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Design and synthesis of novel topoisomerase II[alpha] inhibitors

Material Information

Title:
Design and synthesis of novel topoisomerase II[alpha] inhibitors
Creator:
Abraham, Adedoyin David
Place of Publication:
Denver, CO
Publisher:
University of Colorado Denver
Publication Date:
Language:
English
Physical Description:
xiv, 65 leaves : ill. (some col.) ; 28 cm.

Subjects

Subjects / Keywords:
DNA topoisomerases ( lcsh )
Antineoplastic agents ( lcsh )
Marine pharmacology ( lcsh )
Adenosine triphosphatase ( lcsh )
Adenosine triphosphatase ( fast )
Antineoplastic agents ( fast )
DNA topoisomerases ( fast )
Marine pharmacology ( fast )
Neoamphimedine
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (M.S.)--University of Colorado Denver, 2011. Department of Chemistry
Bibliography:
Includes bibliographical references (leaves 62-65).
Statement of Responsibility:
by Adedoyin David Abraham.

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Source Institution:
University of Colorado Denver
Holding Location:
|Auraria Library
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
782060639 ( OCLC )
ocn782060639

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DESIGN AND SYNTHESIS OF NOVEL TOPOISOMERASE Ila INHIBITORS by Adedoyin David Abraham B.S., University of Ilorin, 2005 A thesis submitted to the University of Colorado Denver in partial fulfillment of the requirements for the degree of Master of Science Chemistry December 2011

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This Thesis for the Masters degree by Adedoyin David Abraham has been approved by Dr. Daniel LaBarbera Dr. Scott Reed

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Abraham, Adedoyin David (M.S. Chemistry) Design and synthesis of novel topoisomerase II a inhibitors Thesis directed by Assistant Professor Daniel V. LaBarbera ABSTRACT Neoamphimedine belongs to the family of compounds known as pyridoacridines, consisting of natural derivatives isolated from marine sponges as well as synthetic derivatives. Neoamphimedine was isolated from the marine sponge Xestospongia sp. and subsequently shown to have potent antitumor activity against a variety of cancer types in vitro and in vivo. Further evaluation of neoamphimedine determined that it is a novel A TP-competitive inhibitor of topoisomerase Ila (Topolla). The main objectives of this research were to; a) synthesize neoamphimedine for further inhibition studies against Topolla; and b) rationally design and synthesize more potent derivatives for the development of novel inhibitors ofTopolla and potential therapeutics for the treatment of cancer. Molecular modeling of neoamphimedine support in vitro inhibition studies with Topolla, which indicate that neoamphimedine binds in the N-terminal domain A TPase site. Using these in silica modeling methods we have rationally designed a series neoamphimedin derivatives, which bind to the ATPase site of Topolla with

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higher predicted binding affinity than the natural product. In addition, we successfully synthesized two novel derivatives of this series. Finally, we conducted in vitro decatenation assay with one of the derivatives, which proved to be as potent as neoamphimedine at inhibiting Topoiia-dependent DNA decatenation validating our rational drug design methodology. Future work will focus on structure activity relationships and drug development of this novel class of Topoiia inhibitors. This abstract accurately represents the content oft its publication.

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ACKNOWLEDGEMENT I wish to express my profound gratitude to God Almighty who in His mercy, love, and guidance saw me through the course of my study (Isaiah 43:2). I would like to express gratitude towards my supervisor, Dr. Daniel LaBarbera, for giving me a tremendous amount of encouragement and inspiration in my endeavor to become a researcher. Without his support and continual encouragement, none of this would have been possible. You are truly an inspiration to me. I would like to extend my sincere gratitude to my co-supervisor, Dr. Douglas Dyckes for his wisdom and guidance throughout my project. I also would like to thank Dr. Philip Regan for his guidance and assistance on the molecular modeling aspect of this project. To the rest of the members of the LaBarbera Lab group--both past and present, Jessica Ponder for her excellent job on the molecular docking of the phenol derivative, and Byong Hoon Yoo, who did excellent job on the DNA decatenation assay. I greatly acknowledge my parents, the late Pa Luke Abraham, and Omolola Grace Abraham for their support throughout my entire life and for giving me the education in order to be a true gentleman and a hard-working individual. I also would like to thank Pastor Temitayo Obigbesan and Marion Obigbesan for their moral and financial support. Finally, my profound gratitude goes to my wife Blessing and my son Daniel for their support, love, and ceaseless words of encouragement.

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TABLE OF CONTENT List of figures ................................................................................... viii List of Tables .................................................................................... xii List of Abbreviations .......................................................................... xiii Chapter 1. Introduction .................................................................................... I 1.1 Topoisomerase II a (Topolla) .............................................................. 1 1.2 Topoisomerase Ila inhibitors from marine origin ...................................... .4 1.2.1 Neoamphimedine .......................................................................... 5 2. Synthesis ofNeoamphimedine ............................................................... 8 2.1 Results and Discussion ..................................................................... 8 2.2 Ethyl-2-nitrobenzoylacetate ................. ......................................................... 9 3. Rational design ofNeoamphimedine derivatives ....... ...................................... 11 3.1 Iminoquinone derivatives ofNeoamphimedine ........................................ 11 3.2 Synthesis of 17 (AA-119) ................................................................. 16 3.3 Phenol derivatives ofNeoamphimedine ................................................ 17 3.4 Synthesis of I(AA-67) ..................................................................... 21 3.5 Decatenation assay with Neoamphimedine and AA-67 .............................. 22 3.6 Synthesis of novel Imidazoquinoline derivatives ofNeoamphimedine ............ 26 vi

