Research Areas
"If medicine was like software, organic chemistry would be the boot code."
General Research Outlook: The main thrusts of research in our laboratory are primarily concerned with developing reactions that enable new strategies to install non-canonical elements into druglike scaffolds, and designing new small molecules with unique biological properties. Our laboratory incorporates themes from physical organic chemistry to develop chemistries, both entirely synthetic as well as those inspired by nature, that expand our chemical toolbox for novel small molecule bioactive agents. This is pursued in three directions: (1) establishing robust chemistry for introducing fluorinated functionalities into complex chemical scaffolds, (2) synthetic derivatization of natural products to more fully probe structure-activity relationships, and (3) development of precision-guided small molecules, including bifunctional probes and chimeric molecular glues, to reprogram cellular logic switches. These are specifically designed in the context of an academic training environment, with a primary motivating goals of building operational competency in modern laboratory techniques in multistep organic synthesis, catalysis, and chemical biology; and building an appreciation for the fundamental role of small molecule synthesis in modern therapeutic science at the chemistry-biology interface.
Expanding Nature's Periodic Table
Remarkably, despite the diversity of function that has been achieved in synthetic manipulation of the 118 elements on the periodic table, most biological systems operate under a much more limited subset of these elements (C, H, O, N, S, P, and a few trace metals). At the same time, given the remarkable metabolic stability and desirable physiochemical properties imbued by fluorinated motifs on biologically active compounds, one in five FDA-approved therapeutics contains one or multiple fluorinated functional groups. (Inoue, ACS Omega 2020; Gillis, J. Med. Chem. 2015). This is achieved in the development of 19F NMR workflows for mechanistic study of complex multicomponent reactions and the development of late stage functionalization strategies for introducing fluorinated motifs on late-stage, highly functionalized chemical architectures.
19F NMR Spectroscopy Enables Real Time Reaction Monitoring and Mechanistic Insight into Complex Reaction Systems
Multicomponent reactions (MCR’s) represent one of the most powerful means of generating chemical complexity from relatively simple and commercially available starting materials. Our laboratory previously reported the usage of 19F NMR spectroscopy for high throughput reaction condition screening in the optimized synthesis of carmofur and its analogs (Wang, Can. J. Chem. 2023) trifluoromethylated pyrazoles (Chyu, et al. Spectroscopy J. 2024) and as a tool to probe the mechanism of the Biginelli multicomponent reaction (Chen, ACS Omega 2023). Current research in the laboratory is centered around extending this workflow to the Ugi four-component MCR and the Hantzsch pyridine synthesis, which we hope to report in the near future. This workflow and approach to MCR’s plays a second role in an academic research laboratory in enabling an inquiry based platform through which students must learn mechanistic organic chemistry.
Photocatalytic C-H Fluorination Through Development of Fluorine Radical Cation Synthons
Strategic incorporation of fluorinated building blocks has demonstrated broad utility in medicinal chemistry, particularly in overcoming metabolism of oxidatively labile C-H bonds, and in fact, nearly one in five FDA approved small molecules today incorporates fluorinated motifs. And yet, there are today very few means of incorporating fluorine into complex structures, despite the much broader range of late-stage C-H functionalization chemistry that has been developed for oxidation, amination, and borylation, among others. Our lab has been interested in strategically taking advantage of the intrinsic reactivity of most oxidatively labile C-H bonds in developing photocatalytic radical anion-cation relay strategies for late-stage installation of C-F bonds.
