4-N-Alkanoyl and 4-N-alkyl gemcitabine analogues with NOTA chelators for 68-gallium labelling.

The conjugation of 4-N-(3-aminopropanyl)-2'-deoxy-2',2'-difluorocytidine with 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bn-NOTA) ligand in 0.1 M Na2CO3 buffer (pH 11) at ambient temperature provided 4-N-alkylgemcitabine-NOTA chelator. Incubation of latter with excess of gallium(III) chloride (GaCl3) (0.6 N AcONa/H2O, pH = 9.3) over 15 min gave gallium 4-N-alkylgemcitabine-NOTA complex which was characterized by HRMS. Analogous [68Ga]-complexation of 4-N-alkylgemcitabine-NOTA conjugate proceeded with high labeling efficiency (94%-96%) with the radioligand almost exclusively found in the aqueous layer (∼95%). The high polarity of the gallium 4-N-alkylgemctiabine-NOTA complex resulted in rapid renal clearance of the 68Ga-labelled radioligand in BALB/c mice.

S-Ribosylhomocysteine Analogues Modified at the Ribosyl C-4 Position.

4-C-Alkyl/aryl-S-ribosylhomocysteine (SRH) analogues were prepared by coupling of homocysteine with 4-substituted ribofuranose derivatives. The diastereoselective incorporation of the methyl substituent into the 4 position of the ribose ring was accomplished by addition of methylmagnesium bromide to the protected ribitol-4-ulose yielding the 4-C-methylribitol in 85% yield as single 4R diastereomer. The 4-C hexyl, octyl, vinyl, and aryl ribitols were prepared analogously. Chelation controlled addition of a carbanion to ketones from the (Si-face) was responsible for the observed stereochemical outcome. Oxidation of the primary alcohol of the 4-C ribitols with the catalytic amount of tetrapropylammonium perruthenate in the presence of N-methylmorpholine N-oxide produced 4-C-alkylribono-1,4-lactones in high yields. Mesylation of the latter compounds at the 5-hydroxyl position and treatment with a protected homocysteine thiolate afforded protected 4-C-alkyl/aryl-SRH analogues as the lactones. Reduction with lithium triethylborohydride and successive global deprotections with TFA afforded 4-C-alkyl/aryl SRH analogues. These analogues might impede the S-ribosylhomocysteinase(LuxS)-catalyzed reaction by preventing ß-elimination of a homocysteine molecule, and thus depleting the production of quorum sensing signaling molecule AI-2.

Investigation of reactions postulated to occur during inhibition of ribonucleotide reductases by 2'-azido-2'-deoxynucleotides.

Model 3'-azido-3'-deoxynucleosides with thiol or vicinal dithiol substituents at C2' or C5' were synthesized to study reactions postulated to occur during inhibition of ribonucleotide reductases by 2'-azido-2'-deoxynucleotides. Esterification of 5'-(tert-butyldiphenylsilyl)-3'-azido-3'-deoxyadenosine and 3'-azido-3'-deoxythymidine (AZT) with 2,3-S-isopropylidene-2,3-dimercaptopropanoic acid or N-Boc-S-trityl-L-cysteine and deprotection gave 3'-azido-3'-deoxy-2'-O-(2,3-dimercaptopropanoyl or cysteinyl)adenosine and the 3'-azido-3'-deoxy-5'-O-(2,3-dimercaptopropanoyl or cysteinyl)thymidine analogs. Density functional calculations predicted that intramolecular reactions between generated thiyl radicals and an azido group on such model compounds would be exothermic by 33.6-41.2 kcal/mol and have low energy barriers of 10.4-13.5 kcal/mol. Reduction of the azido group occurred to give 3'-amino-3'-deoxythymidine, which was postulated to occur with thiyl radicals generated by treatment of 3'-azido-3'-deoxy-5'-O-(2,3-dimercaptopropanoyl)thymidine with 2,2'-azobis-(2-methyl-2-propionamidine) dihydrochloride. Gamma radiolysis of N(2)O-saturated aqueous solutions of AZT and cysteine produced 3'-amino-3'-deoxythymidine and thymine most likely by both radical and ionic processes.

Inhibition of LuxS by S-ribosylhomocysteine analogues containing a [4-aza]ribose ring.

LuxS (S-ribosylhomocysteinase) catalyzes the cleavage of the thioether linkage of S-ribosylhomocysteine (SRH) to produce homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor to a small signaling molecule that mediates interspecies bacterial communication called autoinducer 2 (AI-2). Inhibitors of LuxS should interfere with bacterial interspecies communication and potentially provide a novel class of antibacterial agents. In this work, SRH analogues containing substitution of a nitrogen atom for the endocyclic oxygen as well as various deoxyriboses were synthesized and evaluated for LuxS inhibition. Two of the [4-aza]SRH analogues showed modest competitive inhibition (K(I) ∼40 µM), while most of the others were inactive. One compound that contains a hemiaminal moiety exhibited time-dependent inhibition, consistent with enzyme-catalyzed ring opening and conversion into a more potent species (K(I)(∗)=3.5 µM). The structure-activity relationship of the designed inhibitors highlights the importance of both the homocysteine and ribose moieties for high-affinity binding to LuxS active site.

