The 53rd Annual MIKI Meeting-in-Miniature

April 10th to 12th, 2015 The University of Kansas

School of Pharmacy Lawrence, KS

Welcome to the 53rd Annual MIKI Meeting! On behalf of the Department of Medicinal Chemistry at the University of Kansas, we welcome you to Lawrence, Kansas and the 53rd Annual MIKI Meeting-in-Miniature! This weekend provides a unique opportunity to become reacquainted with old friends and continue to expand your network in a dynamic and collaborative environment. We look forward to hosting such a brilliant group of young scientists. We would also like to thank you for your scientific contributions to the meeting because without those we would not be here! Since its inauguration in 1963, this meeting-in-miniature has provided graduate students from the universities of Minnesota, Illinois at Chicago, Kansas, and Iowa with the opportunity to participate in a larger scientific community by organizing and hosting the conference. The location of the meeting rotates between one of the four participating schools each year, with a full cycle completed every four years. In addition to allowing students to present their research, MIKI allows both faculty and students to exchange ideas, learn about other fields, and expand their network. The MIKI Keynote Address has been presented by world-class scientists, including multiple Nobel Prize winners and ACS Division of Medicinal Chemistry Hall of Fame Inductees. This year we are proud to host Dr. Bruce Roth. Dr. Roth is the inventor of Lipitor®, the best selling drug of all time. Currently, he is the Senior Vice President of Small Molecule Drug Discovery at Genentech Research and Early Development. We hope that you have the opportunity to enjoy the meeting, encourage others, and learn something new! Sincerely, The MIKI 2015 Organizing Committee Chair: Molly Lee Faculty Chair: Ryan Altman, Ph.D.

2015 MIKI Organizing Committee Chairs

Leah Forsberg Manwika Charaschanya Brett Ambler Doug Orsi Rachel Saylor Gavin Gao

Additional Members of the 2015 MIKI Organizing Committee

Elyse Petrunak Vince Crowley Huiyong Ma Anuj Khandelwal Rachel Davis Jessica Hall Rakesh Vekariya Andrew Riley Caleb Vogt

Casey Henderson Solomon Gisemba Stephanie Johnson Jordan Hunt

ACKNOWLEDGEMENTS Generous supporters have contributed greatly to the success of this meeting. Without contributions from the following sponsors, the 53rd Annual MIKI Meeting-in-Miniature would not be possible. Thank you to all of the 2015 MIKI supporters!

This program is supported by a grant from Genentech and an educational grant from Pfizer Inc.

3:00 – 6:00 p.m. Registration Open Best Western HotelHotel Check-in

6:30 – 9:30 p.m Welcome Reception Oread HotelGriffith Ballroom

8:00 – 9:00 a.m. Breakfast School of Pharmacy, (SOP) Lower level

Poster Setup SOP Atrium

Opening Remarks and Welcome

9:00 – 9:20 a.m. Chancellor Bernadette Gray-Little SOP 2020

Professor Gary Grunewald

Session 1 SOP 2020

9:20 – 9:40 a.m. Elbek K. KurbanovThe University of Minnesota

“Structure-Based Inhibitor Design for Anthrax Toxin Lethal Factor”

9:40 – 10:00 a.m. Chaitanya KulkarniThe University of Iowa

“Synthetic Approaches to Novel Quinazoline-2,4-diones”

10:00 – 10:20 a.m. Huiyong MaThe University of Kansas

“Development of Kappa Opioid Receptor Agonists and Antagonists based on Novel Scaffolds”

10:20 – 10:40 a.m. Hitisha PatelThe University of Illinois at Chicago

“Exploiting the Therapeutic Potential of Estrogen for the Treatment of Tamoxifen Resistant Breast Cancer”

10:40 – 11:00 a.m. Break SOP Atrium

Keynote Address SOP 2020

11:00 – 12:00 p.m. Dr. Bruce Roth Overflow: SOP 3020Genentech Research and Early Development

“The Discovery and Development of LIPITOR®. Would anyone make this molecule today?”

Saturday, April 11, 2015

Friday, April 10, 2015

12:00 – 1:30 p.m. Lunch SOP, Lower level

Poster Session SOP, Atrium

1:30 – 2:30 p.m. Odd Numbered Posters Present2:30 – 3:30 p.m. Even Numbered Posters Present

Session 2 SOP 2020

3:30 – 3:50 p.m. Josephine H. SchampThe University of Iowa

“Neurodegeneration: The Role of Dopamine Metabolites, Oxidative Stress, and Antioxidants”

3:50 – 4:10 p.m. Brett R. AmblerThe University of Kansas

“Copper-Catalyzed Methods for Accessing Trifluoromethane-containing Products”

4:10 – 4:30 p.m. Rui XiongThe University of Illinois at Chicago

“Lead Based Development and Evaluation of Partial Agonist Selective Estrogen Mimics (PASEMs)

in Tamoxifen Resistant Breast Cancer”

4:30 – 4:50 p.m. Jingjing ShenThe University of Minnesota

“Design and Characterization of Bispecific T-cells Engaging Chemically Self-assembled Antibody

Nanorings(CSANs) with Potential for Treatment of B Cell Malignancies”

5:30 – 7:00 p.m. Faculty Reception Abe & Jake’s Landing

7:00 – 10:00 p.m. Formal Banquet Dinner Abe & Jake’s Landing

8:00 – 9:00 a.m. Breakfast SOP, Lower levelHotel Check-out Prior to Breakfast

Session 3 SOP 2020

9:00 – 9:20 a.m. Solomon A. GisembaThe University of Kansas

“Peptide Ring Closing Metathesis Reactions: Minimizing Side Reactions in Arodyn Analogs”

Sunday, April 12, 2015

9:20 – 9:40 a.m. Jing LiThe University of Illinois at Chicago

“TDP1 Promotes Assembly of Non-homologous End Joining Protein Complexes on DNA”

9:40 – 10:00 a.m. Adam T. ZarthThe University of Minnesota

“N′-Nitrosonornicotine 5′-Hydroxylation Causes DNA Damage in Rats”

10:00 – 10:20 a.m. Michael P. HayesThe University of Iowa

"Fragment-based Screening Against Regulator of G Protein Signaling 17"

10:20 –10:40 a.m. Break SOP Atrium

Session 4 SOP 2020

10:40 – 11:00 a.m. Chengsong JiaoThe University of Illinois at Chicago

“The Synthesis of Hydroxymethylene Phosphonate Analogs of Polyphosphates”

11:00 – 11:20 a.m. Bo ZhouThe University of Minnesota

“Target Identification and Mechanism Elucidation of Chalcones’ Cytotoxicity via Photoaffinity Probes”

11:20 – 11:40 a.m. Ben WilliamsonThe University of Iowa

"Investigations Toward Overcoming Drug Resistance in Bacteria through the Use of Novel Fluoroquinolones"

11:40 a.m. – 12:00 p.m. Gaurav GargThe University of Kansas

“Development of Biphenylamides as Hsp90 C-Terminal Inhibitors”

12:00 p.m. Concluding Remarks SOP 2020Faculty Meeting

Boxed Lunches Served SOP AtriumBuses Depart

Year Host Speaker Association2015 Kansas Dr. Bruce D. Roth Genentech Inc. 2014 Illinois Dr. Paul A. Wender Stanford University2014 Minnesota Dr. Marvin J. Miller University of Notre Dame2013 Iowa Dr. Heidi E. Hamm Vanderbilt University2012 Kansas Dr. Dennis C. Liotta Emory University2011 Illinois Dr. Tomáš Hudlický Brock University2010 Minnesota Dr. Dale Boger Scripps Research Institute2009 Iowa Dr. Daniel Kahne Harvard University2008 Kansas Dr. Albert Padwa Emory University2007 Illinois Dr. William Fenical University of California, San Diego2006 Minnesota Dr. Christopher Lipinski Pfizer Pharmaceuticals 2005 Iowa Dr. Kenner Rice National Institutes of Health2004 Kansas Dr. C. Dale Poulter University of Utah2003 Illinois Dr. Richard B. Silverman Northwestern University2002 Minnesota Dr. Andrew Hamilton Yale University 2001 Iowa Dr. Michael Marletta University of Michigan2000 Kansas Dr. Roger M. Friedinger Merck Research Laboratories1999 Illinois Dr. Richard A. Lerner Scripps Research Institute1998 Minnesota Dr. John Montgomery Biocryst Pharmaceutical, Inc. 1997 Iowa Dr. David Nichols Purdue University1996 Kansas Dr. Paul Anderson Dupont Merck Pharmaceutical 1995 Illinois Dr. Arthur Patchett Merck Research Laboratories1994 Minnesota Dr. Daniel Rich University of Wisconsin, Madison1993 Iowa Dr. Laurence Hurley University of Texas, Austin1992 Kansas Dr. Julius Rebek Massachusetts Institute of Technology1991 Illinois Dr. Koji Nakanishi Columbia University1990 Minnesota Dr. John Katzenellenbogen University of Illinois, Urbana-Champaign1989 Iowa Dr. Carl Djerassi Stanford University1988 Kansas Dr. William Roush Indiana University1987 Illinois Dr Joseph Fried University of Chicago1986 Minnesota Dr. David Triggle SUNY Buffalo1985 Iowa Dr. Alan Karitzky University of Florida1984 Wisconsin Dr. Paul Bartlett University of California, Berkeley1983 Kansas Dr. Henry Rapoport University of California, Berkeley1982 Illinois Dr. Harry Wasserman Yale University1981 Minnesota Dr. Eugene Jorgensen University of California, San Francisco1980 Iowa Dr. Alan Sartorelli Yale University1979 Kansas Dr. Albert Meyers Colorado State University1978 Illinois Dr. Heinz Floss Purdue University1977 Minnesota Dr. Donald Jerina National Institutes of Health1976 Iowa Dr. Everett May National Institutes of Health1975 Kansas Dr. Marjorie Horning Baylor University1974 Illinois Dr. Arnold Brossi Hoffman-LaRoche1973 Minnesota Dr. Gertrude Ellion Burroughs-Wellcome1972 Iowa Dr. Bernard Belleau University of Ottawa1971 Kansas Dr. Corwin Hansch Pomona College1970 Illinois Dr. Everett Maynert University of Illinois1969 Minnesota Dr. Bernard Baker University of California, Santa Barbara1968 Iowa Dr. Julius Axelrod National Institutes of Health1967 Kansas Dr. Richard Schowen University of Kansas

MIKI KEYNOTE SPEAKERS

53rd ANNUAL MIKI MEETING KEYNOTE SPEAKER

Dr. Bruce Roth

Senior Vice President, Small Molecule Drug Discovery Genentech Research and Early Development

Bruce Roth received his BS in Chemistry in 1976 (St. Joseph’s College), his Ph.D. Organic Chemistry in 1981 (Iowa State University) and after a postdoctoral fellowship at the University of Rochester, joined Warner-Lambert/Parke-Davis Research in 1982, where he rose to the position of Vice President, Chemistry. He is best known as the inventor of Lipitor®, the largest selling drug in the history of the pharmaceutical industry, for which he has received numerous awards, including the 2013 Perkin Medal, the highest award given to industrial chemists in the United States and the 2015 National Academy of Sciences award for Chemistry in Service to Society. Bruce served as Vice President of Chemistry at the Pfizer Global Research and Development, Ann Arbor Laboratories from 2000-2007. He also served as an adjunct associate professor in the Department of Medicinal Chemistry in the School of Pharmacy of the University of Michigan from 1996-2007. In May 2007, Bruce joined Genentech Small Discovery as Senior Director of Discovery Chemistry. On August 10, 2008 he was named one of the American Chemical Society Heroes in Chemistry for the Discovery and Development of Lipitor®. In September 2009, he was promoted to Vice President, Discovery Chemistry, Genentech Research and Early Development (gRED) and in January 2015 he was appointed as Senior Vice President, gRED Small Molecule Drug Discovery. Dr. Roth is the inventor or co-inventor of 54 issued U.S. patents and the author or co-author of 51 manuscripts, 35 published abstracts, 9 book chapters and review articles and has given more than 30 invited lectures. In 2013 he was inducted into the ACS Medicinal Chemistry Division Hall of Fame.

Bruce D. Roth, Ph.D.

KEYNOTE LECTURE

The Discovery and Development of LIPITOR®. Would anyone make this molecule today?

Dr. Bruce D. Roth Senior Vice President, Small Molecule Drug Discovery

Genentech Research and Early Development

The HMG-CoA reductase inhibitor LIPITOR® (atorvastatin calcium), the largest selling drug in the history of the pharmaceutical industry, was designed and synthesized in 1985 at a time when medicinal chemistry was dominated by QSAR analyses and emerging structure-based drug design. This was prior to the dramatic change in the practice of medicinal chemistry caused by the publication of Lipinski’s Rule of 5, which caused the industry to focus on property-based drug design as a way of controlling ADMET properties. Despite this, one of the key aspects of the selection of LIPITOR® as a development candidate involved development of the relationship between lipophilicity and differential drug distribution to liver and peripheral tissues. This talk will explore the development of the understanding of the tissue selectivity of atorvastatin and other HMG-CoA reductase inhibitors in relationship to physicochemical drug properties and place this in the context of the current practice of medicinal chemistry.

ORAL PRESENTATION ABSTRACTS

*Abstracts are produced here with the permission of the author(s) and are not to be construed as publications. Reproduction, quotation, or citation of any data contained herein without approval of the author(s) of said data is prohibited. Distribution of the abstracts is limited to registrants of

the 53rd annual MIKI Meeting.

SESSION I

Structure-Based Inhibitor Design for Anthrax Toxin Lethal Factor

Elbek K. Kurbanov†, Jonathan Solberg‡, Kimberly M. Maize†, Rodney L. Johnson†, Barry C. Finzel†, Jon E. Hawkinson‡, Michael A. Walters‡, Elizabeth A. Amin†

†Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55414 ‡Institute for Therapeutics Discovery and Development, Minneapolis, MN 55414

Anthrax is an infectious disease caused by Bacillus anthracis, a bacterium classified as a Category A pathogen by the CDC. The lethal factor (LF) enzyme secreted by B. anthracis is chiefly responsible for anthrax-related cell death. Although many studies have been conducted toward the design of small-molecule LF inhibitors, no LF inhibitor is yet available as a therapeutic agent. Here I present two projects: (1) the synthesis, experimental evaluation and structural studies of MK-31 analogs (Fig. 1) designed to explore S2′ site in LF and (2) the synthesis, testing, structural studies of series of MK-16 analogs designed to take advantage of a unique conformational change in the S1′ LF subsite (Fig. 2). This conformational change can further be explored to design potent and selective non-hydroxamate LF inhibitors. Project 1.

Figure 1. LF active site with catalytic Zn2+ (blue sphere) co-crystallized with MK-31 (R1=H). Project 2.

Figure 2. LF active site with co-crystallized 33.

NSO

O

F

O

NHOH

Me

33; IC50 = 29.4 µM

Synthetic Approaches to Novel Quinazoline-2,4-diones

Chaitanya Kulkarni, Robert Kerns Division of Medicinal and Natural Products Chemistry, Department of

Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, The University of Iowa, Iowa City, IA 52246

Fluoroquinolones are broad spectrum antibiotics that are widely used to treat bacterial infections. These antibiotics target bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) and inhibit these enzymes by forming a ‘cleaved complex’ consisting of the drug, the DNA and the enzyme. During this process, the C-3, C-4 dicarbonyl moiety of fluoroquinolones makes a critical binding contact with serine and aspartate/glutamate residues of topoisomerases through a magnesium-water bridge. Mutation of either of these residues prevents bridge formation, thereby decreasing the binding of fluoroquinolones in the cleaved complex and leads to resistance. In recent years there has been a rise in the number of bacterial strains that are resistant to fluoroquinolones, thereby emphasizing the need to develop new molecules active against these resistant mutants. Quinazoline-2,4-diones are structurally analogous to the fluoroquinolones. The N-3 amine and C-2 carbonyl groups of the quinazolinediones form a binding contact with a conserved arginine, orienting the diones into a structure similar to that of fluoroquinolones. However, unlike fluoroquinolones, the quinazoline-2,4-diones do not form a magnesium water bridge, and therefore these molecules are active with fluoroquinolone resistant DNA gyrase and/or topoisomerase IV. In our efforts to develop new quinolone class antibiotics that inhibit resistant mutants, we designed a panel of N-1 substituted fluoroquinolones and quinazoline-2,4-diones with an aim to reduce the dependence on the water-magnesium bridge. Two of the quinazolinedione derivatives had two different aromatic rings connected to the N-1 position via a methylene chain. None of the previously reported quinazolinediones had a N-1 benzyl substitution and the methods traditionally used to synthesize this core failed to afford these compounds. This talk outlines the obstacles encountered in the synthesis of these novel N-1 benzyl substituted quinazoline-2,4-diones and the different approaches to overcome these obstacles.

Development of Kappa Opioid Receptor Agonists and Antagonists based on Novel Scaffolds

Huiyong Ma1, Kimberly M. Lovell2, Kevin Frankowski1, Stephen Slauson1, Sarah M. Scarry1, Lei Zhou2, Angela M. Phillips2, Thomas E. Prisinzano1, Laura M. Bohn2 and Jeffrey Aubé1

1Department of Medicinal Chemistry, The University of Kansas, Lawrence, KS 66045 2Departments of Molecular Therapeutics and Neuroscience, The Scripps Research Institute,

Jupiter, FL 33458

The kappa opioid receptor (KOR) regulates a wide range of physiological functions including reward, depression, and pain relief. In pursuit of the therapeutic potential of KOR, several KOR modulators with novel scaffolds have been discovered through a high-throughput screening campaign of the Molecular Libraries compound collection. Here we discuss the optimization of both a bisamide chemotype agonist and sulfonamide chemotpe antagonist scaffolds. The bisamide class of KOR agonist possesses an attractively simple and highly modular peptide-like scaffold. To efficiently explore the potential of this new series, we developed an Ugi multicomponent reaction to afford analogues in a single step. Sixty-two analogues were synthesized and screened in a single round of structure-activity relationship (SAR) study, revealing two examples with single digit nanomolar potency. In addition, several discrete SAR trends emerged from analysis of the data set. The sulfonamide chemotype antagonist is interesting in that preliminary data shows that the lead compound does not possess the long duration of action (up to 3 weeks) in contrast to other KOR antagonists such as norBNI and JDTic. SAR optimization was carried out on sulfonamide scaffold, revealing several analogues with single digit nanomolar potency. The most recent results obtained from this series will be presented.

Exploiting the Therapeutic Potential of Estrogen for the Treatment of Tamoxifen Resistant Breast Cancer

Hitisha K Patel1, Rui Xiong1, Lauren Gutgesell1, Jiong Zhao1, Brad Michaelsen1, Mary Ellen

Molloy2, Debra Tonetti2 and Gregory R. J Thatcher1. 1Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of

Illinois at Chicago, 833 S. Wood St., Chicago, IL 60612. 2Department of Biopharmaceutical Sciences, College of Pharmacy, University of Illinois at

Chicago, 833 S. Wood St., Chicago, IL 60612.

It is a largely forgotten fact that estrogen receptor-α (ERα) agonists, estradiol and diethylstilbesterol (DES), were used effectively for clinical treatment of breast cancer before the advent of tamoxifen. Tamoxifen, an antagonist at ERα in breast tissue, and the prototypical selective estrogen receptor modulator (SERM), is the standard of care for many patients with ER-positive breast cancer. Although 5 year survival rates for DES were superior to tamoxifen, the improved side effect profile of tamoxifen was pivotal. Together with the problematic claims that estrogen replacement therapy (ERT) increases the risk of breast cancer, the early clinical success of estradiol and DES has been forgotten. Tamoxifen increases risk of endometrial cancer and half of ER-positive breast cancer patients do not respond or relapse on treatment with tamoxifen. Tamoxifen-resistant tumors are often ER-positive and endocrine-independent and therefore resistant to aromatase inhibitors. Recent trials have shown the efficacy of estrogen in patients who have undergone exhaustive tamoxifen therapy. Therefore, we propose the development of small molecules that mimic the actions of estradiol in ER-positive breast cancer without the uterotrophic actions of estradiol and tamoxifen: Selective Estrogen Mimics (SEMs). Evidence suggest that a significant subset of ER-positive breast cancer, over-expressing PKCα, and tamoxifen-resistant cancers will respond to SEMs. In order to design and optimize SEMs, tamoxifen-resistant cell lines, MCF-7:5C and T47D: PKCα, were studied. The activation of classical ERα signaling by the estrogen mimics was profiled in MCF-7 cells. Cell viability in 2D culture and spheroid growth in 3D culture was examined as well. Interestingly, it was found that in MCF-7:5C cells, SEM induced cell death was mediated by activation of classical ERα signaling. Two promising SEMs were evaluated in xenograft models of TAM-resistant, PKCα overexpressing breast cancer. While E2 caused regression of these tumors, a significant increase in uterine weight as predicted was observed. More importantly, SEM treated mice had negligible increase in uterine weight underlining the enhanced safety of these molecules. These data suggest that development of SEMs that retain the beneficial properties of estrogen while limiting the side effects is a feasible strategy for the treatment of tamoxifen resistant breast cancer.

SESSION II

Neurodegeneration: The Role of Dopamine Metabolites, Oxidative Stress, and Antioxidants

Josephine H. Schamp1, Eric Emmons2, Nandakumar Narayanan2, and Jonathan A. Doorn1

1 Division of Medicinal & Natural Products Chemistry, College of Pharmacy 2Department of Neurology, Carver College of Medicine

University of Iowa, Iowa City, IA 52242 The progressive degeneration of neurons, as observed for Parkinson’s disease (PD), leads to an inability to control movement. PD is characterized by the selective loss of neurons in the substantia nigra (dopamine-containing) region of the brain. The goal of this work is to test the hypothesis that endogenously produced toxins including: toxic dopamine metabolites, oxidative stress (OxS), and reactive oxygen species (ROS) are key contributors to neurotoxicity relevant to PD and can be regulated by alterations in the antioxidant status of the dopaminergic neurons. Dopamine (DA), a neurotransmitter, undergoes metabolism by monoamine oxidase (MAO) to 3,4-dihydroxyphenylacetaldehyde (DOPAL), and further metabolism by aldehyde dehydrogenases and reductases to 3,4-dihydroxyphenylacetic acid (DOPAC), and 3,4-dihydroxyphenylethanol (DOPET), respectively. Comparing DA and these metabolites, DOPAL displays the greatest toxicity both in vitro and in vivo. DOPAL, a highly reactive aldehyde intermediate, is toxic through a variety of mechanisms: 1) it is able to interact with proteins leading to modification of Lys and Arg residues and forms protein aggregates, 2) DOPAL can autooxidize to subsequent quinone species, which are highly reactive with proteins and generate ROS/OxS. Of note, autooxidation and protein modification by DOPAL results in the generation of ROS (H2O2, O2

-). Production of ROS will impact OxS levels creating an imbalance subsequent to the DOPAL-mediated neurotoxicity. OxS can contribute to cell damage including the increase in DOPAL levels due to the inhibition of carbonyl metabolizing enzymes. To explore the role of antioxidants in DOPAL-mediated neurotoxicity, antioxidant levels were altered (+/-) via addition of N-acetylcysteine (NAC), ascorbate, diamide, and buthionine sulfoxide (BSO) in 4 different cell lines. The data indicate that antioxidants negatively affect DOPAL-protein modification. Antioxidants NAC and ascorbate attenuated the adduction of proteins by DOPAL, and conversely oxidants diamide and BSO increased protein modification by DOPAL. To further support the role of antioxidants in regulating DOPAL-protein interaction and succeeding damage, antioxidant NAC was investigated in an in vivo model. A 6-hydroxydopamine (6-OHDA) rat model was utilized to determine the effects of NAC in a PD model containing a unilateral lesion of the nigrostriatal pathway. The study allows for quantification of behavioral as well as neurochemical changes. This investigation will help in clarifying the roles of toxic dopamine metabolites and elucidating the complex role of OxS, specifically ROS, in DOPAL-mediated neurotoxicity.

