Synthesis Characterization and Antibacterial, Antifungal Activity of N-(Benzyl Carbamoyl or Carbamothioyl)-2-hydroxy Substituted Benzamide and 2-Benzyl Amino-Substituted Benzoxazines

New N-(benzyl carbamothioyl)-2-hydroxy substituted benzamides 13, 20, and 21 were synthesized using sodium bicarbonate and benzyl amine with 2-thioxo-substituted-1,3-benzoxazines 6, 10a, b, 11c, and 12a–n. The 2-thioxo-substituted-1,3-oxazines 6, 10a-b, 11d 12a–n, and 26 were converted to the corresponding 2-methylthio-substituted-1,3-oxazines 14a–l and 24 which were then converted to 2-benzyl amino-substituted-benzoxazines 15a–i by refluxing with benzylamine. Products 15a, b, e, f, and g were also synthesized by boiling the corresponding N-(benzyl carbamothioyl)-2-hydroxy substituted benzamides 13a, b, f, l, and m in acetic acid. 2-Oxo-substituted-1,3-benzoxazines 22 and 25 were prepared by treating the corresponding 2-methylthio-substituted-1,3-oxazines 14 and 24 with dilute HCl. The N-(benzyl carbamoyl)-2-hydroxy substituted benzamide 23 was synthesized from the reaction of 2-oxo-substituted-1,3-benzoxazine 22 with benzylamine. The new products were characterized using IR, 1H, and 13C NMR in addition to microanalysis. Selected compounds were tested in vitro for antibacterial and antifungi activity and the most active compounds were found to be the 4-(substituted-benzylamino)-2-hydroxy benzoic acids 9a and d (M. chlorophenolicum, MIC 50 and 25 µgm L−1, resp.), N1, N3-bis (benzyl carbamothioyl)-4,6-dihydroxy-substituted phthalamides 20a and 20c (B. subtilis MIC 12.5, 50 µgm L−1, resp.) and 21 (M. chlorophenolicum, MIC 50 µgm L−1).


