Identification of a new class of nonpeptidic inhibitors of cruzain

Cruzain is the major cysteine protease of Trypanosoma cruzi, which is the causative agent of Chagas disease and is a promising target for the development of new chemotherapy. With the goal of developing potent nonpeptidic inhibitors of cruzain, the substrate activity screening (SAS) method was used to screen a library of protease substrates initially designed to target the homologous human protease cathepsin S. Structure-based design was next used to further improve substrate cleavage efficiency by introducing additional binding interactions in the S3 pocket of cruzain. The optimized substrates were then converted to inhibitors by the introduction of cysteine protease mechanism-based pharmacophores. Inhibitor 38 was determined to be reversible even though it incorporated the vinyl sulfone pharmacophore that is well documented to give irreversible cruzain inhibition for peptidic inhibitors. The previously unexplored beta-chloro vinyl sulfone pharmacophore provided mechanistic insight that led to the development of potent irreversible acyl- and aryl-oxymethyl ketone cruzain inhibitors. For these inhibitors, potency did not solely depend on leaving group p K a, with 2,3,5,6-tetrafluorophenoxymethyl ketone 54 identified as one of the most potent inhibitors with a second-order inactivation constant of 147,000 s (-1) M (-1). This inhibitor completely eradicated the T. cruzi parasite from mammalian cell cultures and consequently has the potential to lead to new chemotherapeutics for Chagas disease.     

Baylis-Hillman-derived N,N′-disubstituted piperazines: structural and preliminary computational studies

Exploratory studies towards the preparation of potential HIV-1 protease and integrase inhibitors have led to the synthesis of Baylis-Hillman-derived N,N′-disubstituted piperazines. X-ray crystallographic, computer modelling and NMR techniques have been used to elucidate questions concerning configurational preferences, reaction pathways and the apparent difference in susceptibility towards aza-Michael reactions exhibited by methyl acrylate and methyl vinyl ketone (MVK) derived Baylis-Hillman substrates.     

Michael additions to acrylonitrile,methyl acrylate and methyl vinyl ketone to Nb-benzylidenetryptophan methyl ester

Michael addition to methyl acrylate and methyl vinyl ketone of N_b-benzylidene-L-tryptophan methyl ester 1 gave 2-(3-indolylmethyl)glutamic dimethyl ester 2a and α-(3-oxobutyl)tryptophan methyl ester 2b respectively. Addition to acrylonitrile of 1 yielded α,N_a-dicyanoethyltryptophan methyl ester 3 .     

Synthesis of optically active alpha-aminoalkyl alpha'-halomethyl ketone: a cross-Claisen condensation approach

A simple and versatile method was developed for the synthesis of alpha-aminoalkyl alpha'-halomethyl ketone derivatives, which are useful intermediates of protease inhibitors. It involves selective halogenation of the alpha-position on a beta-ketoester, which is prepared by cross-Claisen condensation using N-protected amino acid ester. The title compound is obtained in high yield after decarboxylation of the alpha-halo-beta-ketoester.     

Photochemical electron transfer reactions of tirapazamine

The absorption and fluorescence spectra of 3-aminobenzo-1,2,4-triazine di-N-oxide (tirapazamine) have been recorded and exhibit a dependence on solvent that correlates with the Dimroth ET30 parameter. Time-dependent density functional theory calculations reveal that the transition of tirapazamine in the visible region is pi-->pi* in nature. The fluorescence lifetime is 98+/-2 ps in water. The fluorescence quantum yield is approximately 0.002 in water. The fluorescence of tirapazamine is efficiently quenched by electron donors via an electron-transfer process. Linear Stern-Volmer fluorescence quenching plots are observed with sodium azide, potassium thiocyanate, guanosine monophosphate and tryptophan (Trp) methyl ester hydrochloride. Guanosine monophosphate, tyrosine (Tyr) methyl ester hydrochloride and Trp methyl ester hydrochloride appear to quench the fluorescence at a rate greater than diffusion control implying that these substrates complex with tirapazamine in its ground state. This complexation was detected by absorption spectroscopy.     

