Synthesis of Novel 2-Amino-N-hydroxybenzamide Antimicrobials

Abstract New 2-amino-N-hydroxybenzamide derivatives (3a–j) have been synthesized in good to excellent yields from the reaction of isatoic anhydride with different hydroxamic acids. All the compounds of the series were screened against both Gram-positive and Gram-negative bacteria and fungi, and some of them have antibacterial and antifungal activities compared to the standard drugs. GRAPHICAL ABSTRACT

In view of their potential medical applications, we have synthesized some 2-amino-N-hydroxybenzamide analogs that are capable of acting as multidentate siderophores and thus antimicrobial agents. A solvent-and catalyst-free methodology with mild operating conditions involving simple separation and purification of the products was designed and accomplished. [28][29][30]

Chemistry
The chemical advantages shown by isatoic anhydride as precursor for a variety of medicinally important derivatives lie mainly in its capacity to react at two  fundamental sites: (i) the C-8 position of the aromatic ring through electrophilic aromatic substitution and (ii) the more electrophilic C-4 carbonyl group though nucleophilic substitution reaction by losing a CO 2 group. In addition, it may also react through the N atom due to its nonbonded electrons to give some useful products.
Initially we studied the variations in product yields of 3a and their reaction times at various temperatures with different organic solvents such as EtOH, tetrahydrofuran (THF), CHCl 3 , toluene, and water and then with CH 3 CN by reacting simple isatoic anhydride (1a) and acetohydroxamic acid (2a) ( Table 1). Finally the reaction proceeded well in CH 3 CN at room temperature and hence these conditions are selected as the optimized experimental conditions. Different hydroxamic acids (2a-f) were synthesized from the corresponding carboxylic acids in a two-step reaction procedure [31] and used as substrates to accomplish the title compounds (3a-j) ( Table 2). Both aliphatic and aromatic hydroxamic acids gave good yields with isatoic anhydride and chloro-substituted isatoic anhydrides in CH 3 CN even at room temperature. Scheme 1. Synthesis of 2-amino-N-hydroxy benzamide derivatives (3a-j).  The possible mechanism for the reaction is ring opening of isatoic anhydride through decarboxylation. Hydroxamic acid itself acts as a proton donor and the nitrogen atom present in the hydroxamic acid act as a good nucleophile. Because of the more electrophilic nature of anthranilic C-4 carbonyl carbon, the nucleophilic nitrogen of hydroxamic acid attacks at C-4 instead of C-2 (Scheme 2). Moreover, strong nucleophilicity of hydroxamic acid favors subsequent decarboxylation in a smoother way even at mild conditions and forms the products in good yield. This is a better procedure because the previous reports used either a catalyst or harsh reaction condition.
All the synthesized compounds were fully characterized on the basis of their physical and spectral (infrared [IR], NMR, and mass spectrographic [MS]) data. They showed strong IR absorption bands at 3490-3300, 3230-3170, 1732-1680, 1612-1590, and 2945-2820 cm À1 for the amino, hydroxy, carbonyl, aromatic -C=C-, -CH 2 -, and -CH 3 groups respectively. Their 1 H NMR spectrum gave three multiplets at d 6.78-9.40, which are assigned to aromatic protons, and a singlet at d 2.0-2.6 corresponding to hydroxyl proton. Another singlet at d 6.20-6.70 for two amino protons and the singlet at d 1.

Pharmacology
All the synthesized compounds were screened for antimicrobial activity by the disc diffusion technique. The antibacterial activity of the compounds was evaluated against two Gram-positive bacteria, Staphylococcus aureus and Bacillus subtilis; two Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa; and two fungi, Candida albicans and Candida non-albicans. The test solutions were prepared with a concentration of 10 mg=mL of the compound in dimethylsulfoxide (DMSO) in order to obtain a final concentration of 1 mg=0.1 mL.
Test results are shown in Table 3 and it reveals that among ten synthetic compounds, 3e exhibited the greatest antibacterial activity against three test bacterial species, Bacillus subtilis  The structure-activity relationship correlated from the antimicrobial activity results obtained for the title compounds and their corresponding chemical structures reveals that the compounds having N-benzoyl substitution exhibited greater antimicrobial activity than the compounds having N-acetyl substitution. It is due to the fact that the π electron delocalization enhances proton-donating capacity of the N-hydroxy group, and the multidentate nature of the compound easily facilitates their interaction with the microbes and hence inhibits their growth to act as good antimicrobial agents.

