Introduction

Drug resistance is a natural response that is caused by antibiotic use. Hence, new drugs are needed to struggle with this resistance. Boron compounds are underexploited in medicinal chemistry and have tremendous potential in drug discovery. In the literature, boron-containing compounds have been identified as the agents that have potential biological activities (Ciaravino et al., 2013). Among these, it is worth mentioning the activities of the following compounds: heterocyclic aminoboron compounds (antituberculosis agents) (Campbell-Verduyn et al., 2014), boron-containing GSK2251052 (antimicrobial agent) (Ross et al., 2013), oxaborole compounds (antibacterial prototypes) (Li et al., 2013), α-amino cyclic boronates (inhibitors of HCV NS3 protease) (Li et al., 2010), benzoxaborole compounds (anti-inflammatory agents) (Akama et al., 2009), and boronic acid esters (antibacterial agent with anti-inflammatory activity) (Baker et al., 2006).

Some heterocyclic boron compounds that contain B–N bonds also show biological activity (Das et al., 2012; Jabbour et al., 2004). Oxadiazaboroles possess a B–N bond and are readily obtained from an amidoxime and a boronic acid. On the basis of the biological activity displayed by other heterocyclic systems that contain B–N bonds, oxadiazaboroles should be interesting candidates for biological activity. Considering the structural characteristics of the 1,2,4,5-oxadiazaboroles, and the existence of the oxygen-, nitrogen-, and boron-containing five-membered nonaromatic heterocycles system, it is reasonable to expect physiological activities from these compounds. Therefore, the study of substituent effects on the antimicrobial activity of the oxadiazaboroles was thought to give a better understanding of their structure–antimicrobial activity relationships.

Some oxadiazaboroles were synthesized from amidoximes and phenylboronic acid (Yale, 1971; Dürüst et al., 2007). However, to our knowledge, no antimicrobial activity data of 3,4,5-trisubstituted 4,5-dihydro-1,2,4,5-oxadiazaboroles (4) and 3,5-disubstituted 4,5-dihydro-1,2,4,5-oxadiazaboroles (7) was reported. We synthesized some series of new oxadiazaborole derivatives (Schemes 1 and 2) and determined their antimicrobial activities against some bacteria and fungi. We also applied 1D-quantitative structure–activity relationship (QSAR) and 2D-QSAR analysis to observe the relations of the molecular descriptors with the activities.

Scheme 1
figure 1

Synthesis of compounds (4a–r)

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Scheme 2
figure 2

Synthesis of compounds (7a–s)

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Synthesis

Synthesis of compounds (2–4, Scheme 1) starts with p-chlorobenzaldehyde, which was reacted with hydroxylamine hydrochloride to give p-chlorobenzaldehyde oxime (2a–i). Then, chlorination was followed: p-chlorobenzaldehyde oxime was reacted with chlorine gas in anhydrous chloroform at 0 °C, until a certain weight increase was obtained, to have 4-chloro-N-hydroxybenzimidoyl chloride. The solution was kept in the fridge overnight. Then the solvent was evaporated. The residue, 4-chloro-N-hydroxybenzimidoyl chloride, was reacted with substituted anilines in benzene at room temperature to give N-substituted-p-chlorobenzamidoximes (3a–i). N-(m-tolyl)-substituted benzamidoximes (3j–r) were synthesized by the literature method (Sümengen and Pelter, 1983). Then the compounds (3a–r) were reacted with phenylboronic acid in toluene to yield the corresponding 3,4,5-trisubstituted 4,5-dihydro-1,2,4,5-oxadiazaboroles (4a–r).

Synthesis of compounds 6 and 7, outlined in Scheme 2, started with benzonitrile (5), which was reacted with hydroxylamine hydrochloride to afford benzamidoxime (6). This was followed by cyclodehydration reaction: benzamidoxime (6) was reacted with boronic acid derivatives in benzene to yield the corresponding 3,5-disubstituted 4,5-dihydro-1,2,4,5-oxadiazaboroles (7a–s).

All the synthesized compounds were analyzed by their infrared radiation (IR), 1H and 13C nuclear magnetic resonance spectroscopy (NMR) spectra and the purity of compounds 4a–r and 7a–s was checked by elemental analysis.

Benzamidoxime was synthesized according to the literature methods A (Krüger, 1885) and B (Gosenca et al., 2013). Method B gives less amide impurity and high yield.

Biological activity

The synthesized compounds (4 and 7) have been evaluated for their in vitro antimicrobial activity against a panel of microorganisms, including three gram-positive bacteria (S. aureus, E. faecalis, and S. mutans), two gram-negative bacteria (P. aeruginosa and E. coli) and one fungi (C. albicans) by their minimal inhibitory concentrations (MIC) via broth microdilution susceptibility tests (CLSI, 2002, 2005, 2006). The biological activities of all the compounds are given in Table 1 and shown graphically in Figs. 1 and 2. Compounds 7k, 7l, and 7o have been found to be the most active derivatives against fungi (C. albicans) at MIC value of 25 µg/mL among the tested compounds. All the compounds exhibited antimicrobial activity with MIC values between 25−800 µg/mL against C. albicans. 4i is the most active compound against S. aureus (MIC 25 µg/mL). Additionally, compounds 4f, 4g, 4h, 4i, 4o, and 7n showed activity (MIC 25 µg/mL) against S. mutans. The results show that 3,5-disubstituted 4,5-dihydro-1,2,4,5-oxadiazaboroles (7) are the most active derivatives against P. aeruginosa among the tested compounds (MIC 12.5−25 µg/mL). All compounds exhibited antibacterial activity with MIC values between 25−800 µg/mL against S. aureus, with MIC values between 50−800 µg/mL against E. coli; with MIC values between 12.5−200 µg/mL against P. aeruginosa; with MIC values between 50−800 µg/mL against E. faecalis; and with MIC values between 25−100 µg/mL against S. mutans.

Table 1 Antimicrobial activities of compounds (4a–r) and (7a–s)
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Fig. 1
figure 1

Comparable chart for MIC values (µg/mL) of all compounds against bacterias

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Fig. 2
figure 2

Comparable chart for MIC values (µg/mL) of all compounds against fungi

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QSAR analysis

We have carried out linear regression studies of molecular descriptors against the antimicrobial activity of compounds 4a–r and 7a–s. The results of the biological activities, reported as MIC values (Table 1), were converted to pMIC (− logMIC) on a molar basis and used as dependent variables to obtain the linear relationship. The pMIC values of compounds were first correlated with Hammett polar substituent constant (σ) (Hansch et al., 1991) or lipophilic constant (π) (Hansch et al., 1973), and statistically significant correlations were obtained (Table 2; Eqs. 2, 4, 5, 8, 14, 16, and 18). However, by application of the 2D statistical method, the correlation of pMIC values against σ and π independent variables gave two statistically significant 2D-QSAR models (Table 2; Eqs. 1 and 17).