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3.7 Conclusion .................................................................................. 28 4. Experimental. ........................................ -.......................................... 29 4.1 General experimental procedure ......................................................... 29 4.2 Computational Methods ................................................................... 30 4.3 Syntheses .............................................................................................. 33 4.4 DNA Decatenation Assay with neoamphimedine and with AA-67 ................. .40 Appendix ......................................................................................... 42 A: Supplementary analytical data ............................................................. .42 B: Supplementary molecular modeling data ................................................ 60 References ........................................................................................... 62 vii

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LIST OF FIGURES FIGURE 1.1 CARTOON STRUCTURE OF TO PO ISOMERASE Ilao 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 FIGURE 102 STRUCTURAL ISOMERS OF THE PYRODOACRIDINESO 0 0 0 0 0 0 0 0 0 03 FIGURE 10201: CHEMICAL STRUCTURES OF AA-67, AND AA-26 AND COMPOUND 220 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0006 FIGURE 20101 SYNTHESES OF NEOAMPHIMEDINE INTERMEDIATES 0 0 0 0 0 0 0 0 0 8 FIGURE 20102 SYNTHESIS OF NEOAMPHIMEDINEoooooooooooooooooooOOOOOOOOOOOOoooo9 FIGURE 202 SYNTHESIS OF ETHYL-2-NITRO BENZOYLACETATEoooooooooooo10 FIGURE 3.1 THE CRYSTAL STRUCTURE OF THEN-TERMINAL ATPASE SITES OF TOPOIIA BOUND TO NEOAMPHIMEDINE 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0011 FIGURE 301.1 AA-119 INTERACTIONS WITH THE ATPASE SITE OF TOPOIIaooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo15 FIGURE 302 SYNTHESIS OF AA-119oooooooooooooooooooooooooooooooooooooooo 000000000000016 FIGURE 303 AA-26 INTERACTIONS WITH THE ATPASE SITE OF TOPOIIaooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo19 FIGURE 30301 AA-67INTERACTIONS WITH THE ATPASE SITE OF TOPOIIaooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo20 FIGURE 3.4 SYNTHESIS OF AA-67oooooooooooooooooooooooooooooooooooooooooooooooooooooo22 FIGURE 305 PERCENT INHIBITION CURVES GENERATED FROM DECATENATION ASSAYS 0 0 00 0 0 000 0 0 0 0 0 0 00 0 0 0 0 00 0 00 0 0 0 0 0 00000 0 00 0 0 0 000000 oo 0 0 00 000 Ooo 0 0 0023 FIGURE 306 BENZIMIDAZOLE COUPLED PRODUCToooooooooooooooooooooooooooooo27 FIGURE 30601 3, 6-DIMETHOXY-1, 2,4-TRIACETAMIDOBENZENEooooooooooooo27 viii

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FIGURE Al: 1H NMR SPECTRUM OF ETHYL-2-NITROBENZOYLACETATE .................................................. 42 FIGURE A-2 AROMATIC REGIONS OF ETHYL-2NITROBENZOYLACET ATE .............................................................. .43 FIGURE A-3 1H NMR SPECTRUM OF 7-ACETAMID0-4(-2ACET AMIDOPHENYL)-5,8 DIMETHOXYQUINOLINE ............................. .43 FIGURE A-4 AROMATIC REGIONS OF OF 7-ACETAMID0-4(-2ACET AMIDOPHENYL)-5, 8-DIMETHOXYQUINOLINE ............................ 44 FIGURE A-5:13C NMR SPECTRUM OF 7-ACETAMID0-4(-2ACET AMIDOPHENYL)-5,8-DIMETHOXYQUINOLINE ............................. .44 FIGURE A-6 IR SPECTRUM OF 7-ACET AMID0-4 ( -2-ACET AMIDOPHENYL)-5,8DIMETHOXYQUINOLINE ......................... .45 FIGURE A-7 [M +Ht SPECTRUM OF 7-ACETAMID0-4(-2ACET AMIDOPHENYL)-5,8-DIMETHOXYQUINOLINE ............................ .45 FIGURE A-8 1H NMR SPECTRUM OF AA-67 .......................................... .46 FIGURE A-9 AROMATIC REGIONS FOR THE 1H NMR FOR AA-67 ............ .46 FIGURE A-10 [M +Ht SPECTRUM OF AA-67 ....................................... .47 FIGURE A-ll 13C NMR SPECTRUM OF AA-67 ...................................... .47 FIGURE A-12: IR SPECTRUM OF AA-67 ............................................... .48 FIGURE A-13 IR SPECTRUM FOR 7-ACETAMID0-4(-2ACET AMIDOPHENYL) QUINOLINE-5, 8-QUINOLINEDIONE ................... .48 FIGURE A-14 [M +Ht SPECTRUM FOR 7-ACETAMID0-4(-2ACET AMIDOPHENYL) QUINOLINE-5,8-QUINOLINEDIONE .................... .49 FIGURE A-15 1H NMR SPECTRUM OF AA-119 ...................................... .49 FIGURE A-16 AROMATIC REGIONS FOR THE 1H NMR OF AA-119 ............ 50 ix