Purification and some properties of three serine carboxypeptidases from Aspergillus niger

Three enzymes exhibiting peptidyl-L-amino acid hydrolase and esterase activities have been purified by immobilized metal-ion affinity chromatography and ion-exchange chromatography. The three enzymes were entirely free of the acid protease activity that normally exists along with them in the crude culture filtrates of Aspergillus niger. Although all three exo-peptidases possessed nearly identical molecular weights (ca. 140,000), isoelectric points (ca. 5.0) and other properties, their affinities for the two substrates tested, carbobenzoxy-L-Glu-L-Tyr and benzoyl L-arginine ethyl ester, differed. All three peptidases were inhibited by phenylmethanesulphonyl fluoride, indicating that they are serine carboxypeptidases. They were also inhibited by tosyl phenylalanine chloromethyl ketone, suggesting the presence of a histidyl residue in their active sites. The differences in the number of accessible histidyl residues on the enzyme surfaces could explain the differences in their retentions on Cu2+-iminodiacetate-Sepharose 6B.     

Photoreaction between benzoylthiophenes and N-BOC-tryptophan methyl ester

Drug-induced photoallergy requires as the first step formation of covalent drug-protein photoadducts. One of the key amino acids involved in this process is tryptophan (Trp). In this context, several diaryl ketones, including 2-benzoylthiophene (BT), [2-(5-benzoyl-5-thienyl)]-2-methylpropanoic methyl ester (TPA methyl ester) and 4-(2-thienylcarbonyl)phenyl]-2-methylpropanoic methyl ester (SUP methyl ester) have been irradiated in the presence of N-BOC-(L)-tryptophan methyl ester. Laser flash photolysis has allowed to detect three neutral radicals (ketyl, indolyl and skatolyl radicals) resulting from formal hydrogen-atom abstraction. This correlates well with the isolation of homodimers, as well as with cross-coupling products, in the preparative irradiation. The main cross-coupling products were in all cases lactones arising from the reaction of the Trp-derived skatolyl radicals with the corresponding ketyl radicals. These lactones were obtained as the (4R) stereoisomers with remarkable diasteroselectivity. No coupling products through the phenyl p-position of BT or TPA methyl ester were found. By contrast, ketone homodimers and cross-coupling products arising from reaction through the thienyl 5-position were obtained when using BT and SUP methyl ester; this is very interesting, because stable LAT-derived products are difficult to isolate.     

An extraction-based assay for neutral anionophores: the measurement of high binding constants to steroidal receptors in a nonpolar solvent

The extraction-based protocol for measuring binding constants, developed by Cram and co-workers, has been extended for use with anionic substrates. The method is especially useful for high-affinity receptors, allowing very high binding constants to be measured in nonpolar solvents. Distribution constants K(d) between chloroform and water have been obtained for tetraethylammonium chloride and bromide, thus calibrating the method for these two substrates. Application to steroidal podands 5-9 has confirmed the ability of electron-withdrawing groups to enhance hydrogen-bond donor capabilities. Binding constants of approximately 3 x 10(7) M(-1) have been measured for the most powerful receptor 7. An X-ray crystal structure of 15, the methyl ester analogue of 7, reveals a well-defined binding site preorganised for anion recognition.     

Aqueous phosphoric acid as a mild reagent for deprotection of tert-butyl carbamates, esters, and ethers

Aqueous phosphoric acid (85 wt %) is an effective, environmentally benign reagent for the deprotection of tert-butyl carbamates, tert-butyl esters, and tert-butyl ethers. The reaction conditions are mild and offer good selectivity in the presence of other acid-sensitive groups, including CBZ carbamates, azetidine, benzyl and methyl esters, TBDMS, and methyl phenyl ethers. The mildness of the reaction is further demonstrated in the synthesis of clarithromycin derivative, in which a tert-butyl ester is removed in the presence of cyclic carbamate, lactone, ketal, acetate ester, and epimerizable methyl ketone functionalities. The reaction preserves the stereochemical integrity of the substrates. The reactions are high yielding, and the workup is convenient.     