Table 2 Significant 2D−QSAR models obtained for antimicrobial activity of 3,4,5-trisubstituted 4,5-dihydro-1,2,4,5-oxadiazaboroles (4a–r) and 3,5-disubstituted 4,5-dihydro-1,2,4,5-oxadiazaboroles (7a–s)
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Computer aid ranges from molecular design to architectural design (Agirbas, 2015) and also helps to check the experimental data. In order to add theoretical descriptors to the structure–antimicrobial activity relationship study, the geometrical optimization of all the compounds (4a–r, 7a–s) was done by the ab initio (RHF/3-21G) method incorporated in the Hyperchem package (HyperChem, 2002). Theoretical descriptors, namely, surface area approx (SAA), molecular volume (MV), molar refractivity (MR), polarizability (polar), magnitude of dipolar moment (µ), and the calculated log of octanol–water partition coefficient (clogP) of the compounds were also computed by the Hyperchem software (RHF/3-21G) method (Table 3). E HOMO (energies of the highest occupied molecular orbital) and E LUMO (lowest unoccupied molecular orbital) were calculated by Gaussian 03W software (Frisch et al., 2004), using the DFT (B3LYP) method with 3-21G basis set (Table 3). Several descriptors (Ferreira et al., 2009) were also calculated as shown below:

$$O\left( {{\rm ovality}} \right){=}{{{\rm SA}} \over {4\pi \left( {{{3{\rm MV}} \over {4\pi }}} \right)_{{2/3}} }}$$
$$\chi \left( {{\rm electronegativity}} \right){=}{{E_{{{\rm HOMO}}} {-}E_{{{\rm LUMO}}} } \over {\rm 2}}$$
$$\eta \left( {{\rm hardness}} \right){=}{{E_{{{\rm LUMO}}} {-}E_{{{\rm HOMO}}} } \over {\rm 2}}$$
$$\omega \left( {{\rm electrophilicity}} \right){=}{{\chi ^{{\bf 2}} } \over {{\rm 2}\eta }}$$
$$\eta ^{{{-}1}} \left( {{\rm softness}} \right){=}{{\rm 1} \over \eta }$$
Table 3 Theoretical descriptors of 3,4,5-trisubstituted 4,5-dihydro-1,2,4,5-oxadiazaboroles (4a–r) and 3,5-disubstituted 4,5-dihydro-1,2,4,5-oxadiazaboroles (7a–s) used for the regression analyses
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When these theoretical descriptors were used as independent variables, seven significant correlation with pMIC values was obtained (Table 2; Eqs. 3, 6, 7, 9, 10, 13, and 15). However, with the application of the 2D statistical method, the correlation of pMIC values against the theoretical descriptors (E LUMO and µ) and σ gave two statistically significant 2D-QSAR models (Table 2; Eqs. 11 and 12). The overall quality of 2D-QSAR models was shown by r and r 2 (correlation coefficients), s (standard deviations) of the regression equations, F value (F-statistical analysis; Fischer test), P (probability value), and n (number of data points). The predictability of each model was assessed by using the cross-validated correlation coefficient (r 2 adj). A value of r 2 adj >0.25 was considered for the structure–reactivity models.

To evaluate the predictive power of the model equations, MIC values were split into the training and test sets. The regression equations of the training sets gave fair internal cross-validation (r 2 adj) values and good coefficient of determination (r 2) values (Table 4). Good predicted values of the test sets were also obtained. The plot of the observed pMIC s.a values against predicted ones, using regression equation of the training set (Table 4, Eq. 1), is shown in Fig. 3. This QSAR model has a squared correlation coefficient (r 2) of ~ 0.68. The predicted pMIC s.a. values (determined from Eq. 1) with residuals are given in Table 5.

Table 4 The results of the application of training and test sets to 2D−QSAR models (Eqs. 1–18 in Table 2)
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Fig. 3
figure 3

The plot of observed versus predicted pMIC s.a . using the QSAR model (Table 4; Eq. 1)

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Table 5 Comparison of observed and predicted antibacterial activity obtained by equation (1)
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The correlation matrix for the descriptors is given in Table 6 and, as seen, cross-relations between the descriptors are not observed. Therefore, the values of the descriptors allow its safe use in the multilinear regression relationship (Myers, 1987; Draper and Smith, 1981). The statistical calculations were performed by SigmaPlot program package.

Table 6 Correlation coefficients (r 2) of squared correlation matrix of the theoretical descriptors used in the QSAR study
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Conclusion

A series of eighteen 3,5-disubstituted 4,5-dihydro-1,2,4,5-oxadiazaboroles (7) were synthesized from the reactions of benzamidoxime (6) with boronic acid derivatives. Seventeen 3,4-disubstituted 4,5-dihydro-1,2,4,5-oxadiazaboroles (4) were also synthesized from the reactions of substituted benzamidoximes (3) with phenylboronic acid. The antimicrobial activity studies of these oxadiazaboroles are not present in the literature. Antibacterial and antifungal activities of oxadiazaborole derivatives have been screened against three gram-positive bacteria, two gram-negative bacteria, and one fungi. Interestingly, all the compounds exhibited better results against P. aeruginosa (MIC of 12.5−200 µg/mL) than S. aureus, E. faecalis, E. coli, S. mutans, and C. albicans. These bacteria show the most dramatic resistance problems related to nosocomial infections and multiresistant strains (Kiska and Gilligan, 1999). Additionally, quantitative structure–activity relationship studies were carried out and this allowed us to draw the following conclusions about the antimicrobial activities of these synthesized oxadiazaboroles: (i) all oxadiazaboroles showed fair 2D correlations with some theoretical descriptors (clogP, SAA, E LUMO, and E HOMO) against S. aureus, P. aeruginosa, E. coli, and S. mutans; (ii) 3,5-disubstituted 4,5-dihydro-1,2,4,5-oxadiazaboroles (7a–r) showed fair 2D correlations with the electronic (σ) and lipophilic (π) descriptors against S. aureus and C. albicans; (iii) all oxadiazaboroles gave fair 1D correlations with the theoretical descriptor (SAA) against P. aeruginosa and E. coli; (iv) compounds (4a–i, 4j–r, and 7a–r) showed fair 1D correlations with the electronic (σ) or lipophilic (π) descriptors against S. aureus, E. faecalis, P. aeruginosa, S. mutans, and C. albicans.

The QSAR study has given key information regarding the structural properties of 3,4,5-trisubstituted 4,5-dihydro-1,2,4,5-oxadiazaboroles and 3,5-disubstituted 4,5-dihydro-1,2,4,5-oxadiazaboroles, and may help to design more potent antimicrobial compounds in the future studies.

Experimental

Melting points were determined on the Electrothermal 9200 apparatus and are uncorrected. The FT IR spectra were recorded on the Bruker Alpha-P spectrometer in the region of 4000–400 cm−1. 1H and 13C NMR spectra were recorded on the Bruker DPX-400 (400 MHz) High Performance Digital FT-NMR Spectrometer using CDCl3 and DMSO-d 6 with Me4Si as an internal standard. Silica gel (Fluka or Merck) was used for column chromatography. Bands for the oxime NOH and C=N groups were followed in IR spectrum.

Synthesis of p-chlorobenzaldehyde oxime (2a–i)

A solution of hydroxylamine hydrochloride (53 mmol, 3.7 g) in water (10 mL) and a solution of anhydrous sodium carbonate (27 mmol, 2.8 g) in water (15 mL) were mixed and stirred. p-Chlorobenzaldehyde (53 mmol, 7.5 g) in chloroform (20 mL) was added to this mixture. The reaction was stirred at room temperature for 24 h. After the reaction was complete, the chloroform phase was taken, the water phase was extracted with chloroform (15 mL) three times, and then the chloroform phase was collected and dried with anhydrous CaCl2 overnight. The solvent was evaporated under vacuum. The precipitate was crystallized from ethyl acetate–petroleum ether (1:4) mixture to give p-chlorobenzaldehyde oxime (2a–i) (5.76 g, 70 %). Mp: 107.2–109.3 °C, Lit. (Liu et al., 1980): 105.5–108 °C; IR (ATR), υ (cm−1): 3255 (NOH), 1594 (C=N).