Copper-Catalyzed Methods for Accessing Trifluoromethane-containing Products

Brett R. Ambler, Lingui Zhu, Santosh Peddi, and Ryan A. Altman Department of Medicinal Chemistry, The University of Kansas

Lawrence, KS 66045 Fluorinated functional groups serve important roles in medicinal chemistry due to their ability to modulate the (bio)physical properties of molecules. The ability to access fluorinated substructures from simple functional groups under mild conditions is important for preparing new biological probes and therapeutic candidates. However, gaps in synthetic chemistry restrict access to many useful fluorinated target compounds. For example, the conversion of ubiquitous, readily available alcohols to biomedically relevant trifluoromethanes represents a potentially powerful transformation. However, methods that convert alcohols to trifluoromethanes suffer from several limitations including: 1) harsh reagents that destroy many common functional groups; 2) lengthy reaction sequences that provide low yields of product; 3) (super)stoichiometric equivalents of transition metals and/or superstoichiometric quantities of expensive reagents. Combined, these factors inhibit medicinal chemists from accessing many trifluoromethane-containing target molecules in short sequences and reliable transformations. To circumvent these problems, we present copper-catalyzed decarboxylative deoxytrifluoromethylation reactions, which can be employed to access new fluorinated biological probes and therapeutic candidates. Our work aims to overcome previous limitations in the field by: 1) developing short and direct strategies for converting readily available functional groups into fluorinated analogs; 2) developing methods that provide access to unique and underrepresented fluorinated functional groups; 3) employing mild, inexpensive and environmentally-friendly fluorinated building blocks. This technology tolerates a variety of functional groups that are typically encountered in drug-like molecules, and should be sufficiently mild to modulate natural products with potential therapeutic benefits. Intellectually, the project bridges a gap between two contemporary topics in synthetic chemistry, namely catalytic decarboxylative coupling and fluoroalkylation.

Lead Based Development and Evaluation of Partial Agonist Selective Estrogen Mimics (PASEMs) in Tamoxifen Resistant Breast Cancer

Rui Xiong1, Hitisha K. Patel1, Jiong Zhao1, Xiao Liang1, Brad Michalsen1, Mary Ellen Molloy2,

Debra Tonetti2 and Gregory R. J. Thatcher1 1 Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of

Illinois at Chicago, 833 S. Wood St., Chicago, IL, 60607 2Department of Biopharmaceutical Sciences, College of Pharmacy, University of Illinois at

Chicago, 833 S.Wood St., Chicago, IL, 60607 Tamoxifen is the standard of care for many patients with estrogen receptor positive (ER+) breast cancer and works by antagonizing the actions of estrogen at ER. However it poses an increased risk of endometrial cancer and thrombotic events, and most significantly, 30-50% women develop resistance to tamoxifen therapy, underlining the need for superior therapeutic options. Paradoxically, prior to tamoxifen therapy, estradiol (E2) and the ER agonist, diethylstilbestrol, had been used in breast cancer therapy, though with serious side effects. Development of partial agonist selective estrogen mimics (PASEMs) that cause regression of tamoxifen-resistant breast cancer, but without the side effects of E2, represents a rational therapeutic strategy. Novel PASEMs were developed, which in vitro in 3D cultures and in vivo caused complete regression of tamoxifen-resistant xenografts, with characteristics similar to those of E2. These PASEMs did not fuel growth of estrogen-dependent T47D xenografts and did not cause uterine growth. PASEM mediation of classical ER-signaling was profiled in MCF7 and MDA-MB231:β41 cells. A tamoxifen-resistant cell line, MCF-7:5C, was used to assay induction of cell death by both E2 and the PASEMs and to probe the mechanism of action. Structure-activity relationships were explored, suggesting that partial ERα agonists have the capacity to cause regression of tamoxifen-resistant tumors, without adverse effects associated with estrogenic actions in normal gynecological tissues of the breast and uterus, which might contribute to carcinogenesis. The ER-mediated agonist/antagonist activity, regression efficacy, and pharmacokinetic profiles of these PASEMs were examined to obtain lead compounds that are effective with minimal estrogenic side effects. This research paves the way for use of PASEMs in tamoxifen-resistant breast cancer with enhanced safety profiles.

Design and Characterization of Bispecific T-cells Engaging Chemically Self-assembled Antibody Nanoring(CSANs) with Potential for Treatment of B Cell Malignancies

Jingjing Shen, Adrian Fegan, Daniel A. Vallera, and Carston R. Wagner*

Medicinal Chemistry Department, College of Pharmacy, University of Minnesota Minneapolis, MN 55455

Our laboratory has developed a protocol for the preparation of chemically self-assembled antibody nanorings (CSANs) formed with dimeric dihydrofolate reductase antiCD3 single-chain variable fragment (scFv) fusion proteins (DHFR2-antiCD3) with a bis-methotrexate (bisMTX) ligand. We have previously shown that antiCD3 CSANs displaying multiple copies of scFv maintained the binding behavior of the parental mAb UCHT-1.1,2 This success encouraged us to further develop antiCD3×22 CSAN bispecific T cell engagers (BiTE)3 by self-assembling antiCD3 and antiCD22 fusion proteins, targeting both of the CD3ε subunit of the T-cell-receptor/CD3 complex and the CD22 antigen on malignant B cells such as B-leukemias or lymphomas and redirecting cytotoxic T cells (CTLs) for killing of tumor cells. During our studies, we identified that after cross-linking of T cells and malignant B cells expressing CD22 (Raji cells), redirected T cells upregulate those surface and degranulation markers such as CD25, CD69 and CD107a and secrete the inflammatory cytokines such as IL-2 and IFN-γ. The activated T cells have also shown improved cytotoxic efficacy against B lymphoma cells in vitro. In addition, the in vitro stability of CSANs on T cells was investigated. We further confirmed the ability of an FDA approved DHFR inhibitor trimethoprim to initiate DHFR-oligomer disassembly by incubating bispecific CSANs modified T cells with trimethoprim at micromolar concentrations in vitro. This is one of the distinct advantages of our bispecific CSANs over other BiTEs or Chimeric antigen receptor (CAR)-modified T cells, because disassembly of CSANs abrogating the cell-cell interactions may efficiently reverse side effects including cytokine release syndrome. 4,5 Taken together our approach to design reprogrammable bispecific CSANs can offer an alternative strategy for constructing bispecific T cell engagers for cancer immunotherapy. (1) Li, Q.; Hapka, D.; Chen, H.; Vallera, D. A.; Wagner, C. R. Self-Assembly of Antibodies by

Chemical Induction. Angew. Chem. 2008, 120, 10333–10336. (2) Li, Q.; So, C. R.; Fegan, A.; Cody, V.; Sarikaya, M.; Vallera, D. A.; Wagner, C. R.

Chemically Self-Assembled Antibody Nanorings (CSANs): Design and Characterization of an Anti-CD3 IgM Biomimetic. J. Am. Chem. Soc. 2010, 132, 17247–17257.

(3) Garber, K. Bispecific Antibodies Rise Again. Nat. Rev. Drug Discov. 2014, 13, 799–801. (4) Nagorsen, D.; Kufer, P.; Baeuerle, P. A.; Bargou, R. Blinatumomab: A Historical

Perspective. Pharmacol. Ther. 2012, 136, 334–342. (5) Lee, D. W.; Gardner, R.; Porter, D. L.; Louis, C. U.; Ahmed, N.; Jensen, M.; Grupp, S. A.; Mackall, C. L. Current Concepts in the Diagnosis and Management of Cytokine Release Syndrome. Blood 2014, 124, 188–195.

SESSION III Peptide Ring Closing Metathesis Reactions: Minimizing Side Reactions in Arodyn Analogs

Solomon A. Gisembaa and Jane V. Aldricha,b

aDepartment of Medicinal Chemistry, The University of Kansas, Lawrence, KS 66045. bDepartment of Medicinal Chemistry, The University of Florida, Gainesville, FL 32611.

The acetylated dynorphin A analog arodyn (Ac[Phe1,2,3,Arg4,D-Ala8]Dyn A(1-11)-NH2) exhibits potent and selective kappa opioid receptor antagonism.1 Novel cyclization strategies via ring closing metathesis (RCM) are being pursued to enhance the metabolic stability and potentially stabilize the bioactive conformation of arodyn.2 RCM is compatible with peptide and standard amino acid protecting groups, but side reactions, i.e. olefin isomerization, can compromise the product yield. While RCM involving allylglycine (AllGly) residues showed moderate to high yields, reactions involving protected Tyr(All) resulted in poor yields.2 Initially model RCM precursors were synthesized to probe strategies to minimize side reactions and enhance reaction yields. The optimized synthetic methodology was then applied in the synthesis of arodyn analogs. Results of side reaction minimization strategies, including examination of reagents reported to suppress side reactions,3 and microwave heating, will be presented. This research is supported by NIDA grant R01 DA018832.

1. Bennett, M. A.; Murray, T. F.; Aldrich, J. V. J. Med. Chem. 2002, 45, 5617. 2. Fang, W. J.; Kulkarni, S. S.; Murray, T. F.; Aldrich, J. V. Adv. Exp. Med. Biol. 2009, 611,

279. 3. Hong, S. H.; Sanders, D. P.; Lee, C. W.; Grubbs, R. H. J. Am. Chem. Soc. 2005, 127,

17160.

TDP1 Promotes Assembly of Non-homologous End Joining Protein Complexes on DNA

Jing Li1, Jinho Heo1, Karin Nitiss2,3, Matthew Summerlin1, John L. Nitiss2 and Les Hanakahi1

1. Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Rockford, IL

2. Department of Biopharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Rockford, IL

3. Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford

The repair of DNA double-strand breaks (DSB) is central to the maintenance of genomic integrity. In tumor cells, the ability to repair DSBs predicts response to radiation and many cytotoxic anti-cancer drugs. DSB repair pathways include homologous recombination and non-homologous end joining (NHEJ). NHEJ is a template-independent mechanism, yet many NHEJ repair products carry limited genetic changes, which suggests that NHEJ includes mechanisms to minimize error. Proteins required for mammalian NHEJ include Ku70/80, the DNA-dependent protein kinase (DNA-PKcs), XLF/Cernunnos and the XRCC4:DNA ligase IV complex. NHEJ also utilizes accessory proteins that include DNA polymerases, nucleases, and other end-processing factors. In yeast, mutations of tyrosyl-DNA phosphodiesterase 1 (TDP1) reduced NHEJ fidelity. TDP1 plays an important role in repair of topoisomerase-mediated DNA damage, and mutation of the human TDP1 gene results in an inherited human neuropathy termed SCAN1. We found that human TDP1 physically interacted with and stimulated DNA binding by XLF to form TDP1:XLF:DNA complexes. TDP1 can remove 3’ adducts from DNA, and TDP1:XLF interactions stimulated this activity on dsDNA, but not on ssDNA. TDP1 also promoted DNA binding by Ku70/80 and stimulated DNA-PK activity. Ku70/80 and XLF are recruited to the DSB at the onset of NHEJ, and our data suggest that TDP1 may facilitate assembly of NHEJ factors on damaged and undamaged end. Future experiments will attempt to clarify the role of TDP1 in accuracy and efficiency of NHEJ in human cells. We will use CRISPR/Cas9-mediated genome editing to generate TDP1-deficient cells and use DSB-repair reporter substrates to compare repair between wild type and TDP1-deficient cells. Using an extrachromosomal DSB-repair substrate we will examine the role of TDP1 in repair of a variety of DNA ends. Using a chromosomally integrated DSB-repair substrate we will examine the role of TDP1 in repair of DSBs in the context of chromosomal DNA.

N′-Nitrosonornicotine 5′-Hydroxylation Causes DNA Damage in Rats

Adam T. Zarth,1,2 Pramod Upadhyaya,2 Jing Yang,2 and Stephen S. Hecht1,2

1Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455 2Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455

N′-Nitrosonornicotine (NNN), considered a human carcinogen by the International Agency for Research on Cancer, is present in all tobacco products. Metabolic a-hydroxylation of NNN in vivo forms the activated species 2′-hydroxyNNN and 5′-hydroxyNNN. These intermediates can rearrange to form highly reactive diazonium ions, which can also be produced synthetically from the hydrolysis of a stable precursor (e.g. 5′-acetoxyNNN). DNA adducts arising from 2′-hydroxyNNN have been well-established in target tissues of rats, but adducts arising from 5′-hydroxyNNN have not yet been observed in vivo. 5′-Hydroxylation is the major metabolic pathway in non-human primates, and human P450s can efficiently metabolize NNN to 5′-hydroxyNNN in vitro. For these reasons, we hypothesize that 5′-hydroxylation of NNN is an important source of DNA damage in humans. To investigate this metabolic pathway in vivo, we treated male F-344 rats with NNN and isolated DNA from lung, liver, esophageal mucosa, oral cavity, and nasal cavity to analyze for the formation of DNA adducts including 2-(2-(3-pyridyl)-N-pyrrolidinyl)-2′-deoxyinosine (py-py-dI). Py-py-dI formation was detected in all of these tissues, and a clear dose-response relationship was observed. Adduct formation in the lung and nasal cavity was far greater than in other tissues. This demonstrates for the first time that 5′-hydroxylation of NNN causes DNA damage in vivo and that py-py-dI is the major DNA adduct arising from this pathway. Ongoing work with NNN and 5’-acetoxyNNN in cultured hepatocytes will investigate the formation of py-py-dI at the cellular level. This methodology has established a foundation for further studies on the formation of DNA adducts from NNN in people who use tobacco products.

Fragment-based Screening Against Regulator of G Protein Signaling 17

Michael P Hayes1, C Andrew Fowler2, Liping Yu2, David L Roman1 1Department Pharmaceutical Sciences and Experimental Therapeutics, 2Nuclear Magnetic

Resonance Facility, University of Iowa, Iowa City, IA, 52242 Regulator of G protein Signaling 17 (RGS17) accelerates the GTPase activity of G protein α subunits and is upregulated in prostate and lung cancers. Previous work has shown that inhibition of RGS17 activity via RNAi is sufficient to decrease tumorigenesis and cancer cell proliferation, making RGS17 an attractive target for antineoplastic agents. Here we used NMR-based fragment screening to identify novel chemotypes capable of binding RGS17. 1000 fragments were screened and chemical shift perturbations were monitored via HSQCs. Seven fragments were identified that were capable of binding RGS17, and one of these fragments also showed activity in orthogonal follow-up assays. NMR was then used to determine dissociation constants, with all fragments exhibiting millimolar affinities. Future efforts will focus on exploring the structure activity relationships of these fragments and other compounds nearby in chemical space, with the ultimate goal being to identify specific, high affinity inhibitors of RGS17.

SESSION IV

The Synthesis of Hydroxymethylene Phosphonate Analogs of Polyphosphates

Chensong Jiao, Karol S. Bruzik Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago,

Chicago, IL 60612

Inositol polyphosphates (IPs) and inorganic polyphosphate (polyP) play important roles in bacterial and mammalian cells, yet their protein targets and mechanisms of action are not well understood due to the paucity of proper molecular tools for such studies. In addition, these compounds feature inherently unstable phosphoanhydride bond that undergoes rapid hydrolytic cleavage under biological conditions.

To address these problems, biotin-labeled analogs of inositol hexakisphosphate (IP6), heptakisphosphate (IP7), and polyP featuring hydroxymethylenephosphinophosphate residues as a pyrophosphate replacement were designed and synthesized. To facilitate a pull-down of proteins that interact with polyphosphorylated inositols, we obtained analogs of IP6 and IP7 with biotin attached to 2-hydroxyl group of myo-inositol via a phosphorylated aminohexyl linker. Synthesis of IP6-biotin was accomplished via a 11-step procedure using 3,4,5,6-tetra-O-benzyl-myo-inositol as a starting material, whereas IP7-biotin analog was synthesized using a 12-step procedure starting with myo-inositol orthobenzoate. Synthesis of precise-length analogs of polyP was achieved using an automated DNA synthesizer. The required phosphitylating agent monomer for solid phase synthesis was obtained using a 4-step procedure starting from phosphinic acid. The first automated syntheses showed almost quantitative efficiency of coupling steps, enabling synthesis of isotactic oligomers with 20, 40 and 60 phosphorus atoms, while synthesis of longer polymers remains possible. Synthesis of oligomers with different negative charge density and equipped with fluorescent of biotin tags is also possible.

The analogs of biotin labeled IPs have been used to identify protein targets from mammalian cells via streptavidin affinity chromatography. The polyP analogs of different lengths will be used to help investigate the role of polyP in the blood clotting cascade.

R2OO

OR1

OR3

R2O OR2

PO

HN

O O

O

S

HN

NH

O

IP6 biotin: R1=R2=R3= PO32-

IP7 biotin: R3=R, R1=R2= PO32-

PO P

OO O

O OR:

OBnBnOOBn

BnO OHOH

IP6-biotinO

HOOH

OO

Ph

HO IP7-biotin

P OHH H

O

O P ODMTO

OBnP

BnO

NHO P O P O P O P O

OO

O

OO

OOPOOOH

O n

polyP analog

Target Identification and Mechanism Elucidation of Chalcones’ Cytotoxicity via Photoaffinity Probes

Bo Zhou, Chengguo Xing

Department of Medicinal Chemistry, University of Minnesota Minneapolis, MN 55455

Chalcone-based compounds, both natural and synthetic, have shown a wide spectrum of biological activities with therapeutic potentials, including cytotoxicity, anti-inflammation, anti-microbial, and other. Among these bioactivities, cytotoxicity is of particular interest due to its anti-cancer potential. Although chalcones' bioactivities have been studied extensively, the mechanistic insights and particularly the responsible cellular targets surprisingly remain largely unsettled, which significantly limits the development of chalcone-based compounds into potent and selective therapeutic agents.

In this study, we aimed to identify the cellular target responsible for chalcones' cytotoxicity via photoaffinity labeling approach, and to further elucidate the cytotoxic mechanisms. We first developed a chalcone compound library targeting cytotoxicity. Guided by the sharp structure-activity relationship (SAR) obtained within our library, positive and negative photoaffinity probes were designed and synthesized for direct target labeling and pull-down experiments from cells. Various cell-based photoaffinity labeling experiments unambiguously identified a 52 kD protein as the responsible cellular target for chalcone’s cytotoxicity, which was revealed as β-tubulin by mass spectrometry. Furthermore, cytotoxic chalcones were shown to inhibit tubulin polymerization, disrupt the microtubule network in cells, and cause M-phase cell cycle arrest. These tubulin-related effects not only further supported β-tubulin as chalcones' direct cellular target responsible for cytotoxicity, but also provided a comprehensive view of the cellular mechanisms by which chalcones exert their cytotoxicity.

Altogether, our results provided a target-level mechanistic insight into chalcones' cytotoxicity, which would be important for further development of chalcone-based compounds as potential anti-cancer agents. The methodology applied here could be adapted for the target identification and mechanism elucidation of chalcones' other biological activities.

Investigations Toward Overcoming Fluoroquinolone Resistance in Bacteria

Benjamin H. Williamson and Robert J. Kerns* The University of Iowa, College of Pharmacy, Department of Pharmaceutical Sciences and

Experimental Therapeutics, Division of Medicinal and Natural Products Chemistry, Iowa City, IA 52242.

Fluoroquinolones are highly successful antibiotics that, through widespread use, suffer diminishing clinical utility due to emerging antibiotic resistance. The emergence of fluoroquinolone-resistant bacteria is believed to be caused by evolution-driven changes in the targets of fluoroquinolone action, namely the bacterial type-II topoisomerase enzymes DNA gyrase and topoisomerase IV. The function of type-II topoisomerases is the untangling of knots in bacterial DNA and the separation of newly replicated bacterial DNA. Topoisomerases are crucial to the replication of bacterial DNA, and ultimately cellular reproduction in bacteria. Fluoroquinolones are able to bind into a structure composed of topoisomerase and bacterial DNA. This entire structure composed of the fluoroquinolone, DNA, and topoisomerase is referred to as the ternary complex. The binding of the fluoroquinolone to form ternary complex stops the process of DNA untangling by blocking topoisomerase religating DNA, which in turn halts cellular growth and leads to cell death. Compounds such as the fluoroquinolones that form stable ternary complexes and block DNA relegation are termed topoisomerase poisons. The goal of the research described herein is the development of new fluoroquinolones that are able to poison both non-mutated “wild-type” type-II topoisomerases and mutated, fluoroquinolone-resistant type-II topoisomerases. This was accomplished by the generation of fluoroquinolones that possess novel side chain structures that bind to sites in the ternary complex that are separate from the resistance-causing substitutions within the topoisomerase. The ability of these new fluoroquinolones to 1) poison bacterial topoisomerases and 2) not act on human topoisomerase was tested with purified bacterial and human type-II topoisomerase enzymes. The ability of these compounds to halt bacteria cell growth was tested in wild-type and mutant cell cultures.

Development of Biphenylamides as Hsp90 C-Terminal Inhibitors

Gaurav Garg, Huiping Zhao, and Brian S. J. Blagg* Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045

Heat shock protein 90 (Hsp90) is the master regulator of the protein folding machinery that regulates the conformational maturation, activation, and integrity of more than 200 client proteins. These client proteins play critical roles in several cellular functions, such as signal transduction, protein trafficking, chromatin remodeling, cell growth, differentiation and survival. A large number of these client proteins (e.g., Her2, Raf1, Akt, MET, Src, CDK4 etc.) are often mutated and/or over-expressed in cancer cells and contribute to the hallmarks of cancer. Consequently, Hsp90 inhibition has emerged an attractive strategy for the development of anti-cancer agents. Most drug discovery efforts have focused on the coumarin-containing natural products for development of Hsp90 C-terminal inhibitors. Although, several efficacious analogues have been identified, scarcity of scaffolds has remained a limited. Therefore, new scaffolds that can be easily modified to produce compounds with enhanced activities are sought. Towards this objective, a small library of Hsp90 C-terminal inhibitors containing a biphenyl scaffold was designed, synthesized and evaluated for anti-cancer activity. The preliminary results from these studies will be presented.

POSTER ABSTRACTS

*Abstracts are produced here with the permission of the author(s) and are not to be construed as publications. Reproduction, quotation, or citation of any data contained herein without approval of the author(s) of said data is prohibited. Distribution of the

abstracts is limited to registrants of the 53rd annual MIKI Meeting.

# Name University Abstract Title

1 Rondine Allen Iowa Inhibition of Scavenger Receptors Using Novel PEGylated Polylysine Peptides

2 Mallesh Beesu Kansas Human Toll-like Receptor 8-Selective Agonistic Activities in 1-Alkyl-1H-benzimidazol-2-amines

3 Matthew Bockman MinnesotaTargeting Mycobacterium tuberculosis Biotin Protein Ligase (MtBPL): Synthesis and Evaluation of Nucleoside-based Bisubstrate Adenylation Inhibitors

4 Emily Boldry Minnesota DHB-Lysine: Developing Methods for Detection and Quantitation in Urine

5 Rhea Bovee Illinois Nitric Oxide Regulates DNA Methylation via Direct Inhibition of TET Activity

6 Joseph Buonomo Minnesota β-Lactam Conjugate Prodrugs of D-Cycloserine7 Congmei Cao Kansas Unusual Withanolides from Physalis hispida (Waterf.) Cronquist

8 Erick Carlson Minnesota Exploring CatSper Channel Openers and Binding Site Interactions: Discovery of Steroidal Channel Blockers

9 Giuseppe Caruso Kansas A High-Throughput Screen for the Identification of Novel Innate Immune Stimuli

10 Denise Casemore Minnesota Revised Synthesis of CXL037 and its Use as a Photo Reactive Probe in Identification of Cellular Targets

Andie Cassity KansasNicole Windmon Kansas

12 Manwika Charaschanya Kansas An Improved Schmidt Reaction for the Synthesis of N-Hydroxyalkyl Lactams13 Wei-Lun Chen Illinois Silvestrol Induces Autophagy and Apoptosis in Human Melanoma Cells

14 Sara Coulup Minnesota Design and Synthesis of Stabilized Pironetin Analogs for Resistant Ovarian Cancers

15 Ioana Craciun Iowa Novel Inhibitors of HNE, CatG and Pr3 and Their Evaluation in vitro16 Ming-Hsui Yang Kansas Pd-Catalyzed Decarboxylation Enables Direct Access to α,α-Difluoroketones

17 Rachel Davis Kansas Synthesis and Biological Evaluation of Hsp90 C-Terminal Inhibitors for the Treatment of Cancer

18 Sondra Dean IowaStructural and Dynamic Insight into the Idiosyncratic Nature of Ligand-associated Conformational Changes of Glutamate Racemase at the Atomistic Level

19 Justine Delgado Iowa Effect of Fluoroquinolone N-1 Aryl Substituents on Bacterial Type-II Topoisomerase Inhibition and DNA Binding

20 Skye Doering Minnesota Dual Melanocortin 3 and Melanocortin 4 Receptor Tetrapeptide Antagonists

21 Carter Eiden Minnesota Synthesis and Kinetic Characterization of Mechanism-based Inhibitors of BioA

Saqib Faisal KansasPradip Maity Kansas

23 Leah Forsberg Kansas Hsp90 C-Terminal Inhibitors that Manifest Neuroprotective Activity

24 Zhe Gao Kansas Synthesis and Evaluation of Fluorecent Probes of Prostate Specific Membrane Antigen (PSMA)

25 Sarah Green Illinois Red Clover and Glycyrrhiza inflata Display ERbeta Selectivity, Suggesting Better Safety Profile for Women’s Health

Poster Presentations, School of Pharmacy AtriumOdd Numbered Posters Present: 1:30 p.m. - 2:30 p.m.Even Numbered Posters Present: 2:30 p.m. - 3:30 p.m.