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FIGURE A-17 [M +Hf SPECTRUM OF AA-119 ....................................... 50 FIGURE A-18 IR SPECTRUM OF AA-119 ............................................... 51 FIGURE A-19 1H NMR SPECTRUM OF 2, 5-DIMETHOXY-3, 6DINITROACETANALIDE ................................................................... 51 FIGURE A-20 13C NMR SPECTRUM OF 2, 5-DIMETHOXY-3, 6-DINITROACET ANAL IDE ................................................................... 52 FIGURE A-21 IR SPECTRUM OF 2, 5-DIMETHOXY-3, 6DINITROACET ANALIDE ................................................................... 52 FIGURE A-22 [M +Hf SPECTRUM OF 2, 5-DIMETHOXY-3, 6-DINITROACET AN ALIDE ................................................................... 53 FIGURE A-23 1H NMR SPECTRUM OF 4, 7-DIMETHOXY-2-METHYL-6NITROBENZIMIDAZOLE ................................................................... 53 FIGURE A-24 1H NMR SPECTRUM OF BENZIMIDAZOLE COUPLED PRODUCT ....................................................................................... 54 FIGURE A-25 AROMATIC REGIONS FOR 1H NMR OF COUPLED BENZIMIDAZOLE ............................................................................ 54 FIGURE A-26 IR SPECTRUM OF BENZIMIDAZOLE COUPLED PRODUCT ....................................................................................... 55 FIGURE A-27 13C NMR SPECTRUM OF BENZIMIDAZOLE COUPLED PRODUCT ....................................................................................... 55 FIGURE A-28 [M +H] SPECTROMETRY DATA OF COUPLED BENZIMIDAZOLE ........................................................................... 56 FIGURE A-29 [M -Hr SPECTOMETRY DATA OF COUPLED BENZIMIDAZOLE ........................................................................... 56 FIGURE A-30 1H NMR SPECTRUM FOR 3, 6-DIMETHOXY-1, 2-4TRIACETAMIDOBENZENE ................................................................ 57 FIGURE A-31: AROMATIC REGIONS FOR 3, 6-DIMETHOXY-1,2-4TRIACET AMIDO BENZENE ................................................................ 57 X

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FIGURE A-32 13C NMR SPECTRUM FOR 3,6-DIMETHOXY-1,2-4TRIACETAMIDOBENZENE ............................................................... 58 FIGURE A-33 IRSPECTRUM FOR 3,6-DIMETHOXY-1,2-4TRIACETAMIDOBENZENE ................................................................ 58 FIGURE A-34 [M +Ht FOR 3,6-DIMETHOXY-1,2-4-TRIACETAMIDO BENZENE ....................................................................................... 59 FIGURE B-1 TOPOLlD INTERACTIONS WITH THE ATPASE SITE OF TOPOIIa ........................................................................................ 60 FIGURE B-2 TOPOL2B INTERACTIONS WITH THE ATPASE SITE OF TOPOIIa ........................................................................................ 61 xi

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LIST OF TABLES Table 1: Computational analysis of novel iminoquinone derivatives of neoamphimedine . ... . ... .. . ... .... ...................... . . .......... . 13 Table 2: Computational analysis of AA-67 and AA-26 ......... ... . ....... ............. 18 Table 3: Computational analysis ofnovel Imidazoquinoline derivatives of neoamphimedine ....................................... .................... . 24 xii

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LIST OF ABBREVIATION Ac20: Acetic anhydride ACN: Acetonitrile AcOH: Acetic acid ADP: Adenosine diphosphate Ala: Alanine Arg: Argenine Asn: Asparagine Asp: Aspartic acid A TP: Adenosine triphosphate C: Carbon CAN: Ceric ammonium nitrate DCM: Dichloromethane OEM: Diethyl malonate DMF: Dimethylformamide DMSO: Dimethyl sulfoxide DNA: Deoxyribonucleic acid EtOAc: Ethylacetate GB: Generalized Bor Glu: Glutamic acid xiii

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Gly: Glycine lie: Isoleucine kDNA: Kinetoplastid Deoxyribonucleic acid Lys: Lysine MeOH: Methanol PBSA: Poisson Boltzmann with non-polar Surface Area Pd: Paladium Phe: Phenyl alanine PPA: Poly phosphoric acid pTSA: Para toluene sulfonic acid ROS: Reactive Oxygen Species Ser: Serine TEA: Triethylamine Tf20: Trifluoromethanesulfonic anhydride TLC: Thin Layer Chromatography Topolla: Topoisomerase lla Thr: Threonine Tyr: Tyrosine Vmax Maximum initial rate of reaction xiv

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I. INTRODUCTION 1.1 TOPOISOMERASE lla (TOPOIIa) Topoisomerase II is a ubiquitous enzyme that belongs to the GHKL family (gyrase, Hsp90, histidine kinase, MutL), and has been shown to be evolutionarily conserved in eukaryotes and essential for modulating the topology constrains on DNA (Bates, Berger and Maxwell; Chene et al.; Linka et al.; Wang). In Mammal, topoisomerase II exist as two isoforms, alpha and beta but display differences in expression and in sub-cellular localization especially at the time of mitosis (Bates, Berger and Maxwell; Chene et al.; Hande; Linka et al. ). Topoiia is an enzyme that plays a critical role m cell cycle progression (Nitiss). In addition to relaxing supercoiled DNA, Topoiia catalyzes the catenation (linking) and decatenation of DNA; decatenation must occur before M phase chromosome segregation to prevent chromosome breakage and resulting lethality from mitotic catastrophe (Nitiss). Consequently, Topolla is an important molecular target linked to tumor proliferation and progression in several types of cancer (Azarova et al.; Nitiss). Topolla poisons that inhibit decatenation by stabilizing the cleavable complex with DNA have shown clinical success. However, stabilizing the cleavable complex leads to DNA strand breaks, chromosomal translocations and non specific cytotoxicity, contributing to adverse side effects and secondary malignancies

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(Figure 1.1, modified from Corbett and Berger, and Nitiss) (Corbett and Berger; Nitiss). FIGURE 1.1 CARTOON STRUCTURE OF TOPOISOMERASE lla. Clinically used Topolla poisons etoposide and ICRF -193 and their respective binding sites (left). Topolla-DNA complexs stabilized by ICRF-193 (closed clamp) or etoposide (cleaved complex), leading to non-specific toxicity and adverse side effects (right). Topolla has been shown to interact with numerous proteins that can affect Topolla activity, stability, localization, and multidrug resistance (MDR) (Nitiss; Williamson et at.; Wray, Williamson, Sheema, et at.). Metnase, a recently evolved protein methylase-nuclease fusion protein, interacts with Topolla and promotes 2