EXPERIMENTAL Chemistry
Chemicals were procured from Sigma Aldrich and used as such without further purification. All solvents used for the spectroscopic and other physical studies were reagent grade and were further purified. Melting points were determined using a calibrated thermometer by Guna digital melting-point apparatus. They are expressed in degrees centigrade ( C) and are uncorrected. IR spectra were recorded as neat samples on Bruker Alpha-Eco ATR-FTIR (attenuated total reflection-Fourier transform infrared) interferometer with single-reflection sampling module equipped with ZnSe crystal and reported in reciprocal centimetres (cm À1 ). 1 H and 13 C NMR spectra were recorded as solutions in DMSO-d 6 on a Bruker AMX 500-MHz spectrometer operating at 400 MHz for 1 H and 100 MHz for 13 C NMR. The 1 H and 13 C chemical shifts were expressed in d with reference to tetramethylsilane (TMS) as an internal standard. Electron impact (EI) and high-resolution mass spectrographic (HRMS) data were recorded on a VG 7070 H ev instrument at 70 ev.

General Method for Synthesis of 2-Amino-N-hydroxy-Nacetylbenzamide (3a)
Isatoic anhydride (1 mmol) and acetohydroxamic acid (1 mmol) were taken in a 50-ml round-bottomed flask, and 10 mL of acetonitrile was added. The contents were stirred magnetically for about 3 h at room temperature. After completion of the reaction as indicated by thin-layer chromatography (TLC) using SiO 2 as adsorbent and 4:6 ethyl acetate=n-hexane as eluent, the crude product was separated by filtration and purified by recrystallization from EtOH. The other compounds (3b-j) were prepared by the same procedure.

Pharmacology
In vitro antimicrobial activity. The identified test bacterial strains were retrieved from À80 C freezers, thawed, streaked onto nutrient agar (NA), and checked for purity after incubation for 24 h at 30 C. A loop full of each test strain was inoculated in 10 mL of N-broth separately and was incubated for 24 h in an incubator at 37 C to activate the bacterial strain. [32] Following the pour plate technique, 0.2 mL (10 6 cells per 1 mL) of the activated strain was inoculated into the nutrient agar medium on reaching 40-45 C and allowed to solidify. This complete procedure was done in a laminar airflow to maintain strict sterile and aseptic conditions. After solidification of the media, a well was made in the media with the help of a cup-borer (0.85 cm) and then 0.1 mL of the synthetic compound (dissolved in DMSO) was inoculated into the well. For each test compound against each test strain, three replicas were maintained. Also, controls were maintained for each test bacterial strain where 0.1 ml of only the pure solvent was inoculated into the well. The plates were incubated for 24 h at 37 C. The inhibition zone formed by test compounds against the particular test bacterial strain was observed, and the mean value of the three individual replicates was calculated to determine the zone of growth inhibition of each test compound. [33] The antifungal activity of the synthetic compounds at a concentration of 10 mg=mL in DMSO was tested in vitro against two fungi Candida albicans and Candida non-albicans using the same diffusion technique applied in case of determining antibacterial activity with slight modifications. By following the pour plate technique, 0.1 mL (10 4 cells per 1 mL) of the activated strain was inoculated into the potato dextrose agar medium on reaching 40-45 C and allowed to solidify. This complete procedure was done in a laminar airflow to maintain strict sterile and aseptic condition. After solidification of the media, a well was made in the media with the help of a cup-borer (0.85 cm diameter) and then 0.1 mL of the synthetic compound (dissolved in DMSO) was inoculated into the well. For each test compound against each test strain, three replicas were maintained. Also, controls were maintained for each test bacterial strain where 0.1 mL of only the pure solvent was inoculated into the well. The plates were incubated for 72 h at 30 C. The inhibition zone formed by test compounds against the particular test bacterial strain was observed, and the mean value of the three individual replicates was calculated to determine the zone of growth inhibition of each test compound. Ampotercine B was used in standard antifungal agent. [33] CONCLUSION A series of new 2-amino-N-hydroxybenzamide derivatives (3a-j) has been synthesized in good to excellent yields, and their antimicrobial potentiality has identified 3e and 3 h as potential antibacterial compounds and 3a and 3d as potential antifungal compounds.