Novel Alkyl(aryl)-Substituted 2,2-Difluoro-6-(trichloromethyl)-2H-1,3,2-oxazaborinin-3-ium-2-uides: Synthesis, Antimicrobial Activity, and CT-DNA Binding Evaluations

The synthesis, antimicrobial activity evaluations, biomolecule-binding properties (DNA), and absorption and emission properties of a new series of (Z)-1,1,1-trichloro-4-alkyl(aryl)amino-4-arylbut-3-en-2-ones (4, 5) and 2,2-difluoro-3-alkyl(aryl)amino-4-aryl-6-(trichloromethyl)-2H-1,3,2-oxazaborinin-3-ium-2-uides (6, 7) in which 3(4)-alkyl(aryl) = H, Me, iso-propyl, n-butyl, C6H5, 4-CH3C6H4, 4-CH3OC6H4, 4-NO2C6H4, 4-FC6H4, 4-BrC6H4, 2-naphthyl, is reported. A series of β-enaminoketones (4, 5) is synthesized from the O,N-exchange reaction of some amines (3) with (Z)-1,1,1-trichloro-4-methoxy-4-aryl-but-3-en-2-ones (1, 2) at 61–90% yields. Subsequently, reactions of the resulting β-enaminoketones with an appropriate source of boron (BF3.OEt2) gave the corresponding oxazaborinine derivatives (6, 7) at 50–91% yields. UV-Vis and emission properties of biomolecule-binding properties for the DNA of these new BF2-β-enamino containing CCl3 units were also evaluated. Some compounds from the present series also exhibited potent antimicrobial effects on various pathogenic microorganisms at concentrations below those that showed cytotoxic effects. Compounds 4d, 4e, 6e, and 6f showed the best results and are very significant against P. zopfii, which causes diseases in humans and animals.

Indeed, these properties promote the use of these structures in several applications such as, light emitting diodes (Wang et al., 2013) and solar cells (Kolemen et al., 2010). Nevertheless, besides structure of the BODIPY have high planarity, also shows very small Stokes' shifts (Zhang et al., 2008;Kubota et al., 2010). So, synthesis novel boron analogous compounds with designed structural variations are relevant for interesting fluorescence materials (Poon et al., 2010).
On the other hand, b-enaminoketones are the isosteric analogues of 1,3-enolic ketones and have been used in recent years as N,O double-dentate ligands to form 1,3,2-oxazaborines. These difluoroboron complexes of b-enaminones, which belong to the family of b-iminoenolate boron complexes (Kubota et al., 2011), have attracted attention as a promising class of fluorophores (Josefik et al., 2012). Shankarling et al. described the synthesis and spectral and electrochemical characterization of some boron difluoride complexes of benzoindoline-based b-enaminones (Kumbhar et al., 2015). In 2008, Xia et al. reported excellent solution-state fluorescence for some heterocyclic b-iminoenolates (Xia et al., 2008). Later in 2013, Yoshii et al. described the synthesis of boron-ketoiminate derivatives that showed aggregation-induced emission properties and were prepared by the reactions of benaminoketones derivatives with boron trifluoride-diethyl etherate (BF 3 .Et 2 O) (Scheme 1) (Yoshii et al., 2013).
b-enaminoketones and b-enaminoketo-esters are synthesized through the condensation reactions between carbonyl compounds and primary or secondary amines acid-catalysed (Ferraz and Pereira, 2004;Ferraz and Goncalo, 2007). Due to its acidic nature, citrus juice is considered a good catalyst. Recently, Marvi et al. reported an efficient and green method using citrus juice as natural catalyst (Marvi and Fekri, 2018).
b-enaminones they have been also used to prepare different important antibacterial, (Mahmud et al., 2011). anti-inflammatories (Michael et al., 2001), and antitumor agents (Boger et al., 1989). Moreover, b-enaminones are widely used in the preparation of gamino-alcohols, which are structural units present in various compounds with pharmacological properties and natural products (Harris and Braga, 2004). There are many protocols well-established for the preparation of the g-amino-alcohols. Nevertheless, reduction of 1,3-difunctionalized unsaturated structures [b-enaminoketones (Barluenga et al., 1992;Bartoli et al., 1994;Bartoli et al., 2002), or b-aminoketones (Katritzky and Harris, 1990;Pilli et al., 1990)] in which they present nitrogen and oxygen are more frequently.