Preparation of 1,1-difluorocyclopropanes via Cobalt-Salen Difluorocarbene Transfer Catalysts
While recent advances in metal-catalyzed, organocatalytic, or biocatalytic cyclopropanation have enabled preparatively useful constructions of cyclopropane-containing scaffolds on diverse substrates under fairly mild conditions with stereo- and regiocontrol, there are comparatively few ways to generate gem-difluorocyclopropanes. Here, we propose the preparation of a library of CoII(salen) complexes that might serve as capable difluorocarbene transfer catalysts through transient stabilization of difluorocarbenes as metal-carbene or metal-carbenoid complexes, whose reactivity and selectivity can be controlled by both ligand choice and counteranion additives. Ultimately, the ability to control difluorocarbene reactivity in this fashion might enable access to a broad range of 1,1-difluorocyclopropanes tolerant of a variety of functional groups, and provide opportunities for selective and late-stage installation of difluorocyclopropanes on increasingly complex substrates. We further propose real time reaction kinetic monitoring by 19F NMR spectroscopy on the rate and preference of cyclopropanation on electron-rich and electron-deficient olefins as a means of establishing a mechanistic basis for how these catalysts might operate, with the support of density functional theory calculations. [First disclosed at the 2024 Bay Area Chemistry Symposium]
Editing Nature's Molecular Arsenal
While natural products have been the blueprint for the discovery of a majority of novel bioactive small molecules that are now in clinical use it is often the case that these initial discoveries lend themselves to the development of analogs or derivatives of compounds from nature that are more synthetically accessible, or possess improved or more desirable biological or pharmacokinetic profiles (Newman & Cragg, J. Nat. Prod. 2020; Boger, J. Org. Chem. 2017; Duboc, J. Am. Chem. Soc. Au 2024). To achieve this end, our lab is interested in developing analogs of biologically relevant natural products to probe key questions on pharmacophore design, metabolic stability, and enhanced potency. This is achieved either through semisynthetic modification of natural products or through total synthesis of natural products and their analogs.
Probing the SAR of C-4 analogs of podophyllotoxin, an anticancer lignan natural product
The diversity of lignan small molecules derived from podophyllotoxin, a non-covalent tubulin inhibitor isolated from the Podophyllum family, has led to the clinical development of FDA-approved anticancer agents etoposide and teniposide. While these two compounds share the same tetracyclic core as podophyllotoxin, two subtle structural changes—4’ methylation on the aromatic ring and stereospecific glycosylation at the C-4 hydroxyl—result in an alternate biological mechanism. Given the immense pharmacological importance of these two features, we synthesized and evaluated a systematic library of diversified esters, carbonates, and carbamates to establish a structure-activity relationship regarding modification at C-4 on the properties of podophyllotoxin. We determined the biological activity of these esters through cell viability assays, computer docking models, tubulin polymerization assays, and cell cycle analysis. Altogether, we demonstrated that increasing steric hindrance at C-4 leads to a loss in potency against human cancer cells but has a significantly lesser impact on cell-free tubulin inhibition. (Lu, et al. Natural Product Research 2024, 1-7)
Pharmacophore Editing of Andrographolide Analogs for Modulation of Wnt1 and Nf-kB Signaling Pathways
Andrographolide, a natural product labdane diterpenoid extracted from the plant Andrographis paniculata, is known to have potent anti-cancer activity. The putative mode of action of andrographolide is the inhibition of Nf-kB, which subsequently leads to downregulation of a myriad of cell signaling pathways typically involved in cell cycle regulation. However, previous reports have suggested that chemical modifications to the C19 hydroxyl and C17 alkene may alter the biological target of the andrographolide analog to the Wnt/𝜷-catenin signaling pathway. With this pharmacophore in mind, we prepared a library of targeted andrographolide C19 and C17 analogs with altered polarity and steric profiles to probe the effects of large, hydrophobic silyl and trityl ethers at C19 and epoxidations of C17 on metabolic stability and the primary mechanism of action. After studying the potency of our analogs through MTT and cell migration assays, we assessed the analogs’ downstream transcriptomic effects on key apoptosis-regulating pathways and their potential as a protein inhibitor in the Wnt/𝜷-catenin signaling pathways. (Gu, et al. Bioorganic Medicinal Chemistry Letters 2024, manuscript accepted / ChemRXiv Preprint)
Concise Total Synthesis of the Sporovexin Family of Natural Products
The evolutionary competition between fungal species has yielded an abundance of small-molecule antimicrobial agents. One such compound, Sporovexin A, is a p-hydroxybenzoic acid metabolite of the fungus Sporormiella vexans that was demonstrated in previous studies to exhibit antibiotic properties in preliminary assays. Using computer modeling to overlay a putative structural parallels to p-aminobenzoic acid, a substrate of the enzyme dihydropteroate synthase (DHPS) central to the bacterial synthesis of folate, we hypothesize that the antibacterial behavior of the sporovexin family of natural products arises from its competitive inhibition of DHPS. Here we present the synthesis of two novel des-methyl analogs of the Sporovexin family in two synthetic steps from commercially available starting materials, and a des-hydroxy Sporovexin analog to probe the specific effect of these functional groups on antimicrobial activity. The inhibitory behavior of each compound was determined via Kirby-Bauer testing in bacteria cultures of Bacillus cereus, Escherichia coli, Neisseria sicca, and Staphylococcus epidermidis, providing insight into the antimicrobial properties of these Sporovexin analogs. (C. Zhou, J. McChesney, et al. - manuscript in progress)
Enhancing Modular Molecular Logic
Historically, most medicinal chemistry campaigns have focused on development of single small-molecule modalities to target single biological targets. More recently, the advent of proteolysis targeting chimeras (PROTACs), bioconjugated small molecules, and other bifunctional chemical entities have demonstrated the capacity to achieve an expanded functional logic with with designed small molecules (Crews, Nat. Rev. Drug Disc. 2022). To this end, our laboratory has been interested in expanding the possible logic space by designing small molecules that embody two or more independent functionalities.