Introduction
The search for new antibacterial compounds is a challenging task as bacteria are continuously developing resistance to antimicrobial compounds; however, infections due to such bacterial strains are infrequent although potentially fatal [1][2][3]. This ongoing problem has resulted in the search for newer, more effective antibacterial compounds [1][2][3].
The N-benzoyl-2-hydroxybenzamides [13] are important pharmacophores for antibacterial activity in which the 2hydroxy group (hydrogen bonding donor) contributes to the activity, the imide linker (preferred) or urea linker retains activity and free NH is required for high activity.
The Topliss method [14] was used in the optimization of salicylic acid derivatives for potential use as antibacterial agents. The employment and analysis of physicochemical parameters and molecular electronic surfaces which highlight the electronic, lipophilic, and steric features may be useful guidelines in the continuous search for new, more effective 3-amino-salicylic acid analogs. The synthesis of the urea or thiourea product (3, Scheme 1) was previously achieved from the reaction of benzoylisocyanate or benzoylisothiocyanate 1 (X=O or S, resp.) with amines 2 [15][16][17].
The substitution R on the aromatic ring could be alkyl, alkoxy, -OC=OCH 3 in positions 2, 3, 4, 5, and 6, and the R 1 -N-R 2 in product 3 could be aliphatic, aromatic, or cyclic amine substitutions. Limitation associated with this method is that R, the substitution on the aromatic ring, cannot be -OH or RN-, which is desirable for antimicrobial activity, particularly the hydroxy group [18].   had decreased yield (19%), and none of the cyclic analogue 15a could be isolated. When 2-thio-1,3-benzoxazine 6 was allowed to react with excess benzylamine (4-fold) and the mixture was then heated to reflux in dioxane for 2 hours, the open product 13a was again isolated in 18% yield. To overcome this low yield and possible mixture formation, the reaction procedure was modified in which the benzoxazines 6, 10a-b, 11d, and 12b-m were mixed with NaHCO 3 and suspended in 1 : 1 mixture of methanol, water and the mixture was then heated to 40 ∘ C for a few minutes. Excess (1.5-fold) benzyl amine was added dropwise at room temperature and left stirring for 4 hours. Products 13aq was isolated according to the general procedure B (see Experimental) and the yield were moderate to high (69-87%) (Scheme 3).
With slight modification to the procedure B, by altering the ratio of benzylamine to starting material (3 : 1) and the reaction time to 16 hours, the bis-oxazines 18a,c and 19 were found to react in a similar fashion to give substituted bis(benzyl carbamothioyl) analogues 20a,c (yield 63, 49%) and 21 (yield 63%) (Scheme 4).
The previously prepared N-(benzyl carbamothioyl)-2hydroxybenzamide 13a was characterized by comparison of its physical data (mp, IR, 1 H and 13 C NMR spectra) with values found in the literature [12,19,20]. The structures of new benzyl thiourea compounds 13b-q, 20a,c, and 21 were confirmed using IR, 1 H NMR and 13 C NMR spectroscopy and microanalysis. The 1 H NMR and IR spectra also showed a high correlation with the previously prepared benzyl thiourea 13a [19,20]. In the 1 H NMR spectra, the CH 2 , H-5 of the benzyl amine in compounds 13a-q, 20a,c, and 21 appeared as a doublet at ∼ 4.9 ppm and the 4 -NH, appeared as a triplet at ∼ 11.0 ppm in all cases. Assignment of the carbon-13 chemical shifts was made using the previous reported chemical shifts of 13a [19,20]. The 1 H and 13 C NMR spectra of the parent 2-thioxo-2H-benz[e]-1,3-oxazin-4(3H)-one 12 were also used to aid with structural identification. The simulated 1 H and 13 C NMR spectra using ChemDraw V12 ultra were also used as references to aid the analysis of the observed 1 H and 13 C NMR spectra of the new products.
The reaction of benzylamine with 2-methylthio-benzoxazine took place with no trace of the thiourea analogue 13.
Following their successful synthesis, many of the N-(benzyl carbamothioyl)-2-hydroxybenzamides 13a, b, f, and g were then cyclised by refluxing in acetic acid for 2 hours according to the general procedure D (Scheme 3) and gave the corresponding 2-benzylamino-1,3-benzoxazines 15a, b, f, and g fair to good yields.
Previously prepared 2-benzyl amino-1,3-benzoxazine 15a was characterized by comparison of the physical data (mp, IR, and 1 H and 13 C NMR spectra) with that found in the literature [19,20]. The structures of new 2-benzyl amino-1,3benzoxazine compounds 15b-g were confirmed using IR, 1 H NMR, and 13 C NMR spectroscopy and microanalysis. In the 1 H spectra the CH 2 of the benzyl amine appears as a doublet at ∼ 4.5 ppm. The previously analysed 1 H and 13 C NMR spectra of the parent 2-methylthio-1,3-benzoxazines 14 [35] were used to aid in the analysis of the new products 15b-g s.
Structure Elucidation of Substituted-N-(benzyl carbamoyl)-2-hydroxybenzamides 23a-g. The structures of the newly prepared substituted urea compounds 23a-g were confirmed using IR and 1 H and 13 C NMR spectroscopy and microanalysis. The 1 H NMR and IR spectra supported the proposed structures and showed some correlation with the previously prepared benzyl thiourea 13a-q, 20a, c, and 21. In the 1 H spectra, the methylene CH 2 (H-11 of compounds 23a-g) of the benzyl amine appears to shift up field as a doublet at ∼ 4.3 ppm. The NH (H-10 of compounds 23a-g) appears as Scheme 5: Synthesis of 2-dione-1,3-benzoxazines 22a-I and 25a,b from the 2-methylthio-1,3-benzoxazines 14a,b, d-g, and 24a-b. a broad triplet at ∼ 8.5 ppm. The 1 H and 13 C NMR spectra of the parent 2-oxo-1,3-benzoxazines 22 and 25.  Tables 1 and 2. According to [36], antimicrobial agents are effective on a range of bacterial species at low concentrations, that is, <128 g mL −1 . Therefore, we conducted our MIC experiments using concentrations as high as 300 g mL −1 . We also selected a range of bacterial and fungal species to test our newly synthesised compounds. Some of the species are potentially pathogenic to humans and animals and others are problematic in an environmental setting. Furthermore, the bacterial species selected have different cell wall compositions, that is, some are Gram-negative and some are Grampositive strains. Some antimicrobial agents inhibit bacteria by interacting with components of the cell wall that are absent in Gram-negative bacteria [37]; therefore, the selection of strains was carefully selected with the possibility that an inhibitory compound would also hint to its mechanism of action. Based on the results obtained it is clear that the Gram-negative strains, that is, P. aeruginosa, E. coli, and A. baumannii were least affected by the compounds and when inhibition was observed it was at high levels 200 g mL −1 or higher. Interestingly, compound 9d seemed to have a more dramatic effect Gram-positive strains with the exception of M. smegmatis. Despite the effects that some of the compounds had on the bacterial strains, it appears that these compounds are not so effective when tested on the four fungal cultures chosen with the exception of 9d on A. corymbifera.

