Browsing by Author "Santos, Webster L."

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    1,3-Disubstituted-tetrahydro-β-carbolines: A New Method for Stereochemical Assignment and Synthesis of Potential Antimalarial Agents
    Cagasova, Kristyna (Virginia Tech, 2021-06-21)
    Malaria is a serious mosquito-borne disease affecting the majority of Earth's southern hemisphere. While consistent efforts to curb malaria spread throughout 20th and early 21st century were largely successful, the recent rise in resistance to antimalarial treatments resulted in an increasing incidence rate and stalling mortality rate. This trend clearly signifies the need for the development of novel antimalarial agents able to circumvent current drug-resistance mechanisms. In 2014, in collaboration with Prof. Maria Belen Cassera from the University of Georgia, our group found that compound 1a (1R,3S-MMV008138), discovered from the publicly available Malaria Box, targets an essential biosynthetic pathway (MEP pathway) of malaria-causing parasite Plasmodium falciparum. Analogs of 1a synthesized in our laboratory were found effective against multi-resistant Dd2 strain of P. falciparum which, together with an absence of MEP pathway in humans, suggests that potent analogs of 1a may be safe and efficient antimalarial drug candidates. The initial bioassay studies determined that only one of four possible MMV008138 stereoisomers satisfactorily inhibits the target PfIspD enzyme. Thus a secure determination of stereochemistry in 1a analogs was of utmost importance to the structure-activity relationship studies performed in our group. The second chapter of this work discusses the validation of the previously known empirical stereoassignment method based on analysis of relative shift of 13C NMR resonances between cis and trans diastereomers and compares it to a new method based on 3JHH coupling constants developed in our laboratory. We demonstrate that the new method relying on the analysis of 1H-1H coupling is reliable over large samples of experimental data and suitable even when only a single diastereomer is produced in the synthetic process. Importantly, the origin of 3JHH coupling constants is well understood, unlike the source of relative differences in 13C NMR shifts observed in the older method. The empirical observations for both stereoassignment methods are supported by extensive density-functional theory calculations, which validate the new 1H-1H coupling-based assignment but do not provide a conclusive explanation for the origin of the 13C NMR-based method. In the third chapter, we discuss the replacement of the carboxylic acid moiety in 1a by alternative functional groups promising improved toxicity and bioavailability profile. The total synthesis of tetrazole (trans-23a) and phosphonic acid ((±)-62a) derivatives of 1a is discussed in detail. The tetrazole analog 23a was previously synthesized in the Carlier group as a diastereomeric mixture of cis and trans isomers (dr = 3:7), and it was tested for growth inhibition of multi-resistant P. falciparum with promising results. Later, the synthesis was revisited to obtain a stereochemically pure sample of trans-23a, which was expected to show improved potency compared to the original sample. Furthermore, the synthesis of pure trans-23a confirmed the accuracy of the previous assignment of cis and trans diastereomers in the mixture. Unfortunately, neither analog showed an improvement in potency relative to 1a.
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    Activation of diboron reagents: The development of mild conditions for the synthesis of unique organoboron compounds
    Thorpe, Steven Brandon (Virginia Tech, 2012-03-23)
    The first successful synthesis and isolation of a boronic acid was reported in 1860 by Frankland in the pursuit of novel organometallic compounds. For more than a century, further studies of boronic acids were sparsely published. Suzuki and Miyaura jumpstarted the field in 1979 with an innovative carbon-carbon bond forming reaction employing an organoboronic acid and a carbon halide under palladium catalysis. Indeed, the Nobel Prize in Chemistry was awarded to Professor Akira Suzuki, along with Professors Richard Heck and Ei-ichi Negishi, in 2010 for their important contributions in palladium-catalyzed cross-coupling chemistry. Over the last 30 years, reports on organoboron compounds have increased exponentially. This dissertation describes the author's contributions to the development of preparative methods for organoboronic acid derivatives using transition metal-catalyzed reactions of diboron reagents. A unique "mixed" diboron reagent was developed (PDIPA diboron) that contains sp2- and sp3-hybridized boron atoms, unambiguously confirmed by X-ray crystallography. PDIPA diboron is sufficiently activated internally through a dative-bonding amine to selectively transfer the sp2-hybridized boron regioselectively, in the presence of copper, to electron deficient alkenes including α,β-unsaturated ketones, esters, amides, aldehydes, and nitriles to provide the corresponding boratohomoenolates. A unique β,β-diboration of an α,β-acetylenic ketone was also discovered. The scope of PDIPA diboron reactions was then expanded to a set of substrates with a more complex structural backbone. Allenoates are α,β,γ-unsaturated esters with orthogonal pi systems, which pose several possible difficulties with the regioselectivity of addition, not to mention known isomerizations catalyzed by copper. However, we successfully installed the boron moiety regioselectively on the β-carbon of a variety of allenoates, providing a vinyl boronic ester, and also observed exclusive formation of the (Z)-isomer from racemic starting materials. The resulting vinyl boronic ester was then shown to be an excellent Suzuki-Miyaura cross-coupling partner, affording a diastereopure, trisubstituted alkene in quantitative yield. Commercially available bis(pinacolato)diboron has shown remarkable stability towards hydrolysis and autoxidation. Using this reagent, we developed a copper- and amine-catalyzed boration protocol performed entirely in water and open to air. Using only 1 mol% copper, extraordinary activity was observed. UV-Vis, 11B NMR, and solvent kinetic isotope experiments were employed to gain insight into the mechanism, which showed the possibility of autocatalysis. Attempts to control stereoselectivity were not successful, although these results were rationalized by a dynamic catalyst structure.