Synthesis of N-phenyl-p-chlorobenzamidoxime (3d) (general procedure for 3a–i)

p-Chlorobenzaldehyde oxime (2a–i) (39 mmol, 6.08 g) was dissolved in anhydrous chloroform (100 mL). After cooling the solution in an ice bath, chlorine gas was passed through the solution until the determination of the 3.20-g weight increase. The solution was refrigerated for one night. Then the solution was evaporated under reduced pressure at 40 °C. The formed p-chloro-N-hydroxybenzimidoyl chloride was dissolved in dry benzene (30 mL), and to the solution aniline (78 mmol, 7.28 g) was added dropwise in dry benzene (20 mL) with constant stirring at room temperature for 24 h. After this the mixture was refrigerated for 1 h. The salt precipitated was removed by filtration. The residual solid was subjected to flash column chromatography (eluant:ethyl acetate:petroleum ether). The crude product was crystallized from ethyl acetate:petroleum ether (1:6) mixture to give N-phenyl-p-chlorobenzamidoxime (3d) (5.24 g, 54 %). Mp: 127–130 °C. IR (ATR), υ (cm−1): 3390 (N–H), 3141 (NOH), 1611 (C=N); 1H NMR (DMSO-d 6 ), δ (ppm): 10.67 (s, 1H, NOH); 8.36 (s, 1H, N–H); 7.35 (d, J = 15.5 Hz, aromatic, 4H); 7.05 (d, J = 17.2 Hz, aromatic, 2H); 6.78 (t, J = 7.0 Hz, aromatic, 1H); 6.62 (d, J = 7.9 Hz, aromatic, 2H).

Spectroscopic and analytical data of compounds (3)

N-(p-dimethylaminophenyl)-p-chlorobenzamidoxime ( 3a) yield: 55 %; mp: 205–208 °C; IR (ATR), υ (cm−1): 3369 (N–H), 3198 (NOH), 1636 (C=N); 1H NMR (DMSO-d 6 ), δ (ppm): 10.32 (s, 1H, NOH); 7.89 (s, 1H, N–H); 7.30 (d, J = 19.6 Hz, aromatic, 4H); 6.56–6.46 (m, aromatic, 4H); 2.74 (s, 6H, N(CH3)2).

N-(p-methoxyphenyl)-p-chlorobenzamidoxime ( 3b) yield: 47 %; mp: 185–186 °C; IR (ATR), υ (cm−1): 3408 (N–H), 3137 (NOH), 1632 (C=N); 1H NMR (DMSO-d 6 ), δ (ppm): 10.45 (s, 1H, NOH); 8.11 (s, 1H, N–H); 7.32 (d, J = 19.6 Hz, aromatic, 4H); 6.68–6.58 (dd, J = 20.5 Hz, 12.3 Hz, aromatic, 4H); 3.61 (s, 3H, OCH3).

N-(m-tolyl)-p-chlorobenzamidoxime ( 3c): 38 %; mp: 110–113 °C; IR (ATR), υ (cm−1): 3383 (N–H), 3194 (NOH), 1632 (C=N); 1H NMR (CDCl3), δ (ppm): 7.38–6.37 (m, aromatic, 8H); 2.20 (s, 3H, CH3).

N-(m-methoxyphenyl)-p-chlorobenzamidoxime ( 3e) yield: 42 %; mp: 66–69 °C; IR (ATR), υ (cm−1): 3301 (N–H), 3169 (NOH), 1631 (C=N); 1H NMR (DMSO-d 6 ), δ (ppm): 10.68 (s, 1H, NOH); 8.34 (s, 1H, N–H); 7.40–7.33 (m, aromatic, 4H); 6.96–6.91 (m, aromatic, 1H); 6.35 (d, J = 8.2 Hz, aromatic, 1H); 6.23 (s, aromatic, 1H); 6.15 (d, J = 7.6 Hz, aromatic, 1H); 3.54 (s, 3H, OCH3).

N-(p-bromophenyl)-p-chlorobenzamidoxime ( 3f) yield: 37 %; mp: 186–187 °C; IR (ATR), υ (cm−1): 3395 (N–H), 3085 (NOH), 1632 (C=N); 1H NMR (DMSO-d 6 ), δ (ppm): 10.78 (s, 1H, NOH); 8.55 (s, 1H, N–H); 7.40–7.34 (m, aromatic, 4H); 7.22 (d, J = 6.1 Hz, aromatic, 2H); 6.56 (d, J = 6.7 Hz, aromatic, 2H).

N-(p-chlorophenyl)-p-chlorobenzamidoxime ( 3g) yield: 41 %; mp: 170–172 °C; IR (ATR), υ (cm−1): 3394 (N–H), 3083 (NOH), 1629 (C=N); 1H NMR (DMSO-d 6 ), δ (ppm): 10.76 (s, 1H, NOH); 8.55 (s, 1H, N–H); 7.40–7.36 (m, aromatic, 4H); 7.10 (d, J = 8.7 Hz, aromatic, 2H); 6.61 (d, J = 8.7 Hz, aromatic, 2H).

N-(m-chlorophenyl)-p-chlorobenzamidoxime ( 3h) yield: 44 %; mp: 135–137 °C; IR (ATR), υ (cm−1): 3379 (N–H), 3193 (NOH), 1636 (C=N); 1H NMR (CDCl3), δ (ppm): 10.85 (s, 1H, NOH); 8.63 (s, 1H, N–H); 7.51–7.39 (m, aromatic, 4H); 7.07–7.01 (m, aromatic, 1H); 6.81–6.74 (m, aromatic, 2H); 6.44 (d, J = 7.9 Hz, aromatic, 1H).

N-(m-trifluoromethylphenyl)-p-chlorobenzamidoxime ( 3i) yield: 36 %; mp: 160 °C; IR (ATR), υ (cm−1): 3387 (N–H), 3087 (NOH), 1633 (C=N); 1H NMR (DMSO-d 6 ), δ (ppm): 10.91 (s, 1H, NOH); 8.80 (s, 1H, N–H); 7.40–7.26 (m, aromatic, 5H); 7.08–6.99 (m, aromatic, 2H); 6.79–6.77 (m, aromatic, 1H).

Synthesis of 3-(p-chlorophenyl)-4,5-diphenyl-4,5-dihydro-1,2,4,5-oxadiazaborole (4d) (general procedure for 4a–i)

N-phenyl-p-chlorobenzamidoxime (3d) (1.21 mmol, 0.300 g) and phenylboronic acid (1.36 mmol, 0.165 g) were dissolved in toluene (20 mL) and the solution was refluxed for 30 h in the presence of molecular sieves (4 Ǻ). After extracting with acetone and filtering, the solvent was evaporated under reduced pressure. The residual was crystallized from ethyl acetate–petroleum ether (1:4) mixture to give 3-(p-chlorophenyl)-4,5-diphenyl-4,5-dihydro-1,2,4,5-oxadiazaborole (4d) (0.240 g, 59 %). Mp: 201–202.8 °C. IR (ATR), υ (cm−1): 1599 (C=N), 1371 (B–N), 1125 (B–O); 1H NMR (CDCl3), δ (ppm): 7.55 (d, J = 7.9 Hz, aromatic, 2H); 7.45–7.38 (m, aromatic, 4H); 7.33–7.26 (m, aromatic, 6H); 7.17–7.13 (m, aromatic, 2H); 13C NMR (CDCl3), δ (ppm): 160.0 (C=N); 137.2; 136.7; 134.3; 131.3; 130.5; 129.9; 128.9; 128.3; 128.2; 128.0; 124.7. Anal. calcd. for C19H14BClN2O: C, 68.61; H, 4.24; N, 8.42; found: C, 68.39; H, 4.32; N, 8.35.

Synthesis of 3,5-diphenyl-4-(m-tolyl)-4,5-dihydro-1,2,4,5-oxadiazaborole (4j) (general procedure for 4j–r)

N-(m-tolyl)-benzamidoxime (3j) (1.33 mmol, 0.300 g) and phenylboronic acid (1.49 mmol, 0.181 g) were dissolved in toluene (20 mL) and the solution was refluxed for 30 h in the presence of molecular sieves (4 Ǻ). After extracting with acetone and filtering, the solvent was evaporated under reduced pressure. The residual was crystallized from petroleum ether to give 3,5-diphenyl-4-(m-tolyl)-4,5-dihydro-1,2,4,5-oxadiazaborole (4j) (0.30 g, 72 %). Mp: 191–194 °C. IR (ATR), υ (cm−1): 1601 (C=N), 1370 (B–N), 1125 (B–O); 1H NMR (CDCl3), δ (ppm): 7.57 (d, J = 7.9 Hz, aromatic, 2H); 7.44–7.22 (m, aromatic, 9H); 7.17 (d, J = 7.6 Hz, aromatic, 1H); 6.96 (d, J = 7.0 Hz, aromatic, 2H); 2.30 (s, 3H, CH3); 13C NMR (CDCl3), δ (ppm): 160.9 (C=N); 139.8; 137.4; 134.4; 131.1; 130.3; 129.5; 129.2; 128.9; 128.5; 128.5; 128.2; 126.3; 125.1; 21.5 (CH3). Anal. calcd. for C20H17BN2O: C, 76.95; H, 5.49; N, 8.97; found: C, 76.53; H, 5.37; N, 9.16.

