Immobilization of 3,5-dimethylphenyl carbamate of cellulose and amylose on silica by photochemical and thermal radical processes.

The immobilization of cellulose 3,5-dimethylphenyl carbamate and amylose 3,5-dimethylphenyl carbamate on silica gel carrier was achieved by using photochemical and thermal processes. Both approaches provide an easy access to materials which were applied as chiral stationary phases (CSPs) for the chromatographic resolution of racemic molecules. The influence of parameters such as irradiation time and solvent on immobilization effectiveness was investigated. For the thermal processes, azo-bis-isobutyrontrile and di-tert-butyl peroxide were evaluated as radical initiators. The influence of parameters such as amount of radical initiator, solvent, temperature, and further handling operations on the immobilization rate was examined. The chiral recognition ability and the overall performance of the prepared immobilized phases were evaluated by injection of a series of racemic compounds onto packed HPLC columns. As there is almost no limitation of organic solvent types that can be used as mobile phases with the immobilized CSPs, they can be applied under chromatographic conditions which are prohibited with the corresponding non-bonded CSPs. This extended applicability considerably broadens the options for improving enantioselectivity and resolving chiral compounds which are not or only poorly soluble in the conventional mobile phases.

Polysaccharide Derivatives as Unique Chiral Selectors for Enantioselective Chromatography.

From the beginning of the 1980s, the life science industry increasingly recognized the importance of chirality in biological interaction processes, but the methods for preparing optically pure drugs were still limited. Most of the syntheses of chiral compounds were performed starting from optically active building blocks (chiral pool), mainly from natural sources, or by resolution of the enantiomers via formation of diastereomers. In this context, there was a growing interest for enantioselective processes, such as synthetic methodologies and separation techniques for accessing optically pure substances in an effective manner. Among the separation approaches, enantioselective chromatography looked very promising and a project aiming to explore this option was started in the Central Research Laboratories of former Ciba-Geigy. This article reviews the story of this development which culminated in the discovery of highly efficient polysaccharide-based chiral stationary phases which have now become the gold standard in the world of enantioselective chromatography. It shows also how the technique of enantioselective chromatography has evolved from an analytical tool to a practical preparative technology, up to production scale.

High-Performance Liquid Chromatographic Resolution of Neutral and Cationic Hetero[6]Helicenes.

Cationic hetero[6]helicenes 1+, 2+ and 3+ have been recently disclosed. Herein we report on their enantiomeric separation using high-performance liquid chromatography. Separation of the antipodes can be achieved in preparative scale on neutral adducts with Chiralcel OD-I or Chiralpak ID CSP. Selectivity factors of 1.90, 1.67, and 1.96 were obtained for 1-H, 2-H, and 3-H, respectively. Separation can also be performed on the carbenium ions on regular Chiralpak IA CSP using water-containing eluents, thus allowing for enantiomeric purity determinations in aqueous environments. Resolution of neutral and cationic helicenes is also achieved on more recently developed LARIHC columns. The versatility of the cyclofructan phases allows for baseline separations for both cases and their loading capabilities are demonstrated. Finally, the configurational stability of 1+, 2+, and 3+ was measured. For each replacement of an oxygen atom by an amino group, the racemization barrier increases significantly (ΔG‡ = 29.8, 36.3 and >37 kcal mol(-1) for 1+, 2+, and 3+ respectively).

Photochemically Immobilized 4-Methylbenzoyl Cellulose as a Powerful Chiral Stationary Phase for Enantioselective Chromatography.

A process to immobilize para-methylbenzoyl cellulose (PMBC) on silica gel has been developed and applied to prepare chiral stationary phases (CSPs) for enantioselective chromatography. The immobilization was achieved by simple irradiation of the polysaccharide derivative with ultraviolet light after coating on a silica gel support. The influence of parameters such as irradiation time and solvent on immobilization effectiveness were investigated. The performance of the prepared immobilized phases were evaluated by injection of a series of racemic compounds onto the packed columns and determination of their chiral recognition ability. By contrast to the classical coated phase, the immobilized CSP can be used under various chromatographic conditions without limitation of organic solvent types as the mobile phase. This extended applicability permits to improve selectivity and to resolve chiral compounds which are not or only poorly soluble in the mobile phases which are compatible with the non-immobilized PMBC stationary phase.

trans-(3S,4S)-Disubstituted pyrrolidines as inhibitors of the human aspartyl protease renin. Part I: prime site exploration using an amino linker.

