Modular,efficient synthesis of asymmetrically substituted piperazine scaffolds as potent calcium channel blockers

A novel approach to the synthesis of substituted piperazines and their investigation as N-type calcium channel blockers is presented. A common scaffold exhibiting high activity as N-type blockers is N-substituted piperazine. Using recently developed titanium and zirconium catalysts, we describe the efficient and modular synthesis of 2,5-asymmetrically disubstituted piperazines from simple amines and alkynes. The method requires only three isolation/purification protocols and no protection/deprotection steps for the diastereoselective synthesis of 2,5-dialkylated piperazines in moderate to high yield. Screening of the synthesized piperazines for N-type channel blocking activity and selectivity shows the highest activity for a compound with a benzhydryl group on the nitrogen (position 1) and an unprotected alcohol-functionalized side chain.     

The flexibility in the proline ring couples to the protein backbone

In proteins, the proline ring exists predominantly in two discrete states. However, there is also a small but significant amount of flexibility in the proline ring of high-resolution protein structures. We have found that this side-chain flexibility is coupled to the backbone conformation. To study this coupling, we have developed a model that is simply based on geometric and steric factors and not on energetics. We show that the coupling between phi and chi1 torsions in the proline ring can be described by an analytic equation that was developed by Bricard in 1897, and we describe a computer algorithm that implements the equation. The model predicts the observed coupling very well. The strain in the C(gamma)-C(delta)-N angle appears to be the principal barrier between the UP and DOWN pucker. This strain is relaxed to allow the proline ring to flatten in the rare PLANAR conformation.     

Fasciolicidal potential of proline analogues and proline biosynthesis inhibitors

Sheers Marion, Campbell Anne J., Beames D. J., Edwards S. R., Moore R. J. and Montague P. E. 1982. Fasciolicidal potential of proline analogues and proline biosynthesis inhibitors. International Journal for Parasitology12: 47–52. Hydroxylamine HCl and thiazolidine-4'-carboxylic acid, known inhibitors of important enzymes of proline biosynthesis, inhibited to a similar extent the arginine-dependent proline production by the liver fluke Fasciola hepofica; in vitro there appeared to be no correlation between inhibition of proline synthesis and deterioration of the fluke. Another known inhibitor, thiosemicarbazide, had no effect on arginine-dependent proline production in vitro. None of these compounds was effective in vivo either as a flukicidal agent per se or in the prevention of the establishment of fluke in the bile duct of rats. A variety of proline analogues was also tested for flukicidal activity in vitro and in vivo as well as for their ability to inhibit the establishment of fluke in the bile duct of rats. Only one was effective in vitro and none was effective in vivo. Also continuous administration of l-azetidine-2-carboxylic acid failed to prevent the establishment of an infection of liver fluke in the bile duct. It is concluded that there is little prospect of a successful approach to chemotherapy of fascioliasis in this area.     

Supramolecular chirality induction in bis(zinc porphyrin) by amino acid derivatives: rationalization and applications of the ligand bulkiness effect

The achiral syn conformer (face-to-face) of the ethane-bridged bis(zinc porphyrin) (syn-ZnD) transforms into the corresponding chiral extended anti bis-ligated species (anti-ZnD.L2) in the presence of enantiopure ligands (L: amino acid derivatives). The mechanism of the supramolecular chirality induction is based on chiral ligand binding to zinc porphyrins and subsequent formation of either right- or left-handed screw structures in anti-ZnD.L2. The screw structure formation arises from steric interactions between the bulkiest substituent at the asymmetric carbon of the ligand and the peripheral ethyl groups of the neighboring porphyrin ring directed towards the covalent bridge. The sign and amplitude of the induced circular dichroism (CD) are dependent on the steric bulk of the substituents at the chiral center. The greater difference in size between the chiral center's substituents gives the stronger induced CD signal. Rationalization of the ligand bulkiness effect on chirality induction by amino acid derivatives, application of this supramolecular system for the determination of ligand absolute configuration, and relative bulkiness of the substituents at the asymmetric carbon are discussed.     

Asymmetric Trans-esterification of meso-2,5-Dibromoadipate and Synthesis of Optically Active cis-2,5-Disubstituted Pyrrolidines

Asymmetric trans-esterification of meso-2,5-dibromoadipate to (–)-benzyl methyl 2,5-dibromoadipate by lipase with subsequent chemical reactions afforded optically active cis-2,5-disubstituted pyrrolidines. An equivalent asymmetric transformation was performed by selectively hydrolyzing a cis-2,5-disubstituted pyrrolidine having a chiral N-substituent.     

A library construction of 2,5-disubstituted pyrrole compounds by using solid/solution-phase syntheses

Using solid- and solution-phase synthesis, a library of 2,5-disubstituted pyrrole compounds was constructed. This is the first report that Stetter reaction was applied to the solid-phase synthesis for C-C bond formation. Some of 2,5-disubstituted pyrrole compounds showed the inhibitory activity of LPS-induced mouse B-lymphocyte proliferation.     

