TOTAL SYNTHESIS AND ANTIMICROBIAL ACTIVITY EVALUATION OF NATURAL PRODUCTS AND THEIR ANALOGUES

The need to develop new antimicrobials remains a major driving force as microorganisms continue to develop resistance against existing antibiotics. Natural products are large reservoirs of new biologically active substances and most of the currently used antimicrobials have been developed from naturally occurring lead compounds. Aim of the PhD work was the study of new naturally derived antimicrobial compounds and the development of synthetic sequences which might, in principle, have value in the preparation of the natural products themselves as well as in synthesizing various analogues. Among a number of possible candidates we have selected three natural products, Leopolic acid A, Promysalin and Resormicyn. During the PhD period the synthesis of these natural products has been carried out, and the biological activity of the compounds has been evaluated. Key steps of the synthetic approach to Promysalin encompass an organocatalytic asymmetric \u3b1-hydroxylation of carbonyl compounds employed to fabricate the myristamide framework, and Superhydride\uae mediated reductive elimination of lactam to obtain the salicyldehydroproline fragment. Crucial steps for the leopolic acid synthetic strategy include a Dieckmann cyclization to obtain the 2,3-pyrrolidinedione ring and a Wittig olefination to install the polymethylene chain. Finally, the key steps in resormicyn synthetic approach include the late stage stereospecific dehydration of alcohol to install the Z-olefin and palladium catalyzed one-pot deprotection of allyl and alloc groups. The straightforward and modular nature of the developed syntheses has given easy access to the preparation of structurally related analogues and the biological activity of the compounds has been evaluated. Preliminary SAR have emerged for promysalin and leopolic acid

Total synthesis of the salicyldehydroproline-containing antibiotic promysalin

A convergent total synthesis of promysalin, a metabolite of Pseudomonas putida RW10S1 with antibiotic activity, is described. The synthetic approach is based around a salicyldehydroproline core and a dihydroxymyristamide fragment. Crucial steps include a MacMillan asymmetric \u3b1-hydroxylation applied for the construction of the myristamide framework, and a lactam reduction by Superhydride\uae to obtain the dehydroproline fragment. Because of the modular nature of the synthesis, ready access to analogues for biological evaluation is available

Antibacterial and antifungal activities of 2,3-pyrrolidinedione derivatives against oral pathogens

Among the novel approaches applied to antimicrobial drug development, natural product-inspired synthesis plays a major role, by providing biologically validated starting points. Tetramic acids, a class of natural products containing a 2,4-pyrrolidinedione ring system, have attracted considerable attention for their antibacterial, antiviral, antifungal and anticancer activities. On the contrary, compounds with a 2,3-pyrrolidinedione skeleton have been considerably less investigated. In this work, we established chemical routes to the substituted 2,3-pyrrolidinedione core, which enabled the introduction of a wide range of diversity. In the perspective of a potential application for oral healthcare, a number of analogues with various substituents on the 2,3-pyrrolidinedione core were investigated for their antimicrobial and antifungal activities. The most promising compound showed a significant antimicrobial activity on Streptococcus mutans and Candida albicans, comparable to that of chlorhexidine, the gold standard in oral healthcare

Synthesis of the Tripeptide Antibiotic Resormycin

A short and efficient synthesis of resormycin, a metabolite of Streptomyces platensis MJ953-SF5 with herbicidal and antifungal activity, is described. The key step in our synthetic approach is a late-stage stereospecific dehydration of a \u3b2-hydroxy amino acid to install the Z -olefin. Because of the modular nature of the synthesis, access to analogues for biological evaluation is readily available

Total synthesis and biological investigation of promysalin and analogues

Myristic acid serves as biosynthetic precursor of a plethora of secondary metabolites. These metabolites demonstrate a wide range of antibacterial and antivirulence activity ranging from the inhibition of biofouling and biofilm formation to the disruption of quorum sensing mechanism.1 The metabolites are active against both gram positive and negative bacteria. The rapid rise of antibiotic resistant superbugs justifies the need for identifying new compounds targeting specific pathogenic bacteria. In 2011 Wen Li and coworkers isolated the myristamide metabolite promysalin from rhizosphere of rice root associated Pseudomonas putida RW10S1.2 The natural product exhibits a submicromolar activity against Pseudomonas aeruginosa (PA) and some other gram negative bacteria. The compound also promotes swarming of the producer and its surface colonization. The most recent studies have demonstrated that promysalin disperses established biofilms and inhibits pyoverdine production. The unusual bioactivity, unknown mode of action and structural uniqueness increased the interest for the compound. Here in us report a convergent total synthesis of promysalin.3 The synthetic approach is based around a salicyldehydroproline core and a dihydroxymyristamide fragment. The key steps include a MacMillan asymmetric \u3b1-hydroxylation4 applied for the construction of the myristamide framework and one pot reduction of lactam with Superhydride and base mediated concomitant elimination of lactamol to obtain the dehydroproline fragment.5 The synthetic route can be easily adapted for the rapid generation of analogues

Approach to the total synthesis of new Pseudomonas antifungal metabolites containing a 2-pyrroline-5-carboxyl moiety