Spectroscopic and analytical data of compounds (4)

3-(p-chlorophenyl)-4-(p-N,N-dimethylaminophenyl)-5-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 4a) yield: 75 %; mp: 208 –210 °C; IR (ATR), υ (cm−1): 1601 (C=N), 1371 (B–N), 1126 (B–O); 1H NMR (CDCl3), δ (ppm): 7.59 (d, J = 7.3 Hz, aromatic, 2H); 7.43–7.26 (m, aromatic, 7H); 6.99 (d, J = 8.5 Hz, aromatic, 2H); 6.64 (d, J = 8.5 Hz, aromatic, 2H); 2.99 (s, 6H, N(CH3)2); 13C NMR (CDCl3), δ (ppm): 160.5 (C=N); 149.9; 136.4; 134.4; 131.1; 130.5; 128.8; 128.5; 128.1; 125.6; 125.1; 112.8; 40.6 (CH3). Anal. calcd. for C21H19BClN3O: C, 67.14; H, 5.10; N, 11.19; found: C, 66.48; H, 5.28; N, 11.04.

3-(p-chlorophenyl)-4-(p-methoxyphenyl)-5-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 4b) yield: 61 %; mp: 197–199 °C; IR (ATR), υ (cm−1): 1599 (C=N), 1369 (B–N), 1124 (B–O); 1H NMR (CDCl3), δ (ppm): 7.55 (d, J = 7.0 Hz, aromatic, 2H); 7.44–7.25 (m, aromatic, 7H); 7.05 (d, J = 8.7 Hz, aromatic, 2H); 6.88 (d, J = 8.7 Hz, aromatic, 2H); 3.84 (s, 3H, OCH3); 13C NMR (CDCl3), δ (ppm): 160.2 (C=N); 159.2; 136.6; 134.3; 131.2; 130.5; 129.9; 129.0; 128.9; 128.2; 124.8; 115.0; 55.7 (OCH3). HRMS: m/z (M + H)+ calcd. for C20H17BClN2O2: 363.1072; found: 363.1081 (M + H)+. Anal. calcd. for C20H16BClN2O2: C, 66.24; H, 4.45; N, 7.73; found: C, 65.56; H, 4.76; N, 7.68.

3-(p-chlorophenyl)-4-(m-tolyl)-5-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 4c) yield: 57 %; mp: 182–184 °C; IR (ATR), υ (cm−1): 1599 (C=N), 1369 (B–N), 1124 (B–O); 1H NMR (CDCl3), δ (ppm): 7.55 (d, J = 9.3 Hz, aromatic, 2H); 7.42–7.39 (m, aromatic, 1H); 7.32–7.18 (m, aromatic, 8H); 6.96 (d, J = 6.1 Hz, aromatic, 2H); 2.32 (s, 3H, CH3); 13C NMR (CDCl3), δ (ppm): 160.0 (C=N); 140.0; 137.1; 136.6; 134.4; 131.2; 130.5; 129.7; 129.1; 128.9; 128.5; 128.2; 125.0; 124.8; 21.5 (CH3). Anal. calcd. for C20H16BClN2O: C, 69.30; H, 4.65; N, 8.08; found: C, 68.78; H, 4.58; N, 7.92.

3-(p-chlorophenyl)-4-(m-methoxyphenyl)-5-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 4e) yield: 60 %; mp: 170–172 °C; IR (ATR), υ (cm−1): 1601 (C=N), 1367 (B–N), 1123 (B–O); 1H NMR (CDCl3), δ (ppm): 7.58 (d, J = 7.9 Hz, aromatic, 2H); 7.45–7.25 (m, aromatic, 8H); 6.92 (d, J = 10.8 Hz, aromatic, 1H); 6.75 (d, J = 9.6 Hz, aromatic, 1H); 6.67 (t, J = 2.0 Hz, aromatic, 1H); 3.73 (s, 3H, OCH3); 13C NMR (CDCl3), δ (ppm): 159.9 (C=N); 160.6; 138.3; 136.7; 134.3; 131.3; 130.6; 130.4; 128.9; 128.2; 124.7; 120.2; 113.8; 113.8; 55.6 (OCH3). Anal. calcd. for C20H16BClN2O2: C, 66.24; H, 4.45; N, 7.73; found: C, 65.67; H, 4.53; N, 7.63.

3-(p-chlorophenyl)-4-(p-bromophenyl)-5-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 4f) yield: 59 %; mp: 199–201 °C; IR (ATR), υ (cm−1): 1598 (C=N), 1367 (B–N), 1123 (B–O); 1H NMR (CDCl3), δ (ppm): 7.56–7.42 (m, aromatic, 4H); 7.34–7.23 (m, aromatic, 5H); 7.04–7.0 (m, aromatic, 4H); 13C NMR (CDCl3), δ (ppm): 159.7 (C=N); 137.0; 136.3; 134.3; 133.1; 131.5; 130.5; 129.5; 129.1; 128.3; 124.3; 122.1.11B NMR (CDCl3), δ (ppm): 32.3. Anal. calcd. for C19H13BBrClN2O: C, 55.46; H, 3.18; N, 6.81; found: C, 55.44; H, 3.34; N, 6.96.

3-(p-chlorophenyl)-4-(p-chlorophenyl)-5-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 4g) yield: 59 %; mp: 187–190 °C; IR (ATR), υ (cm−1): 1597 (C=N), 1368 (B–N), 1122 (B–O); 1H NMR (CDCl3), δ (ppm): 7.56–7.52 (m, aromatic, 4H); 7.46–7.24 (m, aromatic, 5H); 7.10–7.07 (m, aromatic, 4H); 13C NMR (CDCl3), δ (ppm): 159.8 (C=N); 136.9; 135.8; 134.3; 134.1; 131.4; 130.5; 130.2; 129.2; 129.1; 128.3; 124.3. Anal. calcd. for C19H13BCl2N2O: C, 62.17; H, 3.57; N, 7.63; found: C, 62.26; H, 3.68; N, 7.66.

3-(p-chlorophenyl)-4-(m-chlorophenyl)-5-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 4h) yield: 60 %; mp: 177.7–179 °C; IR (ATR), υ (cm−1): 1599 (C=N), 1367 (B–N), 1124 (B–O); 1H NMR (CDCl3), δ (ppm): 7.54 (d, J = 8.2 Hz, aromatic, 2H); 7.47–7.25 (m, aromatic, 10H); 7.18 (t, J = 1.7 Hz, aromatic, 1H); 7.04 (m, aromatic, 1H); 13C NMR (CDCl3), δ (ppm): 159.7 (C=N); 138.5; 137.0; 135.5; 134.3; 131.5; 130.9; 130.5; 129.1; 128.7; 128.4; 128.1; 126.3; 124.2. Anal. calcd. for C19H13BCl2N2O: C, 62.17; H, 3.57; N, 7.63; found: C, 62.24; H, 3.52; N, 7.63.

