Substrate-product analogue inhibitors of isoleucine 2-epimerase from Lactobacillus buchneri by rational design.

A rational approach that may be applied to a broad class of enzyme-catalyzed reactions to design enzyme inhibitors affords a powerful strategy, facilitating the development of drugs and/or molecular probes of enzyme mechanisms. A strategy for the development of substrate-product analogues (SPAs) as inhibitors of racemases and epimerases is elaborated using isoleucine 2-epimerase from Lactobacillus buchneri (LbIleE) as a model enzyme. LbIleE catalyzes the PLP-dependent, reversible, racemization or epimerization of nonpolar amino acids at the C-2 position. The enzyme plays an important role in the biosynthesis of branched-chain d-amino acids and is a potential target for the development of antimicrobial agents. 3-Ethyl-3-methyl-l-norvaline (Ki = 2.9 ± 0.2 mM) and 3-ethyl-3-methyl-d-norvaline (Ki = 1.5 ± 0.2 mM) were designed as SPAs based on the movement of the sec-butyl side chain of the substrate l-Ile during catalysis, and were competitive inhibitors with binding affinities exceeding that of l-Ile by 1.3- and 2.5-fold, respectively. Surprisingly, these compounds were not substrates, but the corresponding compounds lacking the 3-methyl group were substrates. Unlike serine, glutamate, and proline racemases, which exhibit stringent steric requirements at their active sites, the active site of LbIleE was amenable to binding bulky SPAs. Moreover, LbIleE bound the SPA 2,2-di-n-butylglycine (Ki = 11.0 ± 0.2 mM) as a competitive inhibitor, indicating that the hydrophobic binding pocket at the active site was sufficiently plastic to tolerate gem-dialkyl substitution at the α-carbon of an amino acid. Overall, these results reveal that amino acid racemases/epimerases are amenable to inhibition by SPAs provided that they possess a capacious active site.

Identification of a 4-fluorobenzyl l-valinate amide benzoxaborole (AN11736) as a potential development candidate for the treatment of Animal African Trypanosomiasis (AAT).

Novel l-valinate amide benzoxaboroles and analogues were designed and synthesized for a structure-activity-relationship (SAR) investigation to optimize the growth inhibitory activity against Trypanosoma congolense (T. congolense) and Trypanosoma vivax (T. vivax) parasites. The study identified 4-fluorobenzyl (1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborole-6-carbonyl)-l-valinate (5, AN11736), which showed IC50 values of 0.15 nM against T. congolense and 1.3 nM against T. vivax, and demonstrated 100% efficacy with a single dose of 10 mg/kg against both T. congolense and T. vivax in mouse models of infection (IP dosing) and in the target animal, cattle, dosed intramuscularly. AN11736 has been advanced to early development studies.

Antifungal dipeptides incorporating an inhibitor of homoserine dehydrogenase.

The antifungal activity of 5-hydroxy-4-oxo-l-norvaline (HONV), exhibited under conditions mimicking human serum, may be improved upon incorporation of this amino acid into a dipeptide structure. Several HONV-containing dipeptides inhibited growth of human pathogenic yeasts of the Candida genus in the RPMI-1640 medium, with minimal inhibitory concentration values in the 32 to 64 µg mL-1 range. This activity was not affected by multidrug resistance that is caused by overexpression of genes encoding drug efflux proteins. The mechanism of antifungal action of HONV dipeptides involved uptake by the oligopeptide transport system, subsequent intracellular cleavage by cytosolic peptidases, and inhibition of homoserine dehydrogenase by the released HONV. The relative transport rates determined the anticandidal activity of HONV dipeptides.

Total syntheses of smenothiazoles A and B.

Concise total syntheses of smenothiazoles A (1) and B (2), two distinguished vinyl chloride containing natural products isolated from the marine sponge S. aurea, have been developed. Silastannation, Stille reaction and a carefully controlled desilylchlorination were employed as key steps to construct unique polyketide acid fragments, and the optimized reaction conditions avoided migration of 2,5-diene to a 2,4-conjugated system. This report unambiguously confirmed the structures of both natural products.

Exploring the Sponge Consortium Plakortis symbiotica-Xestospongia deweerdtae as a Potential Source of Antimicrobial Compounds and Probing the Pharmacophore for Antituberculosis Activity of Smenothiazole A by Diverted Total Synthesis.