11

22

Intramolecular C-Vinylation to Novel Triazole-Fused Vinyl Sultams

Development of High-Load, Immobilized Si-ROMP, Co/C Magnetic Reagents/Scavengers and Ligands

26 Arnie Groehler Minnesota Identification, Characterization, and Quantification of DNA-protein Cross-linking by Cyclophosphamide Metabolite Phosphoramide Mustard

27 Lauren Gutgesell Illinois Analysis of Selective Estrogen Mimics (SEMs) in a 3D Cell Model of Tamoxifen Resistance

28 Atieh Hajirahimkhan IllinoisIn vitro and in vivo Evaluation of NAD(P)H:quinone Oxidoreductase 1 (NQO1) Induction by Licorice Species Used in Botanical Dietary Supplements for Women’s Health

29 Thomas Hanigan Illinois Having a Complex: Implications on HDAC-Ligand Interactions

30 Colin Higgins Iowa RGS Dynamics Determine Sensitivity to Allosteric Inhibition by Small Molecules

31 Abu Gafar M Hossion Kansas Synthesis and in vitro Cytotoxic Evaluation of Analogues of the Tubulin-binding Agent Soblidotin

32 David Huang Minnesota Analogue Synthesis of the α-Tubulin-Binding Natural Product Pironetin as an Ovarian Cancer Chemotherapeutic Agent

33 Atul Jain Illinois A Scaffold-hopping Approach to Discovery of Keap1/Nrf2 Inhibitors

34 Salim Javeed Kansas Phosphate Tether-mediated, One-pot, Sequential Processes towards the Total Synthesis of Lyngbouilloside and Macrocyclic Analogues

Jung Ho Jun KansasMoon Y. Hur Kansas

36 Kelsey Knewtson Kansas Phenotypic Screening of Small Molecules Against Danio rerio Embryos

37 Jill Kyzer Minnesota Design and Synthesis of Nuclear Receptor Antagonist Targeting RARα for Male Contraception

38 Sue Lee Illinois Characterization and Assessment of ALDH2-/- Mice as an Age Related Model of Cognitive Impairment and Alzheimer’s Disease

39 Cody J. Lensing Minnesota Investigating Metabolic Gender Differences with Melanocortin Antagonist SKY 2-23-7

40 Xinyun Liu Kansas Quinone-Promoted Inert Bond Functionalization of Amines

41 Joana Loh Kansas Complementary Pairing: An Aziridine Opening/SNAr Approach to Novel Benzofused Sultams

42 Shams-ul Mahmood Minnesota Synthesis and Biological Evaluation of Azomethine-dihydroquinazolinone Conjugates as Cancer and Cholinesterase Inhibitors

43 Soma Maitra Kansas Phosphate Tether-Mediated Approach towards the C9–C25 Fragment of Spirastrellolide B

44 Kimberly Maize Minnesota Dynamic Anthrax Toxin Lethal Factor

45 Jana Markley Kansas Phosphorus-Based Tether Methods for the Synthesis of 1,3-Anti-diol-containing Natural Products

46 Daniel May Illinois Isolation and Structure Elucidation of Merocyclophane C from the Cultured Cyanbacterium Nostoc sp. (UIC 10110)

47 Sanket Mishra Kansas Structure-Based Design of Grp94-Selective Inhibitors for Glaucoma and Metastasis

48 Michael Mullowney Illinois A Novel Diterpene and M. tuberculosis-active Molecules from a Marine-derived Actinomycete in Vietnam

49 Vanessa M. Nepomuceno Illinois Azaphilone Molecules from Freshwater Fungi

50 Aniekan Okon Minnesota Development of a Pronucleotide Phosphoramidate-based Inhibitor of Eukaryotic Translation Initiation Factor 4E

51 Margaret Olson Minnesota Small Molecule Inhibitors of APOBEC3 DNA Cytosine Deaminases as Leads for HIV-1 and Cancer Therapy

52 Victoria S. Parker IowaThe Inhibition of Human Steroid Sulfotransferases hSULT1E1 and hSULT2A1 by Hydroxylated and Sulfated Metabolites of Polychlorinated Biphenyls

35 An Efficient and Concise Synthesis of Novel β-Keto-Sultams as Tetramic Acid Analogs

53 Jacob Petersburg Minnesota Utilizing Chemically Self-Assembled Antibody Nanorings (CSANs) as Bispecific Targeting Agents for Cell-directed Immunotherapy

54 Elyse Petrunak Kansas Prostate Cancer Target Cytochrome P450 17A1 (CYP17A1): Evaluation and Comparison of Clinically-Relevant Inhibitors

55 Johnny Phan Kansas Development of Quinone-Promoted C-C Bond Oxidative Cleavage

56 Christianna Reedy Kansas CJ-15,208 Analogues: Their Permeability across Biological Barriers and Interaction with P-glycoprotein

57 Benjamin Richardson Illinois Towards the Development of Photoaffinity Probes for Non-covalent Activation of Nrf2

58 Loruhama M. Delgado Rivera Illinois Estrogen Receptor Ligand Photoprobes can Provide Mechanistic Information

of Biomolecular Interactions between the Estrogen Receptor and its Ligands

59 Alex Salyer Kansas Profiling the Immunological Activities of Toll-like Receptor-7 and -8 Agonistic Molecules

60 Jean Leandro dos Santos Minnesota Synthesis and Evaluation of 1,2,5-oxadiazole 2-oxide Derivatives Useful to Treat Sickle Cell Disease Symptoms

61 Pavel Y. Savechenkov Illinois Synthesis and Biological Evaluation of Novel Neurosteroid Photoaffinity Ligand

62 Rachel Saylor Kansas Salvinorin A Analogues for the Development of Addiction Therapies

63 Christopher Seiler Minnesota Epigenetic Regulation of Cytosine Methylation, Hydroxymethylation, Formylation and Carboxylation by Tet Proteins in Normal and Cancer Cells

64 Rachit Shah Minnesota Histidine Triad Nucleotide Binding Protein 1 (HINT1): Potential Target for the Treatment of Chronic Pain

65 Alexander Sherwood Kansas Modular Total Synthesis towards the Development of Salvinorin A Inspired Structures

66 Ernane Souza Iowa Synthesis of Dihydroxyanthracenones and Naphthopyranones through Houser and Stauton-Weinreb Annulations and Oxidative Coupling Reactions

67 Thomas Speltz Illinois Engineering Natural Functional Groups from Leucine and Isoleucine into “Stapling” Amino Acids

68 Nicholas Struntz Minnesota Catch and Release DNA Decoys: Capture and Photochemical Dissociation of Transcription Factors

69 Matthew Summerlin Illinois Synthesis and Biochemical Evaluation of IP6-biotin70 Nicholas Vance Iowa Caspase-7 Allostery Explored with Fragment-Based Drug Discovery

71 Brigitte Vanle Iowa Neuronal Toxicity due to Reactive Dopamine Metabolites and Fungicide Exposure

72 Shuai Wang Illinois Licochalcone A from Glycyrrhiza inflata Modulates P450 1B1-mediated Estrogen Oxidative Metabolism in MCF-10A Cells

73 Chris White Iowa Hepatocyte Targeted Endosomal Escape Agents

74 John Widen Minnesota Natural Product Inspired Cysteine Reactive Probes for Cancer Target Discovery

75 Andi Wisniewski Minnesota Identification of Potential BRDT Inhibitors by Fragment-based Screening Using Differential Scanning Fluorimetry

76 Huaping Zhang Kansas Withanolides from Physalis coztomatl

1 Inhibition of Scavenger Receptors Using Novel PEGylated Polylysine Peptides

Rondine Allen, Nicholas Baumhover, Jason Duskey and Kevin G. Rice

Division of Medicinal and Natural Products Chemistry, College of Pharmacy, The University of Iowa, Iowa City, IA

Scavenger receptors (SR) found on Kupffer and fenestrated endothelial cells of the recticuloendothelial system (RES) in liver are responsible for the swift capture and degradation of both viral1 and non viral gene delivery systems2. Administration of polyinosinic acid (Poly-I) and Poly(guanylic) acid (Poly G) has been shown to improve the gene transfer efficiency by blocking rapid metabolism in the liver. In the present study, we report the discovery of a new class of PEGylated polylysine peptides that are potent and safe SR inhibitors. PEGylated(30kDa)-Cys-Trp-Lys(N) (where N = 10, 15, 20, 25, and 30) were prepared by solid phase peptide synthesis and administered to mice i.v. with 125I-pGL3. Administration of 1 nmol of PEG-peptide with 1 Pg of 125I-pGL3 resulted in capture of 60% of the polyplex dose by the liver. Dose-escalation of the PEG-peptide revealed a dose-dependent decrease in 125I-pGL3 PEG-peptide polyplex capture by the liver, resulting from SR inhibition. A maximum dose of 80 nmols of PEG(30kDa)-Cys-Trp-Lys25 inhibited SR mediated uptake of 125I-pGL3 by the liver to 10%. The potency of PEG-peptide inhibition of SR was also dependent on the length of the Lys repeat, with Lys30=Lys25>Lys20>Lys15>Lys10. Application of delayed hydrodynamic (HD)-stimulation 1-hour after pGL3 polyplex delivery resulted in potent gene expression only when administering a SR inhibitory dose (80 nmol) of PEG-peptide. We believe that these PEG-peptides function by forming 30 nm albumin nanoparticles in the blood that bind to SRs. This new class of safe and potent SR inhibitors may have broad applications by improving both viral and non-viral delivery systems by blocking SR-mediated uptake in the liver. 1. Xu Z, Tian J, Smith JS, Byrnes AP. Clearance of adenovirus by Kupffer cells is mediated

by scavenger receptors, natural antibodies, and complement. J Virol 2008; 82: 11705-13. 2. Khargharia S, Baumhover NJ, Crowley ST, Duskey J, Rice KG. The uptake mechanism

of PEGylated DNA polyplexes by the liver influences gene expression Gene Ther 2014; 21, 1021-28.

2 Human Toll-like Receptor 8-Selective Agonistic Activities in

1-Alkyl-1H-benzimidazol-2-amines

Mallesh Beesu,1 Subbalakshmi S. Malladi,1 Lauren M. Fox,1 Cassandra D. Jones,1 Anshuman Dixit,2 and Sunil A. David1

1Department of Medicinal Chemistry, University of Kansas, Lawrence, KS 66047, USA. 2Department of Translational Research and Technology Development, Institute of Life Sciences,

Nalco Square, Bhubaneswar-751023, India.

Toll-like receptor (TLR)-8 agonists strongly induce the production of T helper 1-polarizing cytokines, and may therefore serve as promising candidate vaccine adjuvants, especially for the very young and the elderly. Earlier structure-based ligand design led to the identification of 3-pentyl-quinoline-2-amine as a novel, human TLR8-specific agonist. Comprehensive structure-activity relationships in ring-contracted 1-alkyl-1H-benzimidazol-2-amines were undertaken, and the best-in-class compound, 4-methyl-1-pentyl-1H-benzo[d]imidazol-2-amine, was found to be a pure TLR8 agonist, evoking strong proinflammatory cytokine and Type II interferon responses in human PBMCs, with no attendant CD69 upregulation in natural lymphocytic subsets. The 1-alkyl-1H-benzimidazol-2-amines represent a novel, alternate chemotype with pure TLR8-agonistic activities, and will likely prove useful not only in understanding TLR8 signaling, but also perhaps as a candidate vaccine adjuvant. TOC Graphic

3

Targeting Mycobacterium tuberculosis Biotin Protein Ligase (MtBPL): Synthesis and Evaluation of Nucleoside-based Bisubstrate Adenylation Inhibitors

Matthew R. Bockman,a Alvin S. Kalinda,a,b Divya Tawari,c Teresa de la Mora,a Barry Finzel,a Dirk Schnappinger,c Courtney C. Aldrich a,b,*

aDepartment of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455

bCenter for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, MN 55455, cDepartment of Microbiology and Immunology, Weill Cornell Medical College, New York, NY 10021

Mycobacterium tuberculosis (Mtb) is responsible for both latent and symptomatic tuberculosis (TB). TB remains the second leading cause of mortality among infectious diseases worldwide, primarily due to the emergence of multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains. To combat this pathogen, the development of new antitubercular agents is essential. Mycobacterial biotin protein ligase (MtBPL) regulates lipid metabolism in Mtb through the post-translational biotinylation of acyl coenzyme A carboxylases that catalyze the initial step in fatty acid biosynthesis. Additionally, MtBPL biotinylates pyruvate coenzyme A carboxylase, a gluconeogenic enzyme integral for lipid catabolism. We will present the synthesis as well as biochemical and structural characterization of a systematic series of bisubstrate biotin-nucleoside inhibitors with modifications to the ribofuranosyl ring. Several of these inhibitors including ribofuranosyl, 3′-deoxy, acyclo and morpholino analogs display sub-nanomolar inhibition of MtBPL as determined by isothermal titration calorimetry (ITC) and potent whole-cell activity against Mtb. While many analogs exhibit less than five-fold decrease in potency or are equipotent to the parent inhibitor containing a ribofuranose ring, certain modifications including the 2′-E-fluoro, 2′-E-azido, 2′-E-methyl and carbocycle completely abolish activity in whole-cell assays. The lack of correlation between biochemical and whole-cell activity suggests certain modifications severely impede intracellular accumulation.

4 DHB-Lysine: Developing Methods for Detection and Quantitation in Urine

Emily J. Boldry,† ‡ Srikanth Kotapati,† ‡ and Natalia Tretyakova†‡ The Masonic Cancer Center† and Department of Medicinal Chemistry‡,

University of Minnesota, Minneapolis, MN 55455

1,3-butadiene (BD) is a known human carcinogen commonly used in the rubber and plastics industries and present in motor vehicle exhaust and cigarette smoke.

BD is metabolically

activated to the following reactive species: 3,4-epoxy-1-butene (EB), hydroxymethylvinylketone (HMVK), 3,4-epoxy-1,2-butanediol (EBD), and 1,2,3,4- diepoxybutane (DEB). EB, HMVK, and EBD can be conjugated with glutathione and excreted in the urine as 2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene and 1-(N-acetyl-L-cystein-S-yl-)-1-hydroxybutene (together referred to as MHBMA), N-acetyl-S-(3,4-dihydroxbutyl)-L-cysteine (DHBMA), and 4-(N-acetyl-L-cystein-S-yl)-1,2,3-trihydroxybutane (THBMA), respectively, which can serve as biomarkers of BD exposure. Of the BD metabolites, DEB is the most genotoxic and mutagenic, but currently there are no DEB-specific urinary biomarkers to evaluate exposure to BD in humans.

In the present work, we have developed such a specific biomarker by focusing on Nε,Nε-(2,3-dihydroxybutan-1,4-diyl)-L-lysine (Nε,Nε-DHB-Lys), an exocyclic amino acid adduct formed via reaction of lysine with DEB. We have synthesized authentic Nε,Nε-DHB-Lys standard and its isotope labeled analog, Nε,Nε-DHB-Lys-d6. Using these standards, we have developed an HPLC-ESI+-MS/MS method for the detection and quantitation of this analyte in urine samples of smokers, nonsmokers, and occupationally exposed workers. Preliminary analysis has revealed the presence of DHB-Lys in urine samples from occupationally exposed workers ranging from 3-7 pmol/mL urine.

5 Nitric Oxide Regulates DNA Methylation via Direct Inhibition of TET Activity

Rhea Bovee, Vy Pham, Divya Vasudevan, Douglas Thomas Department of Medicinal Chemistry and Pharmacognosy,

College of Pharmacy, University of Illinois at Chicago, IL 60607

Nitric Oxide (NO·) is an epigenetic regulator and has an essential role in the progression of many forms of cancer, (specifically at higher levels of NO·) conveys the importance in both disease and non-disease states. Many proteins in the dioxygenase family play a key role in epigenetics. Dioxygenases are primarily responsible for demethylation reactions (histones, nucleic acid, etc.). Previous studies show alterations in methylation contributing to the pathogenesis of multiple disease states, specifically cancer. Dioxygenases commonly require two co-factors: Fe(II) and α-ketoglutarate for optimal function. Ten-eleven-translocation (TET) is in the methylcytosine dioxygenase family comprised of 3 proteins, their major role is cytosine demethylation, which is essential for expression of previously silenced genes. Current literature shows decreased TET activity in various cancer forms, resulting in decreased 5-hydroxymethylated DNA. The known relationship between Fe(II) and NO· in cellular function, along with the catalytic dependency of TETs on Fe(II), will determine the role of NO· regulation of TET function in disease pathogenesis and normal function. This will lead to potential therapeutics to reverse the decreased TET activity found in numerous cancers, to activate genes previously silenced by oncogenic factors. In order to understand the regulatory function of NO· specifically on TET proteins, we are performing biochemical analysis to determine at what level of protein synthesis (DNA, RNA, protein inhibition) can NO· regulate TET activity. We have shown decreased activity of TET proteins in the presence of NO·, hypothesized to be due to interactions of NO with the Fe(II) of the TET proteins catalytic pocket. With qPCR and western blot analysis we are able to demonstrate a tissue specific relationship between TET3 and NO·. These methods will allow us to determine the role NO· plays in regulating TET proteins from de-methylating anti-cancers genes, traditionally seen methylated in the presence of cancer. Reversing the methylation status in disease states to those similar in non-disease states will aid in development of new therapeutics for NO· driven cancers.

6

E-Lactam Conjugate Prodrugs of D-Cycloserine

Joseph A. Buonomo, Christopher D. Brown, Courtney C. Aldrich Department of Medicinal Chemistry, University of Minnesota

Weaver-Densford Hall, 308 SE Harvard St., Minneapolis, MN 55455

Drug-resistant tuberculosis (DR-TB), caused by Mycobacterium tuberculosis (Mtb), represents one of the greatest threats to global public health and new strategies and approaches will be required to tackle this mounting problem. D-Cycloserine (DCS) is highly effective for DR-TB, but its hallmark neurological side effects, including seizures and psychosis, have severely limited clinical use of this important antibiotic. In order to prevent these side-effects, we conceived of a prodrug strategy for DCS through conjugation to a E-lactam promoiety that would simultaneously prevent CNS penetration and result in selective release of DCS within mycobacteria and not within host tissues or serum. The selective release of DCS exploits the chromosomally-encoded extended spectrum beta-lactamase (BlaC) gene in Mtb. Hydrolysis of the E-lactam promoiety by BlaC leads to ejection of DCS via allylic carbamate intermediate, a well-known release mechanism (Figure 1). The key design criteria are: 1) the E-lactam promoiety should be devoid of intrinsic antibacterial activity to prevent disruption of commensal bacteria; 2) the conjugate should be stable and orally bioavailable; and 3) DCS should be selectively released by Mtb and not by the host or other commensal microorganisms.1,2 These three criteria are met in succession with E-lactam conjugates 1–3, which exploit the wealth of knowledge in E-lactam medicinal chemistry. Antibacterial activity is readily eliminated through modification at C7 through incorporation of the E-phenyacylamino moiety of 1–3. Improved chemical stability and oral bioavailability is accomplished through substitution at C2 with the cyclopropyl substituent in 2–3.1 Finally, E-lactamase selectivity can be tuned by installation of the bulky methoxy group at D-C7 as found in 3; a modification that is readily processed by BlaC, but is poorly tolerated by other bacterial E-lactamases.2 Herein, we describe the syntheses of E-lactam conjugates 1–3 and their preliminary biological characterization.

1 Xie, H.; Mire, J.; Kong, Y.; Chang, M.; Hassounah, H. A.; Thornton, C. N.; Sacchettini, J. C.; Cirillo, J. D.; Rao, J. Nat. Chem. 2012, 4, 802-809 2 Cheng, Y.; Xie, H.; Sule, P.; Hassounah, H.; Graviss, E. A.; Kong, Y.; Cirillo, J. D.; Rao, J. Angew. Chem. Int. Ed. 2014, 53, 9360-9364

Figure 1. A) DCS Conjugates and B) The Mechanism of DCS Release from Conjugates.

7 Unusual Withanolides from Physalis hispida (Waterf.) Cronquist

Cong-Mei Cao, Huaping Zhang, Robert J. Gallagher, and Barbara N. Timmermann*

Department of Medicinal Chemistry, University of Kansas, Lawrence, KS 66045

Withanolides are a group of modified C28 ergostane-type steroids with a C-22, C-26 δ-lactone side chain. They are distributed primarily within 25 genera of the Solanaceae, including Datura, Nicandra, Physalis, Salpichroa, and Withania. The genus Physalis has provided an abundant source of withanolides, including a series of these compounds from P. longifolia isolated by our laboratory. As part of a continuing research related to the discovery of novel withanolides from the Solanaceae, a phytochemical study on P. hispida (Waterf.) Cronquist was initiated. The species, commonly known as “prairie groundcherry”, is distributed throughout the central and midwestern United States. Reported herein are the isolation and structure elucidation of nine new withanolides (1−9), withahisolides A−I, as well as nine known compounds (10−18) from the aerial parts. This study represents the first phytochemical study of this species. The structures of 1−9 were elucidated through a variety of spectroscopic techniques, while the structures of 1 and 2 were confirmed by X-ray crystallographic analysis. Eight new withanolides (1-7, 9) were identified to possess an unusual six-membered ring D. Among them, compounds 1−3 are the first withanolides with non-aromatic six-membered ring D moieties. In addition, withanolide 8 represents a novel withanolide skeleton because of the absence of a C-13−C-17 bond within its steroidal nucleus.

1

8 Exploring CatSper Channel Openers and Binding Site Interactions:

Discovery of Steroidal Channel Blockers

Erick J. Carlson, Rawle Francis, Jon Hawkinson and Gunda I. Georg. Institute for Therapeutics Discovery and Development, Department of Medicinal Chemistry,

College of Pharmacy, University of Minnesota, Minneapolis, MN 55414 The cation channel of sperm (CatSper) is the key ion channel involved in Ca2+ regulation in sperm. Composed of 4 subunits, CatSper is expressed exclusively in the testes and disruption of any subunit leads to infertility making CatSper a promising target for potential male contraception. It is known that CatSper is opened by high extracellular potassium concentrations, the steroid hormone progesterone, and prostaglandin E1. The focus of this project is to explore the SAR of steroids acting at the CatSper channel. Hydroxylated and reduced analogues of progesterone, steroids of other classes, and clinically used progestins have been purchased and tested in our FLIPR assay utilizing the calcium-specific dye Fluo-4-AM. From this work, insight into the SAR around the progesterone scaffold has been gleaned in addition to confirming the activity at CatSper of the natural product Sirenin. Furthermore, the first steroidal blockers of the CatSper channel have been discovered in medroxyprogesterone acetate (MPA) and aldosterone. Finally, the mode of inhibition for these steroidal blockers, along with two published T-type calcium channel blockers, mibefradil and ML-218, has been investigated. Whereas MPA reduces the apparent potency of progesterone consistent with competition for the same binding site, mibefradil produces an insurmountable inhibition of progesterone-induced activation, consistent with a noncompetitive interaction. In addition to clarifying the SAR of steroids and confirming the activity of sirenin as activators of Catsper, we have identified steroidal Catsper blockers and have begun to elucidate the interactions between the binding sites on the ion channel.