3-(p-chlorophenyl)-4-(m-trifluoromethylphenyl)-5-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 4i) yield: 46 %; mp: 165–167 °C; IR (ATR), υ (cm−1): 1598 (C=N), 1368 (B–N), 1119 (B–O); 1H NMR (CDCl3), δ (ppm): 7.66 (d, J = 7.9 Hz, aromatic, 1H); 7.55–7.42 (m, aromatic, 5H); 7.34–7.21 (m, aromatic, 7H); 13C NMR (CDCl3), δ (ppm): 159.6 (C=N); 137.9; 137.1; 134.2; 132.6; 132.2; 131.6; 131.3; 130.6; 130.5; 129.2; 128.4; 125.2; 125.1; 125.0; 124.8; 124.0. Anal. calcd. for C20H13BClF3N2O: C, 59.97; H, 3.27; N, 6.99; found: C, 59.81; H, 3.23; N, 6.96.

3-(p-fluorophenyl)-4-(m-tolyl)-5-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 4k) yield: 62 %; mp: 188–190 °C; IR (ATR), υ (cm−1): 1598 (C=N), 1370 (B–N), 1123 (B–O); 1H NMR (CDCl3), δ (ppm): 7.55 (d, J = 8.2 Hz, aromatic, 2H); 7.44–7.39 (t, J = 7.3 Hz, aromatic, 1H); 7.36–7.24 (m, aromatic, 5H); 7.18–6.94 (m, aromatic, 5H); 2.31 (s, 3H, CH3); 13C NMR (CDCl3), δ (ppm): 160.1 (C=N); 165.6; 162.2; 140.0; 137.2; 134.3; 131.4; 131.2; 131.2; 129.6; 129.0; 128.5; 128.2; 125.1; 122.4; 122.4; 115.9; 115.6; 21.5 (s, 3H, CH3). Anal. calcd. for C20H16BFN2O: C, 72.76; H, 4.88; N, 8.48; found: C, 72.88; H, 5.02; N, 8.48.

3-(p-bromophenyl)-4-(m-tolyl)-5-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 4l) yield: 60 %; mp: 192–193 °C; IR (ATR), υ (cm−1): 1598 (C=N), 1367 (B–N), 1122 (B–O); 1H NMR (CDCl3), δ (ppm): 7.55 (d, J = 7.0 Hz, aromatic, 2H); 7.43–7.41 (m, aromatic, 3H); 7.32–7.18 (m, aromatic, 6H); 6.95 (d, J = 6.7 Hz, aromatic, 2H); 2.32 (s, 3H, CH3); 13C NMR (CDCl3), δ (ppm): 160.1 (C=N); 140.1; 137.1; 134.4; 131.8; 131.2; 130.7; 129.7; 129.1; 128.5; 128.2; 125.2; 125.0; 21.5 (s, 3H, CH3). Anal. calcd. for C20H16BBrN2O: C, 61.42; H, 4.12; N, 7.16; found: C, 61.28; H, 4.22; N, 7.12.

3-(m-chlorophenyl)-4-(m-tolyl)-5-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 4m) yield: 75 %; mp: 130–133 °C; IR (ATR), υ (cm−1): 1600 (C=N), 1370 (B–N), 1134 (B–O); 1H NMR (CDCl3), δ (ppm): 7.57 (d, J = 9.3 Hz, aromatic, 2H); 7.44–7.13 (m, aromatic, 9H); 6.96 (d, J = 5.5 Hz, aromatic, 2H); 2.32 (s, 3H, CH3); 13C NMR (CDCl3), δ (ppm): 159.8 (C=N); 140.0; 137.0; 134.5; 134.4; 131.3; 130.5; 129.8; 129.7; 129.4; 129.2; 128.4; 128.2; 128.0; 127.3; 125.0; 21.5 (s, 3H, CH3). Anal. calcd. for C20H16BClN2O: C, 69.30; H, 4.65; N, 8.08; found: C, 69.31; H, 4.46; N, 8.07.

3-(m-bromophenyl)-4-(m-tolyl)-5-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 4n) yield: 56 %; mp: 139–141 °C; IR (ATR), υ (cm−1): 1600 (C=N), 1371 (B–N), 1131 (B–O); 1H NMR (CDCl3), δ (ppm): 7.61–7.55 (m, aromatic, 3H); 7.49–7.41 (dd, J = 18.1 Hz, 10.8 Hz, aromatic, 2H); 7.32–7.09 (m, aromatic, 6H); 6.96 (d, J = 5.5 Hz, aromatic, 2H); 2.32 (s, 3H, CH3); 13C NMR (CDCl3), δ (ppm): 159.6 (C=N); 140.0; 137.0; 134.4; 133.4; 132.2; 131.3; 130.0; 129.7; 129.2; 128.4; 128.2; 127.7; 125.0; 122.5; 21.5 (s, 3H, CH3). Anal. calcd. for C20H16BBrN2O: C, 61.42; H, 4.12; N, 7.16; found: C, 61.37; H, 4.16; N, 7.15.

3-(p-trifluoromethylphenyl)-4-(m-tolyl)-5-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 4o) yield: 36 %; mp: 123–127 °C; IR (ATR), υ (cm−1): 1601 (C=N), 1372 (B–N), 1126 (B–O); 1H NMR (CDCl3), δ (ppm): 7.58–7.40 (m, aromatic, 7H); 7.33–7.19 (m, aromatic, 4H); 6.97 (d, J = 6.7 Hz, aromatic, 2H); 2.32 (s, 3H, CH3); 13C NMR (CDCl3), δ (ppm): 159.8 (C=N); 140.2; 137.0; 134.4; 131.3; 129.8; 129.5; 129.3; 128.4; 128.2; 125.5; 125.5; 125.0; 21.5 (s, 3H, CH3). Anal. calcd. for C21H16BF3N2O: C, 66.35; H, 4.24; N, 7.37; found: C, 66.28; H, 4.32; N, 7.39.

3-(m-nitrophenyl)-4-(m-tolyl)-5-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 4p) yield: 70 %; mp: 125–127 °C; IR (ATR), υ (cm−1): 1599 (C=N), 1349 (B–N), 1138 (B–O); 1H NMR (CDCl3), δ (ppm): 8.24–8.21 (m, aromatic, 2H); 7.71 (d, J = 8.7 Hz, aromatic, 1H); 7.59–7.41 (m, aromatic, 4H); 7.33–7.21 (m, aromatic, 4H); 7.01 (d, J = 7.0 Hz, aromatic, 2H); 2.33 (s, 3H, CH3); 13C NMR (CDCl3), δ (ppm): 159.0 (C=N); 148.1; 140.4; 136.7; 134.9; 134.4; 131.5; 130.0; 129.7; 129.6; 128.4; 128.3; 128.1; 125.1; 125.0; 124.2; 21.5 (s, 3H, CH3). Anal. calcd. for C20H16BN3O3: C, 67.25; H, 4.52; N, 11.76; found: C, 67.55; H, 4.60; N, 11.83.

3-(p-nitrophenyl)-4-(m-tolyl)-5-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 4r) yield: 76 %; mp: 190–192 °C; IR (ATR), υ (cm−1): 1598 (C=N), 1340 (B–N), 1122 (B–O); 1H NMR (CDCl3), δ (ppm): 8.13 (d, J = 10.8 Hz, aromatic, 2H); 7.57–7.53 (m, aromatic, 4H); 7.46–7.41 (m, aromatic, 1H); 7.33–7.21 (m, aromatic, 4H); 6.98 (d, J = 5.8 Hz, aromatic, 2H); 2.33 (s, 3H, CH3); 13C NMR (CDCl3), δ (ppm): 159.2 (C=N); 148.8; 140.4; 136.7; 134.4; 132.6; 131.5; 130.1; 129.9; 129.5; 128.3; 128.3; 124.9; 123.7; 21.5 (s, 3H, CH3). Anal. calcd. for C20H16BN3O3: C, 67.25; H, 4.52; N, 11.76; found: C, 67.48; H, 4.51; N, 11.84.