Substituted lactam and cyclic azahemiacetals modulate Pseudomonas aeruginosa quorum sensing.

Quorum sensing (QS) is a population-dependent signaling process bacteria use to control multiple processes including virulence that is critical for establishing infection. The most common QS signaling molecule used by Gram-negative bacteria are acylhomoserine lactones. The development of non-native acylhomoserine lactone (AHL) ligands has emerged as a promising new strategy to inhibit QS in Gram-negative bacteria. In this work, we have synthesized a set of optically pure γ-lactams and their reduced cyclic azahemiacetal analogues, bearing the additional alkylthiomethyl substituent, and evaluated their effect on the AHL-dependent Pseudomonas aeruginosa las and rhl QS pathways. The concentration of these ligands and the simple structural modification such as the length of the alkylthio substituent has notable effect on activity. The γ-lactam derivatives with nonylthio or dodecylthio chains acted as inhibitors of las signaling with moderate potency. The cyclic azahemiacetal with shorter propylthio or hexylthio substituent was found to strongly inhibit both las and rhl signaling at higher concentrations while the propylthio analogue strongly stimulated the las QS system at lower concentrations.

Site of Azido Substitution in the Sugar Moiety of Azidopyrimidine Nucleosides Influences the Reactivity of Aminyl Radicals Formed by Dissociative Electron Attachment.

In this work, electron-induced site-specific formation of neutral π-type aminyl radicals (RNH·) and their reactions with pyrimidine nucleoside analogs azidolabeled at various positions in the sugar moiety, e.g., at 2'-, 3'-, 4'-, and 5'- sites along with a model compound 3-azido-1-propanol (3AZPrOH), were investigated. Electron paramagnetic resonance (EPR) studies confirmed the site and mechanism of RNH· formation via dissociative electron attachment-mediated loss of N2 and subsequent facile protonation from the solvent employing the 15N-labeled azido group, deuterations at specific sites in the sugar and base, and changing the solvent from H2O to D2O. Reactions of RNH· were investigated employing EPR by warming these samples from 77 K to ca. 170 K. RNH· at a primary carbon site (5'-azido-2',5'-dideoxyuridine, 3AZPrOH) facilely converted to a σ-type iminyl radical (R═N·) via a bimolecular H-atom abstraction forming an α-azidoalkyl radical. RNH· when at a secondary carbon site (e.g., 2'-azido-2'-deoxyuridine) underwent bimolecular electrophilic addition to the C5═C6 double bond of a proximate pyrimidine base. Finally, RNH· at tertiary alkyl carbon (4'-azidocytidine) underwent little reaction. These results show the influence of the stereochemical and electronic environment on RNH· reactivity and allow the selection of those azidonucleosides that would be most effective in augmenting cellular radiation damage.

Probing the catalytic mechanism of S-ribosylhomocysteinase (LuxS) with catalytic intermediates and substrate analogues.

S-Ribosylhomocysteinase (LuxS) cleaves the thioether bond in S-ribosylhomocysteine (SRH) to produce homocysteine (Hcys) and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of the type II bacterial quorum sensing molecule (AI-2). The catalytic mechanism of LuxS comprises three distinct reaction steps. The first step involves carbonyl migration from the C1 carbon of ribose to C2 and the formation of a 2-ketone intermediate. The second step shifts the C=O group from the C2 to C3 position to produce a 3-ketone intermediate. In the final step, the 3-ketone intermediate undergoes a beta-elimination reaction resulting in the cleavage of the thioether bond. In this work, the 3-ketone intermediate was chemically synthesized and shown to be chemically and kinetically competent in the LuxS catalytic pathway. Substrate analogues halogenated at the C3 position of ribose were synthesized and reacted as time-dependent inhibitors of LuxS. The time dependence was caused by enzyme-catalyzed elimination of halide ions. Examination of the kinetics of halide release and decay of the 3-ketone intermediate catalyzed by wild-type and mutant LuxS enzymes revealed that Cys-84 is the general base responsible for proton abstraction in the three reaction steps, whereas Glu-57 likely facilitates substrate binding and proton transfer during catalysis.

Inhibition of S-ribosylhomocysteinase (LuxS) by substrate analogues modified at the ribosyl C-3 position.