9 A High-Throughput Screen for the Identification of Novel Innate Immune Stimuli

Caruso Giuseppe, Subbalakshmi S. Malladi, Karishma K. Khetani, Lauren M. Fox, Alex C. D. Salyer and Sunil A. David.

Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045.

Toll-Like Receptors (TLRs) play a critical role in the early innate immune responses to invading pathogens by sensing microorganisms, and are also involved in sensing endogenous danger signals. Ten human TLRs have been characterized, from TLR-1 to TLR-10. Ligand binding to TLRs result in activation of cellular signaling pathways that regulate expression of genes involved in inflammation and immunity. In order to identify novel innate immune-stimulatory molecules, we utilized a rigorously-standardized multiplexed reporter gene assay with a view to identifying, in a single pass, compounds that would engage any of the major innate immune receptors. We screened a total of 146,432 compounds in our first phase of high-throughput screening (HTS), which was designed to report on TLR-2, -3, -4, -5, -7, -8, -9, NOD-1, or NOD-2 signals. Performance metrics of the assay were carefully monitored throughout the screen, and Z' scores were consistently above 0.62 for each ligand. Preliminary results of the HTS screen, and of ongoing deconvolution studies will be discussed.

10 Revised Synthesis of CXL037 and its use as a Photo Reactive Probe in

Identification of Cellular Targets

Denise Casemore, and Chengguo Xing Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455

A library of CXL compounds have been synthesized by the Xing lab and have shown to be more cytotoxic towards drug-resistant cancer cell lines than their parental cell lines. Photo reactive CXL compounds ((-)-CXL037 and (+)-CXL037) have been developed as probes to identify the cellular targets of these compounds. A racemic mixture of CXL055, the precursor to probe CXL037, was synthesized using a revised Suzuki reaction. The reaction allows for the in situ formation of borylated aniline that can cross couple with bromocoumarin using XPhos Pd G2 as a catalyst, eliminates the need for reduction of a nitro group in the original synthetic scheme, and increases the overall yield. CXL055 enantiomers were then separated by chiral HPLC and subsequently reacted with TMS-azide to form enantiomers of the photo reactive probe CXL037. (-)-CXL037 was more cytotoxic in HL60 and HL60/MX cells than its enantiomer (+)-CXL037. This presentation will also discuss the preliminary application of these probes in identification of the cellular targets in cancer cells.

CXL037

11 Intramolecular C-Vinylation to Novel Triazole-Fused Vinyl Sultams

Andie Jo Cassity, Jung Ho Jun, Naeem Asad, Nicole Marie Windmon, Kasabi Namatama Chinonge, Anna J Diepenbrock and Paul R. Hanson

Department of Chemistry, University of Kansas Lawrence, KS 66045-7582 Synthesis of novel α-exo-methylene-containing triazole-fused G-sultams via utilization of intramolecular C-vinylation of triazoles is reported. The use of intramolecular C-vinylation (and C-arylation) on triazoles is relatively under explored. In this regard, we are investigating a versatile “Click-Click Cyclize” method for rapid assembly of an array of diverse triazole-fused vinyl sultams. Several Pd-catalysts have been screened to optimize the titled intramolecular C-vinylation reaction. Taken collectively, this facile method has been developed for generating novel electrophilic probes for screening.

12

An Improved Schmidt Reaction for the Synthesis of N-Hydroxyalkyl Lactams

Manwika Charaschanya, Hashim F. Motiwala, and Jeffrey Aubé Department of Medicinal Chemistry, University of Kansas

Lawrence, KS, 66045 The Schmidt reaction is commonly used in organic chemistry for the preparation of nitrogenous compounds such as amides, lactams and tetrazoles from carbonyl precursors. The Aubé lab has continuously investigated various Schmidt-type reactions, but the development of such transformations has had a common limitation. Specifically, the Schmidt reaction requires the use of super-stoichiometric amounts of acid catalyst and harsh reaction conditions that renders this transformation unsuitable for acid-sensitive substrates and scale-up. Recently, Motiwala et al. disclosed that a strong hydrogen-bond-donating solvent, hexafluoro-2-propanol (HFIP), promoted catalysis in an intramolecular Schmidt reaction. In this work, the intermolecular Schmidt reaction of ketones and azidoalcohols for the synthesis of N-hydroxyalkyl lactams were optimized in the presence of HFIP, which led to a mild, efficient and improved protocol as compared to previous work. A set of diverse ketones was subjected to the optimized conditions, and afforded N-hydroxyalkyl lactams in good to excellent yields with using only one equivalent of triflic acid under shorter reaction periods than observed previously.

13 Silvestrol Induces Autophagy and Apoptosis in Human Melanoma Cells

Wei-Lun Chen, Joanna E. Burdette and Steven M. Swanson

Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago Chicago, IL, 60612

Silvestrol is a cyclopenta[b]benzofuran that was isolated from the fruits and twigs of Aglaia silvestris, which is indigenous to the island states of Southeast Asia. Previous testing of silvestrol revealed that it is a potent inhibitor of protein synthesis and has cytotoxic activity similar to or more potent than many FDA approved anticancer agents. Silvestrol is currently under preclinical development at the National Institutes of Health Experimental Therapeutic (NExT) program. The purpose of the current study was to determine if inhibition of protein synthesis caused by silvestrol triggers autophagy and apoptosis in solid tumors. By 24h a clear decrease in cyclin B and cyclin D expression was observed in silvestrol-treated cells relative to control. In addition, silvestrol blocks progression through the cell cycle at the G2-phase. Silvestrol treatment also induced caspase-3 activation and apoptotic cell death in a time- and dose-dependent manner. Next, DAPI staining of nuclear chromatin showed nucleosomal fragments. Annexin V staining also showed an increase in apoptotic cells after silvestrol treatment. Furthermore, both silvestrol and SAHA enhanced autophagosome formation in MDA-MB-435 cells. Quantitation of the acidic vacuoles measured by flow cytometry further confirmed these results. MDA-MB-435 cells responded to silvestrol treatment with accumulation of LC3-II and dose-dependent p62 degradation. However, bafilomycin A, an autophagy inhibitor, resulted in the accumulation of LC3 in cells treated with silvestrol. Silvestrol represents a natural product scaffold with the potential for the study of autophagy and apoptosis mechanisms in cancer cells. It also highlights the direction of future drug development.

14

Design and Synthesis of Stabilized Pironetin Analogs for Resistant Ovarian Cancers

Sara K. Coulup, David S. Huang, and Gunda I. Georg Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN, 55455

The natural product pironetin displays potent cytotoxic activity against ovarian cancer cells both sensitive and resistant to first-line chemotherapeutics such as paclitaxel and cisplatin.1 Literature suggests that pironetin covalently binds with α-tubulin, whereas all tubulin-binding agents currently approved by the FDA target β-tubulin.2 α-tubulin is therefore an attractive alternative drug target that would address the critical need for new treatments for drug-resistant ovarian cancers. Despite the potent in vitro activity pironetin was only marginally effective in the single reported in vivo study, which resulted in severe weight loss in the mice, indicating poor pharmacokinetic/pharmacodynamic (PK/PD) properties as well as off target toxicities.3 We have demonstrated that pironetin has a very short half-life in liver microsomes and hypothesized that rapid metabolism could lead to a toxic and less-active agent. Using liver microsomes and LC-MS/MS, we identified the major metabolite and confirmed the identity through semi-synthesis. We are synthesizing pironetin analogs that block metabolism at this site to improve upon the PK/PD properties. These findings and the potential of metabolically stabilized pironetin analogs as novel anti-tubulin agents for resistant ovarian cancers shall be reported herein.

1. Marco, J. A.; Garcia-Pla, J.; Carda, M.; Murga, J.; Falomir, E.; Trigili, C.; Notararigo, S.; Diaz, J. F.; Barasoain, I. Design and synthesis of pironetin analogues with simplified structure and study of their interactions with microtubules. European Journal of Medicinal Chemistry 2011, 46, 1630-1637. 2. Usui, T.; Watanabe, H.; Nakayama, H.; Tada, Y.; Kanoh, N.; Kondoh, M.; Asao, T.; Takio, K.; Watanabe, H.; Nishikawa, K.; Kitahara, T.; Osada, H. The anticancer natural product pironetin selectively targets Lys352 of alpha-tubulin. Chemistry & Biology 2004, 11, 799-806. 3. Kondoh, M.; Usui, T.; Kobayashi, S.; Tsuchiya, K.; Nishikawa, K.; Nishikiori, T.; Mayumi, T.; Osada, H. Cell cycle arrest and antitumor activity of pironetin and its derivatives. Cancer Letters 1998, 126, 29-32.

15 Novel Inhibitors of HNE, CatG and Pr3 and Their Evaluation in vitro

Ioana Craciun and Robert J. Kerns* Department of Pharmaceutical Sciences and Experimental Therapeutics, Division of Medicinal

and Natural Products Chemistry, College of Pharmacy, The University of Iowa, Iowa City, Iowa

Neutrophil serine proteases (NSPs) play an important role in the innate immune system, however when the balance between NSPs and their endogenous protease inhibitors (PIs) is disrupted, they also play a critical role in the pathogenesis of chronic inflammatory lung diseases. Excessive release of NSPs such as human neutrophil elastase (HNE), proteinase 3 (Pr3) and cathepsin G (CatG) leads to destruction of the lung matrix and continued propagation of acute inflammation. Under normal conditions, PIs counteract these effects by inactivating NSPs. However, in chronic inflammatory lung diseases there are insufficient amounts of PIs to mitigate damage. Therapeutic strategies are needed to modulate excessive NSP activity in chronic inflammatory lung diseases. The Kerns laboratory previously demonstrated that heparin derivatives substituted with structurally unique aromatic residues bind with high affinity and selectivity to select Glycosaminoglycan-binding proteins, including NE and CatG. In the work presented here, using a chromogenic peptidolytic assay, we evaluated the members of a recently synthesized panel of N-arylacyl O-sulfonated aminoglycosides for their ability to inhibit NE, CatG and Pr3. We identified O-sulfonated N-carbobenzyloxy Kanamycin as a novel and structurally unique inhibitor of all three neutrophil serine proteases and characterized its type of inhibition with respect to CatG. Finally, we established a high-throughput cell based assay to assess the ability of these aminoglycoside derivatives to mitigate protease induced cell detachment. This assay can be used to screen multiple derivatives in a high-through put manner against each protease individually as well as protease cocktails.

16

Pd-Catalyzed Decarboxylation Enables Direct Access to α,α-Difluoroketones

Ming-Hsiu Yang, Douglas L. Orsi, Niusha Sharifi, and Ryan A. Altman* Department of Medicinal Chemistry, University of Kansas

Lawrence, Kansas 66045

α,α-Difluoroketones possess unique physicochemical properties, which can inhibit serine and aspartyl proteases. Additionally, α,α-difluoroketones can enhance bioactivities for alternate therapeutic targets, and can serve as intermediates for further functionalization. However, current preparation of α,α-difluoroketones either require multi-step transformations or harsh conditions that restrict use of this substructure for medicinal applications. Thus, new strategies are required to access these important substructures.

Decarboxylative coupling transformations provide powerful methods for the construction of C—C bonds under mild conditions. To access the α-allyl-α,α-difluoroketone substructure, complementary Pd-catalyzed decarboxylative allylation reactions were developed to provide linear and branched products. For these orthogonal processes, the regioselectivity was uniquely controlled by fluorination of the substrate and the structure of ligand. Moreover, this strategy enabled access to α,α-difluoroketones at the late-stage of a synthetic sequence. Further refinement of this reaction generated α-benzyl-α,α-difluoroketones, thus broadening the scope of accessible products.

These decarboxylative strategies should be useful for accessing functionalized α,α-difluoroketone-based probes that would otherwise be challenging to prepare.

17 Synthesis and Biological Evaluation of Hsp90 C-Terminal Inhibitors for the

Treatment of Cancer

Rachel Davis, Brian S. J. Blagg Department of Medicinal Chemistry, The University of Kansas

Lawrence, KS 66045

The 90 kDa heat shock protein (Hsp90) is a molecular chaperone responsible for the maturation of nascent polypeptides and rematuration of denatured proteins. Because Hsp90 client proteins are represented in all six hallmarks of cancer, inhibition of Hsp90 function results in the simultaneous disruption of multiple oncogenic pathways, making it an attractive therapeutic target. Hsp90 exists as a homodimer comprised of an N-terminal ATP-binding domain, a middle protein interaction domain, and a C-terminal dimerization domain. Inhibitors targeting the Hsp90 N-terminus induce both the pro-survival heat-shock response and client protein degradation at similar concentrations, resulting in an overall cytostatic effect. Benzamide-containing Hsp90 C-terminal inhibitors do not induce the heat-shock response and therefore manifest cytotoxic activity. In 2000, novobiocin was identified as the first Hsp90 C-terminal inhibitor. Subsequent structure-activity relationship studies with this natural product revealed the piperidine ring as an effective surrogate for the synthetically complex noviose sugar, leading to compounds with enhanced efficacy. Modifications on the piperidine ring will form additional binding interactions by projecting substituents into the proposed phosphate-binding region of the C-terminal ATP-binding site. The syntheses of these analogs and their anti-proliferative activities in Hsp90-dependent cancer cell lines will be presented.

18

Structural and Dynamic Insight into the Idiosyncratic Nature of Ligand-associated Conformational Changes of Glutamate Racemase at the Atomistic Level

Sondra Dean, Katie Whalen, and M. Ashley Spies

Division of Medicinal and Natural Products Chemistry, University of Iowa Iowa City, IA 52246

Glutamate racemase (GR) is a bacterial enzyme responsible for the stereoinversion of L-glutamate to D-glutamate, a vital component of the peptidoglycan layer in both Gram-positive and Gram-negative bacteria. This enzyme has been identified as a promising new antibacterial drug target as it has been proven to be essential to bacterial cell viability, several studies have indicated that inhibition suppresses bacterial cell growth, D-glutamate is not readily available in the environment, and there is no human isozyme. However, this is a highly flexible enzyme, making it a difficult target from a structure-based drug design and discovery perspective. Previous studies have suggested that GR forms distinct complexes with various competitive inhibitors, which has significant effects on the quality of the GR-ligand binding interaction. In this study, a genetically encoded non-natural fluorescent amino acid L-(7-hydroxycoumarin-4-yl) ethylglycine is incorporated into a dynamic region (Tyr53) remote from the active site, which is known to experience changes upon ligand binding and unbinding. The resulting GRY53/7HC enzyme was used to acquire insight into the idiosyncratic nature of ligand-associated conformational changes in GR. The binding of two distinct competitive inhibitor chemotypes, croconate and glucuronate, to GRY53/7HC was studied. Binding of croconate yielded fluorescent quenching while binding of glucuronate yielded fluorescent enhancement, reflecting GR conformational changes occurring upon binding of these ligands. A computational workflow was used to elucidate the ligand-associated conformational phenomena indicated experimentally by this differential fluorescence pattern. This computational workflow proved to be predictive of changes seen in the experimental system.

19 Effect of Fluoroquinolone N-1 Aryl Substituents on Bacterial Type-II Topoisomerase

Inhibition and DNA Binding

Justine Delgado, Chaitanya Kulkarni, Tyrell R. Towle, Robert J. Kerns* Division of Medicinal and Natural Products Chemistry, College of Pharmacy,

University of Iowa, Iowa City, IA 52242 Fluoroquinolones are small molecule broad spectrum antibiotics that target bacterial type-II topoisomerases, DNA gyrase and topoisomerase IV. These enzymes are responsible for maintaining the topology of DNA during replication and transcription. This is accomplished through an ATP driven process that involves relaxing the double helix, introducing supercoils and relieving torsional strain. Fluoroquinolones exert their activity through formation of a ternary complex with DNA and topoisomerase IV and blocking religation of DNA The issue of fluoroquinolone resistance continues to rise as the number of resistant bacterial strains continues to grow every year. One of the main mechanisms of resistance observed is target-mediated resistance. An essential interaction between the fluoroquinolone and the enzyme is through formation of a magnesium-water bridge. A single key mutation in the enzyme can disrupt this interaction leading to resistance. In order to overcome this mechanism of resistance, we have begun probing for novel binding contacts to alleviate the need of the magnesium-water bridge interaction. Novel fluoroquinolones have been developed to have potential new binding interactions within the ternary complex. The ability of these fluoroquinolones to bind DNA, inhibit gyrase and topoisomerase IV activity are described here.

20

Dual Melanocortin 3 and Melanocortin 4 Receptor Tetrapeptide Antagonists

Skye R. Doering,1 Aleksandar Todorovic,2 and Carrie Haskell-Luevano1,2

1Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455; 2Department of Medicinal Chemistry, University of Florida, Gainesville, Florida 32610

Molecular probes with distinct pharmacological profiles are required to identify the in vivo roles of receptors. The melanocortin 3 and 4 receptors (MC3R and MC4R) are both expressed in the central nervous system and are postulated to be involved in maintaining energy homeostasis. An unmet need in the field is the development of selective and potent MC3R competitive antagonists which lack partial agonist activity. We identified a clean MC3R antagonist, Ac-Trp-(pI)DPhe-Arg-Trp-NH2

(Compound 1), via a double substitution strategy on the core melanocortin signaling sequence His-Phe-Arg-Trp. We hypothesized that a single substitution on the fourth

position could alter the pharmacological profile yet retain the unique antagonist profile at the MC3R. We identified two additional tetrapeptides displaying the desired pharmacological profile with modest potency (Ki < 2,000 nM) at the MC3R with additional antagonist activity at the MC4R (Ki < 5 nM); in addition, we identified potent (Ki < 80 nM) and selective antagonists for the MC4R versus the MC3R (> 100-fold). This work was supported by NIH grant R01DK091906 and published in ACS Med. Chem. Lett. 2015, 6, 123−127.

Schild Analysis at Mouse Melanocortin 3 and 4 Receptors

-13 -12 -11 -10 -9 -8 -7 -6 -5 -40.00

0.25

0.50

0.75

1.00

1.25

mMC3R

Log Peptide Concentration (M)

Act

ivity

-13 -12 -11 -10 -9 -8 -7 -6 -5 -40.00

0.25

0.50

0.75

1.00

1.25

MTIIMTII + 10k nM 1MTII + 5k nM 1MTII + 1k nM 1MTII + 500 nM 1Compound 1

mMC4R

Log Peptide Concentration (M)

Act

ivity

21 Synthesis and Kinetic Characterization of Mechanism-based Inhibitors of BioA

Carter Eiden^, Courtney C. Aldrich^, and John D. Lipscomb*.

^Department of Medicinal Chemistry, University of Minnesota *Department of Biochemistry, Molecular Biology, and Biophysics,

University of Minnesota Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains the second leading cause of mortality among infectious diseases worldwide. The emergence of multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains necessitates the development of new antibiotics, ideally with novel mechanisms of action. Mechanism-based inhibitor 1 has been shown to kill Mtb in biotin-deprived conditions through inactivation of BioA, a PLP dependent enzyme in the biotin biosynthetic pathway.1 The rational optimization of mechanism-based inhibitors is challenging since standard equilibrium dissociation constants cannot be used due to the irreversible nature of inhibition. Rather, one must optimize microscopic rate constants of the individual steps involved in ageing the enzyme. The inactivation of BioA by 1 is a four step process, beginning with binding of 1 to the PLP and continuing with three more transformations before an irreversible PLP adduct is formed. Each step shows a unique absorbance signature, allowing us to investigate the kinetics using stopped-flow measurements. Preliminary analysis

suggests the kinetic bottleneck occurs within the second step, suggesting that a new warhead should be designed that can be more easily deprotonated by the lysine of the enzyme. Using the previous information, two new inhibitors that lower the pKa of the alpha proton were designed (2 and 3). The synthesis of each of these is described, with the key step in each synthesis the addition of a lithium acetylide to a Weinreb amide followed by cyclization after quench. Both inhibitors have been shown to be active against BioA.

1 Shi, C.; Geders, T. W.; Park, S. W.; Wilson, D. J.; Boshoff, H. I.; Abayomi, O.; Barry, C. E.; Schnappinger, D.; Finzel, B. C.; and Aldrich, C. C. J. Am. Chem. Soc. 2011, 133, 18194-18201.

Figure 1: Mechanism-Based Inhbitors of BioA

Figure 2: Intermediates in Mechanism-Based Inhibition of BioA by 1

22 Development of High-Load, Immobilized Si-ROMP, Co/C Magnetic

Reagents/Scavengers and Ligands

Saqib Faisal,a Pradip K. Maity,a Agnes Brandhofer,b Patrick C. Kearney,c Qin Zang, a Diana S. Stoianova,d Oliver Reiser,b and Paul R. Hansona*

aDepartment of Chemistry, University of Kansas, Lawrence, KS, 66045. The University of Kansas Center for Chemical Methodologies and Library Development

(KU-CMLD), Lawrence, KS 66047. bInstitute of Organic Chemistry, University of Regensburg, Regensburg, Germany.

cHD Sciences, Lawrence KS 66047, dMateria, Inc., Pasadena, CA 91107.

The development of new ROMP-derived oligomeric soluble/silica/magnetic phosphorus and sulfur based reagents for application in purification-free diversification protocols is reported. Hybrid Si-ROMP benzylic and heterocyclic phosphates and their corresponding derivatives were successfully synthesized as free flowing solids for efficient benzylation and triazolation. Building on the successful development of these ROMP-derived soluble and silica reagents/scavengers, we have further advanced them to supported magnetic Co/C nanoparticles utilizing surface-initiated ROM polymerization. Further developments of new hybrid magnetic reagents are in progress for their utility in synthetic transformations, facilitated synthesis of small molecules, applications in parallel synthesis and potentially automated technologies.

23 Hsp90 C-Terminal Inhibitors that Manifest Neuroprotective Activity

Leah Forsberg1, Sean Emery2, Mercy Anyika1, Rick T. Dobrowsky2, Brian S. J. Blagg1

Department of Medicinal Chemistry1, Department of Pharmacology and Toxicology2, The University of Kansas, Lawrence, KS 66045

Novobiocin, a naturally occurring antimicrobial agent that inhibits DNA gyrase also binds the C-terminus of Hsp90, and exhibits anti-proliferative effects. Through structural changes, the biological effect of novobiocin binding can be modified. Replacement of the benzamide side chain of novobiocin with an acetamide results in neuroprotective activity. A small molecule inhibitor of Hsp90, KU-32, is based on novobiocin and contains the acetamide side chain, which results in neuroprotective activity. In order to further improve the affinity and efficacy of KU-32, molecular modeling studies were investigated. A homology model was used to identify potential key residues that may be responsible and important for inhibitor binding to the C-terminus. The coumarin core of KU-32 was substituted with a biaryl ring system to improve exploration of the binding pocket. A second generation of KU-32 novologues containing this new biaryl ring system was synthesized, and one of the compounds, KU-596, was found to exhibit similar or better activity to KU-32. KU-596 was used as a lead compound for the generation of a new library of small molecule inhibitors, in which modifications were designed to gain additional interactions with the binding pocket. These compounds were synthesized and evaluated. The results from these studies will be presented.