Synthesis of benzamidoxime (6) (general procedure)

Method A: A solution of hydroxylamine hydrochloride (200 mmol, 13.89 g) in ethanol (100 mL) and a solution of anhydrous sodium carbonate (100 mmol, 10.59 g) in boiling water (25 mL) were mixed and stirred. Benzonitrile (200 mmol, 20.62 g) in ethanol (25 mL) was added to this mixture. The reaction was refluxed at 80 °C for 21 h. The solvent was evaporated under reduced pressure. The residue was washed with water and extracted with chloroform. The solution was dried with anhydrous CaCl2 and the solvent was evaporated under vacuum. The precipitate was crystallized from ethyl acetate–petroleum ether to give benzamidoxime (6) (11.88 g, 43 %). Mp: 75–77.5 °C, Lit. (Krüger, 1885): 79–80 °C; IR (ATR), υ (cm−1): 3450, 3358 (NH2), 3181 (N–OH), 1645 (C=N).

Method B (Gosenca et al., 2013): A solution of benzonitrile (3.6 mmol, 0.371 g), hydroxylamine hydrochloride (7.2 mmol, 0.50 g), and potassium carbonate (7.25 mmol, 1.0 g) were suspended in anhydrous ethanol (50 mL). The mixture was refluxed for 8 h. The precipitate was rapidly filtered off before cooling and the solvent was evaporated under vacuum. The crude product was recrystallized from dichloromethane–petroleum ether to give benzamidoxime (6) (0.325 g, 66 %). Mp: 63–65 °C. IR (ATR), υ (cm−1): 3450, 3357 (NH2), 3181 (N–OH), 1642 (C=N).

Synthesis of 3,5-diphenyl-4,5-dihydro-1,2,4,5-oxadiazaborole (7g) (general procedure for 7a–s)

Benzamidoxime (6) (22 mmol, 3.0 g) and phenylboronic acid (22 mmol, 2.86 g) were dissolved in benzene (150 mL) and the solution was refluxed overnight, then the solvent was evaporated under reduced pressure. The residual was crystallized from hexane to give 3,5-diphenyl-4,5-dihydro-1,2,4,5-oxadiazaborole (7g) (4.57 g, 94 %). Mp: 164–164.5 °C, Lit. (Akcan, 2007): 154–156 °C; IR (ATR), υ (cm−1): 3418, 3379 (N–H), 1601 (C=N), 1415 (B–N), 1200 (B–O); 1H NMR (DMSO-d 6 ), δ (ppm): 10.46 (s, 1H, NH); 8.02–7.49 (m, aromatic, 10H); 13C NMR (DMSO-d 6 ), δ (ppm): 159.9 (C=N); 134.6; 131.8; 131.4; 129.6; 128.9; 127.4; 126.9. Anal. calcd. for C13H11BN2O: C, 70.32; H, 4.99; N, 12.62; found: C, 70.26; H, 5.28; N, 12.53.

Spectroscopic and analytical data of compounds (7)

5-(4-N,N-dimethylaminophenyl)-3-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 7a) yield: 91 %; mp: 198–200 °C; IR (ATR), υ (cm−1): 3329 (N–H), 1608 (C=N), 1418 (B–N), 1234 (B–O); 1H NMR (DMSO-d 6 ), δ (ppm): 10.13 (s, 1H, NH); 7.99–7.96 (m, aromatic, 2H); 7.78 (d, J = 8.7 Hz, aromatic, 2H); 7.58–7.55 (m, aromatic, 3H); 6.81 (d, J = 8.7 Hz, aromatic, 2H); 2.97 (s, 6H, N(CH3)2); 13C NMR (DMSO-d 6 ), δ (ppm): 159.6 (C=N); 152.8; 135.8; 131.2; 129.6; 127.7; 126.9; 112.1; 40.2 (CH3). Anal. calcd. for C15H16BN3O: C, 67.95; H, 6.08; N, 15.85; found: C, 68.27; H, 6.28; N, 15.90.

5-(4-hydroxyphenyl)-3-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 7b) yield

82 %; mp: 197–198 °C; IR (ATR), υ (cm−1): 3385 (N–H), 1608 (C=N), 1425 (B–N), 1204 (B–O); 1H NMR (DMSO-d 6 ), δ (ppm): 10.23 (s, 1H, NH); 7.98–7.95 (m, aromatic, 2H); 7.80–7.76 (m, aromatic, 2H); 7.58–7.55 (m, aromatic, 3H); 6.92–6.89 (m, aromatic, 2H); 13C NMR (DMSO-d 6 ), δ (ppm): 160.8 (Ar–O); 159.7 (C=N); 136.4; 131.3; 129.6; 127.5; 126.9; 118.6; 116.1. Anal. calcd. for C13H11BN2O2: C, 65.59; H, 4.66; N, 11.77; found: C, 65.18; H, 5.20; N, 11.41.

5-(4-methoxyphenyl)-3-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 7c) yield

78 %; mp: 145–148 °C; IR (ATR), υ (cm−1): 3358 (N–H), 1603 (C=N), 1414 (B–N), 1236 (B–O); 1H NMR (DMSO-d 6 ), δ (ppm): 10.27 (s, 1H, NH); 7.96–7.85 (m, aromatic, 4H); 7.55–7.52 (t, J = 3.8 Hz, aromatic, 3H); 7.06 (d, J = 8.4 Hz, aromatic, 2H); 3.79 (s, 3H, ArOCH3); 13C NMR (DMSO-d 6 ), δ (ppm): 162.3 (Ar–O); 159.8 (C=N); 136.3; 131.3; 129.6; 127.5; 126.9; 114.6; 55.7 (OCH3). Anal. calcd. for C14H13BN2O2: C, 66.71; H, 5.20; N, 11.11; found: C, 66.75; H, 5.59; N, 11.33.

5-(4-tolyl)-3-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 7d) yield

91 %; mp: 149–149.5 °C; IR (ATR), υ (cm−1): 3377 (N–H), 1614 (C=N), 1416 (B–N), 1211 (B–O); 1H NMR (DMSO-d 6 ), δ (ppm): 10.39 (s, 1H, NH); 8.00–7.97 (m, aromatic, 2H); 7.86 (d, J = 7.6 Hz, aromatic, 2H); 7.58–7.56 (t, J = 3.8 Hz, aromatic, 3H); 7.34 (d, J = 7.6 Hz, aromatic, 2H); 2.37 (s, 3H, ArCH3); 13C NMR (DMSO-d 6 ), δ (ppm): 159.8 (C=N); 141.5; 134.6; 131.4; 129.6; 129.6; 127.4; 126.9; 21.9 (CH3). Anal. calcd. for C14H13BN2O: C, 71.23; H, 5.55; N, 11.87; found: C, 71.43; H, 5.57; N, 11.90.

5-(3-N,N-dimetylaminophenyl)-3-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 7e) yield

82 %; mp: 95–96 °C; IR (ATR), υ (cm−1): 3237 (N–H), 1599 (C=N), 1416 (B–N), 1234 (B–O); 1H NMR (DMSO-d 6 ), δ (ppm): 10.35 (s, 1H, NH); 7.99–7.94 (m, aromatic, 2H); 7.58–7.56 (t, J = 3.5 Hz, aromatic, 3H); 7.39–7.24 (m, aromatic, 3H); 6.91–6.88 (m, aromatic, 1H); 2.96 (s, 6H, N(CH3)2); 13C NMR (DMSO-d 6 ), δ (ppm): 159.9 (C=N); 150.8; 131.4; 129.6; 129.5; 127.4; 126.9; 126.0; 122.3; 118.2; 115.8; 40.4 (CH3). Anal. calcd. for C15H16BN3O: C, 67.95; H, 6.08; N, 15.85; found: C, 67.20; H, 6.34; N, 15.43.

