Rapid and scalable synthesis of innovative unnatural α,ß or γ-amino acids functionalized with tertiary amines on their side-chains.

We report a selective ruthenium catalyzed reduction of tertiary amides on the side chain of Fmoc-Gln-OtBu derivatives, leading to innovative unnatural α,ß or γ-amino acids functionalized with tertiary amines. Rapid and scalable, this process allowed us to build a library of basic unnatural amino acids at the gram-scale and directly usable for liquid- or solid-phase peptide synthesis. The diversity of available tertiary amines allows us to modulate the physicochemical properties of the resulting amino acids, such as basicity or hydrophobicity.

N-ω-chloroacetyl-l-ornithine, a new competitive inhibitor of ornithine decarboxylase, induces selective growth inhibition and cytotoxicity on human cancer cells versus normal cells.

Many cancer cells have high expression of ornithine decarboxylase (ODC) and there is a concerted effort to seek new inhibitors of this enzyme. The aim of the study was to initially characterize the inhibition properties, then to evaluate the cytotoxicity/antiproliferative cell based activity of N-ω-chloroacetyl-l-ornithine (NCAO) on three human cancer cell lines. Results showed NCAO to be a reversible competitive ODC inhibitor (Ki = 59 µM) with cytotoxic and antiproliferative effects, which were concentration- and time-dependent. The EC50,72h of NCAO was 15.8, 17.5 and 10.1 µM for HeLa, MCF-7 and HepG2 cells, respectively. NCAO at 500 µM completely inhibited growth of all cancer cells at 48 h treatment, with almost no effect on normal cells. Putrescine reversed NCAO effects on MCF-7 and HeLa cells, indicating that this antiproliferative activity is due to ODC inhibition.

Clarifying the structure of granadaene: total synthesis of related analogue [2]-granadaene and confirmation of its absolute stereochemistry.

Streptococcus agalactiae is an important agent in the infection of neonates in the first world. One of the most extended methods for its identification is based on the detection of a characteristic red pigment in the patient samples, named [12]-granadaene (1). In this article, we present a modular and flexible approach to simple analogues of this ornithine rhamno-polyene 1 and the elucidation of the most important features of its structure: the absolute configuration at C-27, the stereochemistry of the anomeric center and the link of the amino acid ornithine to the rest of the structure.

Synthesis and application of an Nδ-acetyl-Nδ-hydroxyornithine analog: identification of novel metal complexes of deferriferrichrysin.

Synthesis of Fmoc-protected N(δ)-acetyl-N(δ)-(tert-butoxy)-l-ornithine has revealed it to be a metal-chelating amino-acid precursor. This protected amino acid was compatible with the preparation of ferrichrome peptides by standard Fmoc-based solid-phase peptide synthesis. Evaluation of deferriferrichrysin for metal ion chelation revealed that zirconium(IV) and titanium(IV) formed complexes with deferriferrichrysin.

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.

N5-phosphonoacetyl-L-ornithine (PALO): a convenient synthesis and investigation of influence on regulation of amino acid biosynthetic genes in Saccharomyces cerevisiae.

A scalable four-step synthesis of the ornithine transcarbamylase inhibitor N(5)-phosphonoacetyl-l-ornithine (PALO) is achieved through boroxazolidinone protection of ornithine. Investigations in the model organism Saccharomyces cerevisiae found that, in contrast to a previous report, PALO did not influence growth rate or expression of genes involved in arginine metabolism.

Radiosynthesis and evaluation of N5-(2-18F-fluoropropanyl) ornithine as a potential agent for tumor PET imaging.

OBJECTIVE: Studies have confirmed that tumorigenesis is related to an imbalance of polyamine metabolism and over-expression of oncogenes resulting in the up-regulation of ornithine decarboxylase (ODC, the first rate-limiting enzyme for regulating intracellular polyamines biosynthesis), which has become a target for anti-tumor therapy. In this study, an ornithine derivative, N5-(2-[18F]fluoropropionyl) ornithine (N5-[18F]FPO), has been prepared and its potential utility for tumor PET imaging evaluated. METHODS: N5-[18F]FPO was successfully prepared via a nucleophilic fluorination reaction and a subsequent efficient deprotection step. The in vitro and in vivo stability were determined by HPLC conducted in fetal bovine serum, saline and rat urine. Cellular uptake studies were conducted in HepG2 cells and the biodistribution and micro-PET/CT imaging performed in normal ICR mice and three tumor-bearing mice models, respectively. RESULTS: Total synthesis time of N5-[18F]FPO was about 80 min with a radiochemical yield of 15% ± 6% (uncorrected, based on 18F-, n = 6) and a high radiochemical stability can be seen in vitro and vivo. The N5-[18F]FPO exhibited fast uptake in HepG2 cells and the cellular uptake ability of N5-[18F]FPO can be inhibited by L-ornithine and DFMO, which indicated that the transport pathway of N5-[18F]FPO is similar to that of L-ornithine, interacting with ODC after being transported into the cell. The biodistribution and micro-PET/CT images demonstrate that N5-[18F]FPO was excreted by the urinary system, and excellent tumor visualization with high tumor-to-background ratios can be observed in the three tumor-bearing mice models studied. CONCLUSION: All the above results suggest that N5-[18F]FPO has the potential to be a novel radiotracer for imaging ODC expression in solid tumors.