Fractionation of the bioactive CHCl3-MeOH (1:1) extracts obtained from two collections of the sponge consortium Plakortis symbiotica-Xestospongia deweerdtae from Puerto Rico provided two new plakinidone analogues, designated as plakinidone B (2) and plakinidone C (3), as well as the known plakinidone (1), plakortolide F (4), and smenothiazole A (5). The structures of 1-5 were characterized on the basis of 1D and 2D NMR spectroscopic, IR, UV, and HRMS analysis. The absolute configurations of plakinidones 2 and 3 were established through chemical correlation methods, VCD/ECD experiments, and spectroscopic data comparisons. When assayed in vitro against Mycobacterium tuberculosis H37Rv, none of the plakinidones 1-3 displayed significant activity, whereas smenothiazole A (5) was the most active compound, exhibiting an MIC value of 4.1 µg/mL. Synthesis and subsequent biological screening of 8, a dechlorinated version of smenothiazole A, revealed that the chlorine atom in 5 is indispensable for anti-TB activity.

Evaluation of [18 F]BR420 and [18 F]BR351 as radiotracers for MMP-9 imaging in colorectal cancer.

MMP-9 is a zinc-dependent endopeptidase that is involved in the proteolytic degradation of the extracellular matrix and plays an important role in cancer migration, invasion, and metastasis. The aim of this study was to evaluate the potential of MMP-tracers [18 F]BR420 and [18 F]BR351 for MMP-9 imaging in a colorectal cancer xenograft model. [18 F]BR420 and [18 F]BR351 were synthesized using an automated synthesis module. For [18 F]BR420, a novel and improved radiosynthesis was developed. Plasma stability and MMP-9-targeting capacity of both radiotracers was compared in the Colo205 colorectal cancer model. MMP-9 and MMP-2 expression levels in the tumors were evaluated by immunohistochemistry and in situ zymography. µPET imaging as well as ex vivo biodistribution revealed a higher tumor uptake for [18 F]BR420 (3.15% ± 0.03% ID/g vs 0.94% ± 0.18% ID/g for [18 F]BR351 at 2 hours pi) but slower blood clearance compared with [18 F]BR351. [18 F]BR351 was quickly metabolized in plasma with 20.28% ± 5.41% of intact tracer remaining at 15 minutes postinjection (PI). By contrast, [18 F]BR420 displayed a higher metabolic stability with >86% intact tracer remaining at 2 hours PI. Immunohistochemistry revealed the presence of MMP-9 and MMP-2 in the tumor tissue, which was confirmed by in situ zymography. However, an autoradiography analysis of tracer distribution in the tumors did not correlate with MMP-9 expression. [18 F]BR420 displayed a higher tumor uptake and higher stability compared with [18 F]BR351 but a low tumor-to-blood ratio and discrepancy between tracer distribution and MMP-9 immunohistochemistry. Therefore, both tracers will not be usefulness for MMP-9 imaging in colorectal cancer.

Synthesis of α,ω-polyfluorinated α-amino acid derivatives and δ,δ-difluoronorvaline.

Intending to synthesize ω,ω-difluoroalkyl amino acid derivatives by oxidative desulfurization-fluorination reactions of suitable arylthio-2-phthalimido butanoates and pentanoates, in addition to small amounts of the target products, mainly α,ω-polyfluorinated amino acid derivatives were formed by additional sulfur-assisted α-fluorination. This novel structural motif was verified spectroscopically as well as by X-ray analysis. A plausible mechanism of formation is suggested. Using a different approach, δ,δ-difluoronorvaline hydrochloride was synthesized with at least 36% enantiomeric excess via deoxofluorination of the corresponding aldehyde.

Synthesis and inhibitory activity of substrate-analog fructosyl peptide oxidase inhibitors.

Fructosyl peptide oxidases (FPOXs) play a crucial role in the diagnosis of diabetes. Their main function is to cleave fructosyl amino acids or fructosyl peptides into glucosone and the corresponding amino acids/dipeptides. In this study, the substrate-analog FPOX inhibitors 1a-c were successfully designed and synthesized. These inhibitors mimic N(α)-fructosyl-L-valine (Fru-Val), [N(α)-fructosyl-L-valyl]-L-histidine (Fru-ValHis), and N(ε)-fructosyl-L-lysine (εFru-Lys), respectively. The secondary nitrogen atom in the natural substrates, linking fructose and amino acid or dipeptide moieties, was substituted in 1a-c with a sulfur atom to avoid enzymatic cleavage. Kinetic studies revealed that 1a-c act as competitive inhibitors against an FPOX obtained from Coniochaeta sp., and Ki values of 11.1, 66.8, and 782 µM were obtained for 1a-c, respectively.