The steroid monooxygenase from Rhodococcus rhodochrous; a versatile biocatalyst

The substrate scope of a steroid monooxygenase (STMO) from Rhodococcus rhodochrous DSM 43269 was investigated for a large range of different ketone substrates. These studies revealed that this enzyme not only oxygenates steroids, but also ketone moieties of a series of other open-chain ketones, such as cyclohexyl methyl ketone, cyclopentyl methyl ketone, and 3-acetylindole. Furthermore, the STMO catalyzed the oxygenation of cyclobutanone derivatives. Comparative biotransformations with recombinant Escherichia coli resting cells harboring the STMO, the cycloalkanone monooxygenase (CAMO) from Cylindrocarpon radicicola or the cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus revealed that the STMO is enantiodivergent compared to the CHMO-type. Moreover, the STMO resulted in a higher enantiomeric excess of the product lactones compared to the known BVMOs of the same enantiopreference, such as cyclopentanone monooxygenases.     

A simple gas-liquid chromatographic method with electron capture detection for the determination of progesterone in the blood of humans and domestic animals.

A simple routine method is reported for the determination of progesterone in blood by gas-liquid chromatography. An unlabelled internal standard, testosterone acetate, was added to plasma samples. Following preliminary purification by thin-layer chromatography, progesterone in pregnancy plasma was evaluated as the free steroid with a flame ionization detector. The hormone in the plasma of women during the menstrual cycle and of cycling domestic animals was determined as the 3-enol ester heptafluorobutyrate by electron capture detection. The validity of testosterone acetate as an internal standard was proved by simultaneous processing of [7alpha-3H]progesterone and a [4-14C]testosterone acetate and the determination of the isotope ratio in samples. The results of control experiments and normal values are also presented.     

Synthesis of Tadalafil Analogues

Five novel compounds as potential phosphodiesterase type 5 (PDE‐5) inhibitors were synthesized from D‐tryptophan methyl ester via the Pictet–Spengler reaction and cyclization reaction. The structures of those compounds were confirmed by elemental analyses, IR, ¹H NMR, and ESI‐MS spectra.     

Tricarbonylchromium tryptophan derivatives and their use in peptide synthesis

Incorporation of a Cr(CO)₃ ligand into the indole ring of N-α-t-butoxycarbonyl-tryptophan methyl ester was achieved in 47% yield. The corresponding para-nitrophenyl ester was used in the solid phase synthesis of a peptidic hormone (LHRH) analogue with the aim of decreasing tryptophan alkylation. No improvement was observed.     

Macrocyclic Protease Inhibitors with Reduced Peptide Character

There is a real need for simple structures that define a β‐strand conformation, a secondary structure that is central to peptide–protein interactions. For example, protease substrates and inhibitors almost universally adopt this geometry on active site binding. A planar pyrrole is used to replace two amino acids of a peptide backbone to generate a simple macrocycle that retains the required geometry for active site binding. The resulting β‐strand templates have reduced peptide character and provide potent protease inhibitors with the attachment of an appropriate amino aldehyde to the C‐terminus. Picomolar inhibitors of cathepsin L and S are reported and the mode of binding of one example to the model protease chymotrypsin is defined by X‐ray crystallography.     

Formation of yohimbanones via a novel rearrangement

An investigation of the reaction of tryptophan and its methyl ester with ninhydrin has been conducted. In the reaction of tryptophan with ninhydrin only one product, yohimbanone (1), is isolated. In contrast, an intermediate, Pictet-Spengler product 3, is isolated from the reaction of tryptophan methyl ester with ninhydrin. The isolation of this intermediate provides support for a proposed mechanism of this novel rearrangement, which is presented herein.     