Synthesis of 2-azaspiro[3.3]heptane-derived amino acids: ornitine and GABA analogues.

Synthesis of 6-amino-2-azaspiro[3.3]heptane-6-carboxylic acid and 2-azaspiro[3.3]heptane-6-carboxylic acid was performed. Both four-membered rings in the spirocyclic scaffold were constructed by subsequent ring closure of corresponding 1,3-bis-electrophiles at 1,1-C- or 1,1-N-bis-nucleophiles. The two novel amino acids were added to the family of the sterically constrained amino acids for the use in chemistry, biochemistry, and drug design.

A new non-natural arginine-like amino acid derivative with a sulfamoyl group in the side-chain.

Sulfamoylation of the L-ornithine methyl ester side-chain generates a non-natural arginine isostere which can be coupled with N-Fmoc-L-proline to synthesize analogues which maintain the structural characteristics of the biologically important Pro-Arg dipeptide sequence. As a probe of its biological importance, the sulfamoylated amino acid derivative was also incorporated as P1 residue in tripeptide structures matching the C-terminal subsequence of fibrinogen. The reported results demonstrate that the functionalization of L-ornithine side-chain with a neutral sulfamoyl group can generate an arginine bioisostere which can be used for the synthesis of prototypes of a new class of human thrombin inhibitors.

Inhibitors of N(alpha)-acetyl-L-ornithine deacetylase: synthesis, characterization and analysis of their inhibitory potency.

A series of N (alpha)-acyl (alkyl)- and N (alpha)-alkoxycarbonyl-derivatives of L- and D-ornithine were prepared, characterized, and analyzed for their potency toward the bacterial enzyme N (alpha)-acetyl-L-ornithine deacetylase (ArgE). ArgE catalyzes the conversion of N (alpha)-acetyl-L-ornithine to L-ornithine in the fifth step of the biosynthetic pathway for arginine, a necessary step for bacterial growth. Most of the compounds tested provided IC(50) values in the muM range toward ArgE, indicating that they are moderately strong inhibitors. N (alpha)-chloroacetyl-L-ornithine (1g) was the best inhibitor tested toward ArgE providing an IC(50) value of 85 microM while N (alpha)-trifluoroacetyl-L-ornithine (1f), N (alpha)-ethoxycarbonyl-L-ornithine (2b), and N (alpha)-acetyl-D-ornithine (1a) weakly inhibited ArgE activity providing IC(50) values between 200 and 410 microM. Weak inhibitory potency toward Bacillus subtilis-168 for N (alpha)-acetyl-D-ornithine (1a) and N (alpha)-fluoro- (1f), N (alpha)-chloro- (1g), N (alpha)-dichloro- (1h), and N (alpha)-trichloroacetyl-ornithine (1i) was also observed. These data correlate well with the IC(50) values determined for ArgE, suggesting that these compounds might be capable of getting across the cell membrane and that ArgE is likely the bacterial enzymatic target.

Structure-activity relationships of potentiators of the antibiotic activity of clarithromycin against Escherichia coli.

Several studies that have identified agents that potentiate the antimicrobial activity of antibiotics, but there are limited insights into their structure-activity relationships (SAR). The SAR associated with select N-alkylaryl amide derivatives of ornithine was performed to establish those structural features that were associated with potentiation of the antimicrobial activity of clarithromycin against E. coli ATCC 25922. The data indicate that the N-propyl derivative was slightly more active in reducing the effective MIC of clarithromycin against E. coli ATCC 25922. In addition, the S-enantiomer of compound 9 was somewhat more potent than the R-enantiomer in potentiating clarithromycin activity. No significant enhancement in potentiation activity was observed with the conversion of these secondary amides to their N-methyl tertiary amides. Formation of the N-methyl or N,N-dimethyl derivatives of the primary amine of 9 was associated with the loss of potentiation activity. Conversion of this primary amine to a guanidine was also not associated with an increase in potentiation activity. Among the isomeric diamino pentamides, 15 potentiated the antibacterial activity of clarithromycin to the greatest extent. In addition to these amide derivatives, the desoxy derivatives 16 and 18 were the more potent potentiators within this triamine series. The relative location of the primary amines, as indicated by the relative differences in the potentiation observed with 16 compared to 14, appears to be a critical factor in determining potentiation activity. Cell-based membrane permeabilization and efflux inhibition studies in E. coli ATCC 25922 suggest that the potentiation of clarithromycin activity by 16 reflects its ability to inhibit efflux pump activity and to a lesser extent its actions as a permeabilizer of the outer leaflet of the outer cell membrane.