On this line, fluorinated natural compounds are present in several drug classes. The main progress refers to research in the area of steroids, alkaloids, nucleosides, macrolides, prostaglandins, and amino acids (Kumar et al., 2008). Quinine isolated from chinchona bark led successive and innovative class of antimalarials (Kumar et al., 2003) such as chloroquine, mefloquine, and primaquine. Another example is the fluorocorticoid and fluorouracil derivatives, these drugs are still clinically used (Beǵuéand Bonnet-Delpon, 2006). Following our studies in this area of interest, we decided to investigate the synthesis of novel boron complexes from benaminoketones trihalomethyl that contain the CF 3 or CCl 3 group bonded at the 6-position, N-alkyl(aryl) substituents with electron-donating or electron-withdrawing effect at the 3position and a phenyl group at the 4-position. Furthermore, in view of the biological potential of molecules containing trihalomethyl groups, the aim of this paper was also to report the antimicrobial screening and cytotoxicity analysis of the compounds synthetized here (Scheme 1). Moreover, UV-Vis and emission spectroscopy was employed and DNA-binding properties were investigated of these new BF 2 -b-enamino derivatives in the present work.

EXPERIMENTAL
Unless otherwise indicated, all common reagents and solvents were used as obtained from commercial suppliers and without further purification. 1 H and 13 C NMR spectra were acquired in Bruker Avance III 400 MHz or Bruker Avance III 600 MHz spectrometers for one-dimensional experiments, with 5 mm sample tubes, 298 K, and digital resolution of 0.01 ppm, in CDCl 3 as solvent, and using TMS as the internal reference. The 19 F and 11 B-NMR spectra were acquired in a Bruker Avance III ( 19 F at 564 MHz and 11 B at 192 MHz) equipped with a 5-mm PABBO probe, 5-mm sample tubes at 298 K, and digital resolution of 0.01 ppm, in CDCl 3 , and using CFCl 3 and BF 3 ·OEt 2 , respectively, as the external reference. All spectra can be found at the Supplementary Information File-Figures S1-S30.
All results were reported with the chemical shift (d), multiplicity, integration, and coupling constant (Hz). The following abbreviations were used to explain multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and dd = double doublet. All NMR chemical shifts were reported in parts per million and relative to the internal reference.
All melting points were determined using coverslips in a Microquıḿica MQAPF-302 apparatus and are uncorrected.
The CHN elemental analyses were performed in a Perkin-Elmer 2400 CHN elemental analyzer (University of São Paulo, Brazil).
High resolution mass spectra (HRMS) were obtained for all compounds in an LTQ Orbitrap Discovery mass spectrometer (Thermo Fisher Scientific). This hybrid system combines the LTQ XL linear ion trap mass spectrometer and an Orbitrap mass analyzer. Experiments were performed via direct infusion of the sample (flow: 10 μl min -1 ) in positive-ion mode using electrospray ionization. Elemental composition calculations for comparison were executed using the specific tool included in the Qual Browser module of Xcalibur (Thermo Fisher Scientific, release 2.0.7) software. The spectra can be found in the Single crystals of compounds 4g and 6e were obtained by slow evaporation of EtOH at 25°C. Compounds 4g and 6e was measured using a Bruker D8 QUEST diffractometer using Cu Ka radiation (l = 1.54080 Å) with a KAPPA four-circle goniometer equipped with a PHOTON II CPAD area detector. Absorption corrections were performed using the multi-scan method. Anisotropic displacement parameters for non-hydrogen atoms were applied. Most hydrogen atom positions were calculated geometrically and refined using the riding model, although some hydrogen atoms were refined freely. The structure was solved and refined using the WinGX software package (Farrugia, 2012).
The structures were refined based on the full-matrix leastsquares method using the SHELXL program (Sheldrick, 2008). The ORTEP projections of the molecular structures were generated using the ORTEP-3 program. Crystallographic information files (CIFs) for the novel structures were deposited at the Cambridge Crystallographic Data Centre (CCDC) under identification number 1810806 (4g) and 1855153 (6e). Crystallographic data can be observed in the ESI (Supplementary Information File).