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Topolla-dependent DNA decatenation (Williamson et al.). Recent studies of the Metnase-Topolla interaction revealed a potential mechanism of resistance to Topolla poisons in breast carcinomas and leukemia (Wray, Williamson, Royce, et al.; Wray, Williamson, Sheema, et al.). Specifically, Metanase was shown to stimulate Topolla dependent decatenation and promote tumor cell growth in the presence of the Topolla poisons etoposide and doxorubicin, and may be an important factor in MDR in malignant cancers overexpressing Metnase (Wray, Williamson, Royce, et al.; Wray, Williamson, Sheema, et al.). Therefore identifying novel inhibitors of Topolla, which do not stabilize the cleavable complex, may have greater therapeutic value. In addition, this therapeutic approach may be of clinical benefit where resistance to conventionally used Topolla inhibitors is observed. 0 0 Neoamphimedlne 0 Amphlmedlne Deoxyamphimedine FIGURE 1.2 STRUCTURAL ISOMERS OF THE PYRODOACRIDINES 3

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1.2 TOPOISOMERASE lla INHIBITORS FROM MARINE ORIGIN A number of promising compounds from marine sources have been identified and characterized for cancer treatment. These marine natural products are known to exist as secondary metabolites in various marines' organisms, which includes sponges, tunicates and ascidians, soft corals, sea squirts, and bryozoans (Faulkner; Shinkre et al.). The various types of compounds that have been identified and characterized as being from marine organisms represent many different structural classes, which includes polyethers, terpenoids, alkaloids, macrolides, and polypeptides (Faulkner). In particular, the pyridoacridines are marine alkaloids that contain a planar aromatic skeleton (Matsumoto et al. ). Pyridoacridines have been shown to display potent biological activities such as antitumor, anti-viral, fungicidal, anti-parasitic and bactericidal properties (Marshall). The cytotoxicity of this class have been linked to their DNA binding abilities, inhibition of topoisomerase II and the production of reactive oxygen species (Marshall et al. ). Amphimedine, the parent compound of these manne alkaloids was first reported in 1983, and since then, many additional examples have been described, and a number of papers have appeared describing the synthetic efforts, and the total synthesis of pyridoacridine alkaloids (Echavaren and Stille; Kubo and Nakahara; Daniel V. LaBarbera, Tim S. Bugni and Chris M. Ireland; Nakahara, Tanaka and Kubo; Schmitz et al.). Subsequently, the isolation and characterization of 4

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deoxyamphimedine, neoamphimedine and were reported. These marme pyridoacridines; amphimedine, deoxyamphimedine and neoamphimedine (Figure 1.2) are natural analogues and were isolated from Xestospongia sponges collected in the Philippines and Micronesia (Marshall et at.). Deoxyamphimedine and neoamphimedine have been shown to be cytotoxic, but by differing mechanisms, however, their parent analogue has been shown to be biologically inactive (Marshall et at.). Deoxyamphimedine is a positively charged compound, which differentiates it from other analogues, and has been shown to be a potent DNA intercalating agent that induces significant ROS, which contributes to its overall antitumor activity. Interestingly, neoamphimedine has been shown not to intercalate DNA at relatively low concentrations where antitumor activity is observed (Marshall et at.). This is significant because DNA intercalation and ROS have been shown to cause nonspecific cell death. Therefore, healthy cells are susceptible to these compounds resulting in adverse side effects such as hair loss, cardio toxicity, and secondary malignancies (Azarova et al.). Thus, neoamphimedine offers a unique opportunity to develop a novel and potent antitumor agent with limited adverse side effects. 1.2.1 NEOAMPHIMEDINE Neoamphimedine has been shown to be as effective as etoposide, a clinically used Topolla poison, at inhibiting the growth of xenograft tumors in mice (D. V. LaBarbera, T. S. Bugni and C. M. Ireland; Marshall). Mechanism of action studies with neoamphimedine revealed that it is not a topolla cleaved complex poison, but 5

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neoamphimedine can efficiently induce Topolla-mediated catenation of plasmid DNA (D. V. LaBarbera, T. S. Bugni and C. M. Ireland; Marshall; Ponder et al.). Thus far, neoamphimedine is the only member of the pyridoacridines family known to induce Topolla-mediated catenation ofDNA (Daniel V. LaBarbera, Tim S. Bugni and Chris M. Ireland). Unlike, neoamphimedine, amphimedine was shown to be inactive at all concentrations both in vitro and in vivo (Marshall). This is quite interesting since neoamphimedine only varies structurally from amphimedine by the position of one carbonyl group (Figure 1.2). Based on the collective evidence from the literature and our own preliminary data we hypothesize that neoamphimedine has a unique mode by which it inhibits Toplla. Recently, Ponder, J; Yoo, BH; Abraham, AD; et al. characterized neoamphimedine as anN-terminal ATP-competitive inhibitor ofTopolla, which was supported by molecular modeling (Ponder et al.). Furthermore, they characterized that neoamphimedine circumvents Metnase-mediated MDR, which was shown to completely block the activity of clinically used Topolla drugs. Herein, we report the 0 _)lN H OH AA-67 0 OH AA-26 0 Njl__ H N ---f N H OH 22 FIGURE 1.2.1 CHEMICAL STRUCUTURES OF AA-67, AA-26, AND COMPOUND22 6

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design and synthesis of the first generation of neoamphimedine derivatives (Figure 1.21 ). Thus far, AA-67 shows similar binding affinity to neoamphimedine in silica, and proved to be as potent as neoamphimedine at inhibiting Topolla-dependent DNA decatenation in vitro. 7

