Design and Synthesis of Novel N-Benzylidenesulfonohydrazide Inhibitors of MurC and MurD as Potential Antibacterial Agents

A series of novel N-benzylidenesulfonohydrazide compounds were designed and synthesized as inhibitors of UDP-N-acetylmuramic acid:L-alanine ligase (MurC) and UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) from E. coli, involved in the biosynthesis of bacterial cell-walls. Some compounds possessed inhibitory activity against both enzymes with IC50 values as low as 30 μM. In addition, a new, one-pot synthesis of amidobenzaldehydes is reported.


Introduction
The increasing emergence of pathogenic bacterial strains with high resistance to antibacterial agents constitutes a serious public health threat. Besides established classes of antimicrobial drugs (βlactams, macrolides, and quinolones), drugs considered to be the last line of defence (the glycopeptide vancomycin and the oxazolidinone linezolid) are becoming less effective. This serious situation strongly supports the search for novel antibacterial agents [1][2][3].
One of the most attractive targets for new antibacterial compounds is the bacterial peptidoglycan biosynthetic pathway. Peptidoglycan is an essential component of the bacterial cell wall. It is responsible for a defined cell shape and preserves cell integrity by compensating internal osmotic pressure. Any perturbation of the multi-step peptidoglycan biosynthesis may lead to cell lysis [4]. Peptidoglycan is formed as a linear chain of repeating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) units, interconnected by short peptide chains. Four ADP-forming ligases (MurC, MurD, MurE and MurF) catalyze the assembly of the peptide moiety by the successive additions of L-alanine, D-glutamate, meso-diaminopimelate (or L-lysine) and D-alanyl-D-alanine to UDP-MurNAc. These essential cytoplasmic enzymes are highly specific and are present only in eubacteria, thus making them attractive as targets for the development of new therapeutic agents against bacterial infections [5].
We  Benzylidene rhodanines 1 and 2 (Figure 2), which possess MurC inhibitory activity in the low micromolar range [7], were used as a starting point for further synthetic modification. They were first modified on the thioxothiazolidin-4-one ring, which was replaced with an acyclic aryl-substituted sulfonohydrazone moiety (Figure 3) that still retains the acidic NH group present in rhodanines. Similar acyclic hydrazones have recently been used by other authors for the synthesis of novel antibacterial compounds [8][9][10][11][12]. The p-chlorobenzylthio substituent was then replaced by different moieties to give the corresponding ethers 5a-i, sulfonates 7a-c, carboxamides 11a-j and carboxylic ester 14. In this paper, we report the synthesis and biological evaluation of compounds 5a-i, 7a-c, 11aj and 14.

Results and Discussion
The compounds with general structure 5a-i were synthesized starting from hydroxybenzaldehydes 3a or 3b, which were treated with the corresponding benzyl halides to yield compounds 4 in high yield (75-90%), except for 4b (13%). These were then readily coupled with the appropriate arylsulfonohydrazides [13], to give the corresponding compounds 5a-i in good yield (64-80%) ( Figure  4). Sulfonate derivatives 7a-c were synthesized in a similar manner, starting from the hydroxybenzaldehyde 3b, which was treated with the appropriate phenylsulfonyl chlorides to give 6ac in 57-68% yield, and then coupled with naphthalene-2-sulfonohydrazide hydrochloride to give the targets 7a-c in 46-80% yield.  The synthetic route selected for the preparation of inhibitors 11a-j ( Figure 5) involved the protection of para-(8a) and ortho-(8b) nitrobenzaldehydes as dioxolanes 9a and 9b, which should be deprotectable under mild acidic conditions. The reduction of dioxolanes 9a and 9b led to unstable 1,3dioxolan-2-yl anilines 15, that are prone to form polymers, in a manner similar to that reported for pnitrobenzyl esters by Wakselman and Guibé-Jampel [14]. Different reducing agents (SnCl 2 , Zn dust, sodium sulphides, catalytic hydrogenation) and solvents (water, acetic acid, methanol, THF) were tried, but the polymerization process (seen as the formation of an orange solid) started immediately and was enhanced by the presence of water, acid, or by heating ( Figure 6). The optimal method for reducing 9a or 9b was therefore catalytic hydrogenation in anhydrous THF (2 h, Pd/C as a catalyst, r.t., 5 bar). The product was immediately (without isolation and purification of 15a-b) treated with suitable benzoyl chlorides to give the corresponding amides with simultaneous deprotection of the formyl group. Purification of the crude product by column chromatography yielded amidobenzaldehydes 10a-j in moderate yields for most of the compounds. The condensation of aldehydes 10a-j with 2-naphthalene-sulfonohydrazide hydrochloride afforded compounds 11a-j. The method described in this article was appropriate only for the synthesis of amidobenzaldehydes. When sulfonyl chlorides were used as reagents instead of benzoyl chlorides (i.e. for the synthesis of sulfonamidobenzaldehydes) only the polymerization reaction occurred.
Our synthetic pathway presents a significant improvement in terms of reaction time, cost of reagents and overall yield, when compared to the previously reported synthesis of benzamides (related to 10) with potassium selenocarboxylates via azides [15]. In 2001, the synthesis of 15a from 9a by catalytic hydrogenation was described, however without mention of the polymerization reaction [16].    We also tried to prepare carboxylic ester derivatives in a manner analogous to the preparation of ethers 5a-i and sulfonates 7a-c. Virtually all efforts failed at the last step (i.e. condensation with 2naphthylsulfonohydrazide). We suspect that these aromatic esters hydrolyzed to alcohols and carboxylic acids due to our inability to remove water completely from the reaction mixture during the condensation. Therefore, only compound 14 was synthesized via the acylation of salicylaldehyde with activated 2-(1,3-benzodioxol-5-yl)acetic acid and subsequent condensation of the ester with 2naphthylsulfonhydrazide (Figure 7).

