Chiral Chromatographic Isolation on Milligram Scale of the Human African Trypanosomiasis Treatment d- and l-Eflornithine.

Eflornithine is a recommended treatment against the otherwise fatal parasitic disease late stage human African trypanosomiasis (HAT), also known as Gambian sleeping sickness. It is administered repeatedly as a racemic mixture intravenously (IV) together with oral nifurtimox. Racemic eflornithine has been investigated in clinical trials for oral dosing. However, due to low systemic exposures at a maximum tolerated oral dose, the drug is continued to be administered IV. The eflornithine enantiomers, d- and l-eflornithine, have different affinities to the target enzyme ornithine decarboxylase, suggesting that the pharmacodynamics of the enantiomers may differ. The aim of this study was to develop a method for isolation of d- and l-eflornithine from a racemic mixture. Several chiral stationary phases (CSPs) were evaluated for enantioselectivity using supercritical fluid chromatography (SFC) or high-performance liquid chromatography (HPLC). None of the tested CSPs rendered separation of the enantiomers in SFC mode. Separation of the enantiomers with SFC on the CSP Chiralpak IG was only achieved on an analytical scale after derivatization with ortho-phthalaldehyde (OPA). This was the first reported enantioselective SFC method for an eflornithine derivate. However, due to poor stability, the eflornithine-OPA derivates degraded and no chemically pure enantiomers were obtained. The CSP that showed enantioselectivity in HPLC mode was Chirobiotic R, which resulted in a successful isolation on a semipreparative milligram scale. The isolated eflornithine enantiomers will be tested in nonclinical in vitro and in vivo studies to support and assess the feasibility of a future clinical program with an oral HAT treatment.

Unexpected carbonate salt formation during isolation of an enantiopure intermediate by supercritical fluid chromatography.

The enantiomers of a chiral building block to be used in pre-clinical manufacturing were separated using supercritical fluid chromatography (SFC). Despite an extensive evaluation of different columns and solvent combinations followed by a careful optimization of the chromatographic method, the preparative separation suffered from low throughput and high solvent consumption. Consequently, additional improvements were necessary. By utilizing stacked injections, the chromatographic run time was almost halved, and the high solvent consumption was reduced by recycling of the two mobile phase components, carbon dioxide and methanol. The carbon dioxide was reprocessed by the SFC instrument, whereas methanol was evaporated and recycled from the fractions collected. Hence, the originally inefficient separation method was turned into a more sustainable one, and the desired enantiopure intermediate was delivered to be used in the following synthesis of the selected candidate drug. Unfortunately, when the intermediate was used in the subsequent amide coupling, a surprisingly poor yield was obtained. This was caused by an unexpected formation of a stable carbonate salt of the intermediate under the chromatographic conditions used. By removal of the carbonate prior to the amide coupling reaction, the manufacturing campaign could be saved, and the candidate drug was successfully delivered in time.

Systematic investigations of peak distortions due to additives in supercritical fluid chromatography.

The impact of eluent components added to improve separation performance in supercritical fluid chromatography was systematically, and fundamentally, investigated. The model system comprised basic pharmaceuticals as solutes and eluents containing an amine (i.e., triethylamine, diethylamine, or isopropylamine) as additive with MeOH as the co-solvent. First, an analytical-scale study was performed, systematically investigating the impact of the additives/co-solvent on solute peak shapes and retentions, using a design of experiments approach; here, the total additive concentration in the eluent ranged between 0.021 and 0.105 % (v/v) and the MeOH fraction in the eluent between 16 and 26 % (v/v). The co-solvent fraction was found to be the most efficient tool for adjusting retentions, whereas the additive fraction was the prime tool for improving column efficiency and peak analytical performance. Next, the impacts of the amine additives on the shapes of the so-called overloaded solute elution profiles were investigated. Two principal types of preparative peak deformations appeared and were investigated in depth, analyzed using computer simulation with mechanistic modeling. The first type of deformation was due to the solute eluting too close to the additive perturbation peak, resulting in severe peak deformation caused by co-elution. The second type of deformation was also due to additive-solute interactions, but here the amine additives acted as kosmotropic agents, promoting the multilayer adsorption to the stationary phase of solutes with bulkier aryl groups.

Analytical and preparative separation of phosphorothioated oligonucleotides: columns and ion-pair reagents.