Recent studies on the bio control activity of plant-associated Pseudomonas have revealed their ability to produce a series of antagonistic molecules to compete with other microorganisms, in particular phytopathogenic fungi. In 2011 Wen Li & co-workers isolated a novel type of secondary metabolites produced by Pseudomonas putida RW10S1 to promote its own swarming and biofilm formation, and to selectively inhibit many other Pseudomonas, including multidrug resistant clinical isolates of the opportunistic human pathogen Pseudomonas aeruginosa (IC50 = 1.75 \ub5M). The amphipathic nature of these antibiotics, their role in bacterial quorum sensing and the unprecedented structural features, containing a unique 2-pyrroline-5-carboxyl moiety, enticed us to develop a synthetic sequence to this nucleus, which could be adopted for the construction of variously substituted compounds with the same skeleton. The proposed route is centred on the use of L-pyroglutamic acid as a chiral synthon for the synthesis of 2-pyrroline-5-carboxyl moiety. Key step of the sequence is the reduction of N-protected pyroglutamate by Super-Hydride, followed by dehydratation of the resulting lactamol

Development and validation of a liquid chromatographic enantiomer separation method for the estimation of (S)​-​enantiomer in sitagliptin

A direct chiral separation method development was carried out for sitagliptin and its (S)-enantiomer on ten diverse chiral stationary phases. Chiral stationary phases, which were tested include, R, R Whelk-O1 column, macrocyclic glycopeptides namely, Chirobiotic R (Ristocetin A), Chirobiotic V (Vancomycin), Chirobiotic T (Teicoplanin), Chirobiotic TAG (Teicoplanin aglycone), OJ-H, OJ-RH belonging to tris (4-methylbenzoate) of cellulose. Chiral selectivity was observed on polysaccharide based CSP columns namely Chiralpak IA-3, Chiralpak IC-3 belonging to tris-(3,5-dimethylphenyl carbamate) of amylose, tris-(3,5-dichlorophenyl carbamate) of cellulose respectively and OD-H (tris-3,5-dimethylphenylcarbamate) of cellulose. Better enantioselective separation has been achieved on cellulose tris-(3,5-dichlorophenyl carbamate) column (Chiralpak IC-3), using IPA and n-hexane as mobile phase, both containing 0.05% ethylene diamine and at 0.5 mL/min flow rate. Detection was carried out at 266nm using PDA detector and column maintained at 35\ubaC. The method was validated for precision, accuracy, linearity and robustness. The advantages of the method are rapid equilibration and less solvent consumption due to short column length. Efficient enantio separation (Resolution 3.38) is due to small particle size of 3 \u3bcm. Therefore this method is suitable for chiral purity determination of sitagliptin and its (S)-enantiomer

SAR studies on HDAC inhibitors featured by phenyl-4-yl-acrylohydroxamic acids scaffolds

Epigenetic mechanisms are crucial for normal development and maintenance of tissue-specific gene expression patterns in mammals. Aberrant epigenetic programming can lead to altered gene function and malignant cellular transformation. The main epigenetic modifications in mammals are DNA methylation and posttranslational histone modifications (acetylation, methylation, phosphorylation, etc.). The acetylation status of histones and non-histone proteins is determined by histone deacetylases (HDACs) and histone acetyl-transferases (HATs). Altered expression and mutations of genes that encode HDACs have been linked to tumor development since they both induce the aberrant transcription of key genes regulating important cellular functions (i.e. cell proliferation, cell-cycle regulation and apoptosis). Thus, HDACs are among the most promising therapeutic targets for cancer treatment, and they have inspired researchers to study and develop HDAC inhibitors.1 Inhibition of HDACs causes histone hyperacetylation with transcriptional activation of genes associated with cell cycle arrest or apoptosis in tumor cells. Typically, HDAC inhibitors contain three regions: a 'cap' region or 'surface recognition domain', a 'zinc-binding group', chelating the zinc ion in the active site, and a 'linker' region, connecting the two moieties.2 We have developed a series of hydroxamic acid-based compounds, characterized by a cinnamic spacer capped with a substituted phenyl group. Here we report the SAR studies on these compounds and our efforts to optimize the drug-target interaction by modification of the cap group and the ZBG-functionality. Whereas most of the candidates with alternative Zn-binding groups were less effective than the parent hydroxamic acid, selected compounds modified in the cap region showed an activity towards HDAC2 in the low \ub5M range, assessed through a fluorimetric assay. One of the compounds was further tested in vitro and in vivo in a colon carcinoma model and showed significant proapoptotic and antitumor activity

Highly efficient one-pot amination of carboxylate-substituted nitrogen-containing heteroaryl chlorides via Staudinger reaction

An efficient one-pot method for the synthesis of tert-butyl 6-aminonicotinate (5) is described. The key transformation involves displacement of the chloro group in tert-butyl 6-chloronicotinate (2) with azide followed by a Staudinger reaction. The scope of this methodology is further extended for the synthesis of a series of carboxylate-substituted heteroaryl amines. In particular, we synthesized tert-butyl carboxylate-substituted amino-pyridine, -pyridazine, and -pyrazine. In addition to one-pot conversion, short reaction time, simplicity of operation, ease of purification, and good yields are the key advantages of this methodology