Biological Activity
Results of the in vitro testing of compounds 5a-i, 7a-c, 11a-j and 14 for inhibitory activity against MurC and MurD are presented in Table 1. They are given as residual activities (RA) of the enzyme in the presence of a 100-µM (or less in the case of low solubility) concentration of inhibitory compound.  IC 50 values were determined for the most active compounds. Several compounds possessed inhibitory activity against both ligases MurC and MurD, with the amide series (especially compounds 11f, 11h and 11j) being the most potent low-molecular-weight dual inhibitors of both enzymes reported to date. Lipophilic substituents such as halogens, cyano, nitro and methoxy are preferred on the distal phenyl ring and significantly enhance the activity when compared with compound 11a. Interestingly, the transformation of amide 11j to sulfonate 7c resulted in a significant decrease of inhibitory activity against MurC and in a moderate reduction of inhibitory activity against MurD. In the series of phenylbenzyl ethers not only Ar 1 was changed, but also Ar 2 , in order to gain compounds 5a-i, which are almost equally potent against both enzymes. Compounds 5a and 5b are the most potent inhibitors of MurD from this series. In general, the series of phenylbenzyl ethers had slightly weaker inhibitory activity against MurC and MurD than the amide series. In addition, with exception of 5h, the use of different Ar 2 moieties resulted in lower activity against both enzymes when compared with the analogue with a naphthalene ring, 5a. The optimal distance between the two phenyl rings was established to be two atoms. However, ester 14, which has an additional methylene group, was also active. During the determination of IC 50 values of compounds presented in this paper we noted that some inhibitors displayed high Hill coefficients [17]. Due to this inhibitory characteristic and the lipophilic properties of our inhibitors, there is the possibility that some of these compounds act as nonspecific binders.

Conclusions
In summary, we have described the synthesis and structure-activity relationship of a series of novel N'-benzylidenesulfonohydrazides as inhibitors of MurC and MurD and as potential lead compounds for the development of new antibacterial drugs. In addition, we report a new, rapid and efficient onepot synthesis of amidobenzaldehydes. The amide series provided the most potent dual inhibitors of both MurC and MurD ligases. Replacement of the amide functionality with an ester or benzylether group decreased the activity of compounds. Despite the fact that the series of phenylbenzyl ethers were slightly less active, they served as an indicator that inhibitory activity can be further enhanced with proper tuning of the Ar 1 and Ar 2 substituents. Further optimization of the reported inhibitors and in vitro determination of their MurC and MurD inhibitory activities are in progress and will be reported in due course.