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    Antimalarial Agents: New Mechanisms of  Actions for Old and New Drugs
    Ghavami, Maryam (Virginia Tech, 2018-06-29)
    Worldwide, malaria is one of the deadliest diseases. In 2016 it sickened 216 million people and caused 445,000 deaths. In order to control the spread of this deadly diseases to human, we can either target the mosquito vector (Anopheles gambiae) or the parasite (Plasmodium falciparum). Due to recent emergence of resistance to current insecticides and antimalarial drugs there is a pressing need to discover and develop new agents that engage new targets in these organisms. To circumvent the effect of resistance to pyrethroid insecticides on the efficacy of insecticide treated nets (ITNs), the use of acetylcholinesterase (AChE) inhibitors on ITNs has drawn attention. In the first project, we explored a small library of γ- substituted oxoisoxazole- 2(3H)-carboxamides and isoxazol-3-yl carbamates, and nitriles as AChE inhibitors targeting wild- type (G3) and resistant (Akron) An. gambiae mosquito. In total 23 compounds were synthesized and evaluated. Both carbamates and carboximides with a 2-cyclopropylethyl side chain (1-87a and 1-88a) were extremely toxic to Akron mosquitos, yet these compounds did not exhibit appreciable selectivity between human and An. gambiae AChE. Unfortunately, none of the nitriles showed appreciable toxicity to G3 strain of the mosquitoes, nor did they inhibit An. gambiae AChE. In the second project, conducted in collaboration with Professor Michael Klemba, we focused on the mode of action of an established antimalarial drug, Mefloquine (MQ). Dr. Klemba's recently developed amino acid efflux assay was used to determine the effect of MQ and its open-ring analogs on hemoglobin endocytosis and catabolism in P. falciparum-infected erythrocytes. In total 26 MQ analogs were synthesized and 18 were studied in depth to determine their potency to inhibit leucine (Leu) efflux and parasite growth (SYBR Green). An excellent correlation (R² = 0.98) over nearly 4 log units was seen for these 18 compounds in the two assays. These data are consistent with the hypothesis that the antimalarial action of these compounds principally derives from inhibition of hemoglobin endocytosis. After this observation, a number of photo-affinity probes were designed and synthesized in hopes of isolating the molecular target of MQ. These analogs are currently being used by Dr. Klemba in pull-down experiments. In the third project, conducted in collaboration with Professor Belen Cassera, we sought to optimize a new antimalarial drug lead that would circumvent current resistance mechanisms. In Plasmodium parasites, the methylerythritol phosphate (MEP) pathway is known to be essential for its growth. This pathway is absent in humans, presenting the opportunity to develop potentially safe and effective therapeutic candidates. Previous work in the Cassera and Carlier lab had established that MMV008138 was the only compound in the Malaria Box that targeted the MEP pathway and that it was (1R,3S)-configured. My research expanded previous efforts in the Carlier group and produced synthesis of 73 analogs of MMV008138 (3-21a'1) that were tested for growth inhibition. These analogs featured variation at the A-, B-, C- and D-ring. In the process, a novel Pictet-Spengler ring expansion reaction of ortho-substituted acetphenones was discovered. The ring-expanded products were identified by means of 1D and 2D NMR experiments, HRMS, and X-ray crystallography. Among the 73 analogs prepared, four compounds showed similar growth inhibition potency to the lead 3-21a'1. In particular, the methoxyamide 3-80a, and the fluorinated A-ring analogs 3-124a, 3-124c and 3-124d all showed excellent (500-700 nM) growth IC₅₀ values against P. falciparum. All four showed full rescue upon co-application of IPP (200 μM), confirming that they target the MEP pathway. ADME-Tox evaluation of these new analogs will soon be underway.
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    Antiproliferative Natural Products from the Madagascar Rainforest
    Hou, Yanpeng (Virginia Tech, 2009-09-09)
    As part of an International Cooperative Biodiversity Groups (ICBG) program and a continuing search for anticancer natural products from the Madagascar rainforest, twenty extracts from Madagascar were selected for investigation based on their antiproliferative activity. Bioassay-guided fractionation of five of the extracts yielded sixteen new compounds, and their structures were determined using a combination of 1D and 2D NMR experiments, including COSY, HSQC/HMQC, HMBC, and ROESY/NOESY sequences, mass spectrometry, and chemical conversion. In addition, ten known compounds were obtained from five of the extracts. Studies on the remaining extracts were suspended due to various reasons. A multi-step synthesis of the sesquiterpenoid, (7R*)-opposite-4(15)-ene-1beta,7-diol, was also described. The first chapter of this dissertation reviews the new compounds isolated from Malagasy plants and marine organism in the last two decades. Chapters II to VI discuss the isolation, structure elucidation and bioactivities of new compounds from Scutia myrtina, Cordyla madagascariensis ssp. madagascariensis, Elaeodendron alluaudianum, Cassipourea lanceolata, and Sclerocarya birrea subsp. caffra. Chapter VII describes the synthesis and bioactivity of the sesquiterpenoid,(7R*)-opposite-4(15)-ene-1beta,7-diol. The isolation of known compounds is discussed briefly in the last chapter.