Hydrophobic effects on rates and substrate selectivities in polymeric transaminase mimics

The amination of ketoacids to amino acids by pyridoxamine is greatly accelerated when the pyridoxamine is covalently linked to polyethylenimine carrying N-methyl and N-lauryl groups. Michaelis-Menten kinetics is seen with all substrates, from which the effect of the lauryl groups and the methyl groups can be determined with respect to the strength of binding of the substrate and the rate constant k2 within the complex. The polyamine catalyzes the reaction using acid and base groups, the lauryl groups increase k2 by producing a nonpolar medium in which the reaction occurs, and the lauryl groups promote binding of hydrophobic substrates. The result is that the amination of indolepyruvic acid to produce tryptophan is accelerated by 240000-fold.     

Chiral calix[4]arenes bearing α-hydroxy amide units as membrane carriers for amino acid methyl esters

Novel chiral calix[4]arene derivatives functionalized at the lower rim have been prepared from the reaction of p-tert-butylcalix[4]arene diamine or acylhydrazine derivative with mandelic acid or hydroxyisovaleric acid. The structures of these receptors were characterized by FTIR, ¹H, ¹³C and 2D COSY NMR spectroscopy and elemental analysis. The transport of amino acid derivatives (phenylalanine, phenylglycine and tryptophan methyl ester hydrochlorides) was studied through bulk liquid membrane in the presence of chiral calix[4]arene derivatives. The receptors have been found to act as carriers for transport of aromatic amino acid methylesters from the aqueous source phase to the aqueous receiving phase. The transport rate and L/D selectivity of amino acid esters studied depend strongly upon the structure of the chiral receptors and guests. The best enantioselectivity was obtained in the case of phenylglycine methyl ester for all chiral carriers.     

Two- and three dimensional combinatorial chemistry from multicomponent Grignard reagents

The conjugate addition of five component Grignard reagents to methyl ecgonidine was used to create libraries of 3-substituted tropanes. By variation in the reagent combination in 10 such 5-membered sublibraries, a library of 25 compounds was made in a two-dimensional format. Screening of this library led to identification of two new potent monoamine transporter ligands that were subsequently synthesized. The most potent compound in this library was (1R,2S,3S,5S)-3-(3,4-dimethylphenyl)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylic acid methyl ester, which inhibited dopamine transporter (hDAT) binding and reuptake with a K(i) of 26 and 20 nM, respectively. The conjugate addition to a 5-membered library of methyl ecgonidine analogues with variation of nitrogen substituent was also carried out and used to create 15 sublibraries of 25 compounds, which displayed 125 compounds in a three-dimensional format. From this 3D library, several potent dopamine transport inhibitors were likewise identified and synthesized. The most potent hDAT inhibitor discovered was (1R,2S,3S,5S)-3-(3,4-dimethylphenyl)-8-pentyl-8-azabicyclo[3.2.1]octane-2-carboxylic acid methyl ester. The study also showed that 3-alkyltropanes were poor inhibitors of monoamine transporters.     

Infrared multiple-photon dissociation spectroscopy of tripositive ions: lanthanum-tryptophan complexes

Collision-induced charge disproportionation limits the stability of triply charged metal ion complexes and has thus far prevented successful acquisition of their gas-phase IR spectra. This has curtailed our understanding of the structures of triply charged metal complexes in the gas phase and in biological environments. Herein we report the first gas-phase IR spectra of triply charged La(III) complexes with a derivative of tryptophan (N-acetyl tryptophan methyl ester), and an unusual dissociation product, a lanthanum amidate. These spectra are compared with those predicted using density functional theory. The best structures are those of the lowest energies that differ by details in the π-interaction between La(3+) and the indole rings. Other binding sites on the tryptophan derivative are the carbonyl oxygens. In the lanthanum amidate, La(3+) replaces an H(+) in the amide bond of the tryptophan derivative.