General procedure was used to convert 6-chloro-5H-benzo[a]phenoxazin-5-one and methoxyphenol in 8 h to 710 mg (96%) of the title compound 8a. The compound was a black solid after recrystallization from ethanol-water mixture (2:1). mp <360°C. UV (H₂O): λ_max 269 (1.8261), 389(2.5977), 456 (2.5416) nm. IR (neat, cm⁻¹): 3163 (intramolecular H-bond with C = O), 1626 (C = O), 1582 (C = N, C = C), 1208, 1060 (C–O–C). ¹H NMR (δ): 3.37 (s, 3H, OCH₃), 6.73–6.95 (m, 4H, phenoxy), 6.99–7.29 (m, 4H. Ar–H), 7.41–8.85 (s, 4H, Ar–H).

General procedure was used to convert 6-chloro-5H-benzo[a]phenoxazin-5-one and phenol in 10 h to 750 mg (94%) of the title compound 8b. The compound was a black solid after recrystallization from ethanol-water mixture (2:1). mp >360°C. UV (H₂O): λ_max 270 (2.7050), 390 (2.6160), 490 (2.3979). IR (neat, cm⁻¹): 3181(intramolecular H-bond with C = O), 1631 (C = O), 1563 (C = C, C = N), 1265, 1058 (C–O–C). ¹H NMR (δ): 6.53–6.56 (d, 1H, C2, C4, and C6 of phenoxy), 6.88–7.03 (t, 2H, C3, and C5 of phenoxy) 7.17–7.53 (m, 4H, Ar–H), 7.67–8.53 (s, 4H, Ar–H).

General procedure was used to convert 5-chloro-5H-benzo[a]phenoxazin-5-one and 4-chlorophenol in 8 h to 770 mg (46%) of the title compound 8c. The compound was obtained as a black solid after recrystallization from ethanol-water mixture (2:1). mp. >360°C. UV (H₂O): λ_max 265 (2.7753), 387 (2.6284) nm. IR (neat, cm⁻¹), 3172 (intramolecular H-bond with C = O), 1631 (C = O), 1563 (C = C, (C = N), 1263, 1059 (C–O–C). ¹H NMR (δ): 6.78–6.79 (d, 2H, C2, and C6 of phenoxy), 7.15–7.17 (t, 2H, C3, and C5 of phenoxy), 7.25–7.50 (m, 4H, Ar–H), 7.87–8.68 (1, 4H, Ar–H).

General procedure was used to convert 6-chloro-5H-benzo[a]phenoxazin-5-one and 2-aminophenol in 10 h to 682 mg (94%) of the title compound 8d. The compound was a black solid after recrystallization from ethanol-water mixture (2:1). mp > 360°C. UV (H₂O): λ_max 234 (2.4216), 384 (2.1644), 493 (1.9684). IR (neat, cm⁻¹): 3197 (N–H, intramolecular H-bond with C = O), 1632 (C = O), 1561(C = N), 1459 (C = C), 1323 (C–N), 1269, 1065 (C–O–C). ¹HNMR (δ): 6.37 (s, 2H, NH₂), 6.38 (d, 1H, C₃-H of phenoxy), 6.77–6.78 (d, 1H,C₅-H of phenoxy), 7.17–7.18 (t, 1H, C₆-H of penoxy), 7.24–7.26 (t, 1H, C₄-H of phenoxy), 7.39 (d, 1H, Ar–H), 7.40–7.41 (t, 1H, Ar–H), 7.45–7.51(m, 2H, Ar–H), 7.61–8.25 (m, 4H, Ar–H).

General procedure was used to convert 6-chloro-5H-benzo[a]phenoxazin-5-one and 4-isopropylphenol in 7 h to 765 mg (98%) of the title compound 8e. The compound was a black solid after recrystallization from ethanol-water mixture (2:1). mp >360°C. UV (H₂O): λ_max 260 (3.2448), 384 (2.9294), 485(2.8859) nm. IR (neat, cm⁻¹): 3194 (intramolecular H-bond with C = O), 1641 (C = O), 1563(C = N, C = C), 1266, 1059 (C–O–C). ¹H NMR (δ): 1.61 (d, 6H, 2CH₃), 2.86 (m, 1H, methane–H), 6.66–6.68 (d, 2H, C₂, and C₆ of phenoxy), 6.99–7.00 (m, 2H, C₃, and C₅ of phenoxy), 7.17–7.24 (m, 4H, Ar-H), and 0.55–7.91 (m, 4H, Ar–H).