Antioxidant properties of argpyrimidine.

Argpyrimidine, the product of non-enzymatic protein glycation by methylglyoxal, has been implicated in the pathophysiology of diabetes mellitus and neurodegenerative diseases. Chemically, argpyrimidine is a substituted pyrimidinol with structural features common to known antioxidants. The objective of this study was to investigate the antioxidant properties of argpyrimidine. Argpyrimidine was synthesized by mixing L-arginine with 3-acetoxypentane-2,4-dione under acidic conditions and purified by chromatography. Argpyrimidine inhibited lipid peroxidation of rat brain homogenates catalyzed by hydroxyl radicals, metal ions, and autooxidation in a concentration- and time-dependent manner. In addition, argpyrimidine scavenged superoxide anion, 1,1-diphenyl 2-picryl-hydrazyl-stable free radical, intracellular-hydrogen peroxide, and inhibited free-radical-mediated nicking of plasmid-DNA. Taken together, our data suggest that argpyrimidine has antioxidant properties and may therefore have biological relevance in pathophysiologies associated with diabetes mellitus and neurodegenerative diseases.

Synthesis of two peptide mimetics as markers for chemical changes of wool's keratin during skin unhairing process.

The sheep skins unhairing process with preliminary alkaline treatment of the wool leads to two unnatural dipeptide mimetics lysinoalanine (Lys(*) - Ala) and ornithinoalanine (Orn(*)- Ala) obtaining. They are result from the keratin hydrolysis process. The changes of wool keratin make it resistant to sulphide degradation. We synthesized and characterized these unnatural dipeptides under the experimental conditions. The structures and mechanism of Lys(*) - Ala and Orn(*)- Ala obtaining were elucidated. The using of newly synthesized products as markers for control of wool's keratin changes during skin unhairing process was demonstrated. The developments have also been the result of economic and environmental pressures to meet environmental regulations.

The formation of argpyrimidine in glyceraldehyde-related glycation.

Three major glyceraldehyde-related advanced glycation end products (AGEs) were formed from a mixture of N(alpha)-acetyllysine, N(alpha)-acetylarginine, and glyceraldehyde. Two of the compounds were MG-H1 and GLAP, as previously reported, and the other compound was identified as N(alpha)-acetyl-N(delta)-(5-hydroxy-4,6-dimethyl-pyrimidin-2-yl)-ornithine, argpyrimidine (APN). APN is a modification product of arginine residue, but it did not form from glyceraldehyde with arginine residue. The coexistence of lysine residue was necessary to APN formation.

Design and synthesis of novel sulfonamide-containing bradykinin hB2 receptor antagonists. 2. Synthesis and structure-activity relationships of alpha,alpha-cycloalkylglycine sulfonamides.

Recently we reported on the design and synthesis of a novel class of selective nonpeptide bradykinin (BK) B2 receptor antagonists (J. Med. Chem. 2006, 3602-3613). This work led to the discovery of MEN 15442, an antagonist with subnanomolar affinity for the human B2 receptor (hB2R), which also displayed significant and prolonged activity in vivo (for up to 210 min) against BK-induced bronchoconstriction in the guinea-pig at a dose of 300 nmol/kg (it), while demonstrating only a slight effect on BK-induced hypotension. Here we describe the further optimization of this series of compounds aimed at maximizing the effect on bronchoconstriction and minimizing the effect on hypotension, with a view to developing topically delivered drugs for airway diseases. The work led to the discovery of MEN 16132, a compound which, after intratracheal or aerosol administration, inhibited, in a dose-dependent manner, BK-induced bronchoconstricton in the airways, while showing minimal systemic activity. This compound was selected as a preclinical candidate for the topical treatment of airway diseases involving kinin B2 receptor stimulation.

Discovery of diphenyloxazole and Ndelta-Z-ornithine derivatives as highly potent and selective human prostaglandin EP(4) receptor antagonists.

Two novel classes of diphenyloxazole and Ndelta-Z-ornithine derivatives as highly potent and selective EP(4) antagonists have been discovered. The optimized diphenyloxzole 8 and Ndelta-Z-ornithine 11 effectively competed with [(3)H]PGE(2) binding to human recombinant EP(4), with K(i) values of 0.30 nM and 0.91 nM, respectively, and were selective for all members of the human prostanoid receptor family. 8 was shown to exhibit good pharmacokinetic properties in rats and dogs and potent inhibitory activity toward in vitro PGE(2)-promoted IgE synthesis.