Scaleable catalytic asymmetric Strecker syntheses of unnatural alpha-amino acids.

Alpha-amino acids are the building blocks of proteins and are widely used as components of medicinally active molecules and chiral catalysts. Efficient chemo-enzymatic methods for the synthesis of enantioenriched alpha-amino acids have been developed, but it is still a challenge to obtain non-natural amino acids. Alkene hydrogenation is broadly useful for the enantioselective catalytic synthesis of many classes of amino acids, but it is not possible to obtain alpha-amino acids bearing aryl or quaternary alkyl alpha-substituents using this method. The Strecker synthesis-the reaction of an imine or imine equivalent with hydrogen cyanide, followed by nitrile hydrolysis-is an especially versatile chemical method for the synthesis of racemic alpha-amino acids. Asymmetric Strecker syntheses using stoichiometric amounts of a chiral reagent have been applied successfully on gram-to-kilogram scales, yielding enantiomerically enriched alpha-amino acids. In principle, Strecker syntheses employing sub-stoichiometric quantities of a chiral reagent could provide a practical alternative to these approaches, but the reported catalytic asymmetric methods have seen limited use on preparative scales (more than a gram). The limited utility of existing catalytic methods may be due to several important factors, including the relatively complex and precious nature of the catalysts and the requisite use of hazardous cyanide sources. Here we report a new catalytic asymmetric method for the syntheses of highly enantiomerically enriched non-natural amino acids using a simple chiral amido-thiourea catalyst to control the key hydrocyanation step. This catalyst is robust, without sensitive functional groups, so it is compatible with aqueous cyanide salts, which are safer and easier to handle than other cyanide sources; this makes the method adaptable to large-scale synthesis. We have used this new method to obtain enantiopure amino acids that are not readily prepared by enzymatic methods or by chemical hydrogenation.

Synthesis and characterization of tritium labeled N-((R)-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-oxobutan-2-yl)-3-sulfamoylbenzamide.

N-((R)-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-oxobutan-2-yl)-3-sulfamoylbenzamide is a potent C-C chemokine receptor 1 (CCR1) antagonist. The compound, possessing benzamide functionality, successfully underwent tritium/hydrogen (T/H) exchange with an organoiridium catalyst (Crabtree's catalyst). The labeling pattern in the product was studied with liquid chromatography-mass spectrometry, time-of-flight mass spectrometry, and (3) H-NMR. Overall, multiple labeled species were identified. In addition to the anticipated incorporation of tritium in the benzamide moiety, tritium labeling was observed in the valine portion of the molecule including substitution at its chiral carbon. Using authentic standards, liquid chromatography analysis of the labeled compound showed complete retention of stereochemical configuration.

Homo-Roche ester derivatives by asymmetric hydrogenation and organocatalysis.

Asymmetric hydrogenation routes to homologues of The Roche ester tend to be restricted to hydrogenations of itaconic acid derivatives, that is, substrates that contain a relatively unhindered, 1,1-disubstituted alkene. This is because in hydrogenations mediated by RhP2 complexes, the typical catalysts, it is difficult to obtain high conversions using the alternative substrate for the same product, the isomeric trisubstituted alkenes (D in the text). However, chemoselective modification of the identical functional groups in itaconic acid derivatives are difficult; hence, it would be favorable to use the trisubstituted alkene. Trisubstituted alkene substrates can be hydrogenated with high conversions using chiral analogs of Crabtree's catalyst of the type IrN(carbene). This paper demonstrates that such reactions are scalable (tens of grams) and can be manipulated to give optically pure homo-Roche ester chirons. Organocatalytic fluorination, chlorination, and amination of the homo-Roche building blocks was performed to demonstrate that they could easily be transformed into functionalized materials with two chiral centers and α,ω-groups that provide extensive scope for modifications. A synthesis of (S,S)- and (R,S)-γ-hydroxyvaline was performed to illustrate one application of the amination product.