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    Boron-Mediated Semireduction of Alkynoic Acid Derivatives
    Grams, Robert Justin (Virginia Tech, 2021-04-30)
    Organoboron compounds are commonly used precursors for a variety of reactions in organic synthesis as is exemplified by the Suzuki-Miyaura cross-coupling, which is ubiquitous in industry and academia. Additionally, the Chan-Evans-Lam cross-coupling, lithiation-homologation, allylboration, and many other reactions rely on boron to achieve otherwise difficult chemical transformations. Thus, developing novel methods towards the regio- and/or stereoselective installation of boron into organic molecules remain important for designing new drugs. Boron reagents are also useful in chemical transformations that do not ultimately install a boron moiety on the organic molecule. We have developed several methods that achieve the trans-selective borylation or semireduction of internal alkynes, a process dominated by transition metals and often results in incomplete (E)-stereoselectivity. This dissertation describes three novel uses of a diboron reagent or pinacolborane that reduce propiolic acid derivates selectively to (E)-alkenes and one method that installs pinacolatoboron yielding exclusively (E)-β-borylacrylamides. We investigated the trans-selective hydroboration of primary and secondary propiolamides as reports in the literature accomplish trans-hydroboration via transition metal catalysis, which are limited in substrate scope. We discovered that addition of n-butyllithium to propiolamide and pinacolborane exclusively yields (E)-β-borylacrylamides in good to excellent yield. During the reaction, deprotonation of the amide with a strong base generates an alkoxide that coordinates to pinacolborane and forms a boronate complex. Upon warming to room temperature, a hydride transfer is directed to the α-carbon generating a β-carbanion that subsequently captures boron. Workup protonates the amide, furnishing the (E)-β-borylacrylamide product. As a follow-up from the trans-hydroboration of propiolamides described above, we developed a complementary semireduction of primary and secondary propiolamides. In this reaction, addition of catalytic amounts of potassium tert-butoxide to propiolamides generates an alkoxide that coordinates to pinacolborane and rapidly produces (E)-cinnamamides in 35 – 96% yield and >99:1 E:Z stereoselectivity. This reaction effectively reduces an internal alkyne to afford a product with trans geometry. A deuterium labeling study provided mechanistic insight for the transformation suggesting that the β-proton in the products is derived from the amide nitrogen of the propiolamide. Further, we demonstrated the utility of this reaction by augmenting the total synthesis of FK866, a potent nicotinamide mononucleotide adenyltransferase (NMNAT) inhibitor, and isolated the cinnamamide product in good yield with >99:1 E:Z stereoselectivity. Using a similar strategy, we investigated the ability of bis(pinacolato)diboron and base to mediate the α-borylation of propiolic acids. We observed the formation of a semireduced product, i.e., cinnamic acid. In the presence of a crown ether and cesium carbonate, propiolic acid is deprotonated and activates bis(pinacolato)diboron. Subsequent α-borylation and β-carbon protonation affords a highly unstable α-boronic acid derivative that undergoes rapid protodeborylation yielding predominantly (E)-cinnamic acids. The dual role of the carboxylate in activating the diboron reagent and directing α-borylation was unprecedented and is the first transition metal-free method to reduce propiolic acids to (E)-cinnamic acids. In contrast with boron activation from in situ generated alkoxide, an alternative approach is using phosphine catalysts—a major interest in the development of transition metal-free methodology. As such, we investigated the ability of phosphines to mediate the borylation of primary and secondary propiolamides. Surprisingly, we observed an efficient semireduction of primary and secondary propiolamides to their respective (E)-cinnamamides in the presence of catalytic n-tributylphosphine and stoichiometric pinacolborane. We surveyed the scope and reactivity of substrates bearing N-substitutions, electron-rich or deficient aryls, and aliphatics using optimized reaction conditions and produced a library of (E)-cinnamamides in 31 – 98% yield with >99:1 E:Z stereoselectivity. Deuterium labeling studies suggest that hydrogens on the α- and β-carbon come from pinacolborane and the amide hydrogen, respectively. We also trapped and characterized a key intermediate using tris(pentafluorophenyl)borane that supports a mechanism wherein the phosphine catalyst activates the propiolamide by conjugate addition.