Self-immolating reverse-logic chemical linkers for targeted cancer therapy
The adaptability of personalized dosing regimens of clinically relevant entities is a longstanding challenge in drug delivery. Current methods often rely on intrinsically inactive formulations or prodrugs that are activated upon an environmental trigger. Herein, we present a molecular system with a novel approach to the self-regulating dosing of a therapeutic entity; specifically, we engineered a peptide-based system with a reversal of the aforementioned logic, wherein a drug construct is innately active until neutralized with a trigger only present in healthy cells. We demonstrate the efficacy of this system in the selective destruction of in vitro models of cells in various states of disease progression, while sparing healthy, non-disease state cells. This was evaluated through dose titration toxicity studies and mass-spectrometry and qPCR-based methods to validate the proposed mechanism of action. More broadly, we demonstrate the adaptability of such a system to other disease models. (US Patent App. 011827 / WIPO WO2024155707A1, with Akira Yamamoto)
Targeting ER-positive breast cancer with bifunctional tamoxifen-SN38 hybrids
Several breast cancer isotypes express elevated concentrations of estrogen receptors (ER), progesterone receptor (PR) and HER2. Our laboratory has been working in developing small molecules to target ER+ breast cancer solid tumors that are driven thermodynamically by association of a tethered endogenous ligand to a toxin that localizes small molecules to target tissues that upregulate a particular signaling protein. Specifically, we have developed a library of ER-targeting Regulated Induced Proximity Targeting Chimeras (RIPTACs) that deliver toxins such as topoisomerase I inhibitors SN-38 and camptothecin directly to ER-expresssing breast cancer cells. (Collaboration with Dr. John Wang, ASDRP Biology Department)
Fluorophore-lipid conjugates for selective labeling of the nuclear membrane
Cellular compartmentalization is vital in providing a variety of unique environments within a single cell. While the importance of these compartments and their facilitated functions are well acknowledged, the boundaries between them are poorly understood. Several studies report exciting discoveries in the realm of nucleic acid-lipid membrane systems, which enable processes like proper cytoskeletal and post-mitosis nuclear organization, and regulate cellular development and differentiation. Several heterocyclic lipidic dyes have been used to investigate phospholipid membranes. Thus, dimeric fluorophores containing an amonafide—a heterocyclic DNA-intercalator and topoisomerase II inhibitor—core and lipid tails may be promising imaging agents. Given its biological mechanism, amonafide is typically internalized within cell nuclei. However, upon addition of a hydrocarbon lipid tail, preliminary studies suggest that the fluorophore conjugate begins to localize along extranuclear membranes. Although the specific ramifications of this differentiated localization are unknown, these dimeric fluorophores have the potential to interact with nucleic acids outside of the nucleus, and our results suggest that they may be promising intracellular imaging agents.
Enabling Efficient Medicinal Chemistry
Scalability of synthetic preparations of active pharmaceutical agents are often a rate-determining step in generating clinically-impacting supplies of chemical entities. Our laboratory is interested in optimization of challenging chemical transformations, including asymmetric carbinol synthesis, challenging amide coupling reactions, and preparation of advanced synthetic intermediates for diabetes and cancer research in an atom- and step-economical fashion.