Recently, we reported on the discovery of (3S,4S)-disubstituted pyrrolidines (e.g., 2) as inhibitors of the human aspartyl protease renin. In our effort to further expand the scope of this novel class of direct renin inhibitors, a new sub-series was designed in which the prime site substituents are linked to the pyrrolidine core by a (3S)-amino functional group. In particular, analogs bearing the corresponding sulfonamide spacer (50, 51 and 54a) demonstrated a pronounced increase in in vitro potency compared to compound 2.

Enantioselective Chromatography: From its Emergence to its Successful Implementation in the Pharmaceutical Environment.

The author describes the emergence and development of enantioselective chromatography as a powerful tool for the separation of stereoisomers, on analytical, preparative, and production scale. He summarizes his early contribution to the advance and propagation of this particular technology at the interface between chemistry, separation sciences, and physics, going back to his growing interest in chirality which was stimulated by a postdoctoral stay at the University of Geneva.

Synthesis, resolution, and VCD analysis of an enantiopure diazaoxatricornan derivative.

Using simple organic synthetic transformations, a novel diazaoxatricornan derivative, the 12 c-methyl-12-phenyl-8-propyl-12,12 c-dihydro-8 H-4-oxa-8,12-diazadibenzo[ cd, mn]pyrene ( 6a), was prepared. This novel chiral cup-shaped molecule was isolated in racemic form and in excellent yield after the addition of methyl lithium to the BF 4 salt of a novel unsymmetrical diazaoxatriangulenium cation. Compound 6a was found to be stable under classical laboratory conditions-something not obvious considering the extreme stability of the carbenium ion precursor, the electron-rich nature of the core, and the strain induced by the pyramidalization of the central carbon. The enantiomers were readily separated by chiral stationary phase chromatography, and the absolute configuration of (-)-( S)- 6a was determined by a comparison of the experimental and theoretical vibrational circular dichroism (VCD) spectra. This isolation of (-)-( S)- 6a and (+)-( R)- 6a constitutes thus the first report of a nonracemic closed-capped chiral bowl molecule for which the chirality is due to the intrinsic dissymmetry of the central core of the structure only.

Is adamantane a suitable substituent to pre-organize the acid orientation in E-selectin antagonists?

The selectins play a key role in the inflammatory process, that is, the recruitment of leukocytes from blood vessels into inflamed tissue. Because excessive infiltration of leukocytes can induce acute or chronic reactions, the control of leukocyte extravasation is of great pharmaceutical interest. All physiological ligands of the selectins contain the tetrasaccharide epitope sialyl Lewis(x), which therefore became the lead structure in selectin antagonist research. Previous studies indicated that an important factor for the affinity of sLe(x) is the fact that in solution its pharmacophores are already conformationally pre-organized in the bioactive orientation. In mimics where the GlcNAc- and the NeuNAc-moieties of sLe(x) were replaced by (R,R)-cyclohexane-1,2-diol and (S)-cyclohexyllactic acid, respectively, an optimized pre-organization of the pharmacophores could be realized, leading to antagonists with improved affinities. To further optimize the pre-organization of the carboxylic acid, a pharmacophore essential for binding, the replacement of NeuNAc by bulky (R)- and (S)-adamantyl-lactic acid was studied. Although antagonist (S)-7 showed a slightly reduced affinity, the expected beneficial effect of the (S)-configuration at C-2 of the lactate could be confirmed.

Preparation and evaluation of immobilized 4-methylbenzoylcellulose stationary phases for enantioselective separations.

A photochemical method for immobilizing polysaccharide derivatives on silica gel has been developed and applied to 4-methylbenzoyl cellulose (PMBC). The photochemically immobilized materials have been used as chiral stationary phases (CSPs) for the chromatographic separation of the stereoisomers of chiral molecules. Through to the immobilization which makes the chromatographic material insoluble in almost all organic solvents, there no restriction regarding the kind of solvent used in the mobile phase. This feature permits to considerably extend the possibilities to improve the selectivity of the separations and or the solubility of the solute in the mobile phase. The influence of various parameters such as immobilization process, cross-linker type and amount on the chromatographic properties and chiral recognition ability of the resulting CSPs has been investigated using a set of chiral molecules. The impact of the amount of coated polysaccharide material on chiral recognition ability was also examined.