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    Borylations and Silylations of Alkenyl and Alkynyl Carbonyl Compounds Employing a Mild and Environmentally Friendly Cu(II) Catalyst
    Calderone, Joseph Anthony III (Virginia Tech, 2014-04-25)
    An environmentally friendly, operationally simple copper-amine catalyst system is disclosed. Using this catalyst system, electron deficient alkenes and alkynes with diverse functional groups are borylated and silylated in high yields and with short reaction times. In the case of electron deficient alkynes the identity of the electron withdrawing group controlled diastereoselectivity. Esters and amides exclusively form E-product, while aldehydes and ketones favor Z-product. Mechanistic insights into the catalytic cycle as well as origin of diastereoselectivity are discussed.
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    Branched Peptide Boronic Acids (BPBAs): A Novel Mode of Binding Towards RNA
    Zhang, Wenyu; Bryson, David I.; Crumpton, Jason B.; Wynn, Jessica E.; Santos, Webster L. (The Royal Society of Chemistry, 2013-02-07)
    We report branched peptideboronic acids (BPBAs) that bind to RRE IIB from an on-bead high-throughput screening of a 3.3.4-library (46 656 compounds). We demonstrate that boronic acids are tunable moieties that afford a novel binding mode towards RNA.
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    Branched Peptides Targeting HIV-1 RRE RNA and Structure-Activity Relationship Studies of Spinster Homolog 2 Inhibitors
    Peralta, Ashley N. (Virginia Tech, 2020-06-08)
    Binding of the Rev protein with Rev Response Element (RRE) RNA present in singly- and unspliced mRNA transcripts is necessary for the replication of HIV-1. This interaction transports the mRNA transcripts from the nucleus to the cytoplasm for translation of the necessary structural and enzymatic proteins for the newly budding virus as well as for providing its genetic material. Given the high rate of mutation in HIV-1, the highly conserved and pertinent RRE RNA is of high interest for pharmaceutical intervention. Consequently, a branched peptide library containing unnatural amino acids was developed to target RRE RNA with the goal of increasing stability, potency, selectivity, and in vivo activity for RRE RNA. An unnatural amino acid branched peptide library (46,656 sequences) was synthesized and screened against RRE IIB and several hits in the sub-micromolar regime were found. A number of hits demonstrated selectivity in the presence of other RNAs in addition to two hits, 4A5 and 4B3, significantly inhibiting HIV-1 growth in vitro. These peptides inhibited HIV-1 replication in a concentration dependent manner and were demonstrated to be non-toxic. Further analysis of 4A5 and 4B3 via footprinting and SHAPE-MaP experiments determined that these peptides blocked binding of Rev through binding at the primary and secondary binding sites of RRE RNA. Sphingosine 1-phosphate (S1P) is a signaling molecule that plays a role in various biological processes including immunity, neurogenesis, and angiogenesis. The role S1P plays is largely determined by its location, in which Spinster homolog 2 (spns2) and mfsd2b are the two known transporters. The two transporters exist in different cell types and cellular localizations, with spns2-produced S1P being responsible for trafficking of lymphocytes. As such, spns2 has become of interest for therapeutic targeting in autoimmune and inflammatory diseases. To validate spns2 as a target in pharmaceutical intervention, a series of spns2 inhibitors were developed. A screening of a library of inhibitors found that compound SLP7120922 demonstrated inhibition of spns2 transport activity. The design, synthesis, and biological evaluation of inhibitors based on SLP7120922 is described. Modifications to the lipophilic tail region were performed with one compound 4.40f discovered to be potent, minimally toxic, and active in vivo. A series of modifications to the head region were then conducted that evaluated linear head derivatives with alkyl-, amide-, and amino acid-based groups. A number of compounds are reported that demonstrate good in vitro activity and minimal toxicity with two compounds, 4.48b and 4.52c, showing favorable in vivo activity in mice.
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    Cationic Polythiophenes as Responsive DNA-Binding Polymers
    Carreon, Analyn C.; Santos, Webster L.; Matson, John B.; So, Regina C. (The Royal Society of Chemistry, 2013-09-23)
    A new water soluble cationic polythiophene derivative, poly(N,N,N-trimethyl-3-(2-(thiophen-3-yl)acetamido)propan-1-aminium iodide), was synthesized via two consecutive post-polymerization functionalizations of poly(methyl 2-(thiophen-3-yl)acetate). This conjugated polymer binds DNA at N/P = 5 and forms polyplexes at N/P = 10. Its potential use as a theranostic gene delivery vehicle is investigated here.
