Individual Diastereoisomers Research Articles

Central-to-Folding Chirality Control: Asymmetric Synthesis of Multilayer 3D Targets With Electron-Deficient Bridges.

New multilayer 3D chiral molecules have been designed and synthesized asymmetrically through the strategy of center-to-multilayer folding chirality control and double Suzuki couplings. Individual diastereoisomers were readily obtained and separated via flash column chromatography. The key diastereoisomer was further converted into corresponding enantiomers. These enantiomers possess electron-deficient aromatic bridges layered with top and bottom aromatic scaffolds. X-ray structural analysis has unambiguously confirmed the configuration, and intermolecular packing results in regular planar patterns in solid crystals. The synthesis was achieved in a total of ten steps starting from commercially available starting materials.

Chemical, Analytical and Pharmacokinetic Characterisation of RO7304898, an API Consisting of Two Rapidly Interconverting Diastereoisomers.

Exploration of the chemical, analytical and pharmacokinetic properties of the API, RO7304898, an allosteric EGFR inhibitor, intended to be developed as a mixture of two rapidly interconverting diastereoisomers with composition ratio of approximately 1:1. Assessment of diastereoisomer stereochemistry, interconversion rates, binding to EGFR protein, metabolic stability and in vivo PK in Wistar-Han rats was conducted. The two diastereoisomers of the API undergo fast interconversion at physiologically relevant pH and direct EGFR binding studies revealed diastereoisomer B to be the active moiety. Pharmacokinetic studies in rat revealed a low-moderate total plasma clearance of the API along with similar plasma concentration-time profiles for diastereoisomers A and B, and the diastereoisomeric ratio reached stable equilibrium favoring formation of the potent diastereoisomer B. In in vitro incubations, the API was metabolically stable in plasma and hepatocyte suspension incubations in all species tested except that of rat hepatocytes. Additionally, only small species differences in the A:B composition were observed in vitro with the potent diastereoisomer B being the predominant form. We demonstrated that the API, a mixture of two diastereoisomers; A (impotent) and B (potent), undergoes rapid interconversion which is faster than the apparent distribution and elimination rates of the individual diastereoisomers in vivo in rat, serving to diminish concerns that separate diastereoisomer effects may occur in subsequent pharmacologic and pivotal toxicological studies. Whilst vigilant monitoring of the diastereoisomeric ratio will need to be continued, this data adds confidence on the development pathway for this API to the clinic.

Characterizing the Diastereoisomeric Distribution of Phosphorothioate Oligonucleotides by Metal Ion Complexation Chromatography, In-Series Reversed Phase-Strong Anion Exchange Chromatography, and 31P NMR.

Replacement of a non-bridging oxygen atom of the phosphate diester linkage of an oligonucleotide by sulfur conveys pharmacokinetic benefits, such as increased nuclease resistance and enhanced protein binding. Substitution renders the internucleotide linkages chiral, and so phosphorothioate diester (PS) oligonucleotides comprise complex mixtures of diastereoisomers. Currently, chromatographic separation of individual diastereoisomers is limited to oligonucleotides that contain no more than about four or five PS linkages. The development of therapeutic PS oligonucleotides, which often contain >15 PS linkages, would be greatly aided by methods useful for assessing batch-to-batch stereo-reproducibility. To this effect, the relative sensitivities of metal ion complexation chromatography (MICC), in-series reversed phase-strong anion exchange chromatography (RP-SAX), and 31P NMR toward changes in the diastereoisomeric distributions of therapeutic PS oligonucleotides were compared. Model oligonucleotides synthesized under conditions known to impact PS stereochemistry were used to evaluate the method performance, and all three methods showed excellent sensitivity toward changes in the diastereoisomeric composition. Interactions via the solvent-accessible areas and a combination of hydrophobic and electrostatic forces may be responsible for the selectivity demonstrated by MICC and in-series RP-SAX, respectively.

Polar Organofluorine Substituents: Multivicinal Fluorines on Alkyl Chains and Alicyclic Rings.

This Review outlines the progression, primarily of our own work, but with important contributions from other labs, on the synthesis and properties of multiple vicinally fluorinated alkyl chains and rings. Chain conformations of individual diastereoisomers with -CHF- at adjacent carbons are influenced by stereoelectronic factors associated with the polar C-F bond and the polarised geminal hydrogens. Generally, the chain will prefer a conformation which acts to minimise overall molecular polarity, and where the C-F bonds orient away from each other. However, when vicinal fluorine atoms are positioned on a ring then conformations are more constrained. The ring will adopt optimal conformations such as a chair in cyclohexane and then C-F bonds can be introduced with a stereochemistry that forces parallel (axial) orientations. In the case of cyclohexane, 1,3-diaxial arrangements of C-F bonds impart considerable polarity to the ring, resulting in an electronegative 'fluorine face' and an electropositive 'hydrogen face'. For all-syn 1,2,3,4,5,6-hexafluorocyclohexane, this arrangement generates an unusually polar aliphatic ring system. Most recently the concept has been extended to the preparation of all-syn 1,2,3-trifluorocyclopropanes, a rigid ring system with fluorine atoms on one face and hydrogens on the other. Lipophilicity Log P values of such compounds indicate that they are significantly more polar than their parent alicyclic hydrocarbons and give some positive indication for a future role of such substituents in medicinal chemistry. Expanding to such a role will require access to improved synthesis methods to these motifs and consequently access to a broader a range of building blocks, however some exciting new methods have emerged recently and these are briefly reviewed.

Developmental toxicity of three hexabromocyclododecane diastereoisomers in embryos of the marine medaka Oryzias melastigma

The composition of major hexabromocyclododecane (HBCD) diastereoisomers, i.e. α-, β-, and γ-HBCDs, in marine biota is different from that of the commercially available form (technical HBCD), which is used extensively for toxicological studies. To properly evaluate the impact of HBCDs, the embryos of Oryzias melastigma were used to examine the developmental toxicity of the individual diastereoisomers. Results showed that HBCD diastereoisomers at the environmentally realistic concentrations in the embryos induced malformation rate and heartbeat, and caused the appearance of apoptotic heart. In addition, α-, β-, and γ-HBCDs had similar potency to stimulate the generation of reactive oxygen species, consequently leading to apoptosis in O. melastigma embryos. The order of the developmental toxicity of α-, β-, and γ-HBCDs in O. melastigma embryos was different from that in zebrafish embryos studied previously, which highlighted the importance of using species from both fresh and salt water for toxicity assessment.

ChemInform Abstract: Practical Synthesis of Diastereoisomers by a Temperature‐Guided Reversal of Diastereoselectivity in the Desymmetrization of meso‐Bridged Anhydrides with a Chiral Oxazolidin‐2‐one.

Abstract A perfect temperature-guided diastereoselectivity switch was observed in the desymmetrization of some meso -bridged tricyclic succinic anhydrides with a lithiated chiral 5,5-disubstituted oxazolidin-2-one called SuperQuat. Experimental conditions required for the optimum diastereoselectivity and to obtain the individual diastereoisomers of the SuperQuat linked meso -anhydride opening products with acceptable purity and yield have been established.

Practical synthesis of diastereoisomers by a temperature-guided reversal of diastereoselectivity in the desymmetrization of meso-bridged anhydrides with a chiral oxazolidin-2-one

A perfect temperature-guided diastereoselectivity switch was observed in the desymmetrization of some meso-bridged tricyclic succinic anhydrides with a lithiated chiral 5,5-disubstituted oxazolidin-2-one called SuperQuat. Experimental conditions required for the optimum diastereoselectivity and to obtain the individual diastereoisomers of the SuperQuat linked meso-anhydride opening products with acceptable purity and yield have been established.

Crystallographic, Spectroscopic, and Theoretical Investigation of the Efficiently Separated 21R and 21S‐Diastereoisomers of Argatroban

Argatroban (I), a potent noncovalent reversible thrombin inhibitor, is used as an anticoagulant for the parenteral treatment of heparin-induced thrombocytopenia (HIT) patients. By virtue of its pharmacological properties and the well-balanced risks and benefits, argatroban is now emerging as a clinically relevant antithrombotic agent. The availability of this drug as a mixture of 21R and 21S-diastereoisomers, in a ratio of roughly 64:36, prompted us to design an efficient separation setup of the two epimers. We pursued our efforts on their detailed structural analysis with the aim of understanding their different activity and aqueous solubility. These investigations were accompanied by a modeling study of the two diastereoisomers, with particular attention on the easy interconverting half-chair of the tetrahydroquinoline system and its preferred conformation, which is determined by the configuration at C21. These results, together with the analysis of their physicochemical profiles, provide new useful information for the development of the individual diastereoisomers.

Synthesis and structural characterization of the individual diastereoisomers of a cross-stapled alkene-bridged nisin DE-ring mimic

Herein, we describe the synthesis, structural characterization, and synthetic use as an advanced intermediate of a cross-stapled alkene-bridged hexapeptide to mimic the DE-ring of the lantibiotic nisin. The linear precursor was cyclized by ring-closing metathesis to give the correctly folded bicyclic hexapeptide in a single step, and the four individual diastereoisomers were isolated, structurally assigned and characterized by HPLC, NMR and MS, respectively. The bicyclic hexapeptide was used as a versatile advanced synthon and was modified at its C- and N-terminus, among others, with an azide moiety to access a building block suitable for Cu(I)-catalyzed alkyne-azide cycloaddition-based ligation reactions.

3‐Fluoro‐N‐methyl‐D‐aspartic acid (3F‐NMDA) Stereoisomers as Conformational Probes for Exploring Agonist Binding at NMDA Receptors

N-Methyl-D-aspartate (NMDA) is the prototypical agonist of the NMDA receptor subtype of ionotropic glutamate receptors. Stereogenic placement of a C-F bond at the 3-position of (S)-NMDA generates either the (2S,3S)- or (2S,3R)- diastereoisomers of 3F-NMDA. The individual diastereoisomers were prepared by synthesis in enantiomerically pure forms and it was found that (2S,3S)-3F-NMDA is an agonist with a comparable potency to NMDA itself, whereas the (2S,3R)-diastereoisomer has negligible potency. The difference in potency of these stereoisomers is attributed to a preference of the C-F bond (2S,3S)-3F-NMDA to adopt a gauche conformation to the C-N(+) bond in the binding conformation, whereas the (2S,3R)-3F-NMDA forces these bonds anti, losing electrostatic stabilisation, to achieve the required binding conformation. These observations illustrate the utility of stereoselective fluorination in influencing the molecular conformation of β-fluorinated amino acids and thus probing the active conformations of bioactive compounds at receptors.

Organofluorine Chemistry: Synthesis and Conformation of Vicinal Fluoromethylene Motifs

The C-F bond is the most polar bond in organic chemistry, and thus the bond has a relatively large dipole moment with a significant -ve charge density on the fluorine atom and correspondingly a +ve charge density on carbon. The electrostatic nature of the bond renders it the strongest one in organic chemistry. However, the fluorine atom itself is nonpolarizable, and thus, despite the charge localization on fluorine, it is a poor hydrogen-bonding acceptor. These properties of the C-F bond make it attractive in the design of nonviscous but polar organic compounds, with a polarity limited to influencing the intramolecular nature of the molecule and less so intermolecular interactions with the immediate environment. In this Perspective, the synthesis of aliphatic chains carrying multivicinal fluoromethylene motifs is described. It emerges that the dipoles of adjacent C-F bonds orientate relative to each other, and thus, individual diastereoisomers display different backbone carbon chain conformations. These conformational preferences recognize the influence of the well-known gauche effect associated with 1,2-difluoroethane but extend to considering 1,3-fluorine-fluorine dipolar repulsions. The synthesis of carbon chains carrying two, three, four, five, and six vicinal fluoromethylene motifs is described, with an emphasis on our own research contributions. These motifs obey almost predictable conformational behavior, and they emerge as candidates for inclusion in the design of performance organic molecules.

Single enantiomer synthesis of α-(trifluoromethyl)-β-lactam.

The first synthesis of α-(trifluoromethyl)-β-lactam ((S)-1) is reported. The route starts from α-(trifluoromethyl)acrylic acid (2). Conjugate addition of α-(p-methoxyphenyl)ethylamine ((S)-3b), generated an addition adduct 4b which was cyclised to β-lactam 5b. Separation of the diastereoisomers by chromatography gave ((αS,3S)-5b). N-Debenzylation afforded the desired α-(trifluoromethyl)-β-lactam ((S)-1). The absolute stereochemistry of diastereoisomers 5 was determined by X-ray crystallographic determination of a close structural analogue, (αS,3S)-5c, and then 1H and 19F NMR correlation to the individual diastereoisomers of 5a and 5b.

ChemInform Abstract: Synthesis of Dimeric 1,3,2λ5-Benzoxazaphospholes from Phosphinic Acid Derivatives by Silylation/Desiloxylation.

The reaction of N,N,O-tris(trimethylsilyl)-o-aminophenol with two equivalents of phosphinic chloride yielded dimeric 2,2-disubstituted 1,3,2λ5-benzoxazaphospholes and trimethylsilyl phosphinate. The chlorides having the bulk substituents (o-chlorophenyl or tert-butyl) at phosphorus or containing PN and PO bonds (instead of a PC bond) either didn't react at all or reacted to retain the phosphoryl group. Being stable in solution at 20°C, the individual diastereoisomers of dimeric 1,3,2-benzoxazaphospholes were converted upon warming to an equilibrium mixture of isomers. When reacted with another dimer each gave a mixed dimeric compound having two different phosphorus atoms in the molecule.

Chiral Separation of Diastereoisomers of Cefuroxime Axetil by High-Performance Thin-Layer Chromatography

Conditions for separation and determination of diastereoisomers of cefuroxime axetil by HPTLC on cellulose layers with densitometry were developed. A good separation of the constituents was achieved using a mobile phase composed of 1% aqueous beta-cyclodextrin-methanol (15 + 1, v/v). For detecting spots on the chromatograms, densitometric measurements were used at 285 nm. The method is characterized by high sensitivity; the LOD was 0.04 microg/spot and LOQ was 0.11 microg/spot for both forms of the cefuroxime axetil diastereoisomers. For the individual diastereoisomers, high recovery values from 96.63 to 104.16% were obtained. Identification of the diastereoisomers was performed by 1H-NMR spectrometric analysis.

Synthesis of the Individual Diastereomers of the Cysteine Conjugate of 3-Mercaptohexanol (3-MH)

The individual diastereoisomers of the cysteine conjugate of 3-mercaptohexanol (4) were synthesized with high isomeric purity (>98%). On treatment with Apotryptophanase enzyme, the 3R diastereoisomer of 4 gave an 82% yield of the R enantiomer of 1, with no trace of the 3S enantiomer present. Conversely, the 3S diastereoisomer of 4 gave the 3S enantiomer of 1 (43%) accompanied by a trace of the 3R form (S/R = 98.5:1.5), reflecting the diastereomeric purity of the cysteine conjugate. The same stereochemical outcome was observed when the individual diastereoisomers of 4 were added to fermentations with the Saccharomyces cerevisiae AWRI 1655 yeast strain, which gave 1 in 1% yield. A d(10)-analogue of 1 was synthesized and used as an internal standard to determine, by gas chromatography-mass spectrometry (GC-MS), the amounts of 1 formed in these transformations.

