New Inhibitors of bacterial hyaluronidase - Synthesis and structure-activity relationships

Potent and selective inhibitors of hyaluronidases are not reported in the literature so far. Such compounds are required as pharmacological tools to investigate the physiological and pathophysiological role of the enzymes and their preferred substrate, hyaluronic acid. Moreover, such inhibitors might be of therapeutic value, e.g. in the treatment of arthrosis, as new anti-fertility agents or as an additivie antimicrobial treatment. The main goal of this thesis was the synthesis and identification of lead-like compounds and their structural optimization as inhibitors of bovine testicular hyaluronidase (BTH) and hyaluronate lyase from S. agalactiae (hylB4755). The inhibitory activities on both hyaluronidases were determined by means of a turbidimetric assay and the structure-activity relationships were elaborated. L-Ascorbic acid was found to weakly inhibit hyaluronate lyase from S. pneumoniae (hylSpn). X-ray analysis of the vitamin C-hylSpn complex revealed that vitamin C binds to the active site of the bacterial enzyme. Therefore, as a part of this doctoral project a series of L-ascorbic acid derivatives was synthesized and investigated for inhibitory activity against BTH and hylB4755. Four 6-O-acyl derivatives of L-ascorbic acid were identified as moderate to strong inhibitors of hyaluronidases with preference for the bacterial enzyme. At optimum pH (pH 5) the IC50 values for inhibition of hylB4755 range from 0.9 µM to 475 µM with highest activity for the esters of the long chain alkanoic acids. Among the studied vitamin C derivatives, L-ascorbic acid-6-octadecanoate proved to be the most potent inhibitor of hylB4755 and BTH with IC50 values of 0.9 µM and 39 µM at pH 5, respectively. The octadecanoate is up to 6500 times more potent than the parent compound vitamin C, and is the most potent inhibitor of bacterial and among the most potent inhibitors of bovine testicular hyaluronidase described to date. Additionally, the binding mode of L-ascorbic acid-6-hexadecanoate at hylSpn was determined by X-ray analysis. Starting from a de novo substrate-based inhibitor design 1,3-diacetylbenzimidazole-2-thione was identified as lead compound for hylB4755 (IC50 values of 5 µM and 160 µM at physiological pH and optimum pH, respectively). The investigation of structurally diverse benzimidazoles derived from the lead revealed that N-monoacylated and N,N�-diacylated benzimidazole-2-thiones inhibit the bacterial enzyme hylB4755 but not the BTH. For N-monoacylated derivatives IC50 values in the low µM range were determined at physiological pH, whereas the N,N�-diacylated compounds showed strong inhibition of hylB4755 both at the pH optimum and at physiological pH. Owing to their structural similarity with the aforementioned benzimidazoles corresponding benzoxazoles and indoles were taken into consideration. Based on the X-ray analysis of hylSpn co-crystallized with 1-decyl-2-(4-sulfamoyloxyphenyl)-1H-indol-6-yl-sulfamate the putative binding mode of benzoxazole-2-thiones was suggested. To support this model a series of benzoxazole derivatives was synthesized and investigated for hyaluronidase inhibition. All tested compounds were highly selective for hylB4755 vs. BTH. 3-Acylated benzoxazole-2-thiones inhibited hylB4755 with IC50 values in the µM range. Interestingly, the thiones were much more potent than the corresponding oxo analogues. Within the latter series, 3-phenyl-1-(2-thioxobenzo[d]oxazol-3(2H)-yl)propan-1-one was the strongest inhibitor of hylB4755 with an IC50 value of 15 µM at optimum pH. The 3-phenylpropanoyl group proved to be the optimal substituent at the benzoxazole-N. This is in good agreement with the suggestions from molecular modeling studies. Replacement of the alkanoyl side chain by a sulfonyl or an alkyl group or incorporation of a benzofurane instead of the benzoxazole moiety resulted in a marked decrease in inhibition of hylB4755. Based on the analysis of the binding mode of the aforementioned 2-phenylindole a set of additional indole derivatives was prepared and screened for activity on hylB4755 and BTH. Generally, the investigated indoles showed a preference for the bacterial hyaluronidase with highest activity residing in three N-alkylated 5-amino-2-methyl-indoles (inhibitory activities on hylB4755 in the micromolar range at optimum pH). Different functional groups such as nitro, amino, urea, sulfonamide, carboxylic acid and sulfonic acid were introduced in position 5 of the indole scaffold. As a result high inhibitory activity only resides in indoles substituted with amino or acidic groups. Surprisingly, the N-acylated 5-amino-2-methylindoles had only minor inhibitory activities. The synthesized 1,3-disubstituted 5-amino-2-methyl-indoles weakly inhibited hylB4755. Compared to the benzoxazole-2-thione the indole structure proved to be a less promising scaffold for the synthesis of hyaluronidase inhibitors.