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    Click Chemistry on DNA and Targeting RNA structure with Peptide Boronic Acids
    Crumpton, Jason B. (Virginia Tech, 2012-04-20)
    The utilization of click chemistry to perform inter- and intramolecular ligation on DNA has become ubiquitous in the literature. Advances in copper (I) stabilizing ligands that prevent DNA degradation via redox pathways have provided nucleic acid researchers access to the efficiency and quantitative nature of the click reaction. The majority of ligation procedures in the literature are performed in solution after DNA assembly and modification with alkyne reporter groups. However, without specialty alkyne reagents that can be sequentially and selectively deprotected, the solution phase method requires that the click reaction be performed on all DNA-attached alkynes simultaneously. Therefore, the variability of the azide reagent is limited to a singular R group. However, performing the click reaction on DNA during synthetic elongation (immediately after each alkyne installation) allows for the possibility of performing multiple click reactions with variable azide reagents. Unfortunately, most solid phase click procedures require long reaction times or the utilization of microwave irradiation to accelerate the reaction. The development of methods for the ligation of azides to alkynes without the use of microwave irradiation on solid phase is potentially very useful. Herein, we report a simple, efficient, and robust solid phase synthetic method for the ligation of azido-diamondoids to the alkyne-modified phosphate backbone of DNA with click chemistry using [Cu(CH₃CN)₄]PF₆ without stabilizing ligand. Interestingly, it was found that as the size of diamondoid increased, a corresponding increase in melting temperature of hybridized duplexes was observed. The developed method has the potential to complement existing DNA ligation procedures for applications in biotechnology and diagnostics. Interest in peptides incorporating boronic acid moieties is increasing due to their potential as therapeutics/diagnostics for a variety of diseases such as cancer. The utility of peptide boronic acids may be expanded with access to vast libraries that can be deconvoluted rapidly and economically. Unfortunately, current detection protocols using mass spectrometry are laborious and confounded by boronic acid trimerization, which requires time consuming analysis of dehydration products. These issues are exacerbated when the peptide sequence is unknown, as with de novo sequencing, and especially when multiple boronic acid moieties are present. Thus, a rapid, reliable and simple method for peptide identification is of utmost importance. Herein, we report the identification and sequencing of linear and branched peptide boronic acids containing up to five boronic acid groups by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Protocols for preparation of pinacol boronic esters were adapted for efficient MALDI analysis of peptides. Additionally, a novel peptide boronic acid detection strategy was developed in which 2,5-dihydroxybenzoic acid (DHB) served as both matrix and derivatizing agent in a convenient, in situ, on-plate esterification. Finally, we demonstrate that DHB-modified peptide boronic acids from a single bead can be analyzed by MALDI-MSMS analysis, validating our approach for the identification and sequencing of branched peptide boronic acid libraries. It is well known that RNA ligands incorporating basic and intercalating moieties display high RNA affinity. Unfortunately, these ligands are also often plagued by promiscuous binding to off-target substrates. Due to the potential utility of RNA ligands in biology and medicine, it is imperative to elucidate RNA binders which display high specificity as well as affinity. Boronic acid peptides promise unique RNA binding motifs through the interaction between the empty p-orbital of boron and the 2'-hydroxyl group of RNA. Herein, we describe the incorporation of lysine and phenylalanine boronic acid analogues into a branched peptide combinatorial library in an effort to impart increased selectivity towards the HIV-1 Rev Response Element (RRE). We were able to easily select and deconvolute 6 resulting "hit" peptides from 65,536 unique library members by high throughput screening and de novo sequencing. Although we were unable to evaluate peptide selectivity towards RRE due to general insolubility in aqueous media, we demonstrated the efficient deconvolution of a branched peptide library that incorporates boronic acids.
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    Complex Heterocycles as Mitochondrial Uncouplers
    Murray, Jacob Hadley (Virginia Tech, 2021-04-30)
    Small molecule mitochondrial uncouplers are compounds that dissipate the proton motive force independent of ATP synthase that results in increased energy expenditure. Mild mitochondrial uncoupling has therapeutic potential in treating obesity, diabetes, neurological diseases, non-alcoholic steatohepatitis (NASH), and aging. Our group has previously reported the discovery of a small molecule mitochondrial uncoupler BAM15, which was efficacious in an obesity mouse model. Herein, we describe the design and synthesis of two scaffolds as well as their characterization as mitochondrial uncouplers through a series of in vitro and in vivo assays. Compounds that pass as bona fide mitochondrial uncouplers are administered in mice to determine pharmacokinetic properties and promising compounds are then tested in a mouse model of obesity. The first series of mitochondrial uncouplers are anilinopyrazines. By changing the substitution pattern and electronics on the aniline rings, our investigations reveal the importance of the proximity of aniline rings on the pyrazine core, with the 2,3-positions being crucial for uncoupling activity. We found that mitochondrial uncouplers 2.5g and 2.5l elicited a maximum oxygen consumption rate (OCR) of 260% and 343% with an EC50 of 2.5 and 5.9 µM, respectively. Utilizing the knowledge gained from the anilinopyrazine series, we designed a second novel chemical scaffold based on a related BAM15 analog 6-amino-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol. The new series of 6-amino-[1,2,5]oxadiazolo[3,4-b]pyridin-5-ol derivatives have a pyridine instead of pyrazine core that is decorated with aniline substituents. We found that derivatives with electron withdrawing groups (EWG) substitutions in the 2,5-position on the aniline ring exhibited the greatest uncoupling activity compared to other structural isomers. Strong EWGs CF3/OCF3/SO2CF3 were well tolerated and demonstrated the highest uncoupling activity compared to other EWGs. Our studies indicated that placement of the hydroxyl group in the 2-position of the pyridine moiety was crucial for uncoupling activity. Several of the most promising compounds tested in vitro were examined in vivo and found to have good oral bioavailability in mice with ranges in Cmax of 10-90 µM and t1/2 of 0.9 to >24 hours. We found that analogs that have F/OCF3/SO2CF3 groups on the 4-position exhibited the longest t1/2 compared to other structural isomers, suggesting that this position is a site of metabolic lability. Among the 51 derivatives tested, SHM20519115 demonstrated mild uncoupling activity with 48% BAM15 OCR and an EC50 of 17.1 µM in L6 myoblast cells. SHM20519115 was found to have good oral bioavailability with a Cmax of 57 µM and a t1/2 of 4.4 hours. Additionally, SHM20519115 had significant distribution in adipose tissue where it can promote mitochondrial uncoupling. In a mouse model of obesity, SHM20519115 prevented fat mass gain by 59% compared to the western diet (WD) control group. Importantly, weight loss did not alter lean mass or food intake. Further characterization demonstrated that SHM20519115 prevented glucose and insulin intolerance in mice. Taken together, our investigations support the utility of mitochondrial uncouplers for the treatment of obesity and other metabolic disorders.