Radical migration-addition of N-tert-butanesulfinyl imines with organozinc reagents.

A novel migration-addition sequence was discovered for the reaction of enantioenriched N-tert-butanesulfinyl iminoacetate 1a with functionalized benzylzinc bromide reagents, producing tert-leucine derivatives in excellent diastereoselectivity (dr 98:2). The absolute configurations of two new chiral centers were unambiguously assigned by chemical transformations and X-ray crystallography. In addition, the regio- and diastereoselectivities of this novel reaction were both explained through the key N-sulfinamine intermediate M6 generated by the tert-butyl radical attack on the imine. Computational analysis of this reaction process, which was performed at the B3LYP/6-311++G(3df,2p)//B3LYP/6-31G*-LANL2DZ level, also supported our proposed two-stage mechanism.

Photorelease of amino acids from novel thioxobenzo[f]benzopyran ester conjugates.

Aiming at the enhancement of the performance of (9-methoxy-3-oxo-3H-benzo[f]benzopyran-1-yl) methyl ester as photocleavable protecting group for the carboxylic acid function at long-wavelengths, 9-methoxy-3-thioxo-3H-benzo[f]benzopyran-L-valine and L-phenylalanine model conjugates were prepared through a thionation reaction of the corresponding oxo-benzobenzopyrans. These thioxobenzobenzopyran derivatives were subjected to photocleavage reactions in the same conditions as the parent oxo-benzobenzopyrans at different wavelengths of irradiation, and photocleavage data were obtained. It was found that the exchange of the carbonyl by a thiocarbonyl group enhanced the performance of the heterocyclic protecting group at 419 nm by improving the photolysis rates, making it an appropriate group for practical applications at long wavelengths.

Synthesis, pharmacokinetics, and pharmacodynamics studies of valsartan peptide derivatives.

In order to increase the intestinal permeability of valsartan, 14 esters and peptide derivatives of valsartan were chemically synthesized and their absorption characteristics were described. All derivatives were stable and could be better absorbed into the small intestine than valsartan. There are two barriers for the absorption of valsartan derivatives. The elongated half-life (t(1/2)) and stable blood concentrations for compound 4 due to the hydrolysis of the ester group in the second barrier were demonstrated in pharmacokinetic experiments. Furthermore, compound 4 also displayed modest anti-hypertension activity in vivo, which makes structural modification of valsartan, especially for the purpose of improvement of its intestinal permeability, valuable for further studies.

A further contribution to the study of sagittamide A: synthesis of a pivotal intermediate belonging to a rare L-series.

A key saggitamide intermediate corresponding to a rare sugar framework has been obtained. This approach should help to establish the overall configuration of more complex structures of the sagittamide family.

Bifunctional peptidomimetic prodrugs of didanosine for improved intestinal permeability and enhanced acidic stability: synthesis, transepithelial transport, chemical stability and pharmacokinetics.

Five peptidomimetic prodrugs of didanosine (DDI) were synthesized and designed to improve bioavailability of DDI following oral administration via targeting intestinal oligopeptide transporter (PepT1) and enhancing chemical stability. The permeability of prodrugs was screened in Caco-2 cells grown on permeable supports. 5'-O-L-valyl ester prodrug of DDI (compound 4a) demonstrated the highest membrane permeability and was selected as the optimal target prodrug for further studies. The uptake of glycylsarcosine (Gly-Sar, a typical substrate of PepT1) by Caco-2 cells could be inhibited by compound 4a in a concentration-dependent manner. The Caco-2 cells were treated with 0.2 nM leptin for enhanced PepT1 expression. The uptake of compound 4a was markedly increased in the leptin-treated Caco-2 cells compared with the control Caco-2 cells, both of which were obviously inhibited by 20 mM Gly-Sar. The K(m) and V(max) values of kinetic study of compound 4a transported by PepT1 in Caco-2 cells were 0.91 mM and 11.94 nmol/mg of protein/10 min, respectively. The chemical stability studies were performed in simulated gastric fluid (SGF), phosphate buffers under various pH conditions, rat tissue homogenates and plasma at 37 °C. The concentrations of DDI could not be detected in the two minutes in SGF. But compound 4a could significantly increase DDI acidic stability, and its t(½) was extended to as long as 36 min in SGF. Compound 4a was stable in pH 6.0 phosphate buffer but could be quickly transformed into DDI in plasma and tissue homogenates. The oral absolute bioavailability of DDI was 47.2% and 7.9% after compound 4a and DDI were orally administered to rats at a dose of 15 mg/kg, respectively. The coadministration with antiacid agent could also suggest that compound 4a was more stable under harsh acidic conditions compared with DDI. Compound 4a bioavailability in rats was reduced to 33.9% when orally co-administered with Gly-Sar (100 mg/kg). The In Vivo bioactivation mechanism of compound 4a was investigated by comparing the levels of DDI and compound 4a in the jugular and portal veins in rats. The plasma concentration of intact compound 4a was very low in portal veins and could hardly be detected in the jugular vein. In conclusion, compound 4a could significantly improve the oral bioavailability of DDI in rats through PepT1-mediated absorption and enhanced acidic stability, followed by rapid and mostly intracellular bioactivation, the majority in the intestinal cells but the minority in the liver. Additionally, the prodrug strategy targeted to intestinal PepT1 could offer a promising strategy to improve oral bioavailability of poorly absorbed didanosine.