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2. SYNTHESIS OF NEOAMPHIMEDINE 2.1 RESULTS AND DISCUSSION The syntheses of neoamphimedine and its intermediates were achieved by the synthetic methods described in Figure 2.1.1 and Figure 2.1.2 (D. V. LaBarbera, T. S. Bugni and C. M. Ireland). We synthesized neoamphimedine for further studies on the ATP hydrolysis assay, and for the Topolla dependent decatenation ofkDNA. 'o 'o 1) 10% Pd on C, * cyclohexenelethanol, 15mln. reflux CH2 N 2 2) AcOHIAe:zO 02N N02 0 2 N N02 5 min. reflux OH 03 4 1) 10% Pd on C, cyclohexene/ethanol, "o N02 2hrreflux 0 -...;:::, 2) m-xylenes 160 c, 5hr II I 0 0 0 N02 14 Tf20, DCM, TEA -20"C, 30 min. 0...__ 6 0 ,)l_N li _.....o 8 PPA, 70"C, 5 hrs 99% Formic Acid, TEA, DMF Pd(ll) acetate 'o 0* )lN .& NO H 2 0 _)lN li _.....o 05 0 7 FIGURE 2.1.1 SYNTHESES OF NEOAMPHIMEDINE INTERMEDIATES 8

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0 N H N02 1) AcOH, H20, H2S04 2) NaN02 oc NC 3) CuCN, soc o, EDC,DCM 12rh. rt 1) 10% Pdon C, cyclohexene/ethanol 2) CAN, ACN/H20 10 0 0 2 N02 HzS04 70-120 c FIGURE 2.1.2 SYNTHESIS OF NEOAMPHIMEDINE 2.2 ETHYL-2-NITROBENZOYLACETA TE 14 N02 HO The synthesis of 14 was achieved as described in Figure 2.2 by the method of Okazoe et al. 1999 (Okazoe and Morizawa) as an oily product (88 % yield). We hereby present a detailed procedure for the purification of 14 as there was no earlier report on the procedure for purifying compound 14. Needham et al. 1904 (Needham and Perkin) synthesized compound 14 from crude o-nitrobenzoylacetoacetate, ammonium chloride, and water as an oily product but did not give a vivid account on the purification of 14. Sicker and Mann, reported the synthesis of 14 from the 9

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esterification of ethyl-o-nitrobenzoylacetic acid, but the purification procedure was not reported (Sicker and Mann). Crude 14 can be purified by recrystalization from hexane or continuous extraction method to give white needles. In addition, Sicker and Mann reported the possibility of 14 to be in the keto and in the enol form. Their 1 H NMR is as follows: 8 l.ll(t, 3 H) for the keto form, 8 1.25 (t, 3H) for the enol form, 8 3.69 (s, 2H), no enol form at this region, 8 3.99 (q, 2H) for the keto form, while 8 4.15(q, 2H) for the enol form, 8 5.27 (s, I H) for the enol form, no keto form at this region, 8 7.25-7.80 8 for the aromatic peaks, 812.24(s, I H) for the enol OH, but no evidence of this peak for keto form. However, based on the 1 H NMR spectrum for compound 14 that we synthesized and purified, we could categorically state that, compound 14 is the keto form because there was no enol peak at 5.27 ppm for the methylene proton, and no enol peak for the OH at 12.24pprn, therefore concluded that 14 is a keto form, and that there is possibility of 14 to tautomerize when not in pure form. 1) OEM, magnesium ethoxide, toluene, ethanol, 70 oc, 2 hrs, rt, 2.5hrs 2) p-TSA, H20, reflux 3hrs FIGURE 2.2 SYNTHESIS OF ETHYL-2-NITROBENZOYLACETATE 10

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3. RATIONAL DESIGN OF NEOAMPHIMEDINE DERIVATIVES 3.11MINOQUINONE DERIVATIVES OF NEOAMPHIMEDINE FIGURE 3.1 THE CRYSTAL STRUCTURE (PDB: IZXM) OF THENTERMINAL ATPASE site OF TOPOIIa BOUND TO NEOAMPHIMEDINE. The key interactions are a network of hydrogen bonds with Ser148, Ser149 and Asn91 through an ordered water molecules. Pi-interactions are denoted by the orange lines. The blue sphere represents Mg2+ and the light blue lines represent the coordination with Mg2+. We conducted computational docking studies and found that neoamphimedine binds to the N-tenninal ATPase sites with high affinity (Figure 3.1) (Ponder et al.). These modeling studies are in agreement with our biological studies, which indicate that neoamphimedine is a competitive inhibitor of A TP-hydrolysis. The binding energy of neoamphimedine in the ATPase site of Topolla was calculated to be -61.8 II

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kcaVmol (Ponder et al.). The key interactions observed with neoamphimedine are a network of hydrogen bonds with Serl48, Serl49, and Asnl50, and with Asn9l through an ordered water molecule (Figure 3.1 ). Additionally, neoamphimedine interacts with the A TPase site Mg2+ through charge transfer pi-cation interactions (orange lines in Figure 3.1). Since our computational modeling techniques using neoamphimedine were predictive of our observed biological activity against Topolla in vitro, we utilized this methodology to design novel derivatives of neoamphimedine. In the first set of molecules the iminoquinone and quinoline functionality were retained due to the key interactions observed in the A TPase sites (Table l ). All of these compounds displayed more favorable binding energies. In particular, the sulfonamide derivative binding energy of-216 kcaVmol is considered to be the best among this group of molecules. In addition, the dock scores for this set of molecules were better than or equal to the dock score for neoamphimedine. Interestingly, there was no pi interaction observed with these molecules. The derivative with the secondary amide group (AA-119) was chosen to be the representative of this set of molecule because it was actually synthesized. The molecular docking results revealed that AA-119 has a better affinity with the ATPase site ofTopolla. The key interactions are a network ofhydrogen bonds with Alal67, Asn9l, and Asp94 (Figure 3.1.1 ). Although, there were no pi-interactions observed 12