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    Copper-boryl mediated organic synthesis
    Hemming, David; Fritzemeier, Russell; Westcott, Stephen A.; Santos, Webster L.; Steel, Patrick G. (2018-10-07)
    Organoboron compounds are valuable synthetic intermediates that find application in a diverse variety of processes including both C-X and C-C bond-forming transformations. This has been achieved by using a variety of boron derivatives. Of these, boronate esters are probably the most versatile and, reflecting this, methods for the generation of boronate esters are of considerable current interest. Given the mild reaction conditions, good functional group tolerance, and low cost of the metal catalyst, the use of copper-boryl reagents is particularly attractive. In this review, methodologies in copper-boryl chemistry are discussed and the many different transformations possible are surveyed.
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    Design and modification of rhodium and iridium N-heterocyclic carbene complexes for asymmetric transfer hydrogenation and antimicrobial activity
    Bernier, Chad Michael (Virginia Tech, 2021-01-07)
    The two projects described in this dissertation demonstrate the wide utility of noble metal N-heterocyclic carbene (NHC) complexes. The first project details the design of iridium NHC amino acid complexes for asymmetric transfer hydrogenation (ATH) of prochiral ketones. Iridium(I) bis-NHC complexes were found to undergo oxidative addition with a variety of alpha-amino acids, generating chiral iridium(III) complexes of the form Ir(NHC)2(aa)(H)(X) (aa = amino acid, X = halide). The complexes were screened for ATH of aryl and alkyl ketones, and optimization studies found enantioselectivity in this system was highly sensitive to the reaction temperature, NHC ligand, and amino acid. Incorporation of secondary amino acids was essential to enantioselectivity. Aryl ketones were reduced in high conversion and enantioselectivity when employing the Ir(IMe)2(L-Pro)(H)(I) catalyst in isopropyl alcohol, in some cases giving over 90% ee of the alcohol products. Density functional theory calculations were conducted in order to gain insight into the active catalytic species, and the results suggest that the high enantioselectivity of this system primarily arises from steric effects. The second project details the design of rhodium and iridium NHC piano-stool complexes featuring derivatized tetramethylcyclopentadienyl ligands (Cp*R, R = alkyl or aryl substituent) for antimicrobial applications. Complexes of the form (Cp*R)M(NHC)Cl2 (M = Rh or Ir) were synthesized by transmetallation of the NHC ligand using silver(I) oxide in the presence of the desired noble metal Cp*R dimer. The complexes were screened for biological activity against various bacteria, yeast, and fungi. Many of these compounds were highly active against Mycobacterium smegmatis, displaying minimum inhibitory concentrations (MICs) as low at 0.25 microgram per mL. Analysis of structure-activity relationships found that incorporation of the NHC ligand greatly enhances the antimicrobial properties of rhodium and iridium piano-stool complexes, more so than previously investigated diamine, amino acid, or beta-diketonato ligands. Cytotoxicity studies on one of the rhodium NHC complexes showed this compound was nontoxic towards mammalian cells at low concentrations, which strengthens the potential of these types of compounds as viable drug candidates.
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    Design and Synthesis of Doxorubicin Conjugated Gold Nanoparticles as Anticancer Drug Delivery System
    Xia, Long (Virginia Tech, 2016-06-24)
    Doxorubicin is one of the most widely used and effective anticancer agents to treat a wide spectrum of tumors. But its success in cancer therapy is greatly compromised by its cumulative dose-dependent side effects of cardiotoxicity and tumor cell resistance. For the purpose of addressing these side effects, a gold nanoparticles-based anticancer drug delivery system was designed. Five novel thiolated doxorubicin analogs were designed and synthesized and their biological activities have been evaluated. These doxorubicin analogs and the poly(ethylene glycol) (PEG) stabilizing ligands were conjugated to gold nanoparticles via formation of a gold-thiol bond. The systems were evaluated in vitro and in vivo, and the results show that controlled drug release can be achieved either by acidic conditions or by reducing agents in cancer cells, depending on the design of the thiolated drug construct. The overall drug delivery system should achieve enhanced drug accumulation and retention in cancer cells and favorable drug release kinetics, and should demonstrate therapeutic potential and the ability to address some of the current problems of doxorubicin in cancer therapy.