Highly efficient approach to orthogonally protected (2S,4R)- and (2S,4S)-4-hydroxyornithine.

[reaction: see text] A concise stereoselective approach to both orthogonally protected (2S,4R)- and (2S,4S)-4-hydroxyornithine, key constituents of the biphenomycin- and clavalanine-type antibiotics, respectively, has been developed. The approach is based on bis(oxazoline) copper(II)-complex-catalyzed diastereoselective Henry reactions of nitromethane with the homoserine-derived aldehyde 6. The synthesis of this versatile chiral building block has been markedly improved.

Sagittamides A and B. Polyacetoxy long-chain acyl amino acids from a didemnid ascidian.

[structure: see text]. An unidentified tunicate from Pohnpei, Micronesia, yielded sagittamides A and B-compounds comprising a long-chain C26 dicarboxylic acid that acylates terminal L-valine and L-ornithine groups. The structures, which contain an unprecedented internal O-hexacetyl-1,2,3,4,5,6-hexaol moiety, were determined by combined spectroscopic analysis including mass spectrometry and 1D and 2D NMR and chemical degradation. The partial absolute stereochemistry of the new compounds was addressed by Marfey's analysis.

The utilization of 5-hydroxyl-2-amino valeric acid as a specific marker of oxidized arginine and proline residues in proteins.

Alteration of cellular proteins by oxidative modification could represent an important mechanism leading to cellular dysdifferentiation and age-related diseases. There is difficulty in testing this hypothesis because of a lack of specific assays that can measure the extent proteins are oxidized in nonpurified tissue preparations. Some methods used to measure carbonyl groups in nonpurified samples have serious limitations because of interference from other sources of carbonyl groups not being a product of oxidation-mediated damage. Oxidation of arginine and proline residues has been reported to produce gamma-glutamyl semialdehyde, which on reduction and acid hydrolysis, was predicted to form 5-hydroxy-2-amino valeric acid (HAVA). In this article we confirm this prediction using a GC/MS/SIM technique, and carry out additional experiments to determine if HAVA may be a useful marker of oxidative damage in proteins. These experiments utilized purified preparations of arginine, proline, histidine, and lysine amino acid homopolymers and six different purified proteins preparations in nonoxidized and oxidized states. Results demonstrate that HAVA compares well with the carbonyl group formation as a specific marker of oxidized protein, and that the GC/MS/SIM technique can detect HAVA reliably to 150 femtomoles per injection. Thus, HAVA as a specific marker of oxidized arginine and proline could prove to be a useful assay in pure and nonpurified samples.

alpha-(Fluoromethyl)dehydroornithine and alpha-(fluoromethyl)dehydroputrescine analogues as irreversible inhibitors of ornithine decarboxylase.

(E)-Dehydro analogues of alpha-(fluoromethyl)putrescine and -ornithine derivatives were synthesized and evaluated in vitro as irreversible inhibitors of a preparation of ornithine decarboxylase (ODC, EC 4.1.1.17) obtained from rat liver. The key step in the synthesis of (E)-alpha-(fluoromethyl)dehydroornithine (17) and -putrescine (14) was the addition of propenylmagnesium bromide to fluoroacetonitrile. The resulting unstable conjugated imine salt was reduced regioselectively in situ with NaBH4 or was quenched with a solution of NaCN to give the corresponding unsaturated alpha-(fluoromethyl) amine and alpha-amino nitrile, respectively. These were transformed into 17 and 14 via a four-step sequence involving (a) phthaloyation of the amine function; (b) allylic bromination of the methyl group; (c) Gabriel reaction; and (d) hydrolytic cleavage of the protective groups. (E)-alpha-(Difluoromethyl)dehydroornithine (10) and -putrescine (7) were prepared from ethyl tert-butyl 2-(difluoromethyl)-2-(2-propenyl)malonate and di-tert-butyl 2-(difluoromethyl)-2-(2-propenyl)malonate, respectively, via a sequence similar to that reported previously for the synthesis of the saturated analogues. Compounds 17, 14, 10, and 7 proved to be much more potent enzyme-activated irreversible inhibitors of ODC than the corresponding saturated analogues. The increase in potency is particularly marked in the alpha-fluoromethyl series. The apparent dissociation constants (KI) and the times of half-inactivation of enzyme (tau 50) at infinite concentration of inhibitors are 2.7 microM and 2.6 min for 17 and 42 microM and 0.2 min for 14. The KI and tau 50 of the corresponding saturated analogues are 75 microM and 1.6 min for the ornithine derivative and 56 microM and 4.4 min for the putrescine derivative.