General
Chemicals from Fluka and Sigma-Aldrich Chemical Co. were used without further purification. Anhydrous tetrahydrofuran and Et 3 N were dried and purified by distillation over Na and KOH, respectively. Analytical thin-layer chromatography (TLC) was performed on Merck silica gel (60F 254 ) plates (0.25 mm). Column chromatography was performed on silica gel 60 (Merck, particle size 240-400 mesh). Melting points were determined on a Reichert hot stage microscope and are uncorrected. 1 H-and 13 C-NMR spectra were recorded at 300 and 75 MHz, respectively, on a Bruker AVANCE DPX 300 spectrometer in DMSO-d 6 solutions, unless otherwise indicated, with TMS as internal standard. Chemical shifts were reported in ppm (δ) downfield from TMS. All the coupling constants (J) are in hertz. IR spectra were recorded on a Perkin-Elmer FTIR 1600 spectrometer. Mass spectra were obtained with a VG-Analytical Autospec Q mass spectrometer with EI or FAB ionization (MS Centre, Jožef Stefan Institute, Ljubljana). Elemental analyses were performed by the Department of Organic Chemistry, Faculty of Chemistry and Chemical Technology, Ljubljana, on a Perkin Elmer elemental analyzer 240 C. All reported yields correspond to yields of purified products.

General procedure for the synthesis of 2-(arylmethyloxy)benzaldehydes 4b-d
To a solution of the corresponding arylmethyl chloride (1 mmol) and salicylaldehyde (1 mmol) in THF (5 mL), triethylamine (1.25 mmol) and KF (2 mmol) were added with stirring and the mixture was refluxed under argon for 12 hours. The solvent was removed under reduced pressure. The residue was taken up in ethyl acetate (25 mL), washed sucessively with 10% citric acid (2×5 mL), saturated NaHCO 3 (2×5 mL) and brine (5 mL), and dried over Na 2 SO 4 . After filtration and evaporation of the solvent in vacuo the crude product was purified by column chromatography or recrystallization, as indicated.

General procedure for the synthesis of 2-formylphenyl benzenesulfonate derivatives 6a-c
To a solution of salicylaldehyde (1 mmol) and Et 3 N (1.5 mmol) in THF (1 mL) an appropriate benzenesulfonyl chloride derivative (1 mmol) was added at 0 °C. The solution was stirred for 12 hours at room temperature. The solvent was removed under reduced pressure. The residue was taken up in ethyl acetate (25 mL), washed with 10% citric acid (2×5 mL), saturated NaHCO 3 (2×5 mL) and brine (5 mL), and dried over Na 2 SO 4 . After filtration and evaporation of solvent in vacuo the crude product was purified by recrystallization from Et 2 O.  General procedure for the synthesis of 2-(nitrophenyl)-1,3-dioxolanes 9a and 9b The corresponding nitrophenyl-1,3-dioxolanes 9a and 9b were prepared by a reported method [16]. Suitable nitrobenzaldehyde (5.76 g, 38.1 mmol), 4-toluenesulfonic acid monohydrate (0.24 g, 1.26 mmol) and ethylene glycol (13.2 g, 213 mmol) were dissolved in toluene (40 mL). After the addition of ground 4 Å molecular sieves (1.00 g) the solution was refluxed for 6 hours. After cooling, the mixture was partitioned between toluene and distilled water (50 mL) and the aqueous layer extracted again with toluene (2×20 mL) and ethyl acetate (30 mL). The combined organic layers were washed with brine (40 mL), dried over Na 2 SO 4 , and the solvent removed in vacuo.

General procedure for the synthesis of N-(formylphenyl)benzamide derivatives 10a-j
The corresponding 2-(nitrophenyl)-1,3-dioxolanes 9a or 9b (2.00 g, 10.2 mmol) were dissolved in anhydrous THF (50 mL) and Pd/C (200 mg) was added. The resulting solution was hydrogenated in a Parr apparatus at 5 bar for 2 hours, filtered and immediately used for further reaction. The appropriate benzoyl chloride derivative (10.2 mmol) and K 2 CO 3 (10.2 mmol) were then added and the mixture was stirred for 3 hours. The solvent was removed under reduced pressure. The residue was taken up in ethyl acetate (100 mL), washed with 10% citric acid (2×20 mL), saturated NaHCO 3 (2×20 mL) and brine (20 mL) and dried over Na 2 SO 4 . After filtration and evaporation of the solvent in vacuo the crude product was purified by column chromatography or by recrystallization from Et 2 O, as indicated.