Investigating polymorphism in small molecules using three-dimensional electron diffraction

For scientific, regulatory and intellectual property reasons, the discovery and characterisation of polymorphic systems is an integral aspect of the development process of any solid-formulated drug product. Yet, these studies are often hindered by crystal quality and size, poor yields and the generation of mixtures of phases. Three-dimensional electron diffraction (3D ED) is a technique capable of structure determination from individually selected, nanometre-sized crystals. In this thesis, 3D ED is applied to investigate polymorphism in small molecules. The unique advantages of the method are highlighted across numerous studies to demonstrate how 3D ED can broaden the scope of polymorphism discovery and characterisation in the screening and selection of pharmaceutical crystal forms.  3D ED is first applied to reveal that two crystallisation methods believed for 47 years to produce Form δ of the pharmaceutical compound indomethacin result in two different polymorphs, highlighting the power of the method for polymorphism discovery. The polymorphic crystal structures of a small molecule are then determined directly from melt-grown compact spherulites for the first time to show how 3D ED can widen the application of melt crystallisation in polymorph screening, where polycrystalline spherulites are common products. Furthermore, 3D ED is combined with on-the-grid crystallisation and plunge freezing to follow the polymorph evolution of glycine during crystallisation from an aqueous solution to demonstrate the ability of the method to monitor crystallisation processes in situ. The final part of the thesis explores how a high-throughput method combining 3D ED data collection in batch mode with semi-automated data processing can be applied for the phase analysis of complex melt crystallisation products to improve the efficiency and accuracy of polymorph screening

Investigating polymorphism in small molecules using three-dimensional electron diffraction

For scientific, regulatory and intellectual property reasons, the discovery and characterisation of polymorphic systems is an integral aspect of the development process of any solid-formulated drug product. Yet, these studies are often hindered by crystal quality and size, poor yields and the generation of mixtures of phases. Three-dimensional electron diffraction (3D ED) is a technique capable of structure determination from individually selected, nanometre-sized crystals. In this thesis, 3D ED is applied to investigate polymorphism in small molecules. The unique advantages of the method are highlighted across numerous studies to demonstrate how 3D ED can broaden the scope of polymorphism discovery and characterisation in the screening and selection of pharmaceutical crystal forms.  3D ED is first applied to reveal that two crystallisation methods believed for 47 years to produce Form δ of the pharmaceutical compound indomethacin result in two different polymorphs, highlighting the power of the method for polymorphism discovery. The polymorphic crystal structures of a small molecule are then determined directly from melt-grown compact spherulites for the first time to show how 3D ED can widen the application of melt crystallisation in polymorph screening, where polycrystalline spherulites are common products. Furthermore, 3D ED is combined with on-the-grid crystallisation and plunge freezing to follow the polymorph evolution of glycine during crystallisation from an aqueous solution to demonstrate the ability of the method to monitor crystallisation processes in situ. The final part of the thesis explores how a high-throughput method combining 3D ED data collection in batch mode with semi-automated data processing can be applied for the phase analysis of complex melt crystallisation products to improve the efficiency and accuracy of polymorph screening

A 47-year-old misunderstanding: Indomethacin Polymorph δ revealed to be two plastically bendable crystal forms by 3D electron diffraction

Indomethacin is a clinically classical non-steroidal anti-inflammatory drug that has been marketed since 1965. The third polymorph, Form δ, was discovered by both melt and solution crystallization in 1974. δ-indomethacin cannot be cultivated as a large single crystal suitable for X-ray crystallography and, therefore, its crystal structure has not yet been determined. Here, we report the structure elucidation of δ-indomethacin by 3D electron diffraction and reveal the truth that melt-crystallized and solution-crystallized δ-indomethacin are in fact two polymorphs with different crystal structures. Intriguingly, both structures display plastic flexibility based on a slippage mechanism, making indomethacin the first drug to have two plastic polymorphs. This discovery and correction of a 47-year-old misunderstanding signify that 3D electron diffraction has become a powerful tool for polymorphic structural studies

Direct Structure Determination from Spherulites using 3D Electron Diffraction

The spherulitic morphology is considered to be the most common morphology of crystalline materials and is particularly apparent in melt-crystallized products. Yet, historically, the polycrystalline nature of spherulites has hindered successful crystal structure determination. Here, we report for the first time the direct structure determination of a small molecule organic compound in spherulite form using 3D electron diffraction (3D ED). We employed vemurafenib (VMN), a clinical drug used for the treatment of BRAF-mutant melanoma, as a model compound. VMN has four known polymorphs (α-, β-, γ-, and δ-VMN), three of which were discovered by melt crystallization. We first solved the crystal structures of α-, β-, and γ-VMN from both open and compact spherulite samples using 3D ED, and the resulting structures were highly consistent with those solved by single-crystal X-ray diffraction. We then determined the previously unknown crystal structure of δ-VMN—the least stable polymorph which cannot be cultivated as a single crystal—directly from the spherulite sample resulting from spontaneous nucleation. We unexpectedly discovered a new polymorph during our studies, denoted as Form ε. Single crystals of ε-VMN are extremely thin and are not suitable for study by X-ray diffraction. Again, we determined the structure of ε-VMN from both open and compact spherulite forms. This successful structure elucidation of all five VMN polymorphs demonstrates the possibility of removing the time-consuming step of single crystal growth and directly determining structures from spherulite samples. Thereby, this discovery will improve the efficiency and broaden the scope of polymorphism research, especially within the field of melt-crystallization

Novel spirocyclic systems via multicomponent aza-Diels–Alder reaction

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Novel Broad-Spectrum Antiviral Inhibitors Targeting Host Factors Essential for Replication of Pathogenic RNA Viruses

Recent RNA virus outbreaks such as Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Ebola virus (EBOV) have caused worldwide health emergencies highlighting the urgent need for new antiviral strategies. Targeting host cell pathways supporting viral replication is an attractive approach for development of antiviral compounds, especially with new, unexplored viruses where knowledge of virus biology is limited. Here, we present a strategy to identify host-targeted small molecule inhibitors using an image-based phenotypic antiviral screening assay followed by extensive target identification efforts revealing altered cellular pathways upon antiviral compound treatment. The newly discovered antiviral compounds showed broad-range antiviral activity against pathogenic RNA viruses such as SARS-CoV-2, EBOV and Crimean-Congo hemorrhagic fever virus (CCHFV). Target identification of the antiviral compounds by thermal protein profiling revealed major effects on proteostasis pathways and disturbance in interactions between cellular HSP70 complex and viral proteins, illustrating the supportive role of HSP70 on many RNA viruses across virus families. Collectively, this strategy identifies new small molecule inhibitors with broad antiviral activity against pathogenic RNA viruses, but also uncovers novel virus biology urgently needed for design of new antiviral therapies