S-ribosylhomocysteinase (LuxS) catalyzes the cleavage of the thioether bond of S-ribosylhomocysteine (SRH) to produce homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD), which is the precursor of type 2 autoinducer for bacterial cell-cell communication. In this work, we have synthesized several SRH analogues modified at the ribose C3 position as potential inhibitors of LuxS. While removal or methylation of the C3-OH resulted in simple competitive inhibitors of moderate potency, inversion of the C3 stereochemistry or substitution of fluorine for C3-OH resulted in slow-binding inhibitors of improved potency. The most potent inhibitor showed a K(I)(*) value of 0.43 microM.

Prehydrated One-Electron Attachment to Azido-Modified Pentofuranoses: Aminyl Radical Formation, Rapid H-Atom Transfer, and Subsequent Ring Opening.

Methyl 2-azido-2-deoxy-α-d-lyxofuranoside (1a) and methyl 2-azido-2-deoxy-ß-d-ribofuranoside (2) were prepared from d-xylose or d-arabinose, respectively. Employing ESR and DFT/B3LYP/6-31G* calculations, we investigated (i) aminyl radical (RNH·) formation and (ii) reaction pathways of RNH·. Prehydrated electron attachment to 1a and 2 at 77 K produced transient azide anion radical (RN3·-) which reacts via rapid N2 loss at 77 K, forming nitrene anion radical (RN·-). Rapid protonation of RN·- at 77 K formed RNH· and -OH. 15N-labeled-1a confirmed this mechanism. Investigations employing in-house synthesized site-specifically deuterated derivatives of 1a (e.g., CH3 (1b), C4 (1c), and C5 (1d)) established that (a) a facile intramolecular H atom transfer from C5 to RNH· generated C5· and RNH2. C5· formation had a small deuterium kinetic isotope effect suggesting that this reaction does not occur via direct H atom abstraction. (b) Subsequently, C5· underwent a facile unimolecular conversion to ring-opened C4·. Identification of ring-opened C4· intermediate confirms the mechanism of C5'· mediated unaltered base release associated with DNA-strand break. However, for 2, ESR studies established thermally activated intermolecular H atom abstraction by RNH· from the methyl group at C1. Thus, sugar ring configuration strongly influences the site and pathway of RNH· mediated reactions in pentofuranoses.

S-Ribosylhomocysteine analogs containing a [4-thio]ribose ring.

The [4-thio]-S-ribosylhomocysteine (SRH) analogs containing substitution of a sulfur atom for the endocyclic oxygen were synthesized by coupling of the 4-thioribose substrates with a thiolate generated from the protected homocysteine. Coupling of the protected 1-deoxy-5-O-mesyl-S-oxo-4-thio-D-ribofuranose with homocysteinate salt gave the C4 epimers of [4-thio]-SRH at the sulfoxide oxidation level lacking a hydroxyl group at anomeric carbon. Treatment of these sulfoxides with BF3⋅Et2O/NaI affected simultaneous reduction to sulfide and global deprotection affording 1-deoxy-4-thio-SRH analog. Treatment of the protected 1-deoxy-S-oxo-4-thio-D-ribofuranose sulfoxide with DAST/SbCl3 resulted in the fluoro-Pummerer rearrangement to give 4-thio-ß-D-ribofuranosyl fluoride. Mesylation of the latter at 5-hydroxyl position followed by coupling with homocysteinate salt and subsequent global deprotection with trifluoroacetic acid afforded [4-thio]-SRH thiohemiacetal.

Synthesis and cytostatic evaluation of 4-N-alkanoyl and 4-N-alkyl gemcitabine analogues.

The coupling of gemcitabine with functionalized carboxylic acids (C9-C13) or reactions of 4-N-tosylgemcitabine with the corresponding alkyl amines afforded 4-N-alkanoyl and 4-N-alkyl gemcitabine derivatives. The analogues with a terminal hydroxyl group on the alkyl chain were efficiently fluorinated under conditions that are compatible with protocols for (18)F labeling. The 4-N-alkanoylgemcitabines showed potent cytostatic activities in the low nanomolar range against a panel of tumor cell lines, whereas cytotoxicity of the 4-N-alkylgemcitabines were in the low micromolar range. The cytotoxicity for the 4-N-alkanoylgemcitabine analogues was reduced approximately by 2 orders of magnitude in the 2'-deoxycytidine kinase (dCK)-deficient CEM/dCK(-) cell line, whereas cytotoxicity of the 4-N-alkylgemcitabines was only 2-5 times lower. None of the compounds acted as efficient substrates for cytosolic dCK; therefore, the 4-N-alkanoyl analogues need to be converted first to gemcitabine to display a significant cytostatic potential, whereas 4-N-alkyl derivatives attain modest activity without measurable conversion to gemcitabine.