General procedure was used to convert 6-chloro-5H-benzo[a]phenoxazin-5-one and 1-naphthol in 10 h to 520 mg (68%) of the title compound 8f. The compound was a black solid after recrystallization from ethanol-water mixture (2:1). mp > 360°C. UV (H₂O): λ_max 1263 (3.0078), 385 (2.4314) nm. IR (neat, cm⁻¹): 3171 (intramolecular H-bond with C = O) 1626 (C = O), 1585 (C = C, C = N), 1234, 1059 (C–O–C). ¹H NMR (δ): 6.37 (d, 1H, C₂-H of naphthoxy), 6.62–6.87 (m, 4H, C₃-, C₄-, C₆-, and C₇-H of naphthoxy), 7.18–7.54 (m, 4H, Ar–H), 7.67–8.7 (m, 5H, Ar–H, andC₅ of naphthoxy).

The physicochemical properties, including molecular weight, Log P, hydrogen bond donor (HBD), hydrogen bond acceptor, total polar surface, number of rotatable hydrogen, and volume, of the synthesized compounds were studied using Molinspiration Chemoinformatics softwares. The drug-likeness was evaluated using Lipinski's rule of five.

To have a better understanding on the binding modes of the synthesized derivatives (7a–f and 8a–f); the docking studies were performed. The 3D structures of the three bacteria target proteins were obtained from the Protein Data Bank. They were Glucosamine-6-phosphate synthase (PDB code: 2VF5) at resolution of 2.9 Å (Mouilleron et al., 2008); AmpC beta-lactamase (PDB code: 1KE4) at resolution of 1.72 Å (Powers and Shoichet, 2002), and Lanosterol 14-alpha-demethylase (PDB code: 3JUV) at resolution of 3.12 Å (Strushkevich et al., 2013). We used Discovery Studio Visualizer 4.1 in the preparations of the proteins: the needed chains were selected and multiple ligands and non-protein parts were deleted. The 2D structures of the ligands were drawn using ACD/ChemSketch 2015 version and the 3D structures generated by the same software (ACD/ChemSketch, 2015). OpenBabel GUI version 2.3.2 (O'Boyle et al., 2011) and AutoDock were used to convert the pdb file format to pdbqt. Molecular docking process was done using AutoDock Tools 1.5.6 and AutoDock Vina version 1.1.2 (downloaded from http://autodock.scripps.edu). The docking results were analyzed and visualized with Pymol version 1.7.4.4.