Ultraviolet/visible absorption spectra were recorded using Shimadzu UV2600 spectrophotometer and dichloromethane (DCM), methanol (MeOH), and dimethyl sulfoxide (DMSO) as solvents and concentration solution at 10-5 M range. Steady-state emission fluorescence analysis of samples in the same solvents used in absorption analysis were measured with a Varian Cary 50 fluorescence spectrophotometer (emission; 300-700 nm range; slit 2.0 mm). Due to the absence of emission intensity of all derivatives in all solvents, it was not possible to determine fluorescence quantum yield values.
For DNA interactions, boron complexes titrations with calfthymus DNA (CT-DNA) were performed by UV-vis analysis at room temperature in DMSO (2%)-Tris-HCl buffer mixture (pH 7.2) using DMSO stock solution of derivatives (10 -4 M range). The DNA pair base concentrations of low molecular weight DNA from calf thymus (CT-DNA) were determined by UV-vis absorption spectroscopy using ϵ = 6.600 M −1 cm −1 (per base pair) at l max = 260 nm. Derivatives solutions in DMSO with Tris-HCl were titrated with increasing concentrations of CT-DNA (ranging from 0 to 100 mM). The absorption spectra of the compounds were acquired in the wavelength range of 250-700 nm. The binding constants (K b ) of derivatives were calculated according to the decay of the absorption bands of compounds using Equation 1 through a plot of where [DNA] is the concentration of DNA in the base pairs, ϵ a is the extinction coefficient (A obs /[compound]), and ϵ b and ϵ f are the extinction coefficients of free and fully bound forms, respectively. In the plots of [DNA]/(ϵ a − ϵ f ) versus [DNA], Kb is given by the ratio of the slope for the interception.
Competitive EB-binding studies were performed using steady-state emission fluorescence assay with CT-DNA. Compounds were dissolved in DMSO and through the gradual addition of the stock solution of the boron complexes to the quartz cuvette (1.0 cm path length) containing ethidium bromide dye (EB, 2.0 × 10 -7 M) and DNA (1.0 × 10 -5 M) in a Tris-HCl pH 7.4 buffer solution. Derivative concentration ranged from 0 to 100 μM. All samples were excited at l exc = 510 nm and emission fluorescence spectra were recorded at the range of 550-800 nm, 5 min after each addition of the complex solution in order to allow incubation to occur (incubation time). The fluorescence quenching Stern-Volmer constants (K SV ) of compounds were calculated according to the decay of the emission bands of EB-DNA using Equation 2 through a plot of F 0 /F versus [DNA], where F0 and F are the steady-state fluorescence intensities of EB-DNA adduct in the absence and presence of each derivative, respectively. K sv and k q are the Stern-Volmer quenching constant and the bimolecular quenching rate constant, respectively. The [Q] and t o are the derivative concentration and fluorescence lifetime of EB-DNA without the quencher (23.0 × 10 -9 s) (Nafisi et al., 2007). The standard Gibbs free energy (DG°) of derivative-DNA complex was calculated from the values of binding constant (K b ) using Equation 3: General Procedure for the Synthesis of (Z)-1,1,1-trichloro-4-alkyl(aryl)amino -4-phenylbut-3-en-2-ones (4a-i, 5e) To magnetically stirred solutions of (Z)-1,1,1-trichloro-4-methoxy-4-phenylbut-3-en-2-one (1) (5 mmol) in ethanol (20 ml), the respective amines (3a-i, 5e) (1.5 eq, 7.5 mmol) were added and the resulting mixtures were refluxed for 24 h. After that, the solutions were cooled at -10°C, resulting in yellow or white solids. The solids were filtered under atmospheric pressure, washed with cold ethanol, and dried under reduced pressure.
The products 4 and 5 were obtained by simple precipitation at low temperature. Subsequently, the products were filtered, washed with cold ethanol, and dried under reduced pressure. This procedure allowed to isolate 4a-i and 5e in 61 to 90% of yield (Scheme 2). Compounds 4a (Sosnovskikh et al., 2002), 4c (Hojo et al., 1986), and 4e (Toshiaki and Minoru, 1977)). are already described in the literature, but they were obtained through different precursors and procedures.