Comparison of liquid and supercritical fluid chromatography mobile phases for enantioselective separations on polysaccharide stationary phases.

Analysis and production of enantiomerically pure compounds is a major topic of interest when active pharmaceutical ingredients are concerned. Enantioselective chromatography has become a favourite both at the analytical and preparative scales. High-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) are dominating the scene and are often seen as complementary techniques. Nowadays, for economic and ecologic reasons, SFC may be preferred over normal-phase HPLC (NPLC) as it allows significant reductions in solvent consumption. However, the transfer of NPLC methods to SFC is not always straightforward. In this study, we compare the retention of achiral molecules and separation of enantiomers under supercritical fluid (carbon dioxide with ethanol or isopropanol) and liquid normal-phase (heptane with ethanol or isopropanol) elution modes with polysaccharide stationary phases in order to explore the differences between the retention and enantioseparation properties between the two modes. Chemometric methods (namely quantitative structure-retention relationships and discriminant analysis) are employed to compare the results obtained on a large set of analytes (171 achiral probes and 97 racemates) and gain some understanding on the retention and separation mechanisms. The results indicate that, contrary to popular belief, carbon dioxide - solvent SFC mobile phases are often weaker eluents than liquid mobile phases. It appears that SFC and NPLC elution modes provide different retention mechanisms. While some enantioseparations are unaffected, facilitating the transfer between the two elution modes, other enantioseparations may be drastically different due to different types and strength of interactions contributing to enantioselectivity.

Novel immunomodulator FTY720 is phosphorylated in rats and humans to form a single stereoisomer. Identification, chemical proof, and biological characterization of the biologically active species and its enantiomer.

In vivo phosphorylation of FTY720 (1) in rats and humans resulted exclusively in the biologically active (S)-configured enantiomer, which was proven by an ex vivo o-phthaldialdehyde derivatization protocol especially elaborated for phosphates of 1. Starting from the prochiral amino alcohol 1, racemic and enantiomerically pure phosphates of 1 were synthesized. Pure enantiomers were obtained after purification of a partially protected key intermediate on an enantioselective support. The absolute stereochemistry was determined by X-ray diffraction.

Supercritical fluid chromatography in pharmaceutical analysis.

In the last few years, there has been a resurgence of supercritical fluid chromatography (SFC), which has been stimulated by the introduction of a new generation of instruments and columns from the main providers of chromatographic instrumentation, that are strongly committed to advancing the technology. The known limitations of SFC, such as weak UV sensitivity, limited reliability and poor quantitative performance have been mostly tackled with these advanced instruments. In addition, due to the obvious benefits of SFC in terms of kinetic performance and its complementarity to LC, advanced packed-column SFC represents today an additional strategy in the toolbox of the analytical scientist, which may be particularly interesting in pharmaceutical analysis. In the present review, the instrumentation and experimental conditions (i.e. stationary phase chemistry and dimensions, mobile phase nature, pressure and temperature) to perform "advanced SFC" are discussed. The applicability of SFC in pharmaceutical analysis, including the determination of drugs in formulations and biofluids is critically discussed.

Orally bioavailable competitive CCR5 antagonists.

The chemokine receptor CCR5 plays an important role in inflammatory and autoimmune disorders as well as in transplant rejection by affecting the trafficking of effector T cells and monocytes to diseased tissues. Antagonists of CCR5 are believed to be of potential therapeutic value for the disorders mentioned above and HIV infection. Here we report on the structure-activity relationship of a new series of highly potent and selective competitive CCR5 antagonists. While all compounds tested were inactive on rodent CCR5, this series includes compounds that cross-react with the cynomolgus monkey (cyno) receptor. One of these compounds, i.e., 26n, has good PK properties in cynos, and its overall favorable profile makes it a promising candidate for in vivo profiling in transplantation and other disease models.

Novel Amide-Based Molecular Knots: Complete Enantiomeric Separation, Chiroptical Properties, and Absolute Configuration.