5-(3-tolyl)-3-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 7f) yield

93 %; mp: 158–160 °C; IR (ATR), υ (cm−1): 3239 (N–H), 1591 (C=N), 1422 (B–N), 1200 (B–O); 1H NMR (DMSO-d 6 ), δ (ppm): 10.40 (s, 1H, NH); 7.99–7.94 (m, aromatic, 2H); 7.74–7.70 (t, J = 7.0 Hz, aromatic, 2H); 7.56–7.53 (m, aromatic, 3H); 7.39–7.33 (m, aromatic, 2H); 2.35 (s, 3H, ArCH3); 13C NMR (DMSO-d 6 ), δ (ppm): 159.9 (C=N); 137.8; 135.1; 132.4; 131.6; 131.4; 129.6; 128.8; 128.7; 127.4; 126.9; 21.7 (CH3). Anal. calcd. for C14H13BN2O: C, 71.23; H, 5.55; N, 11.87; found: C, 71.71; H, 5.80; N, 11.94.

5-(3-methoxyphenyl)-3-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 7h) yield

27 %; mp: 115–117 °C; IR (ATR), υ (cm−1): 3228 (N–H), 1604 (C=N), 1423 (B–N), 1233 (B–O); 1H NMR (DMSO-d 6 ), δ (ppm): 10.45 (s, 1H, NH); 8.00–7.97 (m, aromatic, 2H); 7.59–7.41 (m, aromatic, 7H); 3.84 (s, 3H, ArOCH3); 13C NMR (DMSO-d 6 ), δ (ppm): 159.9 (Ar–O); 159.7 (C=N); 131.4; 130.2; 129.6; 127.3; 126.9; 126.7; 119.4; 117.5; 55.7 (OCH3). Anal. calcd. for C14H13BN2O2: C, 66.71; H, 5.20; N, 11.11; found: C, 66.98; H, 5.51; N, 10.88.

5-(4-bromophenyl)-3-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 7i) yield

56 %; mp: 228–229 °C; IR (ATR), υ (cm−1): 3363 (N–H), 1587 (C=N), 1415 (B–N), 1203 (B–O); 1H NMR (DMSO-d 6 ), δ (ppm): 10.50 (s, 1H, NH); 7.98–7.95 (m, aromatic, 2H); 7.89–7.85 (m, aromatic, 2H); 7.75–7.72 (m, aromatic, 2H); 7.59–7.56 (m, aromatic, 3H); 13C NMR (DMSO-d 6 ), δ (ppm): 160.0 (C=N); 136.5; 132.0; 131.5; 129.7; 127.2; 126.9; 125.9. Anal. calcd. for C13H10BBrN2O: C, 51.88; H, 3.35; N, 9.31; found: C, 51.49; H, 3.28; N, 9.25.

5-(4-chlorophenyl)-3-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 7j) yield

84 %; mp: 220–222 °C; IR (ATR), υ (cm−1): 3364 (N–H), 1595 (C=N), 1418 (B–N), 1204 (B–O); 1H NMR (DMSO-d 6 ), δ (ppm): 10.50 (s, 1H, NH); 7.96–7.94 (t, J = 1.7 Hz, aromatic, 4H); 7.59–7.57 (t, J = 3.2 Hz, aromatic, 5H); 13C NMR (DMSO-d 6 ), δ (ppm): 160.0 (C=N); 136.8; 136.3; 131.5; 129.6; 129.1; 127.2; 126.9. Anal. calcd. for C13H10BClN2O: C, 60.87; H, 3.93; N, 10.92; found: C, 60.52; H, 3.89; N, 10.87.

5-(3-chlorophenyl)-3-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 7k) yield

82 %; mp: 155–157 °C; IR (ATR), υ (cm−1): 3381 (N–H), 1592 (C=N), 1408 (B–N), 1207 (B–O); 1H NMR (DMSO-d 6 ), δ (ppm): 10.54 (s, 1H, NH); 7.99–7.96 (m, aromatic, 3H); 7.89 (d, J = 7.3 Hz, aromatic, 1H); 7.60–7.52 (m, aromatic, 5H); 13C NMR (DMSO-d 6 ), δ (ppm): 160.0 (C=N); 134.0; 132.9; 131.6; 131.5; 131.0; 129.7; 127.2; 126.9. Anal. calcd. for C13H10BClN2O: C, 60.87; H, 3.93; N, 10.92; found: C, 60.78; H, 4.11; N, 10.80.

5-(3-bromophenyl)-3-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 7l) yield

77 %; mp: 158–160 °C; IR (ATR), υ (cm−1): 3383 (N–H), 1605 (C=N), 1418 (B–N), 1209 (B–O); 1H NMR (DMSO-d 6 ), δ (ppm): 10.52 (s, 1H, NH); 8.24–7.37 (m, aromatic, 10H); 13C NMR (DMSO-d 6 ), δ (ppm): 160.0 (C=N); 139.9; 134.5; 133.2; 131.5; 131.4; 129.7; 127.2; 126.9; 122.8. Anal. calcd. for C13H10BBrN2O: C, 51.88; H, 3.35; N, 9.31; found: C, 52.00; H, 2.89; N, 9.39.

5-(4-acetylphenyl)-3-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 7m) yield

74 %; mp: 205–207 °C; IR (ATR), υ (cm−1): 3397 (N–H), 1549 (C=N), 1414 (B–N), 1202 (B–O); 1H NMR (DMSO-d 6 ), δ (ppm): 10.57 (s, 1H, NH); 8.04 (s, aromatic, 4H); 7.96–7.93 (m, aromatic, 2H); 7.56–7.54 (m, aromatic, 3H); 2.60 (s, 3H, COCH3); 13C NMR (DMSO-d 6 ), δ (ppm): 198.7 (C=O), 160.1 (C=N); 139.2; 134.8; 131.5; 129.7; 128.7; 128.4; 127.2; 127.0; 27.5 (CH3). Anal. calcd. for C15H13BN2O2: C, 68.22; H, 4.96; N, 10.61; found: C, 68.67; H, 5.07; N, 10.70.

5-(3-cyanophenyl)-3-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 7n) yield

70 %; mp: 236–239 °C; IR (ATR), υ (cm−1): 3381 (N–H), 1599 (C=N), 1429 (B–N), 1221 (B–O); 1H NMR (DMSO-d 6 ), δ (ppm): 10.54 (s, 1H, NH); 8.28 (s, aromatic, 1H); 8.19 (d, J = 7.3 Hz, aromatic, 1H); 7.99–7.90 (m, aromatic, 3H); 7.72–7.67 (t, J = 7.6 Hz, aromatic, 1H); 7.56–7.54 (t, J = 3.2 Hz, aromatic, 3H); 13C NMR (DMSO-d 6 ), δ (ppm): 160.0 (C=N); 138.7; 138.0; 135.1; 131.6; 130.1; 129.7; 128.7; 127.1; 126.9; 119.3; 112.3 (C≡N). Anal. calcd. for C14H10BN3O: C, 68.06; H, 4.08; N, 17.01; found: C, 68.20; H, 4.55; N, 16.52.

5-(3-nitrophenyl)-3-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 7o) yield

53 %; mp: 241–243 °C; IR (ATR), υ (cm−1): 3400 (N–H), 1610 (C=N), 1448 (B–N), 1215 (B–O); 1H NMR (DMSO-d 6 ), δ (ppm): 10.71 (s, 1H, NH); 8.77 (s, aromatic, 1H); 8.39–8.29 (m, aromatic, 2H); 7.99–7.95 (m, aromatic, 2H); 7.83–7.78 (t, J = 7.6 Hz, aromatic, 1H); 7.60–7.58 (t, J = 3.5 Hz, aromatic, 3H); 13C NMR (DMSO-d 6 ), δ (ppm): 160.0 (C = N); 148.3; 140.6; 131.6; 130.6; 129.7; 128.8; 128.7; 127.0; 126.9; 126.3. Anal. calcd. for C13H10BN3O3: C, 58.47; H, 3.77; N, 15.74; found: C, 58.82; H, 3.77; N, 15.69.