Disc Diffusion Susceptibility
Testing. Disc diffusion susceptibility testing was performed on compounds with poor solubility in broth dilution susceptibility testing. The preliminary antimicrobial testing was achieved using the standard agar disk diffusion methods. Compounds that inhibited certain bacteria or fungi are summarized in (Tables  3 and 4). The concentrations of the prepared compounds were 10 −4 g mL −1 (see Experimental). The control data is used to determine if the bacterial strains are resistant (R) or sensitive (S) to the prepared compounds tested. The disc diffusion assay was used as a preliminary guide for all compounds and used in correspondence with the broth dilution method for determining MIC/MFC values. This method is particularly useful when MIC/MFC values are unable to be determined using the broth dilution method due to the compounds insolubility. The insoluble compounds zones of inhibition therefore can be determined in millimeters relative to the control and used as a rough guide. Since the zone of inhibition of clearance may be affected by other parameters, such as, the nutrient agar depth of the plate and solvent used, the results shown using this method therefore should be used as a guide. MIC/MFC values are determined using the broth dilution method. All compounds that showed clearance zones are listed in Tables 3 and 4 and were tested in duplicate with the average given. Any zone of inhibition that was noted around the disc was considered sensitive and the zone of clearly was noted. These results are more useful for compounds that were difficult to dissolve, but equally, these results can indicate resistance if the compound does not diffuse through the agar properly.
Based on the results obtained in Section 2.2.1, it is clear that compound 9d is of interest. Based on the results obtained in Table 3, compound 9d has an inhibitor effect on M. smegmatis but in the MIC study had no effect. This could indicate solubility problems with the compound when in solution; however, this is only speculative; further studies are required to reveal the cause. In addition, compound 9d seems to have no inhibitory effect on S. aureus but in the MIC studies had a dramatic affect. This difference in result is unusual but clearly indicates that different methods could reveal different results and therefore it is important to perform both methods prior to further investigation on their inhibitory effects.
In the MIC studies, we used 300 g mL −1 as the highest cut-off level. If a compound has an inhibitory effect on any strain that is greater than this level, then this should be revealed in the disc diffusion assay. However, further investigation is required as some of these compounds are dissolved in DMSO and when applied to bacterial cultures can come out of solution. The disc diffusion assays seem to indicate some sensitivity to fungal cultures despite the fact that they were undetectable in the MIC studies. Tables 1 and 2 show that the 4-(benzylamino)-2-hydroxybenzoic acid derivative 9d showed the broadest range of activity of the compounds tested, exhibiting activity against the Gram-positive and Gram-negative bacteria and also M. chlorophenolicum. Furthermore, compound 9d showed to be more active than

The Structure Activity Relationships of the Tested Compounds (Broth Dilution). The results in
others against S. aureus with an MIC value of 25 g/mL. Compounds 20a,c and 21 (bis-thiourea products) were found to be particularly active towards Gram-positive B. subtilis at MIC values of 12.5, 25, and 25 g mL −1 . In addition, compound 21 also showed activity towards M. smegmatis (MIC 50 g mL −1 ). Other synthesized compounds which showed an inhibitory effect were 13n which had an inhibitory effect on four bacterial species at 300 g mL −1 and 13d and 13m which had an inhibitory effect on two of the bacterial species at concentration 200 g mL −1 . Interestingly, E. coli was not inhibited by any of the compounds.

The Chemical Compounds Activity and Structural Relationships of the Antimicrobial Assay Results (From Disk Diffusion Assay).
In the presence of a compound, a zone of clearing was greater than the control which was indicative that the strain was sensitive to the compound, whereas a zone of clearing equal to the control indicated resistance. The results reveal that none of the compounds had an inhibitory effect on E. coli at concentration 10 4 g mL −1 ( Table 3). The B. subtilis bacterial species tested showed inhibitory effects to most of the compounds tested, for example, 13d inhibited S. aureus most strongly and compounds 13k, 13f, and 13d inhibited growth of A. baumannii, B. subtilis, and S. agalactiae (Table 3). Some bacterial species that were sensitive to a compound showed similar sized zones of inhibition. One example was 13k which exhibited activity of 2 mm for A. baumannii and B. subtilis and 5 mm for S. aureus and S. agalactiae. The same applies to compounds 8d, 9c, and 9d which had shown a 2 mm clearance zone against P. aeruginosa M. smegmatis M. chlorophenolicum. Similarly to the broth dilution results, compounds 21 and 9d were found to be active against three fungi species, R. oryzae, A. niger, and A. corymbifera, with clearance zones 2-5 mm, respectively. All the compounds tested showed a 2-4 mm zone of clearing for most of the susceptible species with the exception of compound 13b which had a larger 6 mm zone of inhibition. This larger zone indicates a hypersensitive effect on the bacterial species; however, it is specific for the compound and species. Because the mechanism of action of the compound is unknown, it is difficult to explain the reason for the hypersensitive effect. One possible explanation is that B. subtilis encodes a protein that can transport compound 13b into the cell and this has a more toxic effect than those working from outside the cell. A similar phenomenon has been shown with bacterial mercury resistance where the presence of a mercury import protein displays a larger zone of clearing in a disc diffusion assay [38]. 9 The data obtained revealed patterns of inhibition, especially those conducted with the disc diffusion assay. This suggests that a similar mechanism of action could be involved in the inhibition of growth.
We are in the process of synthesising new substituted products by replacing the benzyl group of N-(benzyl carbamothioyl) by 6-aminopenicillanic acid and test their bacteria activity.