Structural modifications of the phenoxazine ring systems using palladium based electron rich bulky aryldialkylphosphine ligand (t-BuXPhos) have given rise to 12 ether derivatives with significant synthetic yields (46–96%) as shown in Table 2. The two key intermediates in the syntheses were 11-amino-9-mercapto-6-chloro-8,10-diaza-5H-benzo[a]phenoxazin-5-one 4 and 6-chloro-5H-benzo[a]phenoxazin-5-one 5, which were prepared by condensation of 4,5-diamino-6-hydroxyl-2-mercaptopyrimidine and 2-aminophenol with 2,3-dichloro-1,4-naphthoquinone respectively in a basic medium (Okafor, 1986). Reaction of compound 4 with various phenols in the presence of a catalyst system consisting of palladium (II) acetate and t–BuXPhos, afforded the ether derivatives 7a–f in good yields (Table 2, entries 1–6). The reaction of compound 5, under similar reaction conditions, gave compounds 8a–f also in good yields (Table 2, entries 7–12). The success of these reactions is attributed to use of the electron-rich, sterically bulky aryldialkylphosphine, t-BuXPhos, as ligand. The results show that using the t-BuXPhos, an electron-rich phosphine ligand, can achieve oxidation of diazabenzo[a]phenoxazine 4 and benzo[a]phenoxazine 5 nuclei to palladium centers. There is a shift in the rate-determining step in the catalytic cycle of the reaction process from the oxidative addition of the diazabenzo[a]phenoxazine 4 and benzo[a]phenoxazine analog 5 to the reductive elimination, which subsequently led to the formation of compounds 7a–f and 8a–f, respectively (Scheme 3). Structure-activity relationship study shows that reaction of compounds 4 and 5 with electronically neutral phenol (C₆H₅OH) gave high yields (96 and 95%, entries 2 and 8) with compound 5 showing a lower yield. In addition, phenols bearing electron donating groups in para-position gave excellent yields (entries 1, 5, 7, and 11). The fact that these electron rich phenols are particularly good reaction partners showed that the presence of compounds 4 and 5 allows the delocalization of the electrons, which may have been generated in the transition state of the reductive elimination of the phenoxazine ether (Mann and Hartwig, 1997; Hartwig, 1998). Furthermore, the reaction of compounds 4 and 5, with electron deficient phenol bearing chlorine at the para-position, gave a better yield with compound 4 (74%, entry 3) than with compound 5 (46%, entry 8). This is in agreement with the observation earlier made by other authors (Mann and Hartwig, 1997; Hartwig, 1998). When an electron rich compound is coupled with electron deficient phenolic compound, it gives higher yield than when lesser electron rich compound is coupled with electron deficient phenol compound. 11-Amino-9-mercapto-8,10-diazabenzo[a]phenoxazin-5-one 4 is more electron rich compound than 6-chlorobenzo[a]phenoxazin-5-one 5, hence the difference in yields in this case. The highest yield was obtained when 1-naphthol was coupled with compound 4 (99%, entry 6). This may be attributed to the presence of the additional benzene ring in naphthol compound, which constituted increase in conjugation resulting in increased electron delocalization. Reaction of compound 5 with 1-naphthol, gave a lower yield (68%, entry 12). This may be because of difference in electronegativity of compounds 4 and 5.

The spectral data are as shown in the experimental section. The electronic spectrum of some of the compounds showed peaks at 260–270 nm which is due to n–π^* transitions. The peaks at 251–258 nm were assigned to n–σ^* transitions. There were also peaks at 217–234 nm which are due to π-π^* transitions. The peaks at the longer wavelengths 451–490 nm are due to extended conjugation in the compounds, which resulted in increase in electron delocalization. Furthermore, all the compounds showed the characteristic absorption of phenoxazine ring systems at 385–390 nm. The compounds were further characterized using infrared data. The asymmetric and symmetric C–O–C stretching of ether compounds in IR spectra were observed at 1,208–1,269, and 1,058–1,065 cm⁻¹, respectively, for all the ether phenoxazine derivatives. The peaks 1,626–1,641 cm⁻¹ observed in all the spectra show the absorption of C = O which forms O-hydroxyl aryl intramolecular hydrogen bonding (Schemes 4a,b). This may account for the deliquescent nature of the synthesized compounds. The intramolecular hydrogen bonding gave peak observed at 3,163–3,197 for compounds 8a–f. This peak was shown for compounds 7a–f at 3,183–3,205 cm⁻¹. The peak 3,183–3,205 cm⁻¹ was also attributed to N-H stretch of primary aromatic amine in compounds 7a–f. Other peaks are in agreement with the assigned structure (Experimental). The NMR spectra further supported the assigned structures. For example, the ¹H NMR spectra of the diazaphenoxazine ether derivatives 7a–f showed singlet peak at δ 2.53–3.56 ppm assigned to SH proton. This peak was not seen in the spectra of the benzo analog 8a–f. Other peaks in the NMR spectra as shown in the experimental section are in agreement with the assigned structures. For the ¹H NMR spectra of compounds 7a–f and 8a–f, see the Supplementary Materials.