Synthesis of <Emphasis Type="Italic">gem</Emphasis>-disubstituted taurines by the regioselective ring-opening of 2,2-disubstituted aziridines with sodium bisulfite and sulfite

The regioselectivity of the ring-opening reactions of 2,2-disubstituted aziridines with sodium bisulfite and sulfite showed significant divergence depending on the nucleophiles and the structure of the substituents of the aziridines. 2,2-Dialkylaziridines were specifically attacked on their less substituted carbon atoms with sodium bisulfite, affording 2,2-substituted taurines, while 2,2-disubstituted aziridines with either one or two aryl substituent(s) were attacked specifically on their more substituted carbon atoms with the same nucleophile, giving rise to aromatic 1,1-substituted taurines. Sodium sulfite attacked the less substituted carbon atoms of all aziridines specifically to afford 2,2-disubstituted taurines. The regioselectivity is governed by the nucleophile and the balance between the steric hindrance and the electronic effect. The current method provides an alternative route to the synthesis of 2,2-dialkyltaurines and aromatic gem-disubstituted taurines.     

The role of proline residues in the dynamics of transmembrane helices: the case of bacteriorhodopsin

Proline residues in transmembrane helices have been found to have important roles in the functioning of membrane proteins. Moreover, Pro residues occur with high frequency in transmembrane α-helices, as compared to α-helices for soluble proteins. Here, we report several properties of the bacteriorhodopsin mutants P50A (helix B), P91A (helix C) and P186A (helix F). Compared to wild type, strongly perturbed behaviour has been found for these mutants. In the resting state, increased hydroxylamine accessibility and altered Asp-85 pK_a and light-dark adaptation were observed. On light activation, hydroxylamine accessibility was increased and proton transport activity, M formation kinetics and FTIR difference spectra of M and N intermediates showed clear distortions. On the basis of these alterations and the near identity of the crystalline structures of mutants with that of wild type, we conclude that the transmembrane proline residues of bacteriorhodopsin fulfil a dynamic role in both the resting and the light-activated states. Our results are consistent with the notion that mutation of Pro to Ala allows the helix to increase its flexibility towards the direction originally hindered by the steric clash between the ring Cγ and the carbonyl O of the i-4 residue, at the same time decreasing the mobility towards the opposite direction. Due to their properties, transmembrane Pro residues may serve as transmission elements of conformational changes during the transport process. We propose that these concepts can be extended to other transmembrane proteins.     

Discovery of novel P2 substituted 4-biaryl proline inhibitors of hepatitis C virus NS3 serine protease

Inhibitors of hepatitis C virus NS3 serine protease often incorporate a large P2 moiety to interact with the surface of the enzyme while shielding part of the catalytic triad. This feature is important in many inhibitors in order to have the necessary potency needed for efficacy. In this Letter we explore some new P2 motifs to further exploit this region of the enzyme. In a continuing effort to replace the often found 4-hydroxyproline P2 core found in the majority of inhibitors for this target, various directly attached aryl derivatives were evaluated. Of these, the 2,4-disubstituted thiazole core proved to be the most interesting. SAR around this motif has lead to compounds with K_i’s in the high picomolar range and provided cellular potencies in the single digit nM range.     

Synthesis and evaluation of in vitro antioxidant properties of novel 2,5-disubstituted 1,3,4-oxadiazoles

2,5-Disubstituted 1,3,4-oxadiazole compounds are one of the most attractive heterocyclic compounds for researchers due to their biological activities. In the undertaken research, a number of potential 2,5-disubstituted 1,3,4-oxadiazole analogues were synthesized through multi step reaction and characterized by FT-IR, ¹H NMR, mass spectra, and also by elemental analysis. Further benzophenone tagged indole acetohydrazides and 2,5-disubstituted 1,3,4-oxadiazoles were evaluated for antioxidant potential, through different in vitro models such as DPPH, nitric oxide and hydrogen peroxide methods. In the series of compounds some of them had shown good to moderate in vitro antioxidant potential compare to the standard drug ascorbic acid in all the above three methods.     