4.1.
Chemistry. Infrared spectra were obtained using a Perkin Elmer FT-IR 1720x spectrometer. 1 H NMR and 13 C NMR spectra were obtained using a Bruker AC 200 NMR spectrometer at 200 and 50 MHz, respectively. All 1 H NMR and 13 C NMR spectral results are recorded as chemical shifts ( ) relative to the internal TMS for proton and 77.0 ppm in CDCl 3 solvent and 39.4 ppm in DMSO-d 6 solvent for 13 C NMR. Microanalysis was performed by Chemical and Micro analytical Services (CMAS), Australia. Melting point determinations were carried out using a Stuart Scientific (SMP3) melting point apparatus and all melting points are uncorrected.

Starting Materials.
The stating reagents benzyl amine, sodium hydrogen carbonate, methyl iodide-amino-2-hydroxybenzoic acid, and dry 1,4-dioxane were purchased from Aldrich Chemical Company and were used as received.
Products 8b, c, and e were not identified and used immediately in the synthesis of compound 9a, b, and d.

Synthesis of 7-N-Substituted-amino-1,3-oxazines 10a, b, and 11a, b, and d
General Procedure B. The substituted-2-hydroxy benzoic acid was allowed to react with the freshly prepared Ph 3 P(SCN) 2 according to previously reported general procedure [22,34]. [1,3]oxazin-7-yl) acetamide 10a. Slightly modified to the previously reported general procedure B [22,34], 4-(acetyl amino)-2hydroxybenzoic acid 8a (1.56 g, 8 mmol) was allowed to react with freshly prepared Ph 3 P(SCN) 2 (10 mmol) at room temperature for 2 hours then under reflux for 16 hours. At the completion of the reaction, the PbBr 2 filter cake was washed by acetic acid (150 mL) to extract the desired product. The acetic acid filtrate was evaporated and minimal toluene was added to dissolve any oil with the product. The crude solid was filtered and recrystallised from ethanol to give 10a (1.  [1,3]oxazin-4(3H)-one 11b. In slight modification to previously reported general procedure B, 2-hydroxy-4-((2-hydroxybenzyl)amino)benzoic acid 9b (1.07 g, 4 mmol) was allowed to react with the freshly prepared Ph 3 P(SCN) 2 (10 mmol) at room temperature for 2 hours then under reflux for 16 hours.  [1,3]oxazin-4(3H)-one 11c. In slight modification to previously reported general procedure [22,34], 4-((3ethoxy-2-hydroxybenzyl)amino)-2-hydroxybenzoic acid 9d (1.21 g, 4 mmol) was allowed to react with the freshly prepared Ph 3 P(SCN) 2 (10 mmol) heated to room temperature for 2 hours then under reflux for 16 hours. The resulting solids (1.94 g) were recrystallised from acetic acid/water to give 11c (0.95 g, 68%) as yellow crystals, mp 227-229 ∘ C decomp.  At the completion of the reaction, the reaction mixture was filtered and the PbBr 2 cake washed with approx 100 mL THF and filtered. Both THF and DCM filtrates were evaporated to dryness under reduced pressure and minimal toluene was added to remove any oil, which may be present. The solid which remained was then recrystallised using THF to give product 18c (1.18 g, 50%). The physical and spectroscopic data is consistent with the literature values [22].

Synthesis of Benzyl Thiourea 13a-q
General Procedure C. The appropriate 2-thio-1,3-benzoxazines (1.7 mmole) 6, 10a,b, 11c, and 12a-m were suspended in a mixture of sodium bicarbonate (1 gm) and water (5 mL)/methanol (5 mL) with stirring, then the reaction mixture was warm to 40 ∘ C for few minutes then benzyl amine (4.25 mmol) was added dropwise, directly from the a pipette, left stirring at room temperature for 4 hours. At the completion of the reaction, the mixture was evaporated to dryness under reduced pressure and the pH was adjusted to 5-6 by using conc. HCl. The resulting solid was collected by vacuum filtration and washed with minimal water and recrystallized from an appropriate solvent.