In Vitro Activity and Structure-Activity Relationship of Dual-Purpose Inhibitors Against Human Acid Ceramidase and the SARS-CoV-2 Main Protease
During the COVID-19 pandemic, carmofur, a 5-fluorouracil derivative initially developed as an antineoplastic agent against colorectal cancer, was identified through drug repurposing as a potent covalent inhibitor of the SARS-CoV-2 main protease (Mpro), making it a promising therapeutic candidate against COVID-19. Previously, we reported the optimization of the synthesis of carmofur through using Benchtop 19F NMR to quantitatively track reaction rates in various reaction conditions. (Wang, et al. Canadian J. Chem. 2023) In this study, we developed an optimized protocol for producing ten novel analogs of carmofur to explore the impact of structural modification on biological activity, bearing diversified alkyl, cycloalkyl, and aryl substituent chains. Additionally, in order to probe the effects of single-atom substitution at the electrophilic carbonyl on the carmofur side chain, we selected a heptyl amide analog and hexyl carbamate analog to synthesize wherein the nitrogen atom in carmofur is substituted with a methylene or an oxygen. Through monitoring reaction rates with 19F NMR, we were able to rapidly determine optimal synthetic conditions for each analog. To evaluate the efficacy of our compounds in inhibiting SARS-CoV-2 main protease activity, we performed a colorimetric assay to evaluate their inhibitory effects on the main protease. To probe the antiproliferative effects of our compounds, we performed MTT assays against colorectal cancer cells. All together, we demonstrate the utility of a Benchtop 19F NMR-enabled workflow in the preparative synthesis of novel 5-fluorouracil analogs, several of which exhibit selective potencies against the main protease of SARS-CoV-2 or as antiproliferative agents that are at times comparable or superior to carmofur. (Gu, et. al. BioRXiv 2024) (+ Collaboration with Dr. Joseph Pazzi, ASDRP Chemistry Dept.)
Discovery, Process Optimized Synthesis, and Anti-Cancer Activity of 5-Phenylisoxazole Based Covalent Inhibitors Targeting G12C Mutant KRAS
Oncogenic mutations in the GTPase protein KRAS are implicated in approximately 25% of human cancers. Specifically, the G12C mutation, which is the most common mutation found in KRAS related pathology, is found in 12% of lung cancers and 3% of colorectal and other solid tumors. This single residue substitution causes irreversible binding to the GTP substrate by inhibiting GTP hydrolysis, thereby forcing the protein into a permanent, activated state. While KRAS has been previously considered an undruggable chemotherapeutic target, the discovery of acrylate based covalent inhibitors of G12C KRAS has led to the development of two FDA approved chemotherapeutic agents: Sotorasib (AMG-510) and Adagrasib (MRTX849). Inspired by this pharmacophore model, we developed a series of isoxazole based covalent inhibitors of G12C KRAS, and evaluated their in vitro potency against two human cancer cell lines. Moreover, we used molecular docking to rationalize the potency of these compounds. (N. Brahan & A. Desarkar, et al. - first disclosed at the American Chemical Society National Meeting, Autumn 2024 @ Denver CO)
Formal synthesis of (+)-etomoxir and analogs, covalent inhibitors of carnitine palmitoyl transferase I for the treatment of diabetes
Etomoxir is a covalent inhibitor of CPT1, a transmembrane mitochondrial protein that acts as the rate-limiting enzyme for fatty acid oxidation. This enzyme plays a major role in metabolic diseases such as diabetes, where regulation of fatty acid biosynthesis and β-oxidation kinetics through CPT1 are effective treatments for such diseases. We demonstrate the scalability of two alternate synthetic routes to intercept a late-stage epoxy alcohol or epoxy acid in route to (+)-etomoxir. Notably, our chosen retrosynthetic disconnection, which either proceeds through a one-flask tandem Aldol Condensation-Luche Reduction sequence, or a one-flask Aldol Condensation-asymmetric conjugate epoxidation using a chiral prolinol catalyst, avoids pyrophoric materials such as n-butyllithium, which is historically used in excess in the previously reported preparation of (+)-etomoxir. We further elaborate the utility of these one-flask sequences on 12 unique substrates containing different functional groups to analyze the scope and possible applications of this transformation. (Y. Xie, N. Kong, R. Raval, et al. manuscript in progress)