5-(4-methanesulfonylphenyl)-3-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 7p) yield

82%; mp: 220–222.5 °C; IR (ATR), υ (cm−1): 3389 (N–H), 1601 (C = N), 1408 (B–N), 1206 (B–O); 1H NMR (DMSO-d 6 ), δ (ppm): 10.69 (s, 1H, NH); 8.21 (d, J = 8.4 Hz, aromatic, 2H); 8.10 (d, J = 8.2 Hz, aromatic, 2H); 8.00–7.97 (m, aromatic, 2H); 7.60–7.58 (t, J = 3.5 Hz, aromatic, 3H); 3.29 (s, 3H, CH3); 13C NMR (DMSO-d 6 ), δ (ppm): 160.1 (C=N); 143.5; 135.4; 135.3; 131.6; 129.7; 128.7; 127.2; 127.1; 126.9; 44.0 (CH3). Anal. calcd. for C14H13BN2O3S: C, 56.02; H, 4.37; N, 9.33; found: C, 55.96; H, 4.38; N, 9.61.

5-(4-nitrophenyl)-3-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 7r) yield

51 %; mp: 257–259 °C; IR (ATR), υ (cm−1): 3417, 3377 (N–H), 1602 (C=N), 1415 (B–N), 1200 (B–O); 1H NMR (DMSO-d 6 ), δ (ppm): 10.73 (s, 1H, NH); 8.36 (d, J = 8.4 Hz, aromatic 2H); 8.20 (d, J = 8.4 Hz, aromatic, 2H); 7.99–7.96 (m, aromatic, 2H); 7.59–7.58 (m, aromatic, 3H); 13C NMR (DMSO-d 6 ), δ (ppm): 160.1 (C=N); 149.8; 135.7; 131.6; 129.7; 127.0; 126.9; 123.6. Anal. calcd. for C13H10BN3O3: C, 58.47; H, 3.77; N, 15.74; found: C, 59.02; H, 3.83; N, 15.80.

5-butyl-3-phenyl-4,5-dihydro-1,2,4,5-oxadiazaborole ( 7s) yield

49 %; mp: 92–94 °C; IR (ATR), υ (cm−1): 3260 (N–H), 2926 (CH2), 1558 (C=N), 1427 (B–N), 1223 (B–O); 1H NMR (DMSO-d 6 ), δ (ppm): 9.82 (s, 1H, NH); 7.88–7.85 (m, aromatic, 2H); 7.49–7.47 (m, aromatic, 3H); 1.53–1.11 (m, 6H, CH2–CH2–CH2); 0.89–0.84 (t, J = 7.0 Hz, 3H, CH3); 13C NMR (DMSO-d 6 ), δ (ppm): 159.2 (C=N); 131.1; 129.5; 127.5; 126.8; 27.3, 25.5 (CH2–CH2–CH2); 14.4 (CH3). Anal. calcd. for C11H15BN2O: C, 65.39; H, 7.48; N, 13.86; found: C, 65.59; H, 8.05; N, 14.25.

Biological assays

The antibacterial activities of 35 oxadiazaboroles (4a–r and 7a–s) have been determined by the broth microdilution susceptibility test, which is outlined by the Clinical and Laboratory Standards Institute M7–A7 (CLSI, 2006). MICs for each compound were determined against S. aureus (ATCC 25983), E. faecalis (ATCC 29212), P. aeruginosa (ATCC 27853), and E. coli (ATCC 25922).

MIC values for each compound were also determined against S. mutans (ATCC 25175). The antibacterial activities of oxadiazaboroles also have been evaluated using Mueller–Hinton broth with 2–5 % lyophilized horse blood for the determination of the wells in the microdilution plate containing the lowest concentration that has completely inhibited visible bacterial growth as recommended by the standards of the Committee Laboratory Standards Institute (CLSI, 2005).

Sterile, disposable, multiwell microdilution plates (96 U-shaped wells) have been used for broth microdilution procedures. The stock solutions were prepared in pure ethanol (Sigma). In the concentrations studied, ethanol had no effect on the microorganisms.

The antifungal activities of the compounds were also determined by using broth microdilution susceptibility test outlined by Clinical and Laboratory Standards Institute M27–A2 (CLSI, 2002). MIC values for each compound were also determined against C. albicans (ATCC 90028). Also, sterile, disposable, multiwell microdilution plates (96 U-shaped wells) have been used for broth microdilution procedures. The stock solutions were prepared in pure ethanol (Sigma) and again ethanol has no effect on the microorganisms in the concentrations studied.

Dilutions of the compounds

For antibacterial activities, all the dilutions of oxadiazaborole solutions were done in the wells of microdilution plates by Mueller–Hinton Broth (Oxoid). For S. mutans antimicrobial activity tests, Mueller–Hinton Broth (Oxoid) with lyophilized horse blood was used. The concentrations of the compounds were 1600, 800, 400, 200, 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.19, 0.09, and 0.04 µg/mL. Ampicillin and ciprofloxacin were used as reference compounds, which were obtained from the manufacturers.

For antifungal activity, all dilutions of the compounds were done with RPMI medium with L-glutamine buffered, pH 7, with MOPS (Sigma) in the wells of microdilution plates. The concentrations of the compounds are the same as above. The fluconazole was used as a reference compound, which was also obtained from the manufacturers.

Inoculum preparation

After the dilutions of oxadiazaborole solutions, standardized inoculum of each bacterium (S. aureus, E. faecalis, P. aeruginosa, and E. coli) (0.5 Mc Farland standard unit, 1  ×  108 CFU/mL; colony forming unit/mL) was prepared. Then, the solutions were diluted once more (1/10), and final concentrations became 1  ×  107 CFU/mL. Five microliters from each dilution was placed into each well containing 100 µL of dilutions of the compounds so that each well contained 5  ×  105 CFU/mL of inoculum. All the inoculated plates were incubated at 35 °C for 16–20 h. The lowest concentration of the compounds that prevents visible growth was considered to be the MIC. To control the reliability of the results, ampicillin and ciprofloxacin were used as reference antimicrobial reagents. The parameters of these reagents were compared with the data obtained from the method applied in this study.

The bacteria (S. mutans) were cultivated on a sheep agar plate for 36–48 h at 37 °C in 5–10 % CO2, and incubation was done in a candle extinction jar. After diluting the compounds, standardized inoculum of each bacterium (0.5 Mc Farland standard unit, 1  ×  108 CFU/mL; colony forming unit/mL) was prepared in Brain-Heart Infusion broth. Then the compounds were diluted once more (1/10), and the final concentrations became 1  ×  107 CFU/mL. Five microliters from each dilution was placed into each well containing 100 μL of dilutions of compounds so that each well contained 5  ×  105 CFU/mL of inoculum. All the inoculated plates were incubated at 35 °C for 36−48 h with 5–10 % CO2. The lowest concentration of compounds that prevents visible growth was considered to be the MIC. Ampicillin was used as reference antimicrobial reagent to compare its parameters with the data that result from the method applied in this work and to control the reliability of the latter.

For antifungal activity, Candida isolates were subcultured in SDA plates, incubated at 35 °C for 24–48 h prior to antifungal susceptibility testing, and passaged at least twice to ensure purity and viability. An inoculum suspension was prepared from individual five colonies (diameter 1 mm). The suspension was adjusted to 0.5 Mc Farland Standard (1–5  ×  106 CFU/mL) and further diluted to 1/20 (1–5  ×  105 CFU/mL), then to 1/50 (0.5–2.5  ×  105 CFU/mL) in RPMI medium. Hundred microliters from each dilution was placed into each well containing 100 µL of dilutions of compounds so that each well contained 1  ×  103 CFU/mL of inoculum. The MIC plates were incubated at 37 °C for 48 h. The end point was determined when the concentration produced optically clear wells (MIC-0) compared with that of drug-free growth control. To control the reliability of the results, fluconazole was used as reference antifungal reagent. The parameter of this reagent was compared with the data obtained from the method applied in this study. Every experiment for the antibacterial and antifungal assays was replicated twice. MIC values for antimicrobial activities are given in Tables 1 and 2, respectively.