24

Synthesis and Evaluation of Fluorescent Probes of Prostate Specific Membrane Antigen (PSMA)

Gavin Zhe Gao, Chamani Perera, and Blake R. Peterson

Department of Medicinal Chemistry, University of Kansas Lawrence, Kansas 66045

In this year, over two hundred thousand new cases of prostate cancer will be diagnosed in the United States, and approximately 30,000 men will die from this disease. One in seven men will be diagnosed with prostate cancer during his lifetime, and this disease represents the second leading cause of cancer death in American men. Five to ten percent of men diagnosed with prostate cancer have advanced disease for which there is no cure. To create novel targeted agents with the potential to selectively accumulate in prostate cancer cells, we are investigating fluorescent probes that bind prostate-specific membrane antigen (PSMA). This folate hydrolase is commonly overexpressed on the surface of prostate cancer cells and is under intense investigation as a target for delivery of cytotoxic and imaging agents in prostate cancer. These probes include a PSMA-binding DUPA moiety linked through peptide-based spacers to fluorescent small molecules. We describe here the use of solid-phase synthesis to prepare these fluorescent probes, the affinity of these probes for PSMA expressed on LNCaP prostate cancer cells, and the use of confocal microscopy to examine their subcellular distribution in living cells. As a novel therapeutic strategy, we are also investigating compounds designed to exhibit synergistic anticancer activity by targeting PSMA, selectively disrupting endosomes of prostate cancer cells, and triggering the release of cytotoxins delivered into endosomes of these cells. This strategy may allow the design of better agents for the treatment of advanced prostate cancer when traditional chemotherapy is ineffective.

25 Red Clover and Glycyrrhiza inflata Display ERbeta Selectivity, Suggesting Better Safety

Profile for Women’s Health Sarah E. Green, Atieh Hajirahimkhan, Charlotte Simmler, Shao-Nong Chen, Guido Pauli, Birgit

Dietz, and Judy L. Bolton, UIC/NIH Center for Botanical Dietary Supplements Research,

Dept. of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago,

Chicago IL, 60612, USA Hormone therapy (HT) used in the treatment of menopausal symptoms has been associated with an increased incidence of hormone dependent cancers. Therefore, many women turned to botanical dietary supplements. Previous studies have shown that estrogen receptor alpha (ERα) activation initiates cell proliferation, potentially leading to the development of cancer. It is believed that ERE agonists do not initiate cell proliferation suggesting a better safety profile. The primary goal of this study was to compare the ERD and ERE activities of popular botanicals such as: hops (Humulus lupulus), red clover (Trifolium pratense), and licorice (Glycyrrhiza glabra (GG), G. uralensis (GU), G. inflata (GI)), along with their respective active compounds, 8-prenylnaringenin, genistein, liquiritigenin(LigF)/isoliquiritigenin (LigC) [GG,GU, GI], and licochalcone A (LicA) [GI]. In ERD Ishikawa endometrial cells, hops and red clover induced estrogen responsive alkaline phosphatase (AP) activity and act as full ERα agonists. The three licorice species also induced AP activity; however, they exhibited partial agonist effects with GI being the most potent. Similarly,8-PN showed the highest ERα potency and full agonist activity followed by genistein, LigF/LigC,and LicA. Among the extracts, red clover and GI showed 10 times better potency in the ERE-luciferase assay in ERE MDA-MB-231/B41 cells compared to ERα cells. While LigF, and LicA were partial ERβ agonists, 8-PN had no ERβ activity. Genistein is the most potent ERβ phytoestrogen and is a full agonist. These data suggest that while hops and 8-PN, have highly potent ERα effects, licorice and its compounds have partial effects for ERα and ERβ. The most potent licorice species, GI, displayed selectivity for ERβ warranting further studies to determine the presence of an unknown ERβ agonist. The ERβ selectivity and the potency of genistein in red clover suggest that red clover may be a relatively safe menopausal remedy. NIH Grant P50 AT00155

26 Identification, Characterization, and Quantification of DNA-protein Cross-linking by

Cyclophosphamide Metabolite Phosphoramide Mustard

Arnold Groehler IV and Natalia Tretyakova Department of Medicinal Chemistry, University of Minnesota

Minneapolis, Minnesota 55455 Cyclophosphamide (CPA) is a nitrogen mustard agent commonly used to treat lymphomas, leukemias, brain cancer, breast cancer, and other solid tumors. Cytochrome P450 monooxygenase mediated bioactivation of CPA yields 4-hydroxy-cyclophosphamide, which spontaneously breaks down to the active metabolite phosphoramide mustard (PM). PM is a bis-electrophile capable of cross-linking cellular biomolecules, yielding DNA-DNA and DNA-protein crosslinks (DPCs). Due to their bulky nature, DPCs can interfere with crucial biological processes such as chromatin folding, DNA replication, and transcription, contributing to the biological effects of CPA. In this present work, HPLC-ESI+-MS/MS analysis was employed to characterize PM-induced DNA-protein cross-linking in human fibrosarcoma (HT1080) cells. Analysis of proteolytic digests has detected N-[2-[cysteinyl]ethyl]-N-[2-(guan-7-yl)ethyl]methylamine (Cys-NOR-N7G) conjugates, indicating that PM forms DPCs between cysteine thiols of proteins and the N-7 position of guanine. Isotope dilution ESI+-MS/MS revealed a concentration-dependent increase in Cys-NOR-N7G in HT1080 cells treated with 0 – 500 µM PM. To identify the DNA repair pathways involved in DPC removal, HT1080 cells, nucleotide excision repair-deficient XPA cells, and homologous recombination-deficient PD20 cells were treated with 250 µM PM and incubated an additional 0 – 24 hours. XPA cells showed no decrease in Cys-NOR-N7G over 8 hours, indicating nucleotide excision repair is involved in removing DPCs.

27

Analysis of Selective Estrogen Mimics (SEMs) in a 3D Cell Model of Tamoxifen Resistance

Lauren M. Gutgesell, Hitisha K. Patel, Rui Xiong and Gregory R.J. Thatcher. Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago

Resistance to tamoxifen, the standard of care for women with estrogen receptor positive (ER+) breast cancer, occurs in about 30-50% of patients. This underlines the need for superior therapeutic options for treatment of these patients. In this study, two tamoxifen resistant cell models, MCF-7:5C and T47D: PKCα, which mimic the regression shown by estradiol in breast cancer in the clinic were used to analyze Selective Estrogen Mimics (SEMs) in 2D and 3D culture. Previously, a matrigel colony formation assay was used to mimic the in-vivo environment of cells and assess SEMs prior to their evaluation in tamoxifen-resistant xenograft mice models. In an effort to increase throughput and mimic the in-vivo environment more closely, we describe the use of a spheroid assay to assess SEMs in 3D culture. To develop the assay, tamoxifen resistant breast cancer cells were plated in two types of plates: the hanging drop plates and the corning ultra-low attachment spheroid plates. Though both of these plates successfully formed spheroids, the corning ultra-low attachment plates were superior due to the ease of handling and assessing the viability of spheroids. The assay was further developed and it was found that both estradiol and SEMs show inhibition of spheroid formation similar to the inhibition of colony formation seen in matrigel. This spheroid assay can now be used to screen and evaluate the SEMs and to understand their mechanism of action in tamoxifen-resistant breast cancer.

28 In vitro and in vivo Evaluation of NAD(P)H:quinone Oxidoreductase 1 (NQO1)

Induction by Licorice Species Used in Botanical Dietary Supplements for Women’s Health

Atieh Hajirahimkhan, Charlotte Simmler, Huali Dong, Dan Lantvit, Guannan Li, Shao-Nong

Chen, Dejan Nikolic, Guido F. Pauli, Richard B. van Breemen, Birgit M. Dietz, Judy L. Bolton*

UIC/NIH Center for Botanical Dietary Supplements Research, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago

With the Women’s health initiative suggesting an increased cancer risk associated with hormone therapy, menopausal women have turned to botanical dietary supplements such as hops (Humulus lupulus) and licorice (Glycyrrhiza spec.) for the alleviation of menopausal symptoms. We have previously shown that hops and its Michael acceptor xanthohumol induce the detoxification enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) in vitro and in vivo. As licorice species contain electrophilic compounds, our current study was designed to evaluate the role of Pharmacopeia approved, chemically distinct licorice species (Glycyrrhiza glabra, GG; Glycyrrhiza uralensis, GU; Glycyrrhiza inflata, GI) and their Michael acceptor isoliquiritigenin (LigC) and the GI marker chalcone, licochalcone A (LicA) in modulating NQO1 activity. GG, GU, and GI as well as LigC and LicA induced NQO1 dose-dependently in murine hepatoma hepa1c1c7 cells.with GI and LigC having the highest efficacy. Induction of antioxidant response element (ARE)-luciferase in human hepatoma (Hep-G2-ARE-C8) cells confirmed these data and suggested the involvement of Keap1-Nrf2 signaling pathway. However; GG and GU as well as LigC were more active than GI and LicA in inducing NQO1 protein in non-tumorigenic breast epithelial MCF-10A cells. NQO1 activity was weakly induced in the mammary tissue of the female Sprague-Dawley rats treated with GG. This observation correlated well with the chemical profile of this extract with higher levels of LigC and its precursors compared to GU. LigC did not induce NQO1 in vivo most likely due to its conversion to the flavonone, liquiritigenin. These data suggest that GG might have potential chemopreventive effects and emphasize on the importance of simultaneous chemical and biological characterization and standardization of the licorice species prior to using them in dietary supplements. Supported by P50 AT00155 provided by ODS and NCCAM.

29 Having a Complex: Implications on HDAC-Ligand Interactions

Thomas Hanigan, Jonna Frasor, Pavel Petukhov

Department of Medicinal Chemistry, University of Illinois at Chicago

Class I HDAC catalytic activity is extensively regulated in vivo through the formation of co-repressor complexes and post translational modification (PTM). We hypothesized that these changes in catalytic activity likely arise due to alteration of the active site of these proteins, which could ultimately affect the binding of small molecule inhibitors or other endogenous ligands. To determine if protein-protein interactions or PTMs could affect ligand binding in a cellular context, we developed a Photoreactive Hydroxamate baseD probe (PHD-probe) that can bind and covalently modify target HDACs, as well as conjugate to a fluorescent substrate for visualization. We show that the PHD-probe is a potent pan-inhibitor of all recombinant class I HDACs, with modest selectivity for recombinant HDAC1, and can bind and specifically label all recombinant class I HDACs. In MCF-7 breast cancer cell lysates, the PHD-probe retains its ability to bind and label all class I HDACs. Despite pan-HDAC activity and labeling efficacy with recombinant HDACs and MCF-7 cell lysates, the PHD-probe binds and differentially labels select nuclear class I HDACs in MCF-7, MCF10A, and BT474 cells in a cell type dependent manner. This is the first experimental evidence that shows the binding of ligands to class I HDACs is cell type dependent and suggests that the network of HDAC protein-protein interactions are responsible for this cell type dependent ligand binding because the PHD-Probe could label all class I HDACs in cell lysates where many of these protein-protein interactions would be disrupted. Taken together, this study suggests a model where HDACs form cell type dependent complexes which determine HDAC ligand binding preference. This model highlights the importance of developing HDAC inhibitors directly in a cellular system where the effects of HDAC complex components are taken into account and it identifies a potentially exploitable avenue for cell type selective inhibition of HDAC complexes.

30 RGS Dynamics Determine Sensitivity to Allosteric Inhibition by Small Molecules

1Colin A Higgins, 2Xu Liu, 1Michael P Hayes, 3C Andrew Fowler, 2,4Ernesto J Fuentes, 3Liping

Yu, and 1,4David L Roman 1Department of Pharmaceutical Sciences and Experimental Therapeutics, 2Department of

Biochemistry, 3Nuclear Magnetic Resonance Facility, University of Iowa, and 4Cell Signaling and Experimental Therapeutics Program, Holden Comprehensive Cancer Center, University of

Iowa Hospitals and Clinics Ischemic stroke morbidity and disability are caused by neuronal death largely driven by glutamatergic excitotoxicity following hypoxia in brain tissue deprived of blood flow. Pharmacological inhibition of RGS4 with chemical probes protects against excitotoxicity in vivo by significantly potentiating neuroprotective signaling by endogenous adenosine through the adenosine A1 receptor. A1R agonists have myriad dose-limiting effects in peripheral tissues, so RGS4 is an attractive drug target because its activity in this context is restricted to the brain by a CNS-specific scaffolding protein, neurabin. Unfortunately, the only known RGS4 inhibitors are not suitable as clinical leads because they are covalent modifiers that inhibit through an uncharacterized, though putatively allosteric, mechanism. We have explored this mechanism and our data indicate that inhibitors bind to and trap a stochastically minor open conformation of RGS4 and shift its population dynamics toward increasingly extended and unstable conformations. We hypothesize that the specificity of RGS inhibition via this mechanism is driven by the accessibility of cysteine residues buried in the hydrophobic interior of RGS4 in its native conformation. We have tested this hypothesis by measuring residue-specific dynamics via nuclear magnetic resonance over timescales from picoseconds to hours for RGS4 and RGS8, a homolog that is much less sensitive to inhibition via this mechanism. These data support our hypothesis, showing that RGS4 lacks a distributed, stable hydrophobic core. Future work will attempt to model in silico the excited states adopted by different RGS proteins in order to enable virtual screening and structure-based drug design to develop improved inhibitors that may be suitable for clinical investigation.

31

Synthesis and in vitro cytotoxic evaluation of analogues of the tubulin-binding agent soblidotin

Abugafar M. L. Hossion and Blake R. Peterson

The University of Kansas, Department of Medicinal Chemistry, 2034 Becker Dr., Lawrence, Kansas 66047

To effectively kill targeted cells, anticancer antibody-drug conjugates generally incorporate highly toxic small molecules. Small molecules with sufficient potency for these applications include analogues of the natural product dolastatin 10 and 15 such as soblidotin and cemadotin (Figure 1). These compounds selectively disrupt tumor vasculature and potently inhibit the polymerization of tubulin. In an effort to create more effective antibody-drug conjugates, we synthesized soblidotin, cemadotin and derivatives, and we are investigating the biological properties of these compounds. Many of these agents are highly cytotoxic against human breast cancer (SKBR3), prostate cancer (PC3) and leukemia (Jurkat) cell lines with IC50 values ranging 0.04 nM to 100 nM. To prepare soblidotin and analogues, we prepared the dolaisoleucine and dolaproline amino acids on gram scales by modifications of existing methods. The synthesis and properties of these compounds will be described.

Figure 1. Chemical structures of dolastatin 10 and 15, soblidotin and cemadotin (left), and an X-ray structure of tubulin bound to soblidotin (right, soblidotin shown as a CPK model, PDB 3E22).

NHN

O

ON N

ON

O OO

NO

O

O

NHN

O

ON N

ON

O O

HN

NHN

O

ON

ONH

NOO

O

SN

Dolastatin 10

Dolastatin 15

Cemadotin

Soblidotin

NHN

O

ON

ONH

NOO

O

32

Analogue Synthesis of the D-Tubulin-Binding Natural Product Pironetin as an Ovarian Cancer Chemotherapeutic Agent

David S. Huang, Sara K. Coulup, Henry L. Wong, Gunda I. Georg Department of Medicinal Chemistry, Institute for Therapeutics Discovery and Development,

University of Minnesota, Minneapolis, MN 55455

The natural product pironetin was isolated in 19931 and 19942,3 and has reported potent anti-proliferative activity against various cancer cell lines including ovarian cancer.4 Pironetin is proposed to inhibit cell division through a novel mechanism of action. It is proposed to disrupt microtubule polymerization dynamics via binding to D-tubulin5 whereas current chemotherapeutics that target tubulin bind E-tubulin. D-Tubulin is an attractive alterative target since cancers can become resistant to E-tubulin inhibitors. In 2011, Nikas et al. reported the overexpression of D-tubulin as a predictor for short-term survival in ovarian cancer patients who did not respond to platinum/taxol chemotherapy.6

While pironetin displays potent in vitro activity, pironetin had poor efficacy in a previous in vivo study in mice bearing P388 murine leukemia cells.7 Animals treated with pironetin also suffered from severe weight loss. We hypothesized the poor in vivo results and observed toxicity could be due to pironetin either having poor pharmacokinetic properties and/or forming covalent adducts in a non-selective manner. Thus, we are in the process of synthesizing pironetin analogues via total synthesis to improve upon these properties and study the structure-activity relationship at various positions of the natural product.

(1) Yoshida, T.; Koizumi, K.; Kawamura, Y.; Matsumoto, K.; Itazaki, H. In Japanese Patenet Kokai; Kokai, J. P., Ed. 1993; Vol. 5-31076. (2) Kobayashi, S.; Tsuchiya, K.; Harada, T.; Nishide, M.; Kurokawa, T.; Nakagawa, T.; Shimada, N.; Kobayashi, K. J. Antibiot. 1994, 47, 697. (3) Kobayashi, S.; Tsuchiya, K.; Kurokawa, T.; Nakagawa, T.; Shimada, N.; Iitaka, Y. J. Antibiot. 1994, 47, 703. (4) Marco, J. A.; Garcia-Pla, J.; Carda, M.; Murga, J.; Falomir, E.; Trigili, C.; Notararigo, S.; Diaz, J. F.; Barasoain, I. Eur. J. Med. Chem. 2011, 46, 1630. (5) Usui, T.; Watanabe, H.; Nakayama, H.; Tada, Y.; Kanoh, N.; Kondoh, M.; Asao, T.; Takio, K.; Watanabe, H.; Nishikawa, K.; Kitahara, T.; Osada, H. Chem. Biol. 2004, 11, 799. (6) Nikas, J. B.; Boylan, K. L. M.; Skubitz, A. P. N.; Low, W. C. Cancer Inform. 2011, 10, 233. (7) Kondoh, M.; Usui, T.; Kobayashi, S.; Tsuchiya, K.; Nishikawa, K.; Nishikiori, T.; Mayumi, T.; Osada, H. Cancer Lett. 1998, 126, 29.

33 A Scaffold-hopping Approach to Discovery of Keap1/Nrf2 Inhibitors

Atul D. Jain, Terry W. Moore

Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612

Ubiquitination and subsequent degradation of the transcription factor Nuclear factor-erythroid2-related factor 2 (Nrf2) is mediated by Kelch-like ECH-associated protein-1 (Keap1). Nrf2 is one of the primary regulators of the antioxidant response, and it directs transcription of detoxifying enzymes, such as glutathione S-transferases (GST), NADPH:quinone oxidoreductase 1 (NQO1), and UDP glucuronosyl transferases (UGTs). Activation of Nrf2 could be a promising therapeutic approach for prevention and treatment of various inflammatory or oxidative stress related diseases. Electrophilic and non-electrophilic activators have been known to interact with Keap1 which in turn leads to disruption of Keap1/Nrf2 complex. Most research is focused on electrophiles that interact covalently with the thiol side chain of Cys residues (e.g. C151) on the IVR region of Keap1. Cysteine is an abundant amino acid, and electrophiles that activate Nrf2 may also react with many of these other cysteine residues, leading to potential off-target toxicity We hypothesize these off-target effects may be avoided with non-electrophilic Nrf2 activators. In this work, we have developed a comprehensive structure-activity relationship around a known, naphthalene-based non-electrophilic activator of Nrf2. The naphthalene moiety may introduce solubility and metabolic liabilities, so we undertook a “scaffold-hopping” approach in which a series of non-naphthalene scaffolds was synthesized to improve the physicochemical and pharmacokinetic properties of the lead target molecule. Preliminary biochemical characterization was performed using fluorescence anisotropy assays and surface plasma resonance assays. Each of these assays measures inhibition of the Keap1/Nrf2 complex. Computational docking analysis of various scaffolds was performed, and the importance of water molecule displacement for affinity was observed. Throughout these studies, we have demonstrated that the naphthalene moiety is not necessary for binding to Keap1 and may be replaced with more drug-like scaffolds.

34 Phosphate Tether-mediated, One-pot, Sequential Processes Towards the Total Synthesis of

Lyngbouilloside and Macrocyclic Analogues

Rambabu Chegondi, Salim Javed, Arghya Ganguly and Paul R. Hanson* Department of Chemistry, University of Kansas, Lawrence, KS, 66045.

The synthesis of 14-membered macrocyclic core present in lyngbouilloside via a phosphate tether-mediated, one-pot, sequential RCM/CM/chemoselective hydrogenation reaction will be discussed. This efficient and pot-economical protocol will enable the synthesis of a variety of simplified analogues of the 14-membered macrolactones for biological screening with our collaborators.

35 An Efficient and Concise Synthesis of Novel β-Keto-Sultams as Tetramic Acid Analogs

Jung Ho Jun, Moon Young Hur, Taylor Rose Atkinson, and Paul R. Hanson*

Department of Chemistry, University of Kansas, Lawrence, KS 66045. The generation of monocyclic and bicyclic sultam analogs of tetramic acids via “Click, Click, Cyclize” methodology with intramolecular Dieckmann-like condensation is reported. This strategy utilizes a “click” mesylation of readily available linear and cyclic amino esters (in case of linear amino esters, a “click” benzylation is performed, sequentially) followed by an intramolecular cyclization via Dieckmann-like condensation to afford β-keto-sultam analogs of naturally-occurring tetramic acids in two to three steps. Continued efforts toward the generation of scaffolds with increased peripheral diversity are currently ongoing in our laboratory.

36 Phenotypic Screening of Small Molecules Against Danio rerio Embryos

Kelsey Knewtson, Casey L. Henderson, Aaron Bender, and Blake R. Peterson

Department of Medicinal Chemistry, University of Kansas Lawrence, KS 66045

Phenotypic drug discovery offers potential advantages over target based drug discovery, especially when searching for first in class drugs. This method obviates the need for knowledge of a mechanism of action, allows screening against multiple pathways at once, and in some cases can provide hits that are immediately active in vivo. Danio rerio (zebrafish) is a powerful model organism that is amenable to relatively high throughput phenotypic screening using microscopy methods. These tropical fresh-water fish have a fully sequenced genome, develop rapidly, and have transparent embryos that can be arrayed in 96-well plates. Additionally, 71% of human genes and 84% of human genes associated with disease have orthologs in zebrafish. For these reasons, we have performed a screen of a library of about 300 small molecules provided by the KU Chemical Methodologies and Library Development Center (CMLD) against zebrafish embryos bred at the KU Molecular Probes Core Facility. From this screen, we identified several compounds including a tetracyclic small molecule that affect the development of zebrafish in a dose dependent manner. We screened forty available analogues of the cytotoxic tetracyclic hit to provide insights into the structure activity relationships associated with this chemotype. The trends in the activity of these compounds are consistent between zebrafish embryos and cultured human cancer cells, indicating that the target may be conserved between these species. Efforts are underway to identify this target and generate more potent analogues.

37

Design and Synthesis of Nuclear Receptor Antagonists Targeting RARD�for Male Contraception�

Jillian L. Kyzer, Rebecca A. D. Cuellar, Trinh A. D. Holth, and Gunda I. Georg*

Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota Minneapolis, MN 55455

The retinoic acid receptor-D (RARD) is a nuclear receptor that has been implicated in male contraception. In the absence of retinoic acid, RARD has been shown to prevent spermatogenesis by inhibiting DNA transcription.1 Previous work by Bristol Myers Squibb identified two RAR antagonists of interest: BMS 189453 and BMS 189532 (Figure 1). While BMS 189453 has been shown to cause reversible infertility in mice when administered orally, it is non-selective over the E and J isoforms; BMS 189532 is selective for RARD, but lacks oral bioavailability. The goal of this project is to design and synthesize selective, bioavailable antagonists of RARD based on the BMS scaffold, which can be broken down into three regions: a hydrophobic ring, a linker, and a carboxylic acid. The antagonists are evaluated using both in silico and in vitro experiments in an iterative design process. The first generation of compounds developed in our lab contained varying aryl substituents on the ring region and amide bioisosteres in the linker region. Unfortunately, these compounds were discovered to be less selective for RARD than anticipated. Following further analysis of the RARD�crystal structure, it was determined that additional lipophilic substituents on the ring portion of the scaffold could increase selectivity. Synthesis and evaluation of these compounds will be discussed.

