SYNTHESIS AND ANTIFUNGAL ACTIVITY OF NEW O-ALKYLAMIDOXIMES

The continuous prospection for molecules that may be useful in the development of new therapeutic agents is a highly relevant issue, mainly because the launch of new drugs on the market does not accompany the emergence of new resistant microorganisms. In this context, this work describes the synthesis of new Oalkylamidoximes and the evaluation of its antifungal activity. The new O-alkylamidoximes were prepared using easy synthetic protocols and tested against three Candida species using the broth microdilution method. The synthesized compounds were obtained in moderate to good yields in high purity and without any observable decomposition. All tested compounds shown moderate antifungal activity against at least one strain of Candida. Despite the moderate activity of the new compounds, this was the first report involving the antifungal activity of O-alkylamidoximes. In view of the low chemotherapy arsenal and the development of fungal strains resistant to traditional antifungal agents, the present study opens new possibilities for the preparation of a new class of more active antifungal agents.


Introduction
Fungi are widespread in the environment and are found in a variety of habitats (Prasad and Kapoor 2004). These microorganisms make up the normal human microbiota and are commonly found in the skin, gastrointestinal, respiratory, and genitourinary tracts (Kapitan et al. 2019). However, in immunodeficient individuals such as transplanted (Eades and Armstrong-James 2019) and HIV-infected (Wang et al. 2017), the contact with pathogenic fungi (Capote et al. 2016) can lead to severe infections. Thus, fungal infections have been a major concern for health regulators, special due to the considerable morbidity and mortality rates, contributing factors to the increased health costs (Silva et al. 2012).
Among the different etiological agents involved in this type of pathology, Candida spp. are the most prevalent, representing approximately 19% of all infections worldwide in intensive care units (Irfan et al. 2017). It is also estimated that around 25 to 75% of healthy individuals to have Candida spp colonies in their normal microbiota (Costello et al. 2009). However, different species of this genus are often identified as the cause of invasive infections in humans, with special attention to C. albicans due to its prevalence in both

General procedure for the synthesis of Amidoximes 2a-h
The synthesis of amidoximes 2a-h was based on the methodology previously described in the literature (Barros et al. 2011). To a flask containing hydroxylamine hydrochloride (2.08 g, 30 mmol) and sodium carbonate (1.6 g, 15 mmol) at 25 o C was added distilled water (30 mL). The mixture was stirred and then a solution of the appropriate nitrile 1a-h (10 mmol) in ethanol (40 mL) was added dropwise. The reaction mixture was placed in an ultrasonic bath at 55±5 °C and monitored by TLC. After completion, the solvents were removed in vacuo. The residual solid was extracted with ethyl acetate (3 x 25 mL) and the combined organic phases dried over anhydrous Na2SO4. Filtration, following removal of the solvent under reduced pressure, provided the corresponding amidoximes 1a-h which were purified by crystallization using hexane/chloroform (10:90) system.

General procedure for preparing O-alkylamidoximes (3a-h)
To the flask was added the appropriate amidoxime 2a-h (0.5 mmol), sodium hydroxide (32 mg, 0.8 mmol), and DMSO (4 mL). The mixture was stirred for 5 minutes and then bromoacetaldehyde diethyl acetal (118.2 mg, 0.6 mmol) was added. The reaction mixture was stirred at room temperature and monitored by TLC [hexane/ethyl acetate (40:60)]. After completion, the reaction mixture was extracted with ethyl acetate (3 x 20 mL). The organic phase was separated, dried over anhydrous Na2SO4, filtered and the solvent removed under reduced pressure. The crude product was purified by a flash column chromatography [hexane/ ethyl acetate (95:5)] to yield compounds 3a-h.

Fungal strains
Candida guilliermondii ATCC 6260, Candida albicans ATCC 76615 and Candida albicans ATCC 76485 were provided by the fungal culture collection from the Laboratório de Micologia da Universidade Federal da Paraíba, Brazil.

Minimum inhibitory concentration (MIC)
The determination of minimum inhibitory concentration (MIC) was performed by broth microdilution according to CLSI (Clinical and Laboratory Standards Institute) document M27-A2 (CLSI 2017). The culture medium used was Sabouraud Dextrose Agar. The inoculum of each of the microorganisms was prepared according to the 0.5 McFarland standard containing approximately 1-5 x 10 6 CFU.mL -1 (colony forming units.mL -1 ). Experiments were conducted at approximately 1-5 x 10 5 CFU.mL -1 in each well. The solutions with compounds 3a-h were prepared at the time of the tests. Dimethylsulfoxide (DMSO) was used to solubilize compounds 3a-h (the concentration of DMSO was always less than 0.5%). Compounds 3a-h was tested at concentrations ranging from 512 to 8 μg.mL -1 at 1:2 serial dilutions. Initially, 100 µl of the culture medium was added to each well of a plate. Then, a 100 µL of the solution of compounds 3a-h (final concentration 512 µg.mL -1 ) was added to the first line of the plate and a 1:2 serial dilution to 1 µg.mL -1 concentration was performed. Finally, 10 µL of the inoculum was added to each well of the plate. The plate was incubated at 35 o C for 24-48 hours. The minimum inhibitory concentration was considered the lowest concentration capable of inhibiting the visible growth of the microorganism. The experiment was performed in triplicate and controls with DMSO (culture medium + inoculum + DMSO) at the same concentration used to solubilize 3a-h were prepared to demonstrate that the growth of the microorganism is not influenced by DMSO.
MIC was considered the lowest concentration of the substances capable of causing visual inhibition of the growth of the strains used in the microbiologic assays, being confirmed by the addition of 20 μL of 2.0% TTC (2,3,5 triphenyl tetrazolium chloride) in each cavity.