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    Design and Synthesis of Orally Bioavailable Sphingosine Kinase 2 Selective Inhibitors
    Sibley, Christopher David (Virginia Tech, 2020-07-16)
    In humans, mammals, and perhaps all vertebrates, sphingolipids exist as a family of cellular signaling molecules and have been shown to be involved in a wide range of biological processes ranging from proliferation to apoptosis. As such, sphingolipid signaling has garnered the attention of numerous researchers as an attractive candidate for pharmacological manipulation. The synthetic pathway of one prominent sphingolipid, sphingosine 1-phosphate (S1P), has been implicated in a variety of disease states such as cancer, sickle cell disease, multiple sclerosis, and renal fibrosis. Formation of S1P is facilitated from the ATP dependent phosphorylation of sphingosine (Sph) through its generative enzyme's sphingosine kinase 1 and 2 (SphK1 and SphK2). Inhibition of SphK1 and SphK2 results in the manipulation of S1P levels, which has been shown to be therapeutic in various animal models of disease. While there are multiple examples of potent SphK1-selective and dual SphK1/2 inhibitors, SphK2-selective inhibitors are scarce. Herein, we describe the design, synthesis and biological testing of SphK2-selective inhibitors. We first describe the discovery that introducing a trifluoromethyl group onto the internal aryl ring of our inhibitor scaffold led to superior selectivity and potency towards SphK2. We demonstrate that the trifluoromethyl moiety is interacting with a previously unknown side cavity in the substrate binding site of SphK2 that is unique and could be exploited in the design of SphK2-selective inhibitors. The synthesis of 21 derivatives with various substituents spanning off the internal aryl ring was completed, therefore characterizing the preferred size and chemical nature of moieties positioned in that portion of the binding site. This work led to the development of the most potent SphK2-selective inhibitor known at the time. We then describe the transformation of our SphK2-selective inhibitors into an orally bioavailable drug. We explain how the guanidine functionality on our inhibitor scaffold hinders our compounds from being orally bioavailable. Consequently, a library of 24 derivatives with various modifications to the guanidine functionality was synthesized and evaluated for improved orally bioavailability. Highlighted in this work is the development of the most potent SphK2-selective inhibitor currently known 3.14 (SLS1081832), which displays a hSphK2 Ki of 82 nM and 122-fold selectivity for SphK2. Chemical modification and in vivo assessment of 3.14 (SLS1081832) prodrugs was explored.
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    Design, synthesis, and biological evaluation of selective sphingosine kinase inhibitors
    Raje, Mithun (Virginia Tech, 2012-04-13)
    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.
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    Design, Synthesis, and Structure-Activity Relationship Investigation of Selective Sphingosine Kinase Inhibitors
    Li, Hao (Virginia Tech, 2019-05-08)
    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).
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    The development and applications of unsymmetrical diboron compounds
    Guo, Xi (Virginia Tech, 2014-12-29)
    Organoboron compounds have shown a wide variety of applications in both organic synthesis and the pharmaceutical field in the past decades. Transition metal-catalyzed boration of unsaturated compounds has been studied extensively as an efficient method to install C-B bonds. Most of the previous examples employed symmetrical diboron reagents such as B₂(pin)₂ (pin = pinacolate) and B₂(cat)₂ (cat = catecholate). There are, however, limited examples of boration using unsymmetrical diboron reagents. This dissertation discloses two transition metal-catalyzed borations of unsaturated compounds with unsymmetrical diboron compounds. A Cu-catalyzed β-boration of electrophilic allenoates with a novel sp²-sp³ hybridized diboron reagent (PDIPA) is described. This unsymmetrical diboron reagent is preactivated and allows the boration to go smoothly under mild reaction conditions. The reaction provides β-borylated β,γ- unsaturated esters with exclusive (Z)-double bond geometry. These borylated products are useful intermediates for subsequent Suzuki-Miyaura cross-coupling reaction. In order to install two C-B bonds in one reaction, a Pt-catalyzed diboration of allenes with a differentially protected diboron reagent (PDAN) is presented. This unsymmetrical diboron reagent is prepared from the sp²-sp³ hybridized diboron compound, and it reacts with a series of 1,1- disubstituted allenes chemo- and regioselectively. Steric control ensures that both boryl moieties add to the terminal double bond, and the pinacol boronate preferentially attaches to the sp-hybridized carbon. The bis-boronyl products can be further converted to other functional groups as well as cross-coupling reactions. A collaborative project with Department of Physics and Department of Chemical Engineering is also discussed. In this project, a series of 𝑜-nitrobenzyl ligands containing a disulfide group as the anchor to gold surfaces are synthesized. The 𝑜-nitrobenzyl group uncages an amine upon photoexcitation. Attempts to make a water soluble analog failed, however, the mixture of methanol and water as the solvent was sufficient to attach them on gold surfaces.