Amine adducts of porphinato carbonylruthenium(II): studies of conformational effects and dynamics

The ¹H chemical shifts for 4-methylpyridine, imidazole, 2,5-dimethylimidazole, 2-undecylimidazole, pyrazole, 3,5-dimethylpyrazole, benzylamine, and α-methylbenzylamine bound to carbonylruthenium mesoporphyrin-IX-dimethyl ester or carbonylruthenium tetra-p-isopropylphenylporphyrin have been determined. The upfield shifts induced in the bound-nitrogen base resonances have been shown to arise principally from the magnetic anisotropy of the porphinato ligand. On this basis, models for calculating magnetic anisotropy in porphyrin systems have been tested and then applied to the determination of adduct geometry. These studies show that steric bulk of substituents ortho to the nitrogen binding determine the stability of various modes and control conformation in ligands. Furthermore, the results suggest adduct geometry and ligand-to-ring plane distances are relatively independent of porphyrin. Correlations between base structure and adduct stability were developed by measuring relative dissociation constants and first-order rate constants for dissociation. These measurements show a direct correlation between thermodynamic and kinetic stability and an inverse correlation between stability and increased steric bulk ortho to the donor atom.     

Novel thiophene derivatives for the treatment of benign prostatic hyperplasia

The syntheses and biological activities of a novel series of 2,4- and 2,5-disubstituted thiophenes are reported. These analogues have shown excellent affinity and selectivity against alpha(1)-adrenoreceptor subtypes.     

Genetically modified cyanobacteriumNostoc muscorum overproducing proline in response to salinity and osmotic stresses

In the parentNostoc muscorum an active proline oxidase enzyme is required to assimilate exogenous proline as a fixed nitrogen source. Cyanobacterial mutants, resistant to growth inhibitory action of proline analogue L-azetidine-2-carboxylate (Ac-R), were deficient in proline oxidase activity, and were over-accumulators of proline. Proline over-accumulation, resulting either from mutational acquisition of the Ac-R phenotype, or from salinity-induced uptake of exogenous proline, confirmed enhanced salinity/osmotic tolerance in the mutant strain. The nitrogenase activity and photosynthetic O₂ evolution of the parent were sensitive to both salinity as well as osmotic stresses than of Ac-R mutant strain. In addition, the mutation to Ac-resistant phenotype showed no alteration in salinity inducible potassium transport system in the cyanobacterium.     

Stereospecific synthesis of α-methylated amino acids

Summary. Both 2,5-trans and 2,5-cis disubstituted 2-tert-butyl-5-(indol-3-yl)methylimidazolidin-4-ones were synthesised and their enolates were prepared using LDA. While the enolate of the 2,5-trans disubstituted derivative could not be methylated, the enolate of the cis-2,5-disubstituted derivative was successfully methylated with methyl iodide to a product which on hydrolysis gave enantiomerically pure α-methyl-L-tryptophan. Received October 31, 1998, Accepted July 23, 1999     

Substituted benzoic and picolinic acids as foliar sprays against potato common scab

Glasshouse tests on potato cv. Arran Banner measured the effects of single early foliar sprays of 18 substituted benzoic and two dichloropicolinic acids on the severity of common scab, caused by soil-borne Streptomyces scabies. The monosubstituted benzoic acids tested (25 mM) did not affect scab. However, anti-scab action was shown by some 2,5-disubstituted acids. Decreases in scab severity were about 35% from 1·6 mM 5-bromo-2-chloro- and 2,5-dimethylbenzoic acids, and over 50% from 1·6 mM 2,5-dichloro-, 2,5-dibromo- and 5-chloro-2-nitrobenzoic acids and from 0·05 mM 3,6-dichloropicolinic acid (clopyralid), which is structurally similar to 2,5-dichlorobenzoic acid. None of the spray treatments affected yield or shape of tubers. No visible effects on foliage were caused by two of the benzoic acids active against scab (5-chloro-2-nitro- and 2,5-dimethyl-), but the other active acids caused some distortion. In tests of the six dichlorobenzoic acids against S. scabies in culture, the 2,5-isomer (which was the most effective against the disease in plants) was one of the least toxic. With other 2,5-disubstituted benzoic acids, it probably decreased scab indirectly by altering the response of the host to infection.     

Regulation and function of proline oxidase under nutrient stress

Under conditions of nutrient stress, cells switch to a survival mode catabolizing cellular and tissue constituents for energy. Proline metabolism is especially important in nutrient stress because proline is readily available from the breakdown of extracellular matrix (ECM), and the degradation of proline through the proline cycle initiated by proline oxidase (POX), a mitochondrial inner membrane enzyme, can generate ATP. This degradative pathway generates glutamate and α‐ketoglutarate, products that can play an anaplerotic role for the TCA cycle. In addition the proline cycle is in a metabolic interlock with the pentose phosphate pathway providing another bioenergetic mechanism. Herein we have investigated the role of proline metabolism in conditions of nutrient stress in the RKO colorectal cancer cell line. The induction of stress either by glucose withdrawal or by treatment with rapamycin, stimulated degradation of proline and increased POX catalytic activity. Under these conditions POX was responsible, at least in part, for maintenance of ATP levels. Activation of AMP‐activated protein kinase (AMPK), the cellular energy sensor, by 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR), also markedly upregulated POX and increased POX‐dependent ATP levels, further supporting its role during stress. Glucose deprivation increased intracellular proline levels, and expression of POX activated the pentose phosphate pathway. Together, these results suggest that the induction of proline cycle under conditions of nutrient stress may be a mechanism by which cells switch to a catabolic mode for maintaining cellular energy levels. J. Cell. Biochem. 107: 759–768, 2009. © 2009 Wiley‐Liss, Inc.     