The physicochemical properties such as molecular weight (MW), Partition coefficient value (Log P), which indicates the Lipophilicity of the ligand; number of hydrogen bond donor (HBD) and number of hydrogen bond acceptor (HBA), total polar surface area (TPSA), number of rotatable bonds (NoRB), and volume for the ligands were obtained as shown in Table 3. For a drug molecule to be orally bioavailable in the systemic circulation, Lipinski's rule of five should apply: “the drug must have molecular weight value of ≤500, hydrogen bond donor ≤5, hydrogen bond acceptor ≤10, and partition coefficient (Log P) value ≤5.” From the results in Table 3, the synthesized compounds are in agreement with the Lipinski's rule of five since there was no violation of more than one parameter studied. In addition, the Polar Surface Area (PSA), which reflects the ligand hydrophilicity, is very vital in protein-ligand interaction. Veber et al. (2002) showed that 10 or less rotatable bonds and polar surface area, PSA ≤140 Ų would have a high probability of good oral bioavailability in rats. Compounds 8a–f were found to have PSA ≤90 Ų. According to van de Waterbeemd et al. (1998), they can cross the BBB and penetrate the CNS, and hence, they are potential candidates for the treatment of cerebral malaria.

From the molecular docking results, modifications of compounds 4 and 5 gave rise to compounds (7a–e) and (8a–e), respectively, which showed better binding affinities with the targets. However, there was no significant difference in the binding affinities of the modified compounds (7a, b, d, f, and 8a–d, f). In addition, there were no significant binding affinities with the receptors between the diazabenzo[a]phenoxazin-5-one and benzo[a]phenoxazin-5-one derivatives. It was also observed that compounds 7c, 7e, and 8e had best interactions with the three target proteins as seen in their appreciable increase in binding affinities (Table 4). This underscores the usefulness of these compounds as potential antimicrobial agents since they can bind to multiple targets in the organism, which will result in the inhibition of several biochemical pathways essential for the survival of the organism and their consequent death. Although all the three target proteins showed good interactions with the compounds, the protein receptor, 2VF5 gave best interaction. Closer docking studies of compounds 7c and 7e with the receptor, 2VF5 have been demonstrated (Figures 1–4) in order to gain further insight. Figure 1 shows the binding mode of 7c with 2VF5; Figure 2 demonstrates its interactions and the nature of interaction with the amino acid residues of the receptor. Figure 3 demonstrates the binding mode of compound 7e with 2VF5, and the types of amino acid residues involved in the interactions, whereas Figure 4 showing compound 7e in the binding cavity of 2VF5, illustrates H-bonds (A) and hydrophobicity (B) regions of the binding site. Compound 7c interacted with the following active amino acid residues of 2VF5: THR:352, THR:302, VAL:605 AND SER:401 forming H-bonds; VAL:605 forming pi-sigma bonds and CYS:300 forming pi-sulfur and pi-alkyl bonds (Figure 2).

Predicted MIC (μg/ml) = 5.68704 + 1.05391 × AM1_dipole + 5.91138 × a_acc + 10.48717 × a_don −1.98295 × b_rotN −5.02794 × logP(o/w)

−1.36589 × rings − 0.45882 × TPSA + 0.25853 × Weight − 2.69962 × b_ar

Regression coefficient (N = 50, R² = 0.7400, R²__(predicted) = 0.5475).

The above-mentioned equation clearly showed that the molecular descriptors, number of rotatable bonds, logP (o/w), number of rings, total polar surface area, and number of aromatic bond showed negative correlation with respect to the biological activity. On the other hand, dipole moment, number of H-donors, number of H-acceptors, number of aromatic bond, and molecular weight showed positive correlation with the biological activity.

QSAR model analysis includes the calculation of cross-validated squared correlation coefficient (R²) for internal validation and the predictive squared correlation coefficient (R²__predicted) for external validation. Here, in this case, the R² was 0.7400, and R²__predicted was 0.5475, which undoubtedly showed the true predictability of the model and that it was not by mere chance. Graphical plot between experimental and predicted activities MIC (μg/ml) of the training and test set compounds are represented in Figures 5, 6 also showed the 3D scatter plot of the three major descriptors (molecular weight, logP (o/w) and TPSA) used.

Each point in the MOE Window corresponds to a molecule, and is colored according to the molecules MIC-value.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.