References 1. Chung, S. S. W.; Cuellar, R. A. D.; Wang, X.; Reczek, P. R.; Georg, G. I.; Wolgemuth, D. J. ACS Med. Chem. Lett. 2013, 4, 446-450.

BMS 189453pan-antagonist

CO2H

ring linker acid

O

NH

CO2H

BMS 189532RAR antagonist

Figure 1. Structures BMS RAR antagonists.

38 Characterization and Assessment of ALDH2-/- Mice as an Age Related Model of

Cognitive Impairment and Alzheimer’s Disease

Sue H. Lee1, Manel Ben Aissa1, Dr. Brian Bennett2, Dr. Gregory R.J. Thatcher1* 1Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of

Illinois at Chicago, Chicago IL 2Department of Biomedical and Molecular Sciences and Centre for Neuroscience Studies,

Queen’s University, Kingston, Ontario, Canada Alzheimer’s disease (AD) currently affects 5.3 million Americans and 30 million people worldwide. However, the therapies available are unable to improve cognition deficits or reverse the degenerating disease. The multifactorial disease is characterized by accumulation of amyloid-β (Aβ) as well as neurofibrillary tangles (NFT) composed of hyperphosphorylated tau. Despite showing promising results in preclinical trials, therapies targeting Aβ continue to fail in late clinical trials calling into question the identification of targets and the transgenic animal models used for preclinical evaluation. Current AD mice models exhibit pathological changes dependent on overexpression of mutations linked with early onset familial AD which only accounts for a small amount of AD cases (1-5%). Therefore, the availability of an animal model that mirrors age-related cognitive impairments could become a valuable tool in assessing therapeutic strategies for improving memory and slowing down, preventing, or reversing AD. Oxidative stress has been suggested to be a driving force in AD pathogenesis. When an enzyme that detoxifies toxic aldehydes, aldehyde dehydrogenase 2 (ALDH2), was knocked down, appearances of AD-like pathologies developed. We exhibited an increase in toxic aldehyde formation, age-dependent cognitive impairments, and dysregulation of AD related biomarkers. Aldh2-/- mice demonstrate key components of AD rarely seen in current mice models such as development of classical characteristics of AD in parallel with biomarkers involved in neuronal and synaptic function and apoptosis. This proposed model for sporadic AD could expand the effectiveness of preclinical models and create greater predictability when shifted to human clinical trials.

39

Investigating Metabolic Gender Differences with Melanocortin Antagonist SKY 2-23-7

Cody J. Lensing, Skye R. Doering, Carrie Haskell-Luevano Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455

The melanocortin 3 (MC3R) and melanocortin 4 receptors (MC4R) have been demonstrated to have implications in food intake and energy homeostasis.1 The intracerebroventricular (ICV) administration of melanocortin agonists is reported to significantly decrease food intake, whereas administration of antagonists is reported to significantly increase food intake.2 Therefore, melanocortin agonist ligands could serve as a potential treatment for obesity, and melanocortin antagonist ligands could serve as a treatment for cachexia. SKY 2-23-7 is a tetrapeptide which was identified in our laboratory with the sequence Ac-Trp-(p-I)Phe-Arg-Trp-NH2. It is a mMC3R and mMC4R antagonist displaying minimal agonist activity at both receptors which is not commonly observed.3 The current study investigated the in vivo effects of SKY 2-23-7 via ICV administration. SKY 2-23-7 displayed a unique pharmacological profile in which it affects male and female mice differently. For example, a 5 nmol dose of SKY 2-23-7 increased male mice feeding but had no effect on female mice. Such gender specific responses could be exploited to overcome some limitations known to melanocortin ligands such as off target effects related to blood pressure and sexual function.4 Future work will further probe the gender differences of the SKY 2-23-7. This work was funded by NIH grant R01DK091906. (1) Huszar, et al.; Cell, 1997, 88, 131-41; Chen, et al.; Nat. Gen., 2000, 26, 97-102. (2) Fan, et al.; Nature, 1997, 385, 165-8; Irani, et al.; Euro. J. Pharm., 2011, 660, 80-7. (3)Doering, S. R. et al. ACS Med. Chem. Lett. 2014, 6, 123-7. (4) Greenfield, J. R. et. al. N. Engl. J. Med. 2009, 360, 44-52.; der Ploeg, V. et. al. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 11381-6.

40

Quinone-Promoted Inert Bond Functionalization of Amines

Xinyun Liu, Martin A. Leon, Johnny H. Phan, Michael D. Clift Departments of Chemistry, University of Kansas

Lawrence, Kansas 66045 Amine-containing compounds are highly important pharmacophores in numerous bioactive natural products and medicinal agents. With the expanding repertoire of biologically relevant nitrogenous molecules, the need for developing efficient synthetic methods to access amines as useful intermediates is attracting significant attention in the field of medicinal chemistry. Existing methods for amine synthesis often require harsh conditions or multi-step synthetic procedures that rely on sensitive carbonyl derivatives. In accordance with our goal to discover novel methodologies for amine synthesis, we have developed a catalytic system utilizing topaquinone mimics inspired by enzymatic catalysis that can promote the formal inert bond functionalization of amines. This method has achieved success in the formal C�H functionalization of simple amine starting materials using a variety of nucleophilic reaction partners. The amine products generated by this method can be further employed to deliver β�amino acid derivatives on a gram scale. In addition to the efficient amine C�H functionalization promoted by the quinone catalyst, ongoing investigation has also revealed that oxidative rearrangement of donor�donor cyclopropanes affords α,β�unsaturated imines under the similar reaction conditions. This strategy is envisioned to constitute a mechanistically distinct and general approach for amine synthesis.

41 Complementary Pairing: An Aziridine Opening/SNAr Approach

to Novel Benzofused Sultams

Joanna K. Loh, Naeem Asad, Thiwanka B. Samarakoon and Paul R. Hanson* Department of Chemistry, University of Kansas, Lawrence, KS 66045.

The University of Kansas Center for Chemical Methodologies and Library Development (KU-CMLD), Lawrence, KS 66047.

The generation of common and stereochemically rich, medium-sized benzofused sultams via the complementary pairing (CP) of o-fluorophenyl-sulfonyl aziridines in a modular one-pot, sequential protocol using an aziridine ring-opening and intramolecular nucleophilic aromatic substitution (SNAr) is reported. The strategy employs a variety of amines/amino alcohols and proceeds with 6+1 and 6+4/6+5 cycloetherification pathways to obtain skeletally and structurally diverse, polycyclic, 7-, 10- and 11-membered benzofused sultams for broad scale screening.

42 Synthesis and Biological Evaluation of Azomethine-dihydroquinazolinone Conjugates as

Cancer and Cholinesterase Inhibitors

Shams-ul Mahmooda,b, Aamer Saeeda, Gunda Georgb

a Department of Chemistry, Quaid-I-Azam University, Islamabad, Pakistan. bUniversity of Minnesota, College of Pharmacy, Department of Medicinal Chemistry,

Minneapolis, MN 55414.

A small library of new azomethine derivatives of 3-aryl-2-thioxo-2,3-dihydroquinazolin-4(1H)-ones was synthesized. The key intermediates 2-thioxo-quinazolinones (3a-e), obtained in 2 steps from the corresponding anilines, were treated with methyl chloroacetate to afford S-substituted esters (4a, d), which were then converted into corresponding acetohydrazides (5a,d). Further, acetohydrazide (5d) was converted to the azomethines derivatives (6a-k) by reacting with a number of suitably substituted Benzaldehydes. FTIR, 1H NMR, 13C NMR, GC-MS, and elemental analyses were used to confirm the assigned structures of the synthesized compounds. Further, compounds 3a, 5d, and 6j were also confirmed by X-ray diffraction data. In order to study anti-cancer activity and their tendency to inhibit potent cholinesterase, the synthesized azomethine-dihydroquinazolinone conjugates were evaluated against lung carcinoma cells and cholinesterases. Several compounds exhibited high cytotoxicity at low micromolar concentrations and were less toxic to normal cells. After 24 hours, 6i showed maximum cytotoxicity. Most of the tested compounds were potent inhibitor of acetylcholinestrease (AChE) and 4-bromo substituted compounds showed highest enzymatic activity. The most potent compound 6c had an IC50 value 209.8 µM against AChE. Tested compounds showed less inhibition against butyrylcholinesterase. Molecular docking studies were performed using the homology models of cholinesterase in order to investigate the plausible binding modes of synthesized compounds. The compounds can be further optimized to treat cancer and Alzheimer’s disease. These derivatives may open new pathways for introducing new therapies for curing cancer and senile dementia.

(6a-k)

O

HN

NS

N

NOCH3

R

6a: R= 2-Br 6b: R= 3-Br 6c: R= 4-Br 6d: R= 2-Cl 6e: R= 3-Cl 6f: R= 4-Cl 6g: R= 3-NO2 6h: R= 4-NO26i: R= 3-OMe 6j: R= 3,4,5-(OMe)36k: R= 2-OBz

43 Phosphate Tether-Mediated Approach

Towards the C9–C25 Fragment of Spirastrellolide B

Soma Maitra, Rambabu Chegondi and Paul R. Hanson* Department of Chemistry, University of Kansas, Lawrence, KS 66045-7582

Synthetic efforts towards the C9–C25 fragment of the polyketide natural product spirastrelolide B is reported. Originally isolated in 2007, spirastrellolide B and its congeners possess interesting biological activity and feature a challenging framework including a 38-membered macrolactone, a bicyclic and a tricyclic spiroacetal subunit. A strategy aimed at developing phosphate tether-mediated regio- and chemoselective reactions to facilitate the synthesis of the C9–C25 fragment in an efficient manner will be discussed.

44 Dynamic Anthrax Toxin Lethal Factor

Kimberly M. Maize, Elbek K. Kurbanov, Elizabeth Ambrose Amin, and Barry C. Finzel

Department of Medicinal Chemistry, University of Minnesota Minneapolis, MN 55455

Lethal Factor protease (LF) is part of a three-component exotoxin produced by Bacillus anthracis, and together with the other toxin constituents, edema factor and protective antigen, it continues to cause damage following the resolution of the primary infection. As a zinc metalloproteinase, LF functions to impair the host immune system by cleaving the N-terminal portions of mitogen-activated protein kinase kinases in macrophages. The development of inhibitors specific to LF is challenging because zinc-chelating inhibitors, while potent, often inhibit endogeneous zinc metalloenzymes. A study of LF crystal structures with diverse ligands reveals that domain 3 of the enzyme has three frequently populated conformational states, where the domain position is influenced by the ligand chemotype. Furthermore, conformationally restrained ligands such as 405 induce a novel solvent-exposed channel in LF as an extension of the S1´ substrate binding site, termed S1´*, facilitated by the movement of Lys656 and Leu677 that may be exploited to increase specificity.

Domain 3 Movement

Leu677

Lys656

S1´

S1´*

F

SO

O

NNH

O

OH

405

45

Phosphorus-Based Tether Methods for the Synthesis of 1,3-Anti-diol-containing Natural Products

Jana L. Markley, Paul R. Hanson* Department of Chemistry, University of Kansas, Lawrence, KS 66045

The use of phosphite-borane triesters as tripodal tether systems to mediate diastereoselective ring-closing metathesis reactions of C2-symmetric, 1,3-anti-diol-containing dienes is reported. Studies focused on the reactivity of these tethers, including deprotection/oxidation transformations and chemoselective cross-metathesis, will also be presented. In particular, this work will touch upon the similarities and differences in the chemical reactivity profile of these phosphite-borane tethers with respect to their phosphate triester relatives, while highlighting the potential power of both tether systems—used separately or in conjunction—to form complex polyols in a facile, manipulable manner.

46

Isolation and Structure Elucidation of Merocyclophane C from the Cultured Cyanbacterium Nostoc sp. (UIC 10110)

Daniel May, Shangwen Luo, Aleksej Krunic, George Chlipala, Jimmy Orjala Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of

Illinois at Chicago, Chicago, IL 60612

Cyanobacteria are known to be prolific producers of biologically active natural products. Initial screening of the extract of UIC 10110, a Nostoc sp. based on 16S rRNA phylogenetic analysis, found it to be cytotoxic against MDA-MB-435 cells and HT-29 cells in an antiproliferation assay. Dereplication by mass spectrometry and NMR indicated the presence of potentially novel constituents. An active constituent was isolated using semipreparative C18 HPLC. Acurate mass spectrometry allowed for the determination of the molecular formula. Analysis of the proton NMR spectrum revealed the active compound to be very similar to Merocyclophane A, a compound recently discovered by our lab. Comparison of the spectra, as well as additional 2-D NMR experiments, confirmed the structure of Merocyclophane C. The absolute configuration of the compound was determined by circular dichroism spectroscopy.

47

Structure-Based Design of Grp94-Selective Inhibitors for Glaucoma and Metastasis

Sanket J. Mishra and Brian S. J. Blagg* Department of Medicinal Chemistry, The University of Kansas

Lawrence, Kansas

Heat shock protein 90 KDa (Hsp90) is a member of molecular chaperone family proteins that serve to fold proteins during the conformational maturation, stability, and trafficking of client proteins. Hsp90 clients play a critical role in the pathogenesis of diseases such as cancer, neurodegeneration and infection. Currently, clinical trials are ongoing for 17 Hsp90 inhibitors, however, all of these inhibitors exhibit pan-inhibition of all four Hsp90 isoforms, which is likely the cause of detrimental side effects observed with these compounds, which include hepatotoxicity, cardiotoxicity, and renal toxicity. Therefore, the development of isoform-selective Hsp90 inhibitors can assign the contribution of each Hsp90 isoform toward the observed toxicities and the development of such therapeutic agents can avoid of aforementioned side effects.

Glucose regulated protein, Grp94, is the endoplasmic reticulum (ER) residing isoform of Hsp90 that is critical for trafficking secreted proteins that mediate cellular communication, adhesion, and proliferation. Recent co-crystal structures of Grp94 have revealed subtle differences between Grp94 and other Hsp90 isoforms that can be exploited for the development of Grp94-selective compounds. In the current study, a structure-based approach has been used to produce efficacious Grp94-selective inhibitors. Binding efficacy and clinical implications of these inhibitors will be presented.

48

1

A Novel Diterpene and M. tuberculosis-active Molecules from a Marine-derived Actinomycete in Vietnam

Michael W. Mullowney,1 Urszula Tanouye,1 Baojie Wan,2 Sanghyun Cho,2 Scott G.

Franzblau,1,2 Van Cuong Pham,4 Brian T. Murphy1,3

1Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, United States

2Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, United States

3Center for Pharmaceutical Biotechnology, College of Pharmacy, University of Illinois at Chicago, Chicago, IL Illinois 60607, United States

4Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, Hanoi, Vietnam

A screening of our actinomycete fraction library against Mycobacterium tuberculosis H37Rv (TB) led to the isolation of a novel diterpene (1), a novel anthraquinone (2), and the previously reported 6-hydroxytetrangulol (3), phananthroviridin aglycone (4), and WS-5995 A (5). These secondary metabolites were produced by a Micromonospora sp. (Strain G039), isolated from sediment collected off the Cat Ba peninsula in the East Sea of Vietnam. They exhibited MIC values ranging from 0.72 µg/mL to 19.4 µg/mL against M. tuberculosis, and proved to be weakly cytotoxic when screened against Vero cells, with IC50 values ranging from 32.07 µg/mL to 42.42 µg/mL. Compound 1 is a novel Δ8,9-pimarane diterpene, representing one of approximately twenty actinomycete-produced diterpenes reported to date. Compounds 2-5 are quinones with newly reported activity against TB. The structure elucidation of 1 and 2 were performed by combined NMR and MS spectral analysis. Stereochemistry for 1 was assigned by combined analysis of 3JHH coupling constants, 2D NMR experiments, and CD spectral data. The isolation and anti-tuberculosis activity of 1-5, as well as the structure elucidation of 1 and 2 will be discussed.

49

Azaphilone Molecules from Freshwater Fungi

Vanessa M. Nepomuceno, Eoghainin O’Hainmhire, Dr. Brian T. Murphy Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago

Ovarian cancer is the most lethal gynecological malignancy and fifth leading cause of cancer death among women. This high mortality rate is due to difficult detection, unclear symptoms, and an absence of known precursor lesions. In addition, tumors that are initially responsive to chemotherapeutics often become resistant. Thus, discovery of drug leads for ovarian cancer treatment is of paramount importance. The aquatic environment contains rich chemical and biological diversity that can be used as a source for these much needed compounds. These aquatic species are a promising source for novel anti-cancer compounds. A fraction library of secondary metabolites produced by aquatic-derived bacteria and fungi were screened for potential selective toxicity against chemo-resistant ovarian cancer cells. Fractions exhibiting biological activity at a median lethal dose value (LC50) of 10 µg/mL or less were analyzed to determine their potential to contain anticancer leads. In the current study, three putatively new molecules of the azaphilone class have been identified from freshwater fungal strain FJ015 in Lake Huron. Herein we will report structure elucidation of the metabolites were elucidated through one- and two-dimensional nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry analyses. Details of the biological activity of these molecules will also be discussed.

50

Development of a Pronucleotide Phosphoramidate-based Inhibitor of Eukaryotic Translation Initiation Factor 4E

Aniekan Okon, Peter Bitterman, Robert Kratzke, Carston R. Wagner* Department of Medicinal Chemistry, University of Minnesota

Minneapolis, MN 55414

The formation of the initiation complex 4F (eIF4F) is a critical step during cap-dependent protein translation. The eukaryotic translation initiation factor 4E (eIF4E) binds the 5´-N7-methyl guanosine (cap) moiety of mRNA and associates with initiation factors 4G and 4A to form the eIF4F complex, which then can associate with other translation factors and the ribosome to facilitate cap-dependent protein translation. Importantly, the formation of a competent 4F complex is regulated by the availability of eIF4E. In cancer biology, several studies have demonstrated that high eIF4E activity is required for tumorigenesis and disease progression. Particularly, cancer cells have been shown to be extremely sensitive to reduced eIF4E activity whereas the growth of normal cells is unaffected by reduced eIF4E activity. Therefore, inhibition of eIF4E activity in cancer cells provides us with a very selective anticancer strategy. The Wagner group has previously demonstrated the synthesis and biological activity of a pronucleotide phosphoramidate-based inhibitor of eIF4E (4Ei-1). Specifically, 4Ei-1 was shown to be nontoxic, cell penetrating, and to sensitize cancer cells to low dose gemcitabine and pemetrexed (in the case of mesothelioma cells). As a result of our work with 4Ei-1, we are currently developing next generation pronucleotide phosphoramidate inhibitors of eIF4E. Initial in vitro evaluation of synthesized inhibitor will be presented.

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51

Small Molecule Inhibitors of APOBEC3 DNA Cytosine Deaminases as Leads for HIV-1 and Cancer Therapy

Margaret E. Olson†, Angela L. Perkins†, Ming Li‡, Reuben S. Harris‡, and Daniel A. Harki†*

†Department of Medicinal Chemistry, ‡Department of Biochemistry, Molecular Biology & Biophysics,

University of Minnesota, 2231 6th Street S.E., Minneapolis, MN 55455, United States APOBEC3 (A3A-A3H) enzymes catalyze DNA cytosine-to-uracil deamination as a function of innate immune defense against foreign, often pathogenic, DNA. Host cellular protection results from A3-catalyzed hypermutation of the foreign genome. When misregulated, however, A3 enzymes have been demonstrated to drive the genetic evolution of HIV-1 and numerous cancers. Specifically, sub-lethal levels of A3D/F/G/H-catalyzed mutation can enable HIV-1 escape from immune defense mechanisms and antiretroviral therapies.1 Moreover, A3B is over-expressed in breast, bladder, cervical, lung, and head and neck cancers.2 A3B over-expression generates high levels of C-to-U mutation, which subsequently drives tumor metastasis and chemotherapeutic resistance. Thus, small molecule inhibitors of A3-catalyzed deamination may not only offer invaluable probes to study the emerging roles of A3s in human disease, but could provide novel strategies for HIV-1 and cancer drug development. Recently, the Harki and Harris laboratories have accomplished the high-throughput (HTS) screening of >500,000 small molecules for A3A/B/G inhibition. Thus far, our efforts have yielded a number of lead A3 inhibitors, which can be divided by mechanism into covalents and non-covalents. At least three unique scaffolds containing reactive electrophilic functionalities are suspected to specifically inhibit A3G by covalently engaging C321, a residue proximal to the active site.3 Conversely, at least two compound classes inhibit multiple A3s through seemingly non-covalent mechanisms of action. This poster will highlight recent work to develop these scaffolds into potent and selective A3 probes through iterative analogue synthesis, biochemical evaluation, and mechanism of action studies. (1) Albin, J. S.; Harris, R. S. Expert Rev. Mol. Med. 2010, 12, e4. (2) Burns, M. B.; Lackey, L.; Carpenter, M. A.; Rathore, A.; Land, A. M.; Leonard, B.; Refsland, E. W.;

Kotandeniya, D.; Tretyakova, N.; Nikas, J. B.; Yee, D.; Temiz, N. A.; Donohue, D. E.; McDougle, R. M.; Brown, W. L.; Law, E. K.; Harris, R. S. Nature 2013, 3, 366-371.

(3) Li, M.; Shandilya, S. M. D.; Carpenter, M. A.; Rathore, A.; Brown, W. L.; Perkins, A. L.; Harki, D. A.; Hook, D. J.; Pandey, K. K.; Parniak, M. A.; Johnson, J. R.; Krogan, N. J.; Somasundaran, M.; Ali, A.; Schiffer, C. A.; Harris, R. S. ACS Chem. Biol. 2012, 7, 506-517.

(4) Olson, M. E.; Li, M.; Harris, R. S.; Harki, D. A. ChemMedChem 2013, 8, 112-117.

52

The Inhibition of Human Steroid Sulfotransferases hSULT1E1 and hSULT2A1 by Hydroxylated and Sulfated Metabolites of Polychlorinated Biphenyls

Parker, Victoria S. [ 1 ] ; Squirewell, Edwin[ 1 ] ; Lehmler, Hans-Joachim[ 2 ] ; Robertson, Larry W.[ 2 ] ; Duffel, Michael W.[ 1 ]

1Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy and 2Department of Occupational and Environmental Health, College of Public Health, the University of

Iowa, Iowa City, IA 52246

Polychlorinated biphenyls (PCBs) are persistent environmental toxins that have associations with multiple adverse health effects. While sulfation is involved in the metabolism and detoxication of many environmental toxins, such as PCBs, it is also an important biological process that results in the normal cellular inactivation of steroid hormones, such as androgens and estrogens. Sulfation therefore regulates the cellular concentrations of the active hormones. Sulfotransferases are enzymes that catalyze the process of sulfation. Inhibiting the catalytic activity of sulfotransferases could lead to abnormally high concentrations of active steroid hormones in certain tissues, and this action will disrupt the balance between active and inactive hormones at those sites. The goal of our project is to determine if hydroxylated and sulfated metabolites of those PCBs that are commonly found in outdoor and indoor air samples are capable of inhibiting the activity of the estrogen and androgen sulfotransferases. Our preliminary studies have shown that the hydroxylated PCB metabolites examined inhibit these enzymes, while the sulfated PCB metabolites are much less effective at this inhibition. Such interactions with these sulfotransferases may have implications for alterations in steroid hormone signaling, and this is a subject for further investigation. (Supported by NIH P42 ES013661 and R25 GM058939)

Figure 1. The Inhibition of hSULT1E1 and hSULT2A1 by 4’-OH-PCB 25

53

Utilizing Chemically Self-Assembled Antibody Nanorings (CSANs) as Bispecific Targeting Agents for Cell-directed Immunotherapy

Jacob Petersburg, Kari Gabrielse, and Carston Wagner

Department of Medicinal Chemistry, University of Minnesota Minneapolis, MN 55455!