Oligonucleotide drugs represent an emerging area in the pharmaceutical industry. Solid-phase synthesis generates many structurally closely related impurities, making efficient separation systems for purification and analysis a key challenge during pharmaceutical drug development. To increase the fundamental understanding of the important preparative separation step, mass-overloaded injections of a fully phosphorothioated 16mer, i.e., deoxythymidine oligonucleotide, were performed on a C18 and a phenyl column. The narrowest elution profiles were obtained using the phenyl column, and the 16mer could be collected with high purity and yield on both columns. The most likely contribution to the successful purification was the quantifiable displacement of the early-eluting shortmers on both columns. In addition, the phenyl column displayed better separation of later-eluting impurities, such as the 17mer impurity. The mass-overloaded injections resulted in classical Langmuirian elution profiles on all columns, provided the concentration of the ion-pairing reagent in the eluent was sufficiently high. Two additional column chemistries, C4 and C8, were also investigated in terms of their selectivity and elution profile characteristics for the separation of 5-20mers fully phosphorothioated deoxythymidine oligonucleotides. When using triethylamine as ion-pairing reagent to separate phosphorothioated oligonucleotides, we observed peak broadening caused by the partial separation of diastereomers, predominantly seen on the C4 and C18 columns. When using the ion-pair reagent tributylamine, to suppress diastereomer separation, the greatest selectivity was found using the phenyl column followed by C18. The present results will be useful when designing and optimizing efficient preparative separations of synthetic oligonucleotides.

Potent and Orally Bioavailable Inverse Agonists of RORγt Resulting from Structure-Based Design.

Retinoic acid receptor related orphan receptor γt (RORγt), has been identified as the master regulator of TH17-cell function and development, making it an attractive target for the treatment of autoimmune diseases by a small-molecule approach. Herein, we describe our investigations on a series of 4-aryl-thienyl acetamides, which were guided by insights from X-ray cocrystal structures. Efforts in targeting the cofactor-recruitment site from the 4-aryl group on the thiophene led to a series of potent binders with nanomolar activity in a primary human-TH17-cell assay. The observation of a DMSO molecule binding in a subpocket outside the LBD inspired the introduction of an acetamide into the benzylic position of these compounds. Hereby, a hydrogen-bond interaction of the introduced acetamide oxygen with the backbone amide of Glu379 was established. This greatly enhanced the cellular activity of previously weakly cell-active compounds. The best compounds combined potent inhibition of IL-17 release with favorable PK in rodents, with compound 32 representing a promising starting point for future investigations.

Saccharin Aza Bioisosteres-Synthesis and Preclinical Property Comparisons.

Saccharin is a well-known scaffold in drug discovery. Herein, we report the synthesis and preclinical property comparisons of three bioisosteres of saccharin: aza-pseudosaccharins (cluster B), and two new types of aza-saccharins (clusters C and D). We demonstrate a convenient protocol to selectively synthesize products in cluster C or D when primary amines are used. Preclinical characterization of selected matched-pair products is reported. Through comparison of two diastereomers, we highlight how stereochemistry affects the preclinical properties. Given that saccharin-based derivatives are widely used in many chemistry fields, we foresee that structures exemplified by clusters C and D offer new opportunities for novel drug design, creating a chiral center on the sulfur atom and the option of substitution at two different nitrogens.

Systematic investigations of peak deformations due to co-solvent adsorption in preparative supercritical fluid chromatography.

Strangely shaped overloaded bands were recently reported using a standard supercritical fluid chromatographic system comprising a diol column as the stationary phase and carbon dioxide with methanol as the mobile phase. Some of these overloaded elution profiles appeared strongly deformed and even had "anti-Langmuirian" shapes although their solute compounds had "Langmuirian" adsorption. To obtain a more complete understanding of the generality of these effects, the investigation was expanded to cover also other common co-solvents, such as ethanol, 2-propanol, and acetonitrile, as well as various stationary phase materials, such as silica, and 2-ethylpyridine. From this expanded study it could be confirmed that the effects of deformed overloaded solute band shapes, due to co-solvent adsorption, is general phenomena in supercritical fluid chromatographic. It could also be concluded that these effects as well as previously observed "solvent effects" or "plug effects" are entirely due to competition between the solute and solvent molecules for the adsorption sites on the stationary phase surface. Finally, guidelines were given for how to evaluate the risk of deformations occurring for a given solvent-column combination, based simply on testing retention times of solutes and co-solvent.

Preparative Scale Resolution of Enantiomers Enables Accelerated Drug Discovery and Development.

The provision of pure enantiomers is of increasing importance not only for the pharmaceutical industry but also for agro-chemistry and biotechnology. In drug discovery and development, the enantiomers of a chiral drug depict unique chemical and pharmacological behaviors in a chiral environment, such as the human body, in which the stereochemistry of the chiral drugs determines their pharmacokinetic, pharmacodynamic and toxicological properties. We present a number of challenging case studies of up-to-kilogram separations of racemic or enriched isomer mixtures using preparative liquid chromatography and super critical fluid chromatography to generate individual enantiomers that have enabled the development of new candidate drugs within AstraZeneca. The combination of chromatography and racemization as well as strategies on when to apply preparative chiral chromatography of enantiomers in a multi-step synthesis of a drug compound can further facilitate accelerated drug discovery and the early clinical evaluation of the drug candidates.