In order to explore the synthetic potential of 4a-i and 5e, (Z)-1,1,1-trichloro-4-phenyl-4-(p-tolylamino)but-3-en-2-one (4d) was employed to evaluate the influence of different reaction conditions such as solvent, temperature, time, and volume of BF 3 .OEt 2 or Et 3 N to obtain the oxazaboron complexes 6, 7 ( Table 1). Optimization of the reaction began based on the methodology described by Bonacorso et al. in 2016. The reaction employs 1 mmol of the ligand precursor, 15 ml of anhydrous CHCl 3 , 4 ml of BF 3 .OEt 2 (~16 mmol), and 4 ml of Et 3 N (~28 mmol) at room temperature, but this attempt did not lead to product formation after 24-48 h (Table 1 -Entry 1). The reactions were monitored by TLC. However, under the same conditions at the reflux temperature of CHCl 3 , product 6d was formed in 68% yield after 18 h of reaction (Table 1 -Entry 2). Based on the work described by Yoshii et al. in 2013(Yoshii et al., 2013, the reaction was refluxed in anhydrous CH 2 Cl 2 and the desired product was not obtained after 24 h of reaction (Table 1 -Entry 3). In this condition, a higher reflux temperature solvent was evaluated, which was ClCH 2 CH 2 Cl (DCE), although the result was only 46% yield (Table 1 -Entry 4).

Rosa et al. Trichloromethylated Difluoro-oxazaborinines: Synthesis and Microbiological Activity
In order to investigate the synthetic reactivity of trifluoromethyl b-enaminoketone 9c against BF 2 .OEt 2 complexation, the optimized reaction condition carried out for the (Z)-1,1,1-trichloro-4-alkyl (aryl)amino-4-phenylbut-3-en-2-ones (4a-i and 5e) was attempted for 9c. However, the formation of trifluoromethylated complex 10c was not possible because the high electron-withdrawing effect of the trifluoromethyl group at position 6, which hinders a stable N-B-O coordination.
The structures of compounds 4a-i, 5e were deduced on the basis of the NMR data of other b-enaminones previously synthesized in literature. 50,51 The 1 H NMR chemical shifts of the b-enaminoketone hydrogens (NH) for 4a-i, 5e showed chemical shifts at an average of 11.87 ppm, which suggests to us that the compounds 4a-i, 5e are in the Z,Z-configuration in solution (CDCl 3 ), which is favored by an hydrogen interaction (N-H···OC). The vinyl hydrogen (H-3) was observed in the form of a singlet at an average of 6.06 ppm for all compounds 4, 5. The 13 C NMR spectra of all compounds 4a-i and 5e showed, on average, chemical shifts at 181.4 ppm C-2, 166.7 ppm for C-4, 90.8 ppm for C-3, and 97.1 ppm for CCl 3 .
A comparison between the 1 H-NMR data of compounds 4a-i, 5e and 6a-i, 7e showed clearly that complexes 6a-i and 7e do not show chemical shift signals (N-H) around at 12 ppm. Furthermore, an alteration in the chemical shifts of the H-3 signals from 6.06 ppm in average for the b-enaminones 4, 5 to 6.53 ppm in the boron complexes 6, 7 was also observed.
Upon comparing the 13 C NMR data of the b-enaminone precursors 4a-i, 5e and the boron complexes 6a-i, 7e, the major evidence for the formation of 6a-i, 7e was the alteration in the chemical shifts at average of the carbons C-4, C-6, CCl 3 , and C-5 for 173.1, 167.3, 96.9, and 92.0 ppm, respectively.
Moreover, in order to demonstrate boron complex formation, 19 F-and 11 B-NMR experiments were also performed. 19 F-NMR spectra of compounds 6a-i, 7e were performed using chloroformd and, the signals were observed in the form of a multiplet for the compounds in the range of -131.7 ppm to -138.7 ppm. For the 11 B-NMR spectra, the chemical shifts were observed in the form of a triplet at 1.26 ppm on average for each compound due to the coupling of both 19 F nuclei. However, for compounds 6a-i and 7e, the 19 F-NMR spectra showed a multiplet for each compound at -134.1ppm and -135.59 ppm, respectively.