Synthesis of 2-Benzyl amino-1,3-benzoxazines 15a-h
General Procedure D. The appropriate 2-methylthio-1,3benzoxazine 14a-h (2.5 mmol) was suspended in dry 1,4dioxane (10 mL) in a 50 mL round-bottomed flask. Benzyl amine (12.5 mmol) was then added dropwise, directly from the pipette, with stirring, and then the reaction mixture was heated to reflux for 4 hours. At the completion of the reaction, the reaction mixture was evaporated to dryness under reduced pressure and triturated with minimal diethyl ether. The resulting solid product 14 was collected by vacuum filtration and recrystallized from an appropriate solvent.
General Procedure E. N-(Benzyl carbamothioyl)-substituted-2-hydroxy-benzamides 13a, b, e, f, and g (0.5 mmol) were suspended in acetic acid (3 mL) in a 25 mL round-bottomed flask. The reaction mixture was heated to reflux for 2 hours then; the acetic acid was evaporated off under reduced pressure. The oily reaction mixture was triturated with minimal diethyl ether and the resulting solid products 15a, b, e, f, and g were collected by vacuum filtration and recrystallized from an appropriate solvent.
Products 15a, b, e, f, and g prepared in this procedure gave identical mp, IR, 1 H NMR and 13 C NMR to the analogues prepared from compound 14 with comparable yields (Scheme 3).

Synthesis of Substituted-1,3-benzoxazine-diones 22a-h and 25
General Procedure F. The appropriate substituted methylthio-1,3-benzoxazine 2.5 mmol was hydrolysed with 10 mL hydrochloric acid (10%) at 80 ∘ C for 4 hours. At the completion of the reaction, the reaction mixture was washed with R.O water, filtered, and recrystallised from an appropriate solvent. Products 22a-h were used in the synthesis of products 23a-h with no further purification.

2H-Benz
[e]-1,3-oxazin-2,4(3H)-dione 22a. 2-(Methylthio)-4H-benz[e]-1,3-oxazin-4-one 14a was allowed to react with hydrochloric acid (10%) according to general procedure F. The crude solid was collected and recrystallised from ethanol to give 22a (75% yield), mp 228 ∘ C. (lit. [19,20]   Each one of the different strains of bacteria were cultured into 10 mL of nutrient broth (NB) or malt extract broth for fungi and incubated overnight at 37 ∘ C except for Mycobacterium smegmatis at 30 ∘ C for 24 hours and Mycobacterium chlorophenolicum for 4 days. All fungal cultures were grown at 25 ∘ C for up to seven days except for Alternaria alternate, 3 days 35 ∘ C. The grown cultures were diluted (1/10 in NB or Malt extract broth) and incubated for a further 2 hours (to reach exponential phase) and then used in the MIC assay. Compound stock solutions of 10 4 g/mL were made up in DMSO ensuring that a maximum of 30 L is used, otherwise inhibitory effects will be shown on some bacterial/fungal cultures). In sterile microcentrifuge tubes, varying amounts of exponential phase culture (0.1 mL) were added and NB (0.9 mL) to make up a total volume of 1 mL. Each culture was then incubated at 37 ∘ C overnight. Control tubes were made using DMSO without the addition of compound. At the completion of the incubation, the microcentrifuge tubes containing culture were vortexed and compared to their respective controls (without compound). Compounds which displayed an absence of turbidity lower than 50 g/mL were subject to further dilutions, while if there is growth (or turbidity) at a particular concentration then the value is recorded as the MIC. The dilution series was carried out in factors of 2 as recommended (i.e., at 200, 100, 50, 25, 12.5, 6.25, and 3.125 g mL −1 resp.). The MIC was determined by the absence of turbidity at the lowest concentration.

Agar Disk Diffusion Method.
Compounds that were insoluble in DMSO or the NB were evaluated for their antimicrobial activity by agar diffusion assays. The surface of an NA or Malt agar plate was flood-inoculated with an overnight NB or malt broth culture of a particular culture adjusted to 10 8 CFU/mL (10 8 colony forming units per millimeter). Each disk contained a specific culture and sterile 12.7-mm paper disk (oxide) was placed onto the dry surface for each compound. The insoluble compound was resuspended in DMSO and a 20 L aliquot was impregnated onto the surface of a sterile paper disc including 20 L of DMSO control. The diameter of the zone of inhibition was measured after incubation for 18 hr and compared to the control zone (DMSO).