Strategy for large-scale isolation of enantiomers in drug discovery.

A strategy for large-scale chiral resolution is illustrated by the isolation of pure enantiomer from a 5kg batch. Results from supercritical fluid chromatography will be presented and compared with normal phase liquid chromatography. Solubility of the compound in the supercritical mobile phase was shown to be the limiting factor. To circumvent this, extraction injection was used but shown not to be efficient for this compound. Finally, a method for chiral resolution by crystallization was developed and applied to give diastereomeric salt with an enantiomeric excess of 99% at a 91% yield. Direct access to a diverse separation tool box will be shown to be essential for solving separation problems in the most cost and time efficient way.

Peak deformations in preparative supercritical fluid chromatography due to co-solvent adsorption.

In supercritical fluid chromatography (SFC) the mobile phase comprises of carbon dioxide (CO2) as main solvent and smaller amounts of an organic polar solvent (often an alcohol) as co-solvent. The co-solvent is considered to function by changing the overall polarity of the eluent, i.e. by acting as a "modifier". However, recent studies indicate that the co-solvent methanol can also adsorb to some common SFC stationary phases. Hence, the co-solvent should also be able to function as an "adsorbing additive", i.e. an eluent component that competes with the injected solutes about the stationary phase surface. In this study it was found by fitting different mechanistic models to systematic experimental data, that the co-solvent methanol can have both functions: at low co-solvent fractions, methanol acts as an additive whereas at larger fractions it acts as a modifier. Moreover, it was found that when the co-solvent adsorbs more strongly to the stationary phase than the solute, "bizarre" deformations of the preparative band shapes can occur. This is illustrated by a solute that converts from a normal "Langmuirian" band shape to an "anti-Langmuirian" shape when changing from neat carbon dioxide (CO2) to an eluent containing co-solvent. This peak shape transition is dependent on both (i) the relative retention of the solute and co-solvent to the stationary phase in eluent containing neat CO2 and on (ii) the relative retention of the additive perturbation peak and the solute peak in eluent containing also co-solvent.

A closer study of methanol adsorption and its impact on solute retentions in supercritical fluid chromatography.

Surface excess adsorption isotherms of methanol on a diol silica adsorbent were measured in supercritical fluid chromatography (SFC) using a mixture of methanol and carbon dioxide as mobile phase. The tracer pulse method was used with deuterium labeled methanol as solute and the tracer peaks were detected using APCI-MS over the whole composition range from neat carbon dioxide to neat methanol. The results indicate that a monolayer (4Å) of methanol is formed on the stationary phase. Moreover, the importance of using the set or the actual methanol fractions and volumetric flows in SFC was investigated by measuring the mass flow respective pressure and by calculations of the actual volume fraction of methanol. The result revealed a significant difference between the value set and the actually delivered volumetric methanol flow rate, especially at low modifier fractions. If relying only on the set methanol fraction in the calculations, the methanol layer thickness should in this system be highly overestimated. Finally, retention times for a set of solutes were measured and related to the findings summarized above concerning methanol adsorption. A strongly non-linear relationship between the logarithms of the retention factors and the modifier fraction in the mobile phase was revealed, prior to the established monolayer. At modifier fractions above that required for establishment of the methanol monolayer, this relationship turns linear which explains why the solute retention factors are less sensitive to changes in modifier content in this region.

Evaluation of scale-up from analytical to preparative supercritical fluid chromatography.

An approach for reliable transfer from analytical to preparative scale supercritical fluid chromatography was evaluated. Here, we accounted for the conditions inside the columns as well as to the fact that most analytical instruments are volume-controlled while most preparative scale units are mass-controlled. The latter is a particular problem when performing pilot scale experiments and optimizations prior to scaling up to production scale. This was solved by measuring the mass flow, the pressure and the temperature on the analytical unit using external sensors. Thereafter, it was revealed with a design of experiments approach that the methanol fraction and the pressure are the two most important parameters to control for preserved retention throughout the scale-up; for preserved selectivity the temperature was most important in this particular system. Using this approach, the resulting chromatograms from the preparative unit agreed well with those from the analytical unit while keeping the same column length and particles size. A brief investigation on how the solute elution volume varies with the volumetric flow rate revealed a complex dependency on pressure, density and apparent methanol content. Since the methanol content is a parameter of great importance to control during the scale up, we must be careful when changing operational and column design conditions which generates deviations in pressure, density and methanol content between different columns.