Influence of proline residues in transmembrane helix packing

Integral membrane proteins often contain proline residues in their alpha-helical transmembrane (TM) fragments, which may strongly influence their folding and association. Pro-scanning mutagenesis of the helical domain of glycophorin A (GpA) showed that replacement of the residues located at the center abrogates helix packing while substitution of the residues forming the ending helical turns allows dimer formation. Synthetic TM peptides revealed that a point mutation of one of the residues of the dimerization motif (L75P) located at the N-terminal helical turn of the GpA TM fragment, adopts a secondary structure and oligomeric state similar to the wild-type sequence in detergents. In addition, both glycosylation mapping in biological membranes and molecular dynamics showed that the presence of a proline residue at the lipid/water interface has as an effect the extension of the helical end. Thus, helix packing can be an important factor that determines appearance of proline in TM helices. Membrane proteins might accumulate proline residues at the two ends of their TM segments in order to modulate the exposition of key amino acid residues at the interface for molecular recognition events while allowing stable association and native folding.     

Moderately increased constitutive proline does not alter osmotic stress tolerance

Proline-overproducing carrot cell lines were isolated by selection in medium containing hydroxyproline, a toxic analogue of proline. During growth of the cells in culture, length of lag phase, doubling time, and maximum fresh weight were the same for the hydroxyproline-resistant cell line (HP) and the wild-type cell line (JW). Proline content and resistance to hydroxyproline in the HP and JW lines were not strictly correlated indicating that another reason besides the constitutive level of proline is involved in hydroxyproline resistance. Tolerance to polyethylene glycol-induced desiccation stress was not different between the two lines except perhaps at the early stages of culture growth when the proline levels of the two cell lines were nearly the same. The complexity of the relationship between proline accumulation and osmotolerance is discussed and strategies to achieve constitutive high levels of proline accumulation in plants are proposed.     

Kinetics of isomerization for the proline helical forms of two oligoproline redox arrays by circular dichroic spectropolarimetry

Summary The kinetics of isomerization of the helical forms of three oligoprolines was determined by far-ultraviolet CD spectropolarimetry and kinetic analysis by singular value decomposition. ZRA (Pro₃-X-Pro₂-Y-Pro₂-Z-Pro₃) and ZRA2 (Pro₇-X-Pro₂-Y-Pro₂-Z-Pro₇) bear large redox-active substituents on proline residues X, Y, and Z, but P9 (Pro₉) does not. All three peptides formed a stable proline-II helix in water. In acetonitrile, both ZRA2 and P9 were converted into a proline-I helical form but ZRA remained predominantly in the proline-II helical form. Evidently, in order to undergo substantial proline II→I isomerization, an oligoproline chain containing large substituents needs to have a segment of consecutive unsubstituted proline residues that is sufficiently long to form a stable proline helix. Biexponential kinetics (A→B, k₁=∼3.3×10⁻⁴s⁻¹; B→C, k₂=∼0.8×10⁻⁴s⁻¹) were observed for the proline II→I isomerization of ZRA2 and P9 in acetonitrile and for the proline I→II isomerization of ZRA2 in water, which provides evidence for the growth and decay of a major kinetic intermediate.     

Kinetics of isomerization for the proline helical forms of two oligoproline redox arrays by circular dichroic spectropolarimetry

The kinetics of isomerization of the helical forms of three oligoprolines was determined by far-ultraviolet CD spectropolarimetry and kinetic analysis by singular value decomposition. ZRA (Pro3-X-Pro2-Y-Pro2-Z-Pro3) and ZRA2 (Pro7-X-Pro2-Y-Pro2-Z-Pro7) bear large redox-active substituents on proline residues X, Y, and Z, but P9 (Pro9) does not. All three peptides formed a stable proline-II helix in water. In acetonitrile, both ZRA2 and P9 were converted into a proline-I helical form but ZRA remained predominantly in the proline-II helical form. Evidently, in order to undergo substantial proline III isomerization, an oligoproline chain containing large substituents needs to have a segment of consecutive unsubstituted proline residues that is sufficiently long to form a stable proline helix. Biexponential kinetics (AB, k1 = 3.3 × 10-4 s-1; BC, k2 = 0.8 × 10-4 s-1) were observed for the proline III isomerization of ZRA2 and P9 in acetonitrile and for the proline III isomerization of ZRA2 in water, which provides evidence for the growth and decay of a major kinetic intermediate.