The molecular structure of the compounds of series 4a-i, 5e, 6a-i, and 7e was determined and confirmed by single-crystal X-ray diffraction of the representative b-enaminone 4g and the boron complex 6e (Figure 1). For the compound 6e was observed that crystallization occurred at the monoclinic solvates-free form ( Figure 1). The boron-atom has a tetrahedral geometry where the B-F, B-N, and B-O distances are 1.372, 1.590, and 1.452 Å, respectively and the bond angles around the B-atom are the 108.7°(F11-B1-N9) to 111.0°(F11-B1-F12). As observed, the O-B bond length is significantly shorter than the N-B one. The complete data of the X-ray diffraction are found in the Supplementary Information File.

Absorption and Emission Properties of Boron Complexes
The UV-Vis analyses of the compounds use dichloromethane, dimethyl sulfoxide, and methanol as solvents are showed in the Figure 2. All absorption spectra are listed in the Supplementary  Information File (Figures S35-37). The UV-Vis data are presented in Table 2, where the solvents are presented according to polarity parameters (Moyon and Mitra, 2011).
In general, all BF 2 -derivatives have absorption bands at UV nm region, with transitions according to the structure of these heterocycles. From the relatively large absorption coefficient ( Table 2) it is plausible that this transition is S 1 (p) ! S 0 (p) in nature. Yoshii et al. also reported and attributed the same nature transition in their compounds, although the authors measured in THF solution (Yoshii et al., 2013). Additionally, slightly solvatochromic behavior indicates no dependence of solvent properties. Unfortunately, all the BF 2 -derivatives studied here did not have luminescent properties in organic solution (Yoshii et al., 2013), regardless of the solvent used in the experiments ( Supplementary Information File-Figure S38).

Rosa et al.
Trichloromethylated Difluoro-oxazaborinines: Synthesis and Microbiological Activity Frontiers in Pharmacology | www.frontiersin.org September 2020 | Volume 11 | Article 1328 All geometrical structures were optimized at the SCRF(PCM)-B3LYP/cc-pVTZ level of theory. The values calculate for the maximum absorption wavelengths were closed and in agreement with the experimental results in DCM, DMSO, and MeOH (solvent effect). As example, Table 3 highlights the electron distribution of the HOMO and LUMO for compound 6c. It is found that the HOMO and LUMO densities in 6c were delocalized over the whole molecule. The same behavior could be observed for all compounds studied ( Supplementary  Information File-Figures S58-S88).

CT-DNA Binding Experiments by Absorption UV-Vis Analysis
To investigate the mentioned interactions, UV-Vis and emission fluorescence analysis are preferred because small molecule-DNA interactions may be experimentally monitored by changes in the intensity and position of the spectroscopic peak responses or changes in the dynamic viscosity of DNA (Chen et al., 1999;Ni et al., 2006).
In the present study, the interaction of the boron complexes 6a-i and 7e against CT-DNA (Calf Thymus DNA) was studied by UV-Vis at 250-600 nm region using DMSO (2%)/ Tris-HCl pH 7.2 buffer mixture solution. The effect of different concentrations of CT-DNA on the UV-Vis spectra of derivative 6a is presented in Figure 3. Other BF 2 -derivatives absorption spectra are shown in Figures S39-S47 in the Supplementary Information File and the binding parameters are listed in Table 4.

CT-DNA by absorption EB-DNA by emission
Comp.  shift in some cases was also observed, indicating a weak or nonelectrostatic interaction observed between the cited compounds and CT-DNA ( Table 4). The transition change of the derivatives may be a result of the interaction of the BF 2 moiety with the nucleobase residues of the DNA (Figure 3), which are possible via hydrophobic forces, as previously in the literature (Bonacorso et al., 2018b).
Furthermore, the binding constant (K b ) data for the BF 2compounds were listed in Table 4. These K b values are associated to the BF 2 -DNA complex stability, while the free energy indicates the spontaneity/non-spontaneity of derivative-DNA binding process, indicating the spontaneity of the interaction between the organoboron complexes and DNA.