Synthesis of angiotensin II receptor blockers by means of a catalytic system for C-H activation.

A highly efficient catalytic system for C-H activation has been worked out that involves inexpensive RuCl(3)·xH(2)O and a specific amount of PPh(3). This procedure has been successfully applied to a practical synthesis of angiotensin II receptor blockers (ARBs). The residual ruthenium that existed in the reaction mixture was thoroughly removed by treatment with properly selected metal scavengers. The new process permits ready access to the important class of drugs in a highly atom-economical and sustainable manner.

Synthesis and biological evaluation of new salvinorin A analogues incorporating natural amino acids.

The synthesis and in vitro evaluation of a new series of salvinorin A analogues substituted at the C(2) position with natural amino acids is reported. Compound 12, containing Val, displayed high affinity and full agonist activity at the kappa-opioid receptor. Analogues with bulky and/or aromatic residues were inactive, showing the importance of size and electronegativity of C(2)-substituents for binding affinity of salvinorin A derivatives.

Design, synthesis, and in vivo characterization of a novel series of tetralin amino imidazoles as γ-secretase inhibitors: discovery of PF-3084014.

A novel series of tetralin containing amino imidazoles, derived from modification of the corresponding phenyl acetic acid derivatives is described. Replacement of the amide led to identification of a potent series of tetralin-amino imidazoles with robust central efficacy. The reduction of brain Aß in guinea pigs in the absence of changes in B-cells suggested a potential therapeutic index with respect to APP processing compared with biomarkers of notch related toxicity. Optimization of the FTOC to plasma concentrations at the brain Aß EC(50) lead to the identification of compound 14f (PF-3084014) which was selected for clinical development.

A new general pathway for synthesis of reference compounds of N-terminal valine-isocyanate adducts.

Adducts to Hb could be used as biomarkers to monitor exposure to isocyanates. Particularly useful is the measurement of carbamoylation of N-terminal valines in Hb, after detachment as hydantoins. The synthesis of references from the reactive isocyanates, especially diisocyanates, has been problematic due to side reactions and polymerization of the isocyanate starting material. A simpler, safer, and more general method for the synthesis of valine adducts of isocyanates has been developed using N-[(4-nitrophenyl)carbamate]valine methylamide (NPCVMA) as the key precursor to adducts of various mono- and diisocyanates of interest. By reacting NPCVMA with a range of isocyanate-related amines, carbamoylated valines are formed without the use of the reactive isocyanates. The carbamoylated products synthesized here were cyclized with good yields of the formed hydantoins. The carbamoylated derivative from phenyl isocyanate also showed quantitative yield in a test with cyclization under the conditions used in blood. This new pathway for the preparation of N-carbamoylated model compounds overcomes the above-mentioned problems in the synthesis and is a general and simplified approach, which could make such reference compounds of adducts to N-terminal valine from isocyanates accessible for biomonitoring purposes. The synthesized hydantoins corresponding to adducts from isocyanic acid, methyl isocyanate, phenyl isocyanate, and 2,6-toluene diisocyanate were characterized by LC-MS analysis. The background level of the hydantoin from isocyanic acid in human blood was analyzed with the LC-MS conditions developed.