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Doctor of Philosophy White blood cells comprise a significant portion of the body's natural defense mechanisms. In healthy individuals, these white blood cells identify and destroy foreign materials and organisms. However, in patients with multiple sclerosis, immune cells can become sensitized to protein fragments lining the myelin sheath of neurons. These autoreactive immune cells recognize the body's natural neuronal proteins as antigens. Damage exerted by autoreactive cells leads to the development of neurological impairments (i.e., fatigue, muscle weakness, and slurred speech) as nerve impulses are disrupted before reaching their target. First-line treatment of multiple sclerosis often centers on the administration of immunosuppressive drugs to curtail the progression of the disease and mitigate immune cell-directed demyelination. A driving factor in white blood cell localization is the lipid sphingosine-1-phosphate (S1P). Concentrations of S1P are often not static in the body, with different tissue types and fluids possessing variable levels. Immune cells, and lymphocytes in particular, use this natural S1P gradient to dictate their movement within the body. Lymphocytes will track with the S1P gradient, going from areas of lower S1P concentration (lymph tissue) to areas of higher S1P concentration where synthetic enzyme expression is upregulated (multiple sclerosis lesions). Consequently, the development of drugs that can alter this S1P gradient represents an ideal avenue to achieve immunosuppression. One key mediator of S1P release is the transmembrane transport protein Spinster homolog 2 (Spns2). This protein directs the secretion of intracellular S1P into the extracellular space and is necessary for lymphocytes to enter circulation. However, little effort has been devoted to the development of Spns2 inhibitors. As such, the inhibition of this protein represents a novel and underexplored target for the treatment of autoimmune disorders. In this disclosure, the structures of several highly potent Spns2 inhibitors are revealed. The work around these structures led to the discovery of 4.11i. This compound proved highly potent in biological assays and animal models. Mice treated with 4.11i experienced a reduction in circulating lymphocyte counts and demonstrated less symptom manifestation in multiple sclerosis disease models.

Organoboron compounds are ubiquitous in organic chemistry. Fundamental transformations utilizing organoboron compounds are a necessary addition to any organic chemist's synthetic toolbox. In addition to their extensive use as synthetic intermediates, organoboron compounds are being increasingly studied for their material and medicinal properties. Excitingly, significant advances have been made over the years towards the synthesis of a wide variety of organoboron substrates. In the case of vinylboronic acids, synthesis primarily occurs through cis addition of boron reagents across triple bonds. However, methods affording trans addition products are scarce. Furthermore, many current methods rely on the use of expensive and toxic transition-metal catalysts. Herein, we describe the development of trans addition of boron reagents across polarized triple bonds to afford novel vinylboronic acids. Emphasis is placed on the transition metal-free nature of the reactions as well as the regio- and stereoselectivity observed in the products. In addition, the synthetic utility of the resulting trans addition products is demonstrated in the synthesis of biologically relevant molecules. We first describe the Brønsted base-mediated trans silaboration of propiolamides in which two functional groups with orthogonal reactivity are simultaneously installed. We then go on to describe an organocatalyzed trans hydroboration of propiolate esters as well as a complementary Brønsted base-mediated trans hydroboration reaction of propiolamides. To conclude this portion, we demonstrate how the products from the previous methods can be used to synthesize difluoroborylacrylamides which possess unique and versatile reactivity. Herein we disclose the first small-molecule inhibitors of the sphingosine-1-phosphate (S1P) transporter spinster homolog 2 (SPNS2). While little is known in regard to the structure and function of SPNS2, previous studies have demonstrated the vital role SPNS2 plays in S1P mediated processes and have identified SPNS2 as a potential clinical target. For example, SPNS2 is critical to S1P-mediated lymphocyte egress from primary lymphoid tissues. Thus, small molecule inhibition of SPNS2 represents a novel therapeutic strategy for the treatment of autoimmune disorders such as multiple sclerosis. In this study, we report the discovery of small molecule inhibitors that display low micromolar activity using a novel yeast-based SPNS2 assay. Inhibitor structure-activity-relationship studies led to the discovery of the imidazole-based amine inhibitor 7.54. Furthermore, administration of 7.54 to mice recapitulates the lymphopenic phenotype observed in previous SPNS2 knockout studies. Doctor of Philosophy Boron-containing compounds are important in organic chemistry and are involved in the synthesis of a variety of materials and medicines used in everyday life. As such, the ability to efficiently and sustainably prepare boron-containing compounds has far reaching consequences. Access to an important class of boron-containing compounds known as vinylboronic acids has previously been established; however, product selectivity is often limited to what is referred to as cis addition products. Furthermore, access to the corresponding trans addition products is often limited to processes involving expensive transition metal catalysts that produce environmentally toxic waste. Herein, novel transition metal-free trans addition processes are described for preparing vinylboronic acids. In addition, the application of the resulting products is demonstrated through the synthesis of biologically relevant compounds. Sphingosine-1-phosphate (S1P) is an important signaling lipid that is involved in a variety of physiological processes. Improper balance in the amount of S1P in the body is associated with a variety of disease states such as autoimmunity and cancer. Two drugs that inhibit S1P-mediated processes have been approved by the FDA, fingolimod (Gilenya®) and siponimod (Mayzent®). However, there are drawbacks to targeting the S1P receptor directly, including dose-limiting side effects that are associated with these drugs. Consequently, recent efforts have focused on developing new ways to control the effects of S1P. Herein, we describe the discovery and development of the first reported inhibitors of the S1P transporter, spinster homolog 2 (SPNS2). A library of compounds was synthesized and tested for SPNS2 inhibition. The resulting structure-activity-relationship studies led to the discovery of the imidazole-based propanamine derivative 7.54. Furthermore, we demonstrate the potential of SPNS2 inhibition to control the effects of S1P in mice. These studies provide a foundation for future SPNS2-based drug discovery that will hopefully lead to the development of improved therapies for the treatment of autoimmune disease and cancer.

