Hinton medium (MCD Lab) with a concentration of 0.4 × 10 4 -5 × 10 4 conidia/mL, and the disks were impregnated with each of the 24 compounds at a concentration of 1.0 mg/mL. Inhibition halos were compared to VRC. Subsequently, the most active compounds, 7, 11, 13, 22 and 27, were evaluated at different concentrations using the liquid medium susceptibility method against the three Lomentospora strains previously mentioned (11-2242, 10-1167, 09-1125). Then, 5 mL snap tubes and 900 mL of nutrient broth (BD Bioxon) were added at a concentration of 0.4 × 10 4 -5 × 10 4 conidia/mL, and 100 mL of the compounds was added at concentrations between 100 and 1000 mg/mL. The tubes were vortexed and incubated at 35 • C for 72 h, and the concentration range was determined for subsequent evaluation by the CLSI method M38-A2.
Finally, the in vitro antifungal susceptibility of the five strains of Lomentospora species was determined by the macrodilution method of the CLSI M38-A2 protocol. The reference antifungal agent used was VRC, which was obtained as pure reagent grade powder. The final drug concentrations ranged from 0.125 to 64 µg/mL for VRC and from 650 to 1000 µg/mL for compounds 7, 11, 13, 22 and 27. The tubes were incubated at 37 • C for 72 h, and the minimal inhibitory concentration (MIC) for all antifungals was registered visually. The MIC was defined as the drug concentration for which a 100% reduction in turbidity compared with the drug-free control for voriconazole and the compounds 7, 11, 13, 22 and 27 were observed. Assays were carried out in duplicate using Candida parapsilosis ATCC 22019 as quality control organisms.

Cell Culture
The monkey kidney cell line (COS-7) used for biological tests was obtained from Centro Médico Siglo XXI.
The cell line was cultured in RPMI-1640 culture medium supplemented with fetal bovine serum (10%) and an antibiotic-antifungal mixture (1%).

MTT Assay
Active compounds (7, 11, 13, 22 and 27) against L. prolificans and VRC were evaluated by an MTT assay to evaluate their cytotoxicity in healthy COS-7 cells. Briefly, the cells were harvested with EDTA trypsin solution, already detached from the base of the culture flask, harvested and diluted with supplemented medium to inactivate trypsin. An aliquot of cells was taken to perform the viability count through the trypan blue technique using an electronic counter. The inoculum density was adjusted to 10 × 10 4 cells/mL, and they were deposited at a volume of 100 µL in 96-well plates. The cell cultures were incubated for 24 h to promote their adherence to the substrate at the bottom of the well. Next, the compounds were added in the solution with supplemented medium and their corresponding solvent at a volume of 100 µL. The compounds were prepared at a concentration of 40 mM in DMSO and/or ethanol; the final concentration was 1000 µM for the test drugs and ≤1% for DMSO. The etoposide was prepared at 20 µM in DMSO. At 24 h, the cell was removed from the medium and washed with a phosphate buffer, 100 µL of an MTT solution (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) was immediately added and they were left for 4 h in the incubator. Afterwards, the MTT solution was removed, and 100 µL of DMSO was added to favor the solubility of formazan produced during the cell metabolism; optical density (OD) was measured in a microplate reader at a wavelength of 470 nm.
Cell viability was calculated from the following expression% Viability = (OD treatment/OD vehicle) × 100. Data are processed individually or by an independent experiment, obtaining the average of these, plus the standard error of the mean.

Conclusions
The microwave-assisted KF reaction proceeded in 10 min with moderate to good yields and without employing catalysts. The characteristic H-P and C-P coupling constants corroborated the formation of the 12 new α-aminophosphonates and 12 new α-aminophosphonic acids. When evaluating the antifungal activity of the 24 synthesized compounds against L. prolificans by the CLSI protocol M38-A2, compounds 7, 11, 13, 22 and 27 exhibited in vitro antifungal activity against five strains of L. prolificans, and the molecular docking studies with the enzyme lanosterol-14-alpha-demethylase suggest that these compounds interact in an allosteric cavity. From the docking and SAR analysis, it was concluded that, in the design of new α-aminophosphonates and α-aminophosphonic acids with possible antifungal activity, it is recommended to consider the inclusion of certain structural features such as the phosphate group, aromatic rings substituted with electron-donating and sterically hindered groups at the R 1 position and electro-withdrawing groups at the R 2 position. Data Availability Statement: Data available on request due to restrictions privacy. The data presented in this study are available on request from the corresponding author.