Refinements to the diastereoisomer‐specific method for the analysis of hexabromocyclododecane

The emergence of hexabromocyclododecane (HBCD) as a bromine-based flame retardant of concern is partly attributable to recent measurements on the environmental occurrence of the individual diastereoisomers (alpha, beta and gamma). These measurements were fuelled by a newly developed liquid chromatography/tandem mass spectrometric (LC/MS/MS)-based analytical method. However, in the course of our recent studies on the environmental fate and behaviour of the diastereoisomers of HBCD, some interesting features of the LC/MS/MS method became apparent. For example, the ion signal of the native ions was found to be dependent on the final extract volume. This was true for both biotic and sediment samples and was found to arise from the suppression of the ion signal due to endogenous material in the extracts that escape clean-up. We have also found differences in the stability of the diastereoisomers in different solvents. If left unaccounted for, both factors can compromise analytical measurement data. By way of a series of controlled experiments conducted at our two laboratories [Department of Fisheries & Oceans Canada (DFO) and Environment Canada (EC)], we illustrate these features and demonstrate that use of newly synthesized labelled HBCD isomers [(13-carbon (13C) and deuterium (d18)] can minimize and often circumvent matrix-related effects.

Configurations of diastereomeric hydroxyethylene isosteres strongly affect biological activities of a series of specific inhibitors of human-immunodeficiency-virus proteinase.

Human immunodeficiency virus (HIV) proteinase (PR) represents an important target for antiviral chemotherapy. We present an analysis of inhibitory activities of a series of pseudopeptide inhibitors of HIV-1 PR. All inhibitors were N-protected tetrapeptides with the scissile bond replaced by a nonhydrolysable hydroxyethylene or hydroxyethylamine isostere. To elucidate subtle structural requirements of the PR binding cleft, we synthesised inhibitors with four combinations of configurations at the asymmetric carbons of the isostere. Compounds were tested in vitro using purified recombinant enzyme and a chromogenic peptide substrate. The differences in inhibition constants between individual diastereoisomers reached three orders of magnitude. The most active hydroxyethylene-containing inhibitor possessed the 2R,4S,5S configuration at the isostere. Inhibitor activity was also tested in mammalian cell culture by analysing reduction of viral polyprotein processing and virus infectivity. The results obtained in tissue culture were generally in agreement with the in vitro data, giving a similar order of potency for the individual diastereoisomers. The most active compounds completely blocked production of infectious virus. A simulation method for interaction was employed to build a model of the inhibitors in the PR active site, to identify the interactions responsible for the differences in activities of individual stereoisomers, and to estimate the relative contribution of individual structural features to the overall inhibitory activity.

The photo-epimerisation of gossypol Schiff's bases

1H NMR studies show that the diastereoisomeric adducts 3 of gossypol 1 and (L)-phenylalanine methyl ester undergo epimerisation, in hexane/ethyl acetate or deuteriochloroform, in sunlight. The individual diastereoisomers 3 can be equilibrated to a 50:50 mixture of diastereoisomers, thus allowing an efficient procedure for the recycling of the biologically-inactive D-gossypol. 1H NMR studies show that the diastereoisomeric adducts 3 of gossypol 1 and (L)-phenylalanine methyl ester undergo epimerisation, in hexane/ehtyl acetate or deuteriochloroform, in sunlight thus allowing the recycling of the biologically inactive enantiomer of gossypol.

ChemInform Abstract: Asymmetric Total Synthesis of the Individual Diastereoisomers of Hypoglycin A.

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Asymmetric total synthesis of the individual diastereoisomers of hypoglycin A.

The individual diastereoisomers that constitute the unusual methylenecyclopropane containing α-amino acid hypoglycin A have been synthesised utilising the Sharpless epoxidation to permit an asymmetric methylenecyclopropane synthesis.