Chemically Self-Assembled Nanorings (CSANs) are formed by engineering fusion proteins that contain a single-chain variable fragment (scFv) fused to two E. Coli dihydrofolate reductase (DHFR2) molecules that spontaneously self-assemble into octameric nano rings upon the addition of a chemical dimerizer, bis-methotrexate (BisMTX). We have designed a system in which two different scFv-displaying fusion proteins can be combined into a single nanoring, thereby targeting multiple cell surface biomarkers simultaneously. More specifically, the combination of an anti-CD3 fusion protein and anti-EpCAM fusion protein generates the formation of bispecific, multivalent, CSANs that specifically target both the CD3 T cell receptor and EpCAM receptor expressed on multiple carcinoma and cancer stem cells. Our results show that upon incubation, bispecific CSANs direct selective cell-cell interactions between CD3+ T cells and EpCAM+ breast cancer cells. Furthermore, these directed cell-cell interactions led to the selective cell killing of EpCAM+ breast cancer cells, with up to 90% cell death over 24 hours. Thus far, a majority of the attention in immunotherapy has centered solely on the utilization of T cell CD3 receptors and the CD8+ cytotoxic T lymphocyte subpopulation. However, recent work performed in our lab to separately functionalize both CD4+ T helper cells and CD8+ T effector cells, with bispecific CSANs, has highlighted the importance of understanding the function and synergism of both subpopulations in adoptive immunotherapy. By adjusting the ratio of CD4+ and CD8+ T cells prior to incubation with bispecific CSANs we were able to augment the cell killing of EpCAM+ cells as well as better understand the interpatient variability of immunotherapy. Taken together our results demonstrate that bispecific CSANs provide a promising platform for future use in cell-directed cancer immunotherapy.

54

Prostate Cancer Target Cytochrome P450 17A1 (CYP17A1): Evaluation and Comparison of Clinically-Relevant Inhibitors

Elyse M. Petrunak and Emily E. Scott

Department of Medicinal Chemistry, The University of Kansas, Lawrence, Kansas 66045 Prostate cancer is the second leading cause of cancer-related deaths in American men. One new treatment is inhibition of the steroidogenic enzyme cytochrome P450 17A1 (CYP17A1). CYP17A1 hydroxylation and lyase reactions generate tumor-promoting androgens. However hydroxylation is also necessary for corticosteroid biosynthesis, so selective lyase inhibition is desirable for prostate cancer treatment. Thus, both potency and hydroxylase vs. lyase selectivity are desirable features for CYP17A1 inhibitors. Of three inhibitors evaluated with recombinant human CYP17A1 (shown below), FDA-approved abiraterone had the highest potency for all CYP17A1 reactions (progesterone and pregnenolone hydroxylation and 17α-hydroxypregnenolone lyase). Galeterone was ~2-fold less potent than abiraterone for all reactions, whereas orteronel was >30-fold less potent. Abiraterone and galeterone demonstrated little to no selectivity for inhibition of the lyase reaction vs. hydroxylation reactions. Orteronel, previously reported to be 5-fold selective for lyase inhibition, was herein found to be only 2-3-fold selective. This lack of selectivity is consistent with the major binding mode observed for these inhibitors, which coordinate to the active site heme iron required for catalysis of both reactions. The minimal selectivity and lower potency of orteronel is consistent with recent phase 3 trials results leading to termination of development. The current studies suggest that lyase-selective CYP17A1 inhibitors might be better designed without direct iron coordinating features.

Above: Inhibitors of prostate cancer target CYP17A1.

HO

N

O

NH

N

NOH

HO

N

N

abiraterone galeterone orteronel

55

Development of Quinone-Promoted C-C Bond Oxidative Cleavage

Johnny Phan, Benjamin J. Haugeberg, Samuel Kier, Thomas J. O’Connor, Xinyun Liu, and Michael D. Clift

Department of Chemistry, University of Kansas Lawrence, KS 66045

Amine-containing compounds are abundant in materials, synthetic, and medicinal chemistry. Given the high value of amine-containing compounds across a wide variety of scientific disciplines, the development of novel methods for their preparation is of paramount importance. Traditional methods of amine synthesis involve sensitive carbonyl functionalities that must be carried throughout multi-step protocols. To this end, our work has developed a new quinone-promoted method that enables the formal C−C bond functionalization of amines via oxidative decarboxylation of α-amino acids to form valuable synthetic intermediates. Ongoing work involves coupling of the intermediates with a variety of nucleophilic reaction partners and applying the oxidative bond cleavage to amino alcohols.

56

CJ-15,208 Analogues: Their Permeability across Biological Barriers and Interaction with P-glycoprotein

Christianna Reedya, Archana Mukhopadhyaya, Sanjeewa Senadheeraa, Tanvir Khaliqa,

and Jane V. Aldricha,b aDepartment of Medicinal Chemistry, University of Kansas, Lawrence, KS, 66045; bDepartment of Medicinal Chemistry, University of Florida, Gainesville, FL, 32611

Ligands for the κ-opioid receptor (KOR) represent attractive pharmacological targets for the treatment of pain and drug abuse. While peptide KOR ligands can display desirable durations of action, peptide oral delivery can be hindered by poor permeability of biological barriers. Both the macrocyclic tetrapeptide natural product CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]) and its D-Trp isomer are active after oral administration, but exhibit different opioid activity profiles in vivo.1,2 We studied the interactions of CJ-15,208 and [D-Trp]CJ-15,208 in an in vitro biological barrier model using Madin-Darby canine kidney (MDCK) cells transfected with MDR1, the gene coding P-glycoprotein (P-gp), an efflux protein important at the intestinal and the blood-brain barriers. The peptides’ inhibition of P-gp was assessed using rhodamine 123 uptake and transport across MDCK-MDR1 monolayers, and their efflux by P-gp was assessed by analyzing bidirectional transport of the lead macrocyclic peptides across MDCK-MDR1 monolayers. Initial results suggest that while both CJ-15,208 and [D-Trp]CJ-15,208 are inhibitors of P-gp, only CJ-15,208 is a P-gp substrate. Funding was provided by the NIGMS Biotechnology Training Program and NIDA grants R01 DA023924 and R01 DA032928. 1 Aldrich, J. V.; Senadheera, S. N.; Ross, N. C.; Ganno, M. L.; Eans, S. O.; McLaughlin, J. P. J. Nat. Prod. 2013, 76, 433-438. 2 Eans, S. O.; Ganno, M. L.; Reilley, K. J.; Patkar, K. A.; Senadheera, S. N.; Aldrich, J. V.; McLaughlin, J. P. Br. J. Pharmacol. 2013, 169, 426–436.

57

Towards the development of photoaffinity probes for non-covalent activation of Nrf2

Benjamin G. Richardson, Atul D. Jain, Terry W. Moore Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of

Illinois at Chicago, Chicago IL 60612 The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the expression of detoxification enzymes involved in protecting cells from oxidative and electrophilic stress. It is regulated by Kelch-like ECH-associated protein 1 (Keap1), which binds both Nrf2 and the E3 ubiquitin ligase Cul3. In this arrangement, Cul3 polyubiquitinates Nrf2, leading to proteasomal degradation of Nrf2. When the body is stressed by electrophiles or oxidants, Nrf2 is released, translocates to the nucleus, and binds to antioxidant response elements (AREs) in gene promoter regions, initiating expression of detoxification enzymes. This Keap1-Nrf2 pathway has become an attractive target for the prevention and treatment of multiple oxidative-stress related diseases and conditions, with FDA-approved drugs like Tecfidera® (dimethyl fumarate) in use as an Nrf2 activator. Most research into the pathway has uncovered reactive electrophiles that covalently modify reactive cysteines leading to release of Nrf2. Because we are interested in the pharmacological outcome of selectively activating Nrf2, we have investigated an alternative pathway of activation—directly inhibiting the Nrf2/Keap1 interaction with a small molecule—and we are preparing a proteomic tool compound that will allow us to interrogate the selectivity of these non-electrophilic compounds. Recently, reports in the literature found such a non-electrophilic activator of Nrf2 based on a 1,4-bis-sulfonamido naphthalene parent structure. One compound in this series was found to have extremely high potency for the binding pocket, IC50 = 23 nM. Using this molecule as a starting point, we have carried out a structure-activity relationship to allow us to determine allowable modifications for preparing photoaffinity probes, which will ultimately allow us to address the selectivity of non-electrophilic Nrf2 activators.

58

Estrogen Receptor ligand Photoprobes can Provide Mechanistic Information of Biomolecular Interactions between the Estrogen Receptor and its Ligands

Loruhama M. Delgado Rivera, Rui Xiong, Gregory Thatcher

Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 S Wood Street, Chicago, IL, 60612

Estrogen receptors (ERs) are a class of ligand-activated transcriptional factors whose function is mediated by binding of estrogen agonists. Canonical signaling posits ligand binding, ER dimerization and translocation to the nucleus where binding to estrogen response elements (EREs) located in the promoter region of ER genes recruits a multi-protein complex, which is transcriptionally active. The plasticity of ER and dynamic nature of the complex suggest that different ligands might evoke agonist, antagonist or partial agonist activity. These molecules can stabilize different conformations, therefore modulating the activity and localization of ER within the cell. The mechanistic aspects of ligand binding to ER are believed to be well known; however the hypothesis is mostly based upon two extreme conformations of truncated ERα ligand binding domain (LBD) bound to full agonist or antagonist. Our lab is interested in exploring partial and full agonists that have more diverse structures which may stabilize non-traditional or multiple ER complexes. To define the binding mode of such ligands, we will use photo-induced cross-linking (PIC), a technique that would allow us to identify transient or stable interactions between ER and its ligands. This project focuses on the development of an ER partial agonist that incorporates as a photoprobe an aryl azide moiety. This functionality allows the formation of singlet nitrenes, which are able to react with nearby amino acids, providing temporal as well as spatial information of agonist binding to ER. Here we will present the synthesis and characterization of a 2-(4-azidophenyl)benzo[b]thiophen-6-ol photoprobe. The information obtained from future studies of this probe with ER will be used to identify mechanistic differences between conformations stabilized by agonists, antagonists and partial agonists, allowing design of new ligands and providing mechanistic information about the biomolecular interactions of ER and its ligands.

59

Profiling the Immunological Activities of Toll-like Receptor-7 and -8 Agonistic Molecules

Alex C. D. Salyer, Sunil A. David Department of Medicinal Chemistry, University of Kansas

Lawrence, Kansas 66045

Toll-like receptor-7 and -8 (TLR) agonists are thought to drive Th1 adaptive immune responses by induction of Type I interferons (IFN-α/β) and Type II interferons (IFN-γ), mediated through the engagement of plasmacytoid dendritic cells and myeloid dendritic cells, respectively. In rabbit immunization models, dual TLR-7/8 agonists were shown to be superior adjuvants in respect to antibody titers when compared to either pure TLR-7 or TLR-8 agonists. However, the mechanisms of adjuvanticity for these molecules at the cellular level remains poorly understood. In whole blood ex vivo stimulation assays dual TLR-7/8 agonists appeared to induce cell death (Annexin V+, DAPI-) in monocytes, resulting in the formation of microparticles. The resulting monocyte-derived microparticles were between 1-5 µm in size and CD142+, which showed similarity to microparticles derived from apoptotic cell death. Interestingly, all three classes of TLR agonists appear to activate monocytes as measured by CD80 expression, but only dual TLR-7/8 agonists appear to cause monocyte death. Unlike pure TLR-8 agonists, pure TLR-7 and dual TLR-7/8 agonists also showed the capacity in activate B lymphocytes in whole blood. Monocyte activation by pure TLR-7 and dual TLR-7/8 agonists was accompanied by increased uptake of soluble antigens in monocytes, but pure TLR-8 agonists did not promote antigen uptake. The mechanisms underlying differences between upregulation of co-stimulatory molecules and antigen uptake remain to be elucidated. The differences in cellular activity profiles of the three classes of TLR agonists examined likely contribute to the differences observed in adjuvanticity and are being investigated to determine the optimal activity profiles for adjuvanticity.

60

Synthesis and Evaluation of 1,2,5-oxadiazole 2-oxide Derivatives Useful to Treat Sickle Cell Disease Symptoms

Thais Regina Ferreira Melo2, Karina Pereira Barbieri2, Carolina Lanaro2 and Jean Leandro dos

Santos1,2 1Department of Medicinal Chemistry, University of Minnesota

Minenapolis, MN 55455; 2School of Pharmaceutical Science, State University of São Paulo, Araraquara, SP 14801902

Sickle cell disease (SCD) is one of the most common genetic disorders worldwide. The only available drug approved by FDA is hydroxyurea (HU); however long-term treatment with it is associated with several deleterious effects1. After metabolism, HU is converted to nitric oxide (NO) that induces of γ-globin gene expression and fetal hemoglobin2. In this work, we synthesized and evaluate 1,2,5-oxadiazole 2-oxide compounds as NO-donors useful to treat SCD. Four compounds were synthesized (yields ranged 25-55%) and characterized by analytical methods. All compounds demonstrated ability to induce nitrite formation between 6.7-12.1%, superior to that of isosorbide dinitrate, used as control. All compounds inhibited tumor necrosis factor (TNFα) production in levels superior to that of thalidomide. For TNFα inhibition, compound I demonstrated IC50 of 1.15µM. In addition, compound I was able to induce gamma-globin gene expression two times more than HU at concentrations 6 times inferior of this reference drug. All 1,2,5-oxadiazole 2-oxide derivatives have shown promising useful to treat sickle cell disease symptoms. References: 1 Dos Santos, J.L et al., J. Med. Chem. 54(16):5811-9, 2011. 2 Dos Santos J. L. et al., J. Med. Chem. 55(17):7583-92, 2012.

61

Synthesis and Biological Evaluation of Novel Neurosteroid Photoaffinity Ligand

aPavel Y. Savechenkov, cDavid C. Chiara, bRooma Desai, bDouglas E. Raines, bAlexander T. Stern, bXiaojuan Zhou, cJonathan B. Cohen, bStuart A. Forman, b,cKeith W. Miller

and aKarol S. Bruzik

aUniversity of Illinois at Chicago, Chicago, IL, 60612; bMassachusetts General Hospital, Boston, MA 02114, cHarvard Medical School, Boston, MA 02115

Alfaxalone 1 is a neuroactive steroid and a general anesthetic exerting its biological activity by allosteric GABAA receptor binding and modulation of its ion-channel function. To identify the neurosteroid binding sites on the GABAA receptor, we synthesized a photoreactive azidotetrafluorobenzoate analog of alfaxalone 3, starting from 11α-hydroxyprogesterone 2 via a linear 9-step sequence. To enable sequence-specific determination of amino acids that compose the binding site, we also synthesized the ditritiated analog 3 with a very high specific radioactivity (45 Ci/mmol).

The preliminary biological evaluation indicated that 3 is a potent general anesthetic with EC50 = 0.72 µM (panel A), it is modulating binding of GABA-antagonist, muscimol (B), with EC50 = 0.57 µM, and shifts the dose-response curve for GABA-binding to the left more strongly than the parent alfaxalone (C), indicating an allosteric agonism. The photolabeling experiments showed high incorporation of the probe into the receptor protein, however, the protein modification was not inhibitable by either alfaxalone or the “cold” 3. The results of photolabeling for will be discussed in the context of the neurosteroid anesthetic mechanism. Further work aiming at site-specific determination of the labeled amino acids is currently underway. A B C

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62

Salvinorin A Analogues for the Development of Addiction Therapies

Rachel Saylor and Thomas E. Prisinzano Department of Medicinal Chemistry, University of Kansas

Lawrence, KS 66045 Over 23 million Americans require treatment for drug or alcohol addiction each year. Limited medication options are currently available for the treatment of opioid and alcohol abuse, but no FDA approved medications exist for stimulant abuse. Development of a therapy for reaching these hundreds of thousands of Americans suffering from stimulant abuse is essential.

Opioid receptors have been the focus of addiction research for many years, mainly due to their role in regulating the reward pathway. Currently approved addiction treatments act through the µ opioid receptor (MOR), but two other opioid receptors, the κ opioid receptor (KOR) and the δ opioid receptor (DOR), are also potentially useful in treating addiction. While activation of the MOR has a rewarding effect that can lead to addiction, activation of the KOR has an anti-rewarding effect. KOR agonists have shown success in treating animal models of cocaine abuse. Thus KOR activation may be a viable option for treating drug addiction.

Many KOR agonists suffer from undesirable side effects common to other opioids due to their structural similarity to morphine. The potent and selective KOR agonist salvinorin A (SVA) is a structurally-unique natural product, lacking a basic nitrogen, and a chemically complex molecule, with a variety of functional groups and stereocenters. While SVA has some interesting and desirable pharmacological effects, it still possesses some undesirable pharmacokinetic (PK) properties such as poor water solubility and bioavailability. By developing analogues of SVA to probe the structure-activity-relationship at the KOR, we hope to identify a point on the molecule that can be modified to address these PK shortfalls without loss of KOR activity.

Previous investigation of the SVA structure has identified the lactone as being tolerant to modifications (Munro, et. al. J. Med. Chem. 2005, 48, 3445). Preliminary results have verified these findings, demonstrating that the C17 position can be selectively modified and that small modifications do not drastically affect KOR activity. Therefore we hypothesize that further exploration at this position may validate the lactone as a point on the molecule through which structural changes can be made that manipulate PK properties while maintaining KOR activity. More diverse C17 analogues have been synthesized and evaluated for KOR activity, and these results suggest that polar substitutions capable of hydrogen-bonding are preferred.

63

Epigenetic Regulation of Cytosine Methylation, Hydroxymethylation, Formylation and Carboxylation by Tet Proteins in Normal and Cancer Cells

Christopher L. Seiler,¥,‡ Jungmin Song,¥,† Molly Andersen,¥ Jenna Fernandez,¥,‡

Fekadu Kassie,¥,† and Natalia Tretyakova¥,‡

Masonic Cancer Center,¥ College of Veterinary Medicine,† and Department Medicinal Chemistry‡ University of Minnesota

Tumors are characterized by profound changes in gene expression, resulting in silencing of tumor suppressor genes, activation of oncogenes, and decreased chromosomal stability. Epigenetic changes are critically important in the development of cancer. The presence of 5-methyl-2′-deoxycytidine (meC) within gene promoter regions typically down-regulates gene expression. In contrast, little is known about epigenetic effects of related cytosine modifications, 5-hydroxymethyl-2′-deoxycytidine (hmC), 5-formyl-2′-deoxycytidine (fC), and 5-carboxyl-2′-deoxycytidine (caC), which are formed through Ten Eleven Translocation (Tet) mediated oxidation of the methyl group on 5mdC. While these modifications are expected to be intermediates in an active demethylation process, there is increasing evidence for their own function. In the present work we employed HPLC-ESI-MS/MS based methodologies to determine the kinetic parameters of DNA methyltransferase 1 (DNMT1) mediated methylation of hmC-, fC-, and caC-containing DNA duplexes. Our results indicate that hmC, fC, and caC interfere with maintenance methylation by DNMT1, potentially leading to passive cytosine demethylation. Additionally, hmC, fC, and caC were quantified in mouse lung tumors induced by tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). We have developed and implemented quantitative proteomics to identify the proteins which recognize meC and caC. Finally, to identify compounds to probe the activity of Tet proteins, a computation screen was completed using NCI Diversity Set IV and an in vitro gel based assay.[figure1]

64

!

Histidine Triad Nucleotide Binding Protein 1 (HINT1): Potential Target for the Treatment of Chronic Pain

Rachit Shah†, Pilar Sànchez-Blàzquez‡, Javier Garzòn‡ and Carston R.Wagner†*

†Department of Medicinal Chemistry, University of Minnesota, USA ‡Cajal Institute of Neuropharmacology, Spain

In periaqueductal grey (PAG) matter of the brain, cross talk between µ-opioid receptors and N-methyl D-aspartate Receptors (NMDAR) results in the development of analgesic tolerance to morphine. Histidine Triad Nucleotide Binding protein 1 (HINT1) mediates this cross talk by co-associating with both receptors. Consequently, HINT1-deficient mice exhibit enhanced morphine-induced analgesia as well as decreased morphine tolerance. Moreover, HINT1 negatively regulates NMDAR activity, which is implicated in neurodegeneration and neuropathic pain modulation. Despite the current evidence of HINT1 involvement in regulating µ-opioid receptor and NMDAR function, the correlation between the role of the HINT1 active site in regulating pain remains unclear. We hypothesize that the HINT1 active site is involved in the development of morphine tolerance and regulating neuropathic pain. To address our hypothesis, we evaluated the effect of a small molecule inhibitor (i-HINT1) and substrate (s-HINT1) of HINT1 in a mouse model against the development of analgesic tolerance to morphine. Following intra-cerebroventricular injection (i.c.v), both i-HINT1 and s-HINT1 were found to enhance the analgesic effect of morphine. In addition, the analgesic effect of morphine was significantly enhanced by the intravenous injection (i.v) of i-HINT1 (at higher doses). Moreover, i-HINT was able to restore the effect of morphine in animals rendered tolerant to morphine (acute tolerance). Next we evaluated the effect of i-HINT1 in neuropathic pain using a Chronic Constricted Injury mouse model (CCI). Unexpectedly, we observed that i-HINT1 was able to reduce sensitivity to neuropathic pain for several hours. In summary, we have shown that the active site of HINT1 is involved in the development of morphine tolerance and modulating neuropathic pain. Targeting HINT1 with small molecule inhibitors provides a potential new approach for the treatment of chronic pain.

65

Modular Total Synthesis Towards the Development of Salvinorin A Inspired Structures

Alexander M. Sherwood, Anil Yilmaz, and Thomas E. Prisinzano Department of Medicinal Chemistry, University of Kansas

Lawrence, KS 66045 The natural product salvinorin A is a hallucinogenic neoclerodane diterpenoid and is a potent agonist at the κ-opioid receptor. As part of our continued efforts to probe its exact mode of binding at the κ-opioid receptor, a modular total synthesis has been designed in order to provide access to previously unattainable compounds inspired by salvinorin A. The synthesis has been designed to specifically explore the role of the C19 and C20 methyl groups on the biological activity of salvinorin A. Our proposed approach also permits other functionality to be explored elsewhere within the molecule. We envision access to the desired products by application of previously established methodology, which has demonstrated that the tricyclic neoclerodane core can be efficiently assembled via transannular Michael reaction cascade of a bisenone 14-membered macrolide. The work presented here represents the current state of our ongoing effort towards the synthesis of the key macrolide intermediate as well as the proposed future strategy to affording the desired salvinorin A inspired compounds.

O

O

O

Ar

R1

O

OTES

Ar

CHO

CO2Me

R1O O

P O

OEtO

EtO

O

O

Ar

OR2O

R1H

H

H H

OOO

MeO2C

O

O

O

H H

Salvinorin A

(This Work)

H

H

19

20

66

Synthesis of dihydroxyanthracenones and naphthopyranones through Houser and Stauton-Weinreb annulations and oxidative coupling reactions

Ernane Souza, Victor Calvario and Horacio F. Olivo Division of Medicinal & Natural Products Chemistry, University of Iowa

Iowa City, IA 52242

The hydroxyanthracenone and naphthopyranone moieties constitute important scaffolds for the total synthesis of naturally occurring secondary metabolites with diverse biological activities, including anticancer, antibiotic, antimalarial, antioxidant and neuroprotection. For instance, the bioxanthracene (-)-ES-242-4, which has been isolated from fungi of the genus Verticillium, exhibits potent activity as N-methyl-D-aspartate (NMDA) receptor antagonist, being a potential chemotherapeutic agent for the treatment of neurodegenerative diseases. The aim of this work is to develop a convergent synthesis of dimeric hydroxyanthracenone derivatives with therapeutic interest. The monomeric hydroxyanthracenones and naphthopyranones were built through two different strategies. The first one involved a Houser annulation, where a phthalide anion underwent 1,4-addition to an α,β-unsaturated ketone, followed by a Dieckmann-type cyclization in one pot and subsequent dehydration of the corresponding alcohol formed. The second approach involved a Stauton-Weinreb annulation, where an ortho-toluate anion underwent a tandem 1,4-addition to an α,β-unsaturated ketone and posterior aromatization. The monomeric anthracenones were dimerized through oxidative coupling reactions. The results presented in this work represent different approaches towards the total synthesis of hydroxyanthracenones and naphtopyranones with eminent pharmacological relevance. Future perspectives rely on developing novel synthetic derivatives for further biological evaluation and determination of structure-activity relationships.