Competitive Experiments With DNA by Steady-State Emission Fluorescence
In the emission assays, EB-DNA (Ethidium Bromide-DNA) analysis were also conducted to determine the displacement of the intercalating agent ethidium bromide (EB) dye from CT-DNA. As example, the fluorescence emission of compound 6a was monitored by increasing the compound concentration at a fixed concentration of CT-DNA pre-treated with EB dye ( Figure  4). The EB-DNA adduct emission fluorescence spectra of other derivatives are listed in Figures S48-S56 in the Supplementary Information File.
The EB-DNA competition experiment shows an intense emission band located at l em = 649 nm by excitation at l exc = 510 nm. After adding derivative 6a to the EB-DNA solution, the EB-DNA emission appeared to decrease in emission intensity. This fact demonstrates a weak fluorescence quenching of the EB-DNA adduct, as estimated by the K SV values ( Table 4). This behavior can be assigned to the competition of the BF 2 complexes with EB over binding to the base pair of CT-DNA (intercalation mode). High values were obtained for the quenching constant rate (kq), which were higher than the diffusion rate constant (k diff ≈ 7.40 × 10 9 L mol -1 s -1 at 298K) (Montalti et al., 2006). These observed values indicate a possible static interaction mechanism between the BF 2 -complexes and EB-DNA (Montalti et al., 2006), which takes place via ground state association.

Antimicrobial Activity
Twenty of the newly synthesized compounds 4a-i, 5e, 6a-i, and 7e were evaluated for them in vitro antimicrobial activity against a panel of microorganisms including yeasts, filamentous fungi, bacteria, and alga by determining their MIC and minimal fungicidal/bactericidal/algacidal concentrations using broth microdilution methods according to CLSI standard protocols In order to classify the antimicrobial activity, antibacterial and two antifungal agents currently employed in therapeutics were compared (Supplementary Information File- Table S13). Therefore, the range of MICs until 10 mg/ml was considered significant activity for yeast-like fungi, with MIC-ranges between 20 and 40 mg/ml considered moderate activity and concentrations beyond not being considered. For filamentous fungi and P. zopfii, the MICs until 1.0 mg/ml were considered strong activity, with MIC ranges from >1 to 4 mg/ml considered moderate activity and above this range not being considered. For bacteria, all MICs under 4.0 mg/ml were considered as active and above they were not considered (Supplementary Information File- Table S13). The comparisons among MICs and MCC (minimal "cidal" concentrations) revealed that they were similar in 66% of cases (48/73) and showed that the MCC were higher by one or more concentration in 34% (25/73). The comparisons are important because they show the differences between compounds that are only inhibitory from those able to inhibit and kill pathogenic microorganisms.
Considering the antimicrobial activity of the synthesized compounds, the algaecidal action against P. zopfii stands out. Compounds 4a, 4b, 4d, 4e, 6c, 6e, 6f, and 6h were strongly effective in both inhibiting growth and causing algae death at a concentration below 1.0 μg/ml, while the other compounds shows moderate activity against this alga (except 4i and 5e). These results are encouraging, since P. zopfii is an environmental agent of bovine mastitis that causes many losses resulting from the compromised quality and production of milk and, in addition, it presents high antimicrobial resistance and the optimal treatment strategy for this infection has not yet been well established. In addition, P. zopfii can cause cutaneous and serious systemic infections in humans (Lass-Flörl and Mayr, 2007). The compounds that showed higher inhibitory and algaecidal activity were 4d, 4e, 6e, and 6f (MIC = 0.31 μg/ml). These results showed that changes in the basic structure as well as in the substituent slightly increased activity against P. zopfii.
In relation to the evaluated pathogenic yeasts (Candida spp. and Cryptococcus gattii) only compound 6i showed moderate activity for both, the other compounds of series 6 (6b-h) were moderately effective in inhibiting only the growth of C. gattii, as well as compounds 4a, 4b, 5e, and 7e. Compounds 4a-i and 5e have the same substituents as compounds 6a-i and 7e although the basic structure is comprised only by a b-enaminoketone for the former, whereas for compounds 6a-i and 7e a basic structure is constituted by a boron complex. Compounds 6a-j and 7e showed moderate activity against C. neoformans, and remained inactive against S. cerevisiae. Considering the difficulty in treating meningitis caused by C. gattii, which is normally resistant to one of the main antifungal drugs used in this pathology, fluconazole, increasing the mortality rate, potential treatment alternatives are extremely necessary.