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    Development of Methods for Boron Reagents
    Gates, Ashley Michelle (Virginia Tech, 2020-03-19)
    Boron reagents are known to be valuable in the field of organic chemistry due to their abilities to undergo a variety of transformations, resulting in useful pharmaceuticals and synthetic intermediates. It has also been shown that diboron reagents can act as reaction mediators due to the unique properties of the boron atom. To that end, this dissertation discloses three novel methods of employing boron reagents. Chapter 1 describes a method of utilizing a diboron reagent mediator in the palladium-catalyzed hydrogenation of allenes. In the presence of a palladium catalyst, tetrahydroxydiboron and stoichiometric water, allene semireduction proceeds in good yield. This semireduction is regioselective for the terminal alkene and results in the selective formation of Z-alkenes when used with unsymmetrical allenes (>80:20 Z:E). It is also compatible with more sterically hindered 1,1-diarylallenes, resulting in tri-substituted alkenes in good yields (63-88%). A borylation, defluorination of alpha-trifluoromethyl-alpha,beta-unsaturated esters is described in Chapter 2. The borylation is copper-catalyzed (10 mol %) and proceeds in the presence of stoichiometric bis(pinacolato)diboron and sodium tert-butoxide. The reaction affords compounds that contain two potentially useful functional handles: boronic esters and gem-difluoroalkenes. The products are obtained in moderate to good yield (up to 75%) with a large substrate scope including compounds with electron-donating, electron-withdrawing, heteroatom, and aryl substituents. In addition, the utility of the products in further transformation is demonstrated. A proposed reaction mechanism that provides rationale for the formation of products is described along with experimental evidence. Finally, Chapter 3 describes a transition-metal-free trans hydroboration of alkynoate esters and amides. The reaction is phosphine-catalyzed and proceeds with pinacolborane to afford (E)-beta-borylacrylates and (E)-beta-borylacrylamides in good to excellent yields. The reaction products are converted into novel oxaboroles through reduction with sodium borohydride. Theoretical calculations provide mechanistic insight for the transformation. The formation of a key phosphonocyclobutene intermediate is responsible for the observed stereoselectivity.
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    Development of Novel anti-estrogens for endocrine resistant Breast Cancer
    Rajalekshmi Devi, Sarika (Virginia Tech, 2016-06-27)
    ER+ breast cancer raises a significant diagnostic challenge since resistance invariably develops to the current endocrine therapies. 70% of breast cancers are ER+, which results from the overexpression of estrogen receptor. ER mediates strong anti-inflammatory signaling in ER+ tissues. Once activated with estradiol (E2), ER inhibits inflammatory gene expression via protein-protein interactions that block NF-kappa B transcriptional activity. Importantly, NF-kappa B is a primary mediator of resistance in many cancers, including breast cancer. All current endocrine suppressive treatments block this palliative signaling pathway, along with the desired proliferative pathway. Thus, there is a significant unmet clinical need for novel endocrine treatments for breast cancer that can ameliorate patient outcome in resistant populations, be less prone to resistance development, retain anti-inflammatory action, and cause fewer side effects. Following the hypothesis driven approach, the work described here introduces structural analogs of an innovative ligand scaffold, 5,6-bis-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonic acid phenyl ester, termed OBHS, which reduces gene activation through ligand-induced shifts in helices 8 and 11, thereby indirectly modulating helix 12 of ER (hence, indirect antagonists). This new class of ligands with a bicyclic hydrophobic core retains strong anti-inflammatory effects while dialing out the proliferative effects of E2 (similar to Selective Estrogen Receptor Modulators, SERMS), and could potentially replace the current endocrine therapies of breast cancer. In this work, we carried out rational design and syntheses of two series of OBHS analogs, namely OBHS-A (for acetamido derivatives), and OBHS-P (for propargyl derivatives), while we explored a synthetic methodology for a third series of OBHS compounds. Many analogs from the OBHS-A series exhibited high binding affinity. For example, the exo diastereomer of 2.11a, 2.11b, 2.11c, 2.11d, and 2.11e exhibited Relative Binding Affinities (RBAs) of 22.6%, 10.5%, 19.5%, 12.1%, and 14.4%, respectively. As observed before, endo OBHS compounds exhibited lower binding affinities than exo compounds. The RBA values with acetamide, and isobutyramide (i.e. short hydrophobic chains) were very comparable to each other. However, unexpectedly the propionamide compound showed lower binding affinity than butyramide. Nevertheless, we consider OBHS analogs with RBA values greater than 1% (Kd = 20 nM) to be very potent. This data is only the first step in a battery of assays that will be conducted eventually on these compounds. In particular, our emphasis is in ascertaining and improving the NF-kappa B mediated anti-inflammatory property, where these compounds have shown promising activity in conjunction with their anti-proliferative activity.