67

Engineering Natural Functional Groups from Leucine and Isoleucine into “Stapling” Amino Acids

Thomas Speltz and Terry Moore

Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612

The solvent-exposed surfaces of proteins comprises unique conformations, such as alpha helices that can stabilize protein-protein interactions by binding to specific grooves on the surface of partner proteins. Peptides mimicking the secondary structure of interacting regions can be used for therapeutic purposes when they are designed to bind at the interface where two proteins interact and disrupt important pathways of signal transduction. One well developed approach for constraining peptides into alpha-helical conformations is to chemically install an all hydrocarbon “staple” via ring closing metathesis of two non-natural alkene-containing amino acids. A common implication when incorporating a chemical staple is that the introduced alkyl chain—meant to be just a constraint—can itself interact with hydrophobic regions on the target protein’s surface and augment the affinity and selectivity characteristics of the amino acid residues from the natural sequence. A specific example of this has been shown in a crystal structure of a stapled peptide derived from steroid receptor coactivator 3 that binds to the estrogen receptor. In this example the hydrocarbon staple interacts with the receptor in place of isoleucine and leucine residues. We hypothesize that enhanced selectivity and potency can be achieved by better reproducing the natural binding surface of SRC3, and we have prepared novel stapling amino acids that incorporate functionality from isoleucine and leucine for use in developing peptides that inhibit the estrogen receptor/steroid receptor coactivator interaction. !

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68

Catch and Release DNA Decoys: Capture and Photochemical Dissociation of Transcription Factors

Nicholas B. Struntz and Daniel A. Harki*

Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455 DNA decoys have been used to regulate transcription of genes in eukaryotes and work by sequestering transcription factors (TFs) through mimicking the DNA binding site that is present in genomic DNA. Recently, through the use of caged molecules, DNA decoys have been used to regulate gene expression in both a spatial and temporal manner. We have developed a ‘catch and release’ mechanism for precise control of transcription factor sequestration using depurination nucleotides. Herein, we demonstrate the ability to capture and photochemical release an endogenous transcription factor heterodimer for the first time using a novel photo-responsive catch and release DNA decoy (CRDD). Photolysis of the CRDD results in formation of abasic sites and truncated products, which abolishes all affinity for TF binding. These results demonstrate that CRDDs can be used to effectively capture and spatiotemporally release TFs, which, until this work, was unattainable. The specificity to the TF is determined by the sequence of the decoy, therefore, it can also be applied to other TFs, which could be used to characterize and find therapeutic targets within many different biochemical pathways and diseases.

69

Synthesis and biochemical evaluation of IP6-biotin

Matthew Summerlin*, Chensong Jiao*, Karol S. Bruzik and Leslyn A. Hanakahi The purpose of this study was to synthesize a biotinylated analogue of inositol hexakisphosphate (IP6-biotin) and assess its ability to bind the Ku protein complex and facilitate DNA repair via non-homologous end joining (NHEJ). IP6-biotin was synthesized starting from myo-inositol by a linear 12-step synthesis. The biotin moiety was attached to the 2-hydroxyl position of inositol via a phosphorylated aminohexyl linker. We found that IP6-biotin was able to stimulate NHEJ relatively similar to unlabeled IP6. We tested its ability to capture the Ku protein and showed that it was able to both out of whole cell extracts as well as recombinant purified protein. We further investigated the role of IP6 in NHEJ by showing that in homogenous whole cell extract, core factors XLF and XRCC4 can be recruited to bind IP6-biotin, but recombinant protein cannot bind specifically to the probe indicating an indirect contact through the Ku protein. Using the probe to manipulate the NHEJ complex, we also discovered that IP6-biotin could be used to pull-down and identify specific proteins that bind polyphosphorylated inositols. Streptavidin dynabeads were ligated with IP6-biotin and used in capture experiments involving the whole HeLa cell homogenate. In addition to Ku, we were able to identify another known IP6 binding protein, CK2, as interacting with the probe. Literature suggested nucleolin as also being an IP6 binding protein. We further explored this idea by expressing epitope-tagged nucleolin (HA-Nuc) and identified nucleolin as the only protein with the epitope tag that was able to bind to the IP6-biotin and be competed off by unlabeled IP6. Overall, our results indicate that IP6-biotin is a useful tool in studies of the role of IP6 in NHEJ, and for identification of cellular proteins that bind inositol polyphosphates.

70

Caspase-7 Allostery Explored with Fragment-Based Drug Discovery

Nicholas R. Vance and M. Ashley Spies Division of Medicinal and Natural Products Chemistry, Department of Pharmaceutical Sciences

& Experimental Therapeutics, College of Pharmacy, The University of Iowa, Iowa City, IA 52242

Apoptosis is executed by a tightly regulated family of proteins known as caspases. This family of cysteine-dependent, aspartate-specific proteases are stored as zymogens (procaspases), which require upstream activation in order to carry out the final steps of apoptosis. The apoptotic (-3, -6, -7, -8, -9) caspases have garnered widespread attention from pharmaceutical companies and academics alike. Executioner (-3, -6, -7) caspases are activated by initiator (-8, -9) caspases to hydrolyze key peptides in structural and regulatory proteins, leading to the characteristic membrane blebbing and DNA fragmentation characteristic of apoptotic cells.1 Models of neurodegenerative disease have shown that caspase-7 knockout and irreversible pan-caspase inhibition can prevent neuronal cell death.2

Executioner caspases (-3, -7) have a preference for the negatively charged, synthetic, tetra-peptide substrate, DEVD, and molecules targeting this active site will have difficulty penetrating the blood-brain barrier. Thus, we hypothesize that small molecules targeting an allosteric site on caspase-7 will impart caspase specificity and more favorable physicochemical properties. Furthermore, since high-throughput screening campaigns have failed to produce drug-like, lead molecules capable of allosterically inhibiting caspase enzymes, we propose that Fragment-Based Drug Discovery will provide the diversity necessary to discover lead scaffolds binding this allosteric site. Fifteen commercially available fragment libraries were aggregated and evaluated for their diversity, cost, and adherence to the ‘Rule of 3’ physicochemical properties. A fragment library was screened against caspase-7 using the thermal shift assay, also known as differential scanning fluorimetry. Twenty hits were selected from this primary screen and were further characterized using surface-plasmon resonance and biochemical assays. Thus far, two novel, non-competitive inhibitors of caspase-7 have been discovered.

1. McIlwain, D.R.; Berger, T.; Mak, T.W. Caspase Functions in Cell Death and Disease. Cold Spring Harb. Perspect. Biol. 2013, 5, 1-28.

2. Burguillos et al. Caspase signaling controls microglia activation and neurotoxicity. Nature. 2011, 473, 319-324.

71

Neuronal Toxicity due to Reactive Dopamine Metabolites and Fungicide Exposure

Brigitte Vanle, Virginia Florang, Jonathan Doorn Parkinsons disease (PD) is a slow-progressive neurodegenerative disorder affecting 5-6 million people around the globe. The disease is manifested by the rapid deterioration of dopaminergic cells in the substantia nigra portion of the brain; however, the pathological mechanism of this selective dopaminergic neuronal death is unknown. Dopamine is metabolized by monoamine oxidase to form the endogenous neurotoxin 3,4-dihydroxyphenylacetaldehye (DOPAL). This DOPAL metabolite is of great interest to our group as the reduction in levels of DOPAL is biologically critical, given this aldehyde is highly toxic to dopaminergic cells and is a highly reactive to proteins and DNA Investigating neuronal protein targets is essential in determining the cause of toxicity. An essential protein-GAPDH (e.g., glyceraldehyde-3-phosphate dehydrogenase) is an abundantly expressed enzyme known for its glycolytic activity and recent research has implicated its role in oxidative stress-mediated neuronal death. This work positively shows GAPDH as a target for DOPAL modification and is a potent inhibitor for GAPDH enzymatic activity. This enzyme inhibition is also time and DOPAL dose-dependent. In addition to finding a novel enzyme inhibited and modified by DOPAL, This work has also confirmed linking DOPAL levels to a fungicide associated with PD risk. This benzimidazole fungicide, benomyl was preliminarily shown to inhibit ALDH2 in vitro enzyme assays. However, inhibition of ALDH2 by benomyl has not been tested in immortalized cell lines or metabolites measured within cells. The ratios of DOPAL and product of ALDH were measured by HPLC-ECD, and found that benomyl does inhibit ALDH2 in relevant cell models. The cytotoxicity of benomyl, DA, DOPAL and the combination of DA or DOPAL with benomyl was assessed by MTT assay. It is of interest to exogenously treat cells with DA as DA is readily transported into cells and metabolizes into DOPAL within the cells. Surprisingly, the only toxic combination was the combination of DA or DOPAL with benomyl. In fact, this toxicity appears to be synergistic as none of the single treatments are significantly toxic to the cells. This synergistic effect also affects GAPDH aggregation. When cells are treated with the combined treatment, there is greater intracellular GAPDH aggregation, highlighting further that GAPDH is a relevant protein targeted by DOPAL. Exposure to environmental toxins such as pesticides and fungicides have long been linked to PD risk, and only recently to DOPAL levels. This work provides a novel mechanism by which fungicide exposure may stimulate PD pathogenesis.

72

!

Licochalcone A from Glycyrrhiza inflata Modulates P450 1B1-mediated Estrogen Oxidative Metabolism in MCF-10A Cells

Tareisha Dunlap, Shuai Wang, Charlotte Simmler, Guido Pauli, Birgit Dietz, and Judy L. Bolton Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of

Illinois at Chicago, Chicago, IL 60612

The risk of developing breast cancer increases with cumulative exposure to estrogens. Estrogens are known to stimulate cell proliferation (hormonal carcinogenesis) and are metabolized by P450 1B1 to 4-OH metabolites that are oxidized to genotoxic quinones (chemical carcinogenesis), both contributing to breast cancer risk. Furthermore, inflammation potentiates estrogen oxidative metabolism and P450 1B1 expression (inflammation-stimulated estrogen carcinogenesis). In normal breast cells (MCF-10A), TNF-alpha and IFN-gamma up-regulate P450 1B1 mRNA expression, which is inhibited by either a nuclear factor-kappaB (NF-kB) inhibitor or aryl hydrocarbon receptor (AhR) antagonist. We tested three licorice species, Glycyrrhiza glabra (GG), inflata (GI), and uralensis (GU), to determine their effect on inflammation and P450 1B1-mediated estrogen metabolism. We first compared their anti-inflammatory activity; GI more significantly inhibited iNOS activity than GG and GU. A constituent in all three licorice species isoliquiritigenin (LigC) and the marker compound from GI licochalcone A (LicA) inhibited iNOS activity also. We then investigated the effect of licorice on inflammatory-driven P450 1B1 expression; GG, GU, and LigC in combination with cytokines additively increased expression, yet GI and its marker compound LicA reduced P450 1B1 expression. Similarly, GG, GU, and LigC increased estrogen metabolism to 4-OH metabolites, yet GI and LicA reduced estrogen oxidative metabolism. LicA also inhibited XRE-luciferase reporter activity in the presence of TCDD, indicating that it is an antagonist of AhR. Our data strongly suggest that GI and LicA inhibit inflammation and P450 1B1-mediated estrogen oxidative metabolism, and may protect women against estrogen carcinogenesis.

Supported by P50 AT00155 and T32 AT007533 from the Office of Director, National Institutes of Health (OD) and the National Center for Complementary & Integrative Health (NCCIH).

73

Hepatocyte Targeted Endosomal Escape Agents

Christopher W. White, Nicholas J. Baumhover, Samuel T. Crowley, and Kevin G. Rice Division of Medicinal and Natural Products Chemistry, College of Pharmacy, The University of

Iowa, Iowa City, IA

Endosomal escape is a current bottle neck in the development of non-viral gene delivery vectors. We propose the use of novel endosomal escape agents to enhance transfection efficiency of our peptide-based vector, potentially leading to unstimulated gene expression. Three major strategies have emerged, one that utilizes membrane lytic peptides, a second that uses proton sponge polymers, and a third that uses an enzyme to catalyze the cleavage of phospholipids. Each novel endosomal escape agent is designed to target liver hepatocytes through the asialoglycoprotein receptor. The present study compares these approaches by conducting 384-well in vitro transfections on primary hepatocytes, as previously described.1

Melittin (Mel), a 26-mer membrane lytic peptide, was modified to contain an N-terminal Cys. Triantennary N-glycan (Tri), a potent high affinity asialoglycoprotein receptor ligand, was purified from bovine fetuin and chemically modified to contain a free thiol. Following bioconjugation with melittin, the membrane lytic activity of the resulting disulfide linked Tri-S-S-Mel (Figure 1) is abrogated. Therefore, the molecule can be safely i.v. dosed in mice and only upon bioactivation does the peptide regain activity, as determined in RBC hemolysis assays. A second strategy involves the synthesis of a proton sponge polymer based on the 25 kDa branched polyethylenimine (PEI) backbone. The many 1° and 2° amines of PEI were modified with D-Lactose by reductive amination and with 4-imidazoleacetic acid using EDC/NHS coupling, see Figure 2. These modifications serve many functions including: reduction of electrostatic potential at pH 7.5, targeting to the asialoglycoprotein receptor, reduction of toxicity, and increased buffering capacity. Early generation polymers demonstrated improved lethality profiles when i.v. dosed via mouse tail vein in comparison to PEI.

Our final strategy utilizes the membrane disruptive enzyme Phospholipase A2 (PLA2). PLA2 is a 15.2 kDa enzyme that cleaves the sn-2 acyl bond of phospholipids, making it a potential endosomal escape agent. To effectively target PLA2 to liver hepatocytes, multiple triantennary residues were covalently linked to PLA2 through disulfide bond linkage. These novel endosomal escape agents are being compared for their ability to mediate in vitro gene transfer of glycan targeted DNA and mRNA in miniaturized primary hepatocyte transfection assays.

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!1. Li J, Crowley ST, Duskey J, Khangharia S, Wu M, Rice KG. Miniaturization of gene transfection assays in 384- and 1536-well microplates. Analytical Biochem. 2015; 470: 14-21.

74

Natural Product Inspired Cysteine Reactive Probes for Cancer Target Discovery

John C. Widen, Aaron M. Kempema, Joseph K. Hexum, and Daniel A. Harki* Department of Medicinal Chemistry, University of Minnesota, 2231 6th St. SE, Minneapolis, MN

55455 Helenalin and parthenolide are two plant-derived sesquiterpene lactone natural products that contain an exocyclic methylene butyrolactone. This moiety reacts with cysteines within a cell proteome forming a covalent linkage between the target protein and the natural product via a hetero-Michael addition. Our lab and others have demonstrated that the exocyclic methylene butyrolactone pharmacophore is necessary for their biological activity, which includes anti-inflammatory, anti-proliferative, bactericidal, and fungicidal effects. Both compounds have also been shown to target cancer stem cell and cancer stem-like cells, which have been proposed to cause drug resistance and relapse in patients. Multiple molecular targets have been suggested to interact with both compounds, but none of these targets explain the unique cancer stem cell targeting effect. We have taken advantage of the covalent reactivity of helenalin and parthenolide to develop alkyne-functionalized probes for the discovery of protein targets that are responsible for cancer stem cell survival.

75

Identification of Potential BRDT Inhibitors by Fragment-based Screening Using Differential Scanning Fluorimetry

Andi Wisniewski, Kristen John, Jon Hawkinson, Gunda I. Georg

University of Minnesota Department of Medicinal Chemistry and Institute for Therapeutics Discovery and Development

Minneapolis, MN 55414 Bromodomain (BRD) containing proteins are essential for acetylated lysine (Kac) recognition on histone proteins during transcriptional activation. BRDT is a testis-specific BRD protein required for male germ cell differentiation and proves an interesting target for non-hormonal male contraception. Differential scanning fluorimetry (DSF), a thermal shift assay, was used to detect protein binding in an initial screen of a fragment library, with a hit identified when the transition temperature (Tm) was shifted ≥2 °C above the baseline (ligand free) Tm, indicating increased protein stability. Several confirmed hit compounds were purchased and sent to collaborators for crystallographic studies. Only one fragment resulted in a crystal structure (TL00757), indicating a need to revisit the DSF assay conditions. Optimization of several assay parameters, including the concentrations of protein, fragments, and DMSO, was performed. Assay validation was executed using BET inhibitors reported in the literature as positive control compounds and to create dose response curves, such as (+)-JQ-1. Using the optimized assay conditions, two fragment libraries were screened, identifying a number of compounds that met the new hit criteria: shifting the Tm ≥ 1 °C (a positive hit) or ≤ 5 °C (a negative hit). Of these hits, 93 were chosen for confirmation via dose response using the DSF assay; encouragingly, the hit fragment identified during the initial screen (TL00757) was shown to be the most active positive hit. The ten positive hits and the best performing negative hits were repurchased and will be retested in the DSF assay before being confirmed using protein-observed 19F-NMR spectroscopy with fluorine-labeled BRDT. Confirmed hit compounds will be co-crystallized with BRDT to provide data on binding interactions. Selected hits will undergo rounds of hit-to-lead optimization and structure activity relation manipulations to refine lead compounds to create potent and selective BRDT inhibitors. Dose Response of TL00757 (GPHR-00278821)

-4 -3 -2

-1

0

1

2

3

4

5

Log (M)

Del

ta T

m

O

NH

O

GPHR-00278821TL00757

Avg. ∆Tm at 5 mM = 3.8 °C

76

Withanolides from Physalis coztomatl

Huaping Zhang,† Cong-Mei Cao,† Robert J. Gallagher,† Victor W. Day,‡ Kelly Kindscher,! and Barbara N. Timmermann*,†

† Department of Medicinal Chemistry, School of Pharmacy, University of Kansas ‡ The Small-Molecule X-ray Crystallography Laboratory, University of Kansas

! Kansas Biological Survey, University of Kansas Lawrence, KS 66047

Six new withanolides (1-6), as well as two known withanolides (physachenolide D 7 and withanoside VI 8), were isolated from the aerial parts of Physalis coztomatl (Solanaceae). Structural elucidations of 1-6 were achieved through 2D NMR and other spectroscopic techniques, while the structure of 1 was confirmed by X-ray crystallographic analysis. In addition, the stereochemical orientation of the 17-hydroxy group in withanolides was discussed in relation to 13C NMR shifts of C-12. Such analysis revealed that coagulansin A in the literature contains a 17α-hydroxy moiety rather than the reported 17β-hydroxy functionality, and this structure has been revised accordingly.

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53rd Annual MIKI Attendees Minnesota Evan Alexander Elizabeth Ambrose Amin Matthew Bockman Emily Boldry Joseph Buonomo Jin Cai Erick Carlson Denise Casemore Sara Coulup Dibyendu Dana Skye Doering Carter Eiden Bassem Elsherbini Jenna Fernandez Barry Finzel Katlyn Fleming Gunda Georg Arnie Groehler Xianghong Guan Daniel Harki David Huang Shaofei Ji Jiewei Jiang Oxgun Kilic Elbek Kurbanov Jill Kyzer Cody Lensing Feng Liu Shams-ul Mahmood Kimberly Maize Aniekan Okon Margaret Olson Fatemeh Oroojalian Kellan Passow Jacob Petersburg Jean Leandro dos Santos Christopher Seiler Rachit Shah Jingjing Shen Tom Shier Kristen Stoltz Alex Storm Nicholas Struntz Zinayyera Subhani Trent West John Widen Andi Wisniewski Adam Zarth Bo Zhou Yu Zhu

Illinois Manel Ben Aissa Judy Bolton Rhea Bovee Karol Bruzik Joanna Burdette Skylar Carlson Luying Chen Wei-Lun Chen Camila Manoel Crnkovic Brian David Maryam Elfeki Michael Federle Sarah Green Brian Guo Lauren Gutgesell Atieh Hajirahimkhan Thomas Hanigan Caitlin Howell Lingyi Huang Atul Jain Ammar Jastaniah Chensong Jiao Brian Lallier Sue Lee Jing Li Guannan Li Yunlong Lu Daniel May Obinna Mbachu Terry Moore Michael Mullowney Brian Murphy Vanessa Nepomuceno Daniel Nosal Jimmy Orjala Hitisha Patel Pavel Petukhov Benjamin Richardson Loruhama Delgado Rivera Michael Rush Pavel Savechenkov Anam Shaikh Thomas Speltz Peter Sullivan Matthew Summerlin Greg Thatcher Emily Thayer Douglas Thomas Sarah Tincher Shuai Wang Charles Woodbury Rui Xiong

Kansas Ryan Altman Brett Ambler Jeff Aubè Mallesh Beesu Brian Blagg Jane Buttenhoff Katherine Byrd Congmei Cao Guiseppe Caruso Andie Cassity Manwika Charaschanya Kasabi Chinonge Michael Clift Kevin Criscione Vincent Crowley Sunil David Rachel Davis Melissa Denler Apurba Dutta Saqib Faisal Leah Forsberg Gavin Zhe Gao Gaurav Garg Solomon Gisemba Suman Ghosh Gary Grunewald Robert Hanzlik Ben Haugeberg Casey Henderson Abu Gafar Hossion Jordan Hunt Moon Hur Prashi Jain Salim Javeed Stephanie Johnson Jung Ho Jun Anuj Khandelwal Kelsey Knewtson Molly Lee Martin Leon Xinyun Liu Joana Loh Huiyong Ma Soma Maitra Pradip Maitry Jana Markley Mary McDonald James Meinig Sanket Mishra Lester Mitscher Hashim Motiwala Douglas Orsi Chamani Perera Blake Peterson Elyse Petrunak Johnny Phan Sahishna Phaniraj Thomas Prisinzano

Iowa Arturo Aguirre Rondine Allen Laura Folly da Silva Constantino Ioana Craciun Sondra Dean Justine Delgado Jon Doom Mike Duffel Michael Hayes Colin Higgins Aaron Kern Robert Kerns Chaitanya Kulkarni Ellisa Mullen Victoria Parker Dave Roman Josephine Schamp Ernane Souza Ashley Spies Nicholas Vance Brigitte Vanle Chris White Benjamin Williamson Kansas (cont.) Mike Rafferty Digamber Rane Christianna Reedy Andrew Riley Sudeshna Roy Alex Salyer Rachel Saylor Sarah Scarry Alexander Sherwood Gurpreet Singh Barbara Timmermann Rakesh Vekariya Caleb Vogt Nicole Windmon Peter Wirth Christina Wong Ming-Hsui Yang Anil Yilmaz Qin Yin Euna Yoo Zheng Zhang Hank Huaping Zhang Qian Zhang

Conference Locations

A Hotel Best Western Lawrence 2309 Iowa Street B All oral/poster sessions and keynote lecture University of Kansas School of Pharmacy 2010 Becker Drive C Friday night mixer The Oread Hotel 1200 Oread Avenue D Saturday night banquet Abe and Jake’s Landing

8 East 6th

Street

Directions from Best Western to KU School of Pharmacy

Start at Best Western (A) Head North on South Iowa

(0.3 miles)

← Turn left on 21st Street

(334 ft)

→ Turn right on Becker Drive (0.2 miles) Continue to School of Pharmacy (B) on left

Location

P.M. A B

11:30

A.M. 12:05 12:10

12:45 12:50

1:25 1:30

1:35 1:40

Map of Downtown Lawrence/Late Night Shuttle Service

Shuttle service will be provided between downtown Lawrence and the Best Western for all registrants on Saturday Night from 11:30 PM to 2 AM. Shuttle will pick up MIKI registrants only at points A & B.

The MIKI Late Night Shuttle Bus will display “PRIVATE SHUTTLE” on the front of the bus.

KU/Lawrence buses 52/53/54/55 WILL NOT take you to the Best Western.

See below for scheduled pick-up times.

* Indicates Abe & Jake’s Landing