Sphingosine kinase 1 (SphK1) is the key enzyme catalyzing the formation of sphingosine-1-phosphate (S1P), which is an important signaling molecule that regulates multiple biological process including inflammatory responses. Elevated SphK1 activity as well as upregulated S1P levels is linked to various diseases such as cancer, fibrosis and sickle cell disease. Therefore, there is a growing interest in studying SphK1 as a potential target for these diseases. Through high-throughput screening, various SphK1 inhibitors have been discovered, among which PF-543 is the most potent and selective inhibitor reported to date (Ki=3.6 nM, >100 fold selectivity for SphK1). Previous research indicated that SphK1 inhibitor PF-543 is effective in reducing S1P levels and slowing down the development of sickle cell disease in vivo. However, the lack of in vivo stability of PF-543 still makes it necessary to develop inhibitors with an improved pharmacokinetic profile. In this study, PF-543 was employed as the lead compound, and the influence of different tails groups and head groups on binding affinity and in vivo stability were investigated. In brief, (R)-prolinol-based derivatives with various tail groups including alkyl, alkoxy and biphenyl groups were synthesized. Their inhibition potency was tested in a broken-cell assay, and hit compounds were further evaluated in a yeast cell assay to determine EC50 values. The U937 cell line and mice model were utilized for hit compounds to quantify S1P reduction in vitro and in vivo. Our preliminary results indicated compound 2.14d was the best hit discovered, with 88% SphK1 inhibition at 1 μM. In addition, compound 2.14d with a Ki of 0.68 μM and an EC50 of 0.15 μM, reduced the S1P of U937 cells by 90% at 1 μM. Its analog with a shorter tail group, 2.14a, reduced plasma S1P levels by 20% in mice (10 mg/kg, 3 h). Further modification of the head group of 2.14d produced compound 3.14c bearing a secondary benzylamine head group, with an EC50 value of 0.39 μM and less in vivo activity (14% plasma S1P reduction at 10 mg/kg, 6 h). Doctor of Philosophy Sphingosine-1-phosphate (S1P) is a molecule related to various diseases, such as cancers and inflammatory diseases. Elevated levels of S1P promote the development of these diseases, thus making it necessary to reduce the production of S1P in patients. Since S1P is generated in human body by an enzyme called sphingosine kinase (SphK), inhibiting the activity of SphK can be beneficial for reducing S1P levels. Developing inhibitors for SphK is also a promising strategy for curing such diseases. A very potent inhibitor has been reported, but it could be metabolized quickly into other inactive metabolites in human, which renders it ineffective. To develop better drug candidates, a series of compounds with similar structure has been synthesized and tested for their potency and metabolic stability. Based on analysis of the relationship between the compound structures and activities, several compounds with less potency and different metabolic stability has been prepared and their efficacy in reducing S1P levels has been tested.

Sphingosine kinase (SphK) has emerged as an attractive target for cancer therapeutics due to its role in cell proliferation. SphK phosphorylates sphingosine to form sphingosine-1-phosphate (S1P) which has been implicated as a major player in cancer growth and survival. SphK exists as two different isoforms, namely SphK1 and SphK2, which play different roles inside the cell. The dearth of isoenzyme-selective inhibitors has been a stumbling block for probing the exact roles of these two isoforms in disease progression. This report documents our efforts in developing SphK2-selective inhibitors. We provide the first demonstration of a SphK inhibitor containing a quaternary ammonium salt. We developed highly potent and moderately selective inhibitors that were cell permeable and interfered with S1P signaling inside the cell. In an effort to improve the selectivity of our inhibitors and enhance their in vivo stability, we designed and synthesized second generation inhibitors containing a heteroaromatic linker and a guanidine headgroup. These inhibitors were more potent and selective towards SphK2 and affected S1P signaling in cell cultures and various animal models. Ph. D.