Short and Efficient Synthesis of Alkyl- and Aryl-Ortho-Hydroxy-Anilides and their Antibiotic Activity

Abstract Ortho-hydroxy-anilides are part of natural products like the new antibiotics platencin (A) and platensimycin (B). An important step in the total synthesis of these antibiotics or their derivatives is the preparation of the o-hydroxy-anilide partial structure. The presented method allows the preparation of o-hydroxy-anilides and o-dihydroxy-anilides from 2-nitrophenol esters in a one-step synthesis without protecting the hydroxy group. Aryl- and alkyl-anilides were prepared following this method as simple analogues of platensimycin (A). The resulting compounds were tested in an agar diffusion assay for their antibiotic potency.


Introduction
The 3-amino-2,4-dihydroxybenzoic acid core is an essential part of the new antibiotic drugs platencin (A) and platensimycin (B) [ Figure 1], which show a high activity against The total synthesis of both natural products is long and expensive. In the last few years, a lot of total syntheses of platencin (A) and platensimycin (B) were published. Even the synthesis of the 3-amino-2,4-dihydroxy benzoic acid partial structure, which is essential for binding to the enzyme FabF ( Figure 2), takes several steps in these total syntheses [5][6][7][8][9][10][11][12][13]. In continuation of our work on simple platensimycin analogues [14,15], we hereby present the short and effective preparation of the o-hydroxy-anilide partial structure without the requirement of a protecting group.

Results and Discussion
In the first series, commercially available 4-hydroxy-3-nitrobenzoic acid (1) was esterified with methanol / H 2 SO 4 following a standard protocol to give the methyl ester 2 [14]. The phenol group of the methyl ester 2 was esterified with several aromatic or aliphatic carboxylic acid chlorides to give the phenol esters 3a-h. The following hydrogenation of the nitro group with Pd/C (5%) in methanol led to the amino group which reacted in the same procedure under aminolysis of the phenol ester to the resulting anilides 4a-g [14][15][16][17][18]. The olefin partial structures of 3a and 3f were hydrogenated under these conditions but the halogen substituent of 3b was stable. Reaction of 3h led only to a mixture of the products that couldn´t be separated by flash column chromatography. Exemplarily, two of the methyl esters were hydrolyzed to give the benzoic acid derivatives 5a and 5g as found in the natural products platencin (A) and platensimycin (B).
In the second series, 2-nitroresorcinol (6) was esterified with a half equivalent of acyl chloride to give the monoesters 7a-h. As a byproduct, a remarkable amount of the diesters was observed even when using an excess of 2-nitroresorcinol (6), but the diesters could be separated clearly by preparative flash column chromatography. The resulting esters 7a-h were hydrogenated in the way described above to give the anilides 8a-h in high yields (80-90% The mechanism of the aminolysis of the phenol esters is an intramolecular rearrangement as shown by the hydrogenation of an equimolar mixture of 3c and 7h and a subsequent GC-MS analysis. The gas chromatogram showed only two peaks of the products 4c and 8h and no peak of 8c or methyl 3-benzamido-4-hydroxybenzoate.

Tab. 1.
Agar diffusion assay 100 µg / disc, (te: tetracycline, cl: clotrimazol 30 µg/disc); GI (growth inhibition), nt: not tested, zone of inhibition [mm] The resulting compounds were tested in an agar diffusion assay [21] against several bacteria (Gram-positive and Gram-negative) and fungi, but showed only weak or no antibiotic activities in this assay as shown exemplarily for some compounds in Table 1.
Only the precursor 2 showed an interesting activity against bacteria and fungi.

Conclusion
The presented synthesis describes a simple and efficient method to prepare o-hydroxyanilides directly from o-nitrophenol esters under mild conditions and without affording any protecting group. Thus it is a helpful tool in preparing platensimycin analogues or other natural products containing this partial structure.
The tested compounds themselves showed no or only weak antibiotic activity as shown exemplarily for some compounds (Table 1). This indicates that the o-hydroxy-anilide partial structure is not the determining factor alone for the interaction with the FabF enzyme of platencin or platensimycin. The complex cyclic part is also essential for the high antibiotic activity of these natural products.

General Methods
All solvents used were of HPLC grade or p.a. grade and/or purified according to standard procedures. Chemical reagents were purchased from Sigma Aldrich (Schnelldorf, Germany) and Acros (Geel, Belgium).

General Procedure 1 (Preparation of Phenol Esters)
One mmol to 2.0 mmol of the acid were dissolved in 20 mL dry toluene or dry 1,2-dimethoxyethane and 1.0 mL to 2.0 mL (11.5 mmol to 27.5 mmol) SOCl 2 were added. The solution was refluxed for 1 h, the solvent was evaporated, and the residue was dissolved in 25 mL toluene or 1,2-dimethoxyethane. Alternatively, 0.5 to 1.5 mmol of the commercially available acid chlorides were used. One mmol of the phenol or 1 mmol of the 2-nitroresorcinol and 5 mL N-ethyl-N-methyl-ethanamine or 5 mL pyridine were added and the solution was stirred for 12 h at room temperature. The solvent was evaporated and the residue was taken up in 30 mL water (for the methyl esters in 30 mL 10% aqueous NaOH) and 30 mL ethyl acetate or diethyl ether. The organic layer was separated and the aqueous layer was again extracted with 30 mL ethyl acetate or diethyl ether, the combined organic layers were dried over Na 2 SO 4 , the solvent was evaporated and the residue was purified by flash column chromatography (isohexane/ethyl acetate 8-10:1).
Alternatively, the reaction could be carried out in a microwave reactor at 80°C for 15 minutes and 235 W, but in most cases with lower yields. Sci Pharm. 2014; 82: 501-517

General Procedure 2 (Hydrogenation)
One mmol of the phenol ester was dissolved in 30 mL methanol and 50 mg 5% Pd on charcoal were added. The suspension was stirred for 14 h under H 2 atmosphere at room temperature, the catalyst was filtered off (over silica gel 60), and the solvent was evaporated. If necessary, the residue was purified by flash column chromatography.

General Procedure 3 (Ester Hydrolysis)
An amount of 0.5 mmol of the ester were dissolved in 30 mL methanolic K 2 CO 3 solution (5%) and refluxed for 24 h. The solvent was evaporated, the residue dissolved in aqueous HCl (10%), and extracted with ethyl acetate (3 × 30 mL). The combined organic layers were dried over Na 2 SO 4 and the solvent was evaporated. If necessary, the residue was purified by flash column chromatography.