Results and Discussion
The general strategy for the synthesis of the new antifungal agents involved two reaction steps, the preparation of the amidoximes 2 from different nitriles followed by the alkylation of the obtained compounds to afford the O-alkylamidoximes. The synthesis started from the reaction of commercially available nitriles 1a-h and hydroxylamine hydrochloride under sonication (Barros et al. 2011;Andrade et al. 2016). In all cases, the corresponding amidoximes 2a-h were obtained in short reaction times and moderate to high yields after purification by crystallization (Figure 1). The characterization data of the obtained compounds are in accordance with the literature (Li et al. 2013;Ozcan et al. 2013;Andrade et al. 2016;Tarasenko et al. 2017). Thus, compound 2a was prepared at the shortest reaction time probably due to its high solubility in the reaction medium and lower steric hindrance. Steric factors are also important, the ortho isomer 2b was obtained in lower yield and longer reaction time when compared to the meta isomers and para isomers, 2c and 2d, respectively. When halogens were present in the aromatic ring the corresponding compounds 2e and 2f were obtained in higher yields. The best result was observed when the nitro group, a strong electronwithdrawing group was used, where compound 2g was obtained in 91% yield. The method proved also to be efficient for the synthesis of heteroaromatic compounds, where 2h was obtained in good yield after a half-hour.
The O-alkylation of amidoximes has few examples described in the literature (Coviello 1964;Veerman et al. 2016). In this way, the synthesis of alkylated amidoximes was revisited to find the best condition for this reaction. The desired compounds, 3a-h were obtained in moderate to good yields after 2 to 7 hours depending on the group present in the aromatic ring ( Figure 2).

Figure 2. Synthesis of O-alkylated-amidoximes.
Compounds 3a-h were then submitted to antifungal activity against three Candida species due to their prevalence in the epidemiology of fungal infections (Whaley et al. 2017) using the broth microdilution method (CLSI 2017). Thus, the Minimum Inhibitory Concentration (MIC) (Espinel-Ingroff et al. 2005) capable of visually inhibiting 100% of fungal growth for the synthesized compounds was evaluated. The results are depicted in Table 1. In general, all compounds shown antifungal activity against at least one strain of Candida. As described by Morales et al. (2008) a compound with poor antimicrobial activity shows MIC values above of 1500 μg.mL -1 , moderate activity in the range of 500-1500 μg.mL -1 , and good activity with MIC values in the range of 50-500 μg.mL -1 . It is also interesting to note that the position of the substituents attached to the aromatic ring did not imply significant changes in MIC values. For example, compounds 3b-d which contain a methyl group at the ortho, meta, or para positions, respectively, exhibited similar MIC values (Table 1, entries 2-4). Aromatic rings containing a halogen as substituent 3e and 3f exhibited better activities against all strains tested (Table 1, entries 5 and 7). These findings are in accordance with the commercially available antifungal agents chlorotrimazole and econazole. Structure-activity studies have revealed that the presence of a halogen is critical for drug activity for both compounds (Kasper et al. 2015).
The best result was observed for compound 3g, which contains the strongly electron-withdrawing group nitro group (Table 1, entry 7). This is an interesting result, while a number of antibiotics bearing nitro groups were reported from bacteria, such as chloramphenicol and pyrrolnitrin (Al-Zereini et al. 2007) but not for antifungal agents. Finally, compound 3h exhibits an antifungal activity similar to 3a, which has no substituents at the aromatic ring (Table 1, entries 1 and 8).
One of the most important steps in the process in the development of new drugs is the prospecting of new prototypes that can be optimized based on planned molecular modifications for viable therapeutic options (Pinzi and Rastelli 2019). Further studies in the mode of action of the synthesized compounds are underway in our laboratories.

Conclusions
New O-alkylamidoximes were obtained in moderate to good yields (68-81%) without any observable decomposition using a simple, reproducible, and non-expensive method. All O-alkylamidoximes showed in vitro antifungal activity against at least one of the tested strains of Candida. These findings are the first step in the development of new drugs, providing important information for future research to obtain new classes of antifungal agents. Synthesis and antifungal activity of new O-alkylamidoximes conception and design, acquisition of data, and drafting the article; FREITAS, J.C.R.: conception and design, acquisition of data, and drafting the article. All authors have read and approved the final version of the manuscript.

Conflicts of Interest:
The authors declare no conflicts of interest.
Ethics Approval: Not applicable.