By comparison of experimental and theoretical CD curves the absolute configurations of chromatographically baseline-separated enantiomers of new trefoil molecular knots could be determined. From the results of syntheses with differently substituted starting materials, conclusions can be drawn about the knot-formation mechanism.

The discovery of novel potent trans-3,4-disubstituted pyrrolidine inhibitors of the human aspartic protease renin from in silico three-dimensional (3D) pharmacophore searches.

The small-molecule trans-3,4-disubstituted pyrrolidine 6 was identified from in silico three-dimensional (3D) pharmacophore searches based on known X-ray structures of renin-inhibitor complexes and demonstrated to be a weakly active inhibitor of the human enzyme. The unexpected binding mode of the more potent enantiomer (3S,4S)-6a in an extended conformation spanning the nonprime and S1' pockets of the recombinant human (rh)-renin active site was elucidated by X-ray crystallography. Initial structure-activity relationship work focused on modifications of the hydrophobic diphenylamine portion positioned in S1 and extending toward the S2 pocket. Replacement with an optimized P3-P1 pharmacophore interacting to the nonsubstrate S3(sp) cavity eventually resulted in significantly improved in vitro potency and selectivity. The prototype analogue (3S,4S)-12a of this new class of direct renin inhibitors exerted blood pressure lowering effects in a hypertensive double-transgenic rat model after oral administration.

A novel class of oral direct renin inhibitors: highly potent 3,5-disubstituted piperidines bearing a tricyclic p3-p1 pharmacophore.

A small library of fragments comprising putative recognition motifs for the catalytic dyad of aspartic proteases was generated by in silico similarity searches within the corporate compound deck based on rh-renin active site docking and scoring filters. Subsequent screening by NMR identified the low-affinity hits 3 and 4 as competitive active site binders, which could be shown by X-ray crystallography to bind to the hydrophobic S3-S1 pocket of rh-renin. As part of a parallel multiple hit-finding approach, the 3,5-disubstituted piperidine (rac)-5 was discovered by HTS using a enzymatic assay. X-ray crystallography demonstrated the eutomer (3S,5R)-5 to be a peptidomimetic inhibitor binding to a nonsubstrate topography of the rh-renin prime site. The design of the potent and selective (3S,5R)-12 bearing a P3(sp)-tethered tricyclic P3-P1 pharmacophore derived from 3 is described. (3S,5R)-12 showed oral bioavailability in rats and demonstrated blood pressure lowering activity in the double-transgenic rat model.

Stereoselective synthesis of configurationally stable functionalized ethano-bridged Tröger bases.

New functionalized ethano-bridged Tröger bases are readily prepared using a simple alkylation/rearrangement sequence which affords configurationally stable derivatives as single stereoisomers (de > 96%).

Conformational refinement of hydroxamate-based histone deacetylase inhibitors and exploration of 3-piperidin-3-ylindole analogues of dacinostat (LAQ824).

Inspired by natural product HDAC inhibitors, we prepared a series of conformationally restrained HDAC inhibitors based on the hydroxamic acid dacinostat (LAQ824, 7). Several scaffolds with improved biochemical and cellular potency, as well as attenuated hERG inhibition, were identified, suggesting that the introduction of molecular rigidity is a viable strategy to enhance HDAC binding and mitigate hERG liability. Further SAR studies around a 3-piperidin-3-ylindole moiety resulted in the discovery of compound 30, for which a unique conformation was speculated to contribute to overcoming increased lipophilicity and attenuating hERG binding. Separation of racemate 30 afforded 32, the R enantiomer, which demonstrated improved potency in both enzyme and cellular assays compared to dacinostat.

Spirotetrahydro beta-carbolines (spiroindolones): a new class of potent and orally efficacious compounds for the treatment of malaria.

The antiplasmodial activity of a series of spirotetrahydro beta-carbolines is described. Racemic spiroazepineindole (1) was identified from a phenotypic screen on wild type Plasmodium falciparum with an in vitro IC(50) of 90 nM. Structure-activity relationships for the optimization of 1 to compound 20a (IC(50) = 0.2 nM) including the identification of the active 1R,3S enantiomer and elimination of metabolic liabilities is presented. Improvement of the pharmacokinetic profile of the series translated to exceptional oral efficacy in the P. berghei infected malaria mouse model where full cure was achieved in four of five mice with three daily doses of 30 mg/kg.