Among filamentous fungi selected to study the activity of benaminoketone boron complex, the four most frequent agents of the life-threatening disease aspergillosis (A. fumigatus, A. flavus, A. niger, and A. terreus) were chosen. It is important to note that the antifungal therapy for these microorganisms is difficult and therapeutic failures are frequent (Leimann et al., 2004). Immunocompromised and neutropenic patients require fungicidal agents to treat their infections due to immunologic system failures (Dignani et al., 2003;Nucchi et al., 2004;Walsh et al., 2004).
Significant results were observed for compounds 4c, 4g, and 4h, which exhibited the best activities against Aspergillus. A. niger growth was completely inhibited at the concentration of 2.5 μg/ml. For boron complexes (6a-i and 7e), concentrations two or more times higher were required to inhibit A. niger growth. Compound 4g showed moderate activity against A. flavus (MIC = 20 μg/ml), which is the second most important agent of aspergillosis.
Another interesting point is the lack of activity of 4i against all the filamentous fungi studied when compared with 6i. The incorporation of BF 2 resulted in the acquisition of antifungal activity. This shows that substituents in the boron complexes can bring significant differences in antimicrobial activities.
The activity of the series of compounds against a panel of bacteria clinically important was poor, with the best activities observed with 6c (MIC = 5.0 μg/ml) and 6i (MIC = 5.0 μg/ml), which were both against K. pneumoniae, and an opportunistic gram-negative rod. This is curious because, in general, the grampositive cocci, which is represented here by S. aureus, is more sensible than gram negative rods (Kiska and Gilligan, 1999;Pfaller and Diekema, 2004).
The cytotoxicity of the b-enaminoketones and boron complexes were assessed using in vitro cell-based assay with 3T3 fibroblasts as the cell model, and MTT as endpoints to determine cell viability. As shown in Table S13 ( Supplementary  Information File), all compounds tested at concentrations between 1 and 100 μg/ml exhibited low or negligible cellular toxicity, as determined by MTT assay.
The results of the DNA-binding studies by spectroscopic analysis of the new BF 2 -b-enaminoketone compounds indicated that the greatest interaction with nucleic acids.
Multidrug-resistant microorganisms are increasingly common. These types of microorganisms can resist the effects of conventional antimicrobial drugs, increasing the mortality rates in both humans and animals affected by infections that are difficult to treat. In this context, new and more potent compounds are required. The series presented here showed to be able to inhibit the growth of several tested microorganisms, with outstanding algaecidal activity, however weak or no antibacterial activity, and moderate antifungal activity. We believe that these compounds could be chemically modified to improve their antimicrobial activity. Some compounds from the present series exhibited potent antimicrobial effects on various pathogenic microorganisms at concentrations below those that showed cytotoxic effects. Notably 4d, 4e, 6e, and 6f showed the best results and were very significant against P. zopfii, which is an agent that causes diseases in humans and animals.
Finally, the new molecules presented here open good prospects for the development of analogue structures with possible application in microbiology and studies involving interactions with biomolecules. The introduction of other known chromophore substituents in similar chelates to those reported here is under initial development and will be published in due course.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

AUTHOR CONTRIBUTIONS
WR, IR, MR, NZ, and HB: These researchers and students were responsible for the development of the part concerning the synthesis, purification, NMR, GC-MS and X-ray diffractometric data, and TD-DFT calculations for the new compounds. HC, LD, and PL: These researches were responsible for the development of the part related to the evaluation of the minimum inhibitory concentration (MIC) of the series of compounds 5-7. Twenty of the newly synthesized compounds 4a-i, 5e, 6a-i, and 7e were evaluated for their in vitro antimicrobial activity against a panel of microorganisms including yeasts, filamentous fungi, bacteria, and alga by determining their MIC and minimal fungicidal/bactericidal/algaecidal concentrations using broth microdilution methods according to CLSI standard protocols. TA and BI: This researcher and the student were responsible for the development of the part concerning to the study of the optical properties of the new compounds (UV-vis, fluorescence, quantum yield calculations, Stokes shift) and the interaction of the boron complexes 6a-i and 7e against CT-DNA, which was studied by UV-Vis absorption spectroscopy.