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with this molecule, but this derivative has a better binding energy ( -139.8 kcaVmol) and a better dock score than neoamphimedine. Table I Computational analysis of novel iminoquinone derivative of neoamphimedine. The sulfonamide derivative has the best binding energy,-215.9kcaVmol. The amine with the binding energy; -84.0kcaVmol is the highest binding energy of the group. Generally, these molecules have better binding energies and better docks than neoamphimedine. Molecule Number Binding LigandInteraction Dock of poses Energy Protein with Mg Score (kcaVmol) interactions (A) (3.5A) 50 -169.7 Asn91-021 2.66 38.94 N Ala167-021 I I I H2N // N 0 0 50 -151.9 Alal67-021 2.91 50.06 Lysl68-020 N H29-llel41 I I I HO ..-: N 0 0 50 -139.8 H30-Asp94 1.09 126.8 I N Asn91-018 I 0 I I ) _)lN Asn91-N7 N H 0 13

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Table I Con 't. Molecule Number Binding LigandInteraction Dock of poses Energy Protein with Mg Score (kcaVmol) interactions (A) (3.5A) 50 -215.9 Asni50-NI3 1.30 341.6 N ::::.... I Glyl66-021 0 I I "-'.:: ....... ,, Asn91-018 ,;;,N .....: N 0 H 0 Asn91-N7 I 8 -84.0 H28-Asnl50 5.95 30.30 N "-'.:: Seri48-0 18 I I I "-'.:: .....: H28-Serl49 H2N N 0 I 4 -109.7 Ser149-NI3 2.19 31.21 ::::.... N Seri48-N20 I I I "-'.:: .....: N:c N 0 14

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FIGURE 3.1.1 AA-1191NTERACTIONS WITH THE ATPASE SITE OF TOPOIIa. The key interactions are a network of hydrogen bonds (black dash lines) with Ala167, Asn91, and Asp94. Coordination interaction is denoted by the light blue line, the blue sphere represent the magnesium metal cation. 15

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3.2 SYNTHESIS OF AA-119 The first step in the synthesis of 17 (AA-119) was to functionalize 15 to quinolinedione intermediate 16 through mild direct oxidation hours (Blanco et al.; Cherif, Cotelle and Catteau) using 5 equivalent of CAN in equal volume ofwater and ACN (Figure 3.2). The 1H NMR data was not conclusive enough due to resonance effect, but the peaks at o 4.15 and o 3.5, which correspond to the methoxy groups of 15, were no longer present. It is therefore evident that the methoxy groups have been demethylated respectively. The data from Mass Spectrometry; revealed that compound 16 was actually synthesized, giving m/z [M+Hf = 350, which corresponds to molecular mass of compound 16 in a protonated form. Next, treatment of 16 under alkaline condition for 48 resulted in cyclization to yield 17 as an orange solid (68% yield) (Brahic et al. ). 0 __)l._N H 15 0 N)l___ H Seq CAN, ACNIH20 0 ---_.)l_N H FIGURE 3.3 SYNTHEESIS OF AA-119 16 0 Nj(__ H CHCI3 1 N NaOH, rt, 48 hr. 0 _)lN H 0 17

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3.3. PHENOL DERIVATIVES OF NEOAMPHIMEDINE Iminoquinone moieties have been shown to cause non-specific toxicity (Guo et al.; Park et al.; Saltiel and McGuire), which result in adverse side effects with clinically used drugs. Therefore, in this set of derivatives we designed out the 1mmoqumone functionality. Instead we utilized a 5-methoxy, 8-hydroxy functionality, which maintains key hydrogen bonds in the ATPase active site (Figures 3.3 and 3.3.1). AA-67 and AA-26 bind in the ATPase site like neoamphimedine (Table 2). AA-67 has the lowest non-solvent binding energy of -138 kcaVmol and a dock score of 129; AA-26 has a non-solvent binding energy of -106 kcal/mol, while neoamphimedine has a non-solvent binding energy of -62. kcaVmol and a dock score of 28, with pi-cation interactions observed with AA-26 and AA-67 as found with neoamphimedine. AA-26 has the highest number of pi-interactions (Figure 3.3), including: pi-cation interaction with Mg2+, pi-cation interaction with Lys 168, and pi sigma interaction with Tyr34, while AA-67 has one pi-cation interaction with Lys 168 (Figure 3.3.1 ). 17

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Table 2 Computational of AA-67, and AA-26. This Analysis predicts that AA-67 and AA-26 are better inhibitors ofTopolla. Compound Binding Ligand-protein Pi-interactions energy (H-bonds) (Kcal/mol) Interactions AA-67 -138.8 Lysl68 I Pi-cation with Asnl50 Arg98 Asn91 AA-26 -106.1 Serl49 I Pi-cation with Mg.!+ Glyl64 I Pi-cation with Lysl68 Lysl68 l Pi-sigma with Tyr34 18

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.. I FIGURE 3.3 AA-26 INTERACTIONS WITH THE ATPASE SITE OF TO POll a Non-solvent binding energy= I 06 kcal/mol. The key interactions are a network ofhydrogen bond (black dash lines) with Ser149, Lys 168, and G ly 166. Coordination interaction is shown as light blue line while orange lines denote pi-cation attraction. 19

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Lys168 FIGURE 3.3.1 AA-67 INTERACTIONS WITH THE ATPASE SITE OF TOPOIIa. Binding energy= -138 kcaVmol. The key interactions are a network of hydrogen bond (black dash lines) with Asn91, Lys 168. The Active site amino acid residues are shown as white carbon atoms and Mg2+ as a blue sphere. Coordination interactions are shown as blue lines while orange lines denote pi cation attraction. 20

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3.4 SYNTHESIS OF 1 (AA-67) The nitro group of compound 9 was reduced to the amine product by refluxing the suspension containing 9, cyclohexene, ethanol, and I 0% Pd on carbon (Figure 3.4). It is important to note that catalytic hydrogenation was unsuccessful giving multiple products. The amine product was acetylated without purification at room temperature for four hours to give 15 (92 % yield) as a yellow compound that also fluoresces yellow. Also important to note is that the reaction must be covered with a drying tube containing drierite; an attempt to carry out this experiment in the absence ofthe drying tube resulted in a blue fluorescent compound with many side products. Finally, compound 1 was prepared by selective demethylation of 15 using two equivalents of lithium iodide in refluxing 2, 6-lutidine for 8 hours to produce a tan colored solid (53% yield). 21

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0 _)lN H _......0 9 1)10%PdonC, cyclohexene/ethanol, reflux, 1.5 hr 2) AcOH/Ac20, 4hr. FIGURE 3.4 SYNTHESIS OF AA-67 3.5 DECA TENA TION ASSAY WITH AA-67 0 __)lN H 0 _)lN H _......0 15 0 Njl____ H l Lil (2 eq) 2, 6-Lutidine, reflux Bhrs. 0 N)l___ H OH 1 (AA-67) In vitro decatenation assays (Figure 3.5) show that AA-67 inhibits Topolla by 25% at I !J.M, 50% at 2 !J.M, 60% at 5 !J.M, 78% at I 0 !J.M, 85% at 30 !J.M, and 80%. Figure 3.5 also revealed that neoamphimedine displayed similar in vitro activity as AA-67 at the same concentrations tested, giving 32% inhibition at l !J.M, 56% inhibition at 2 !J.M, 65% inhibition at 5 !J.M, 66% inhibition at I 0 !J.M, 85% inhibition at 30 !J.M, and 84% inhibition at 50 !J.M. Therefore, AA-67 displays similar inhibitory 22

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activity as neoamphimedine over a concentration range of 1-50 J.!M. This result validates our drug design methodology and further designed derivatives are summarized in Table 3. -...., cu c: Q) ...., cu (.) Q) c c: 0 -...., .c 0 D Neo AA-67 1 2 5 10 30 50 F I GURE 3 .5 PERCENT fNHIBITION CURVES GENERA TED FROM DECATENATION ASSAYS 23

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Table 3: Computational analysis of novel lmidazoquinoline derivatives of neoamphimedine. These derivatives have better binding energies and dock scores than neoamphimedine. ProteinBinding ligand interactions energy plDock Molecule Label (NS) interactions (3.5 A) score Asnl50-017 I .0 Glyi64-N23 H 'o N_..... 3 pi-cation Tyri65-N23 I H with Mg2* N 7" Ala167-018 --{ I ...-:: Lysi68-N7 N N OH TopoLID -107.6 I pi-cation Lysl68-018 60.49 with Arg98 I Asn91-018 'o .0 N/ Asn91-N23 H I H Ala 167-N23 N 7" I lle141-H25 N ...-:: N Asn91-H36 OH TopoL2B -179.4 I pi-pi with Asp94-H32 88.39 Phel42 I .0 H N 4 pi-cations Serl48-0 17 I I Seri49-N7 N with (, I I Asn150-N7 N ...-:: N Lys168-N20 0 TopoL3A -72.04 I p1-s1gma 43.19 (Asn91) 24

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Table 3 Con t. I '-':::: 'o _.-::. NH2 Asn91-022 Asnl50-019 1'-':::: '-':::: Asni50-N7 HO _.-::. /. Asp94-H33 N 0 OH TopoL4B -193.4 I pi-cation Seri49-H31 92.16 with Arg98 Tyr34-H32 I '-':::: 0 _.-::. N)l_ 'o H H N 1'-':::: '-':::: Asnl50-023 --<\ N _.-::. /. Thr215-024 N OH TopoL5A -166.6 I Pi-cation Seri49-H34 47.85 with Mg2+ Asn95-H38 I N,l o Asn91-024 H H N Asnl50-025 r-<_, "" Lys168-024 HO N N OH TopoL6A -171.5 I Pication lie 141-H39 68.23 with Arg98 Glu87-H45 Arg162-029 I "-'::: ..Jl Glyl64-030 o Tyrl65-031 N H H Lysi68-NI8 N "-'::: O H ,N-{ lob ..-: Lys168-024 N N '"o HO OH TopoL7B -173 2 I Pi-cation Asn91-H38 118.1 with Arg98 Ilei41-H43 25

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3.6 SYNTHESIS OF NOVEL IMIDAZOQUINOLINE DERIVATIVES The synthesis of 22 and the other imidazoquinoline derivatives has not been completed at this moment. However, the syntheses of various novel intermediates to prepare 22 are hereby reported (Figure 3.6). Compound 5 was dissolved in nitric acid/sulfuric acid (4:1) at room temperature and warmed between 30-40C for ten minutes giving 18 as a lemon yellow solid in 90% yield. Next, 18 was selectively reduced using cyclohexene, with I 0% palladium on Carbon as the catalyst. The amine was converted to the benzimidazole via Philips condensation (Phillips) using acetic acid and acetic anhydride to give 19 ( 60% yield). An attempt to synthesize 19 through Philip condensation of 21 (Figure 3.6.1 ), did not work. The nitro group of 19 was reduced via hydrogenation with palladium on carbon under 50 psi of Hydrogen. The amine was then converted to 20 via amide formation with 14 using m-xylene as a solvent and with continual removal of ethanol (Figure 3.6). However, this method gave 20 with poor yield (26%). When DMF was used as a solvent % yield was significantly improved to 48%. 26

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HN03 / Acetic acid 30-40"C, 15 min. 5 Pd on C, cyclohexene reflux 15 min. reflux 4hr. 1) Pd on C, methanol H2 50 psi, 18 hr. 2) 14, DMF, 160"C, 7hr. 'o llAN02 /0 20 FIGURE 3.6 BENZIMIDAZOLE COUPLED PRODUCT 18 --{0 0/ Pd on C, ACOH I HN* H2 50 pal, 6 hr. 0 I '-':: 0 )lN h N)J......_ H O H ...... 21 Reflux 5hr. )( FIGURE 3.6.1 3, 6-DIMETHOXY-l, 2, 4-TRIACETAMIDOBENZENE 27

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3.7 C O NCLUSI O N Using computer-aided structure based drug design we have successfully identified the ftrst generation of neoamphimedine derivatives ln addition we have synthesized AA-67, which displays potent activity against topoiiA-dependent DNA decatenation in vitro that is comparable to the natural product neoamphimedine. Furture studies will focus on further design synthesis, and biological evaluation of neoamphimedine derivatives as potential novel antitumor agents. 28

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4. EXPERIMENTAL 4.1 GENERAL EXPERIMENTAL PROCEDURES Except where otherwise indicated, all chemicals were purchased from chemical suppliers and used as received. All computational simulations were performed within the Discovery Studio software package (Version 2.5.5; Accelrys Inc., San Diego, CA), and all crystallographic coordinates were obtained from the RCSB protein databank (http://www.pdb.org). Melting points were taken with a digital melting point apparatus and All TLCs were performed on silica gel plates using a variety of solvents and fluorescent indicator for visualization. IR spectra were recorded on Avatar FT-IR 360 spectrometer. NMR data were collected using a Bruker Avance Ill 400 (1H 400 MHz, 13C 100 MHz). LC-MS analyses were conducted using a Micromass QTOF-2 Micromass spectrometer equipped with lock spray and an Agilent 1100 HPLC capillary pump fitted with a C18 Mass Spec column from Vydac (I X 150mm). The MS was run in positive mode with a capillary voltage of 2.5kV and a cone voltage of 30V. Accurate mass measurements were performed using leucine enkephalin as a lock mass and data were processed using MassLynx 4.1. The methods of LaBarbera et al, 2007 (D. V. LaBarbera, T. S. Bugni and C. M. Ireland) were employed for the synthesis of neoamphimedine, and the intermediates 3 to 13, while the methods of Okazoe et al, 1999 (Okazoe and Morizawa) was employed for the synthesis of 14. 29

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4.2 COMPUTATIONAL METHODS The preparation of protein and ligand model: The crystal structure of Topolla with a resolution of 1.87 A was retrieved from the Protein Data Bank with the entry code l ZXM. From the PDB file, the protein chain A and B were extracted, water molecules were removed, and the magnesium ions were retained in both chains. The protein chains A and B are presented in different color codes to show their distinct interactions. Accelrys Discovery studio 2.5.5 was employed to prepare the protein by setting the "tool" command to generate conformations. In order to avoid identical conformations, a root mean square deviation threshold of l.5A and a score threshold of 20.00 kcaVmol were used. The protein conformation with the lowest energy was retained out of several poses (outputs) generated. Once the protein is prepared molecular docking of neoamphimedine and derivatives were performed as described in Ponder et al. (Ponder et al.). Binding Energy Calculation: The non-solvent binding energy calculation for the three poses generated was set to run using the Accelrys Discovery studio software. The protocols for binding energy calculation: The results for the docking were opened and made visible in the graphic window. This was made possible by selecting all the poses generated with simultaneous selection of the "show" command. Note, the report window must always be closed when not in use to prevent duplication of molecules and selection of molecules of non-interest. Next, the "input receptor" 30

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command, (1 ZXM: 1 ZXM) was selected, while for the "input ligands" command ( 1 ZXM: Selected) was chosen. The "in situ ligand minimization" was set as false; "ligand conformation entropy" was set as false, "implicit solvent model" was set as none. The "none bond list radius" was set to 14 (default), and "parallel processing" was set as false. The "protocol" command was selected, which allowed the in silica calculation of binding energies. Additionally, using similar methodology three binding energy calculations were run, including: non-solvent binding energy, Generalized Born (GB), and Poisson Boltzmann with non-polar Surface Area (PBSA). Protein-ligand interactions: To determine the number of hydrogen bond interactions between the amino acid residues of the protein and the ligands, the "structure icon" was selected, the monitor under the structure was selected, and "H bonds" was selected to show the hydrogen bonds. The Hydrogen bond was selected on the graphic window, and with simultaneous selection of the "attribute" command, the default distance was changed from 2.5 A to 3.3 A. The longest hydrogen bonds were removed (this was done when two hydrogen from different residue bonds to the OH or OCH3 group). 31

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General methods for ligand scoring: The molecular modeling on the imidazoquinoline and the phenol group (Table 4) of molecules revealed that there were at least 50 poses generated during the docking and the binding energies calculation. To avoid complications, we initially chose the best four poses (labeled A to D) out of the 50 poses, based on their binding energies and their dock scores, for further computational studies. However, to make the computational studies easier, we decided to select one pose out of the four initial poses selected, and came up with a label formula that allows easy identification. An example of the label formula is TopoLI D, which is simply Topolla ligand number I, pose D. Below is the rationale behind the selections ofthe poses generated. Example 1 (TopoL 1 D): This pose (Figure 8-1) was selected among the four best poses for this particular compