The 4-aza-S-ribosyl-L-homocysteine derivatives and the related gamma-lactam and azahemiacetal analogs: Synthesis, inhibition and quorum sensing activity

Quorum sensing (QS) is a population-dependent signaling process bacteria use to control multiple processes including virulence, critical for establishing infection. There are two major pathways of QS systems. Type 1 is species specific or intra-species communication in which N-acylhomoserine lactones (Gram-negative bacteria) or oligopeptides (Gram-positive bacteria) are employed as signaling molecules (autoinducer one). Type 2 is inter-species communication in which S-4,5-dihydroxy-2,3-pentanedione (DPD) or its borate esters are used as signaling molecules. The DPD is biosynthesized by LuxS enzyme from S-ribosylhomocysteine (SRH). Recent increase in prevalence of bacterial strains resistant to antibiotics emphasizes the need for the development of new generation of antibacterial agents. Interruption of QS by small molecules is one of the viable options as it does not affect bacterial growth but only virulence, leading to less incidence of microbial resistance. Thus, in this work, inhibitors of both N-acylhomoserine lactone (AHL) mediated intra-species and LuxS enzyme, involved in inter-species QS are targeted. The γ-lactam and their reduced cyclic azahemiacetal analogs, bearing the additional alkylthiomethyl substituent, were designed and synthesized targeting AHL mediated QS systems in P. aeruginosa and Vibrio harveyi. The γ-lactams with nonylthio or dodecylthio chains acted as inhibitors of las signaling in P. aeruginosa with moderate potency. The cyclic azahemiacetal with shorter propylthio or hexylthio substituent were found to strongly inhibit both las and rhl signaling in P. aeruginosa at higher concentrations. However, lactam and their azahemiacetal analogs were found to be inactive in V. harveyi QS systems. The 4-aza-S-ribosyl-L-homocysteine (4-aza-SRH) analogs and 2-deoxy-2-substituted-S-ribosyl-L-homocysteine analogs were designed and synthesized targeting Bacillus subtilis LuxS enzyme. The 4-aza-SRH analogs in which oxygen in ribose ring is replaced by nitrogen were further modified at anomeric position to produce pyrrolidine, lactam, nitrone, imine and hemiaminal analogs. Pyrrolidine and lactam analogs which lack anomeric hydroxyl, acted as competitive inhibitors of LuxS enzyme with KI value of 49 and 37 µM respectively. The 2,3-dideoxy lactam analogs were devoid of activity. Such findings attested the significance of hydroxyl groups for LuxS binding and activity. Hemiaminal analog of SRH was found to be a time-dependent inhibitor with IC50 value of 60 µM.

Synthesis of multisubstituted halo -olefins via Pd-catalyzed cross -coupling reactions. Applications in nucleoside chemistry

The enzyme S-adenosyl-L-homocysteine (AdoHcy) hydrolase effects hydrolytic cleavage of AdoHcy to adenosine (Ado) and L-homocysteine (Hcy). The cellular levels of AdoHcy and Hcy are critical because AdoHcy is a potent feedback inhibitor of crucial transmethylation enzymes. Also, elevated plasma levels of Hcy in humans have been shown to be a risk factor in coronary artery disease. ^ On the basis of the previous finding that AdoHcy hydrolase is able to add the enzyme-sequestered water molecule across the 5',6'-double bond of (halo or dihalohomovinyl)-adenosines causing covalent binding inhibition, we designed and synthesized AdoHcy analogues with the 5',6'-olefin motif incorporated in place of the carbon-5' and sulfur atoms. From the available synthetic methods we chose two independent approaches: the first approach was based on the construction of a new C5'-C6' double bond via metathesis reactions, and the second approach was based on the formation of a new C6'-C7' single bond via Pd-catalyzed cross-couplings. Cross-metathesis of the suitably protected 5'-deoxy-5'-methyleneadenosine with racemic 2-amino-5-hexenoate in the presence of Hoveyda-Grubb's catalyst followed by standard deprotection afforded the desired analogue as 5' E isomer of the inseparable mixture of 9'R/S diastereomers. Metathesis of chiral homoallylglycine [(2S)-amino-5-hexenoate] produced AdoHcy analogue with established stereochemistry E at C5'atom and S at C9' atom. The 5'-bromovinyl analogue was synthesized using the bromination-dehydrobromination strategy with pyridinium tribromide and DBU. ^ Since literature reports on the Pd-catalyzed monoalkylation of dihaloalkenes (Csp2-Csp3 coupling) were scarce, we were prompted to undertake model studies on Pd-catalyzed coupling between vinyl dihalides and alkyl organometallics. The 1-fluoro-1-haloalkenes were found to undergo Negishi couplings with alkylzinc bromides to give multisubstituted fluoroalkenes. The alkylation was trans-selective affording pure Z-fluoroalkenes. The highest yields were obtained with PdCl 2(dppb) catalyst, but the best stereochemical outcome was obtained with less reactive Pd(PPh3)4. Couplings of 1,1-dichloro-and 1,1-dibromoalkenes with organozinc reagents resulted in the formation of monocoupled 1-halovinyl product. ^

Design and synthesis of S-ribosylhomocysteine analogues

Bacteria are known to release a large variety of small molecules known as autoinducers (AI) which effect quorum sensing (QS) initiation. The interruption of QS effects bacterial communication, growth and virulence. ^ Three novel classes of S-ribosylhomocysteine (SRH) analogues as potential inhibitors of S-ribosylhomocysteinase (LuxS enzyme) and AI-2 modulators of QS were developed. The synthesis of 2-deoxy-2-bromo-SRH analogues was attempted by coupling of the corresponding 2-bromo-2-deoxypentafuranosyl precursors with the homocysteinate anion. The displacement of the bromide from C2 rather than the expected substitution of the mesylate from C5 was observed. The synthesis of 4-C-alkyl/aryl-S-ribosylhomocysteine analogues involved the following steps: (i) conversion of the D-ribose to the ribitol-4-ulose; (ii) diastereoselective addition of various alkyl or aryl or vinyl Grignard reagents to 4-ketone intermediate; (iii) oxidation of the primary hydroxyl group at C1 followed by the intramolecular ring closure to the corresponding 4-C-alkyl/aryl-substituted ribono-1,4-lactones; (iv) displacement of the activated 5-hydroxyl group with the protected homocysteinate. Treatment of the 4-C-alkyl/aryl-substituted SRH analogues with lithium triethylborohydride effected reduction of the ribonolactone to the ribose (hemiacetal) and subsequent global deprotection with trifluoroacetic acid provided 4-C-alkyl/aryl-SRHs. ^ The 4-[thia]-SRH were prepared from the 1-deoxy-4-thioribose through the coupling of the &agr;-fluoro thioethers (thioribosyl fluorides) with homocysteinate anion. The 4-[thia]-SRH analogues showed concentration dependent effect on the growth on las (50% inhibitory effect at 200 µg/mL). The most active was 1-deoxy-4-[thia]-SRH analogue with sufur atom in the ring oxidized to sulfoxide decreasing las gene activity to approximately 35% without affecting rhl gene. Neither of the tested compounds had effect on bioluminescence nor on total growth of V. harveyi, but had however slight inhibition of the QS.^

Synthesis of multisubstituted halo-olefins via Pd-catalyzed cross-coupling reactions : applications in nucleoside chemistry

The enzyme S-adenosyl-L-homocysteine (AdoHey) hydrolase effects hydrolytic cleavage of AdoHcy to adenosine (Ado) and L-homocysteine (Hcy). The cellular levels of AdoHcy and Hcy are critical because AdoHcy is a potent feedback inhibitor of crucial transmethylation enzymes. Also, elevated plasma levels of Hcy in humans have been shown to be a risk factor in coronary artery disease. On the basis of the previous finding that AdoHcy hydrolase is able to add the enzyme-sequestered water molecule across the 5',6'-double bond of (halo or dihalohomovinyl)-adenosines causing covalent binding inhibition, we designed and synthesized AdoHcy analogues with the 5',6'-olefin motif incorporated in place of the carbon-5' and sulfur atoms. From the available synthetic methods we chose two independent approaches: the first approach was based on the construction of a new C5'- C6' double bond via metathesis reactions, and the second approach was based on the formation of a new C6'-C7' single bond via Pd-catalyzed cross-couplings. Cross-metathesis of the suitably protected 5'-deoxy-5'-methyleneadenosine with racemic 2-amino-5-hexenoate in the presence of Hoveyda-Grubb's catalyst followed by standard deprotection afforded the desired analogue as 5'E isomer of the inseparable mixture of 9'RIS diastereomers. Metathesis of chiral homoallylglycine [(2S)-amino-5-hexenoate] produced AdoHcy analogue with established stereochemistry E at C5'atom and S at C9' atom. The 5'-bromovinyl analogue was synthesized using the brominationdehydrobromination strategy with pyridinium tribromide and DBU. Since literature reports on the Pd-catalyzed monoalkylation of dihaloalkenes (Csp2-Csp3 coupling) were scarce, we were prompted to undertake model studies on Pdcatalyzed coupling between vinyl dihalides and alkyl organometallics. The 1-fluoro-1- haloalkenes were found to undergo Negishi couplings with alkylzinc bromides to give multisubstituted fluoroalkenes. The alkylation was trans-selective affording pure Zfluoroalkenes. The highest yields were obtained with PdCl 2(dppb) catalyst, but the best stereochemical outcome was obtained with less reactive Pd(PPh3)4 . Couplings of 1,1- dichloro-and 1,1-dibromoalkenes with organozinc reagents resulted in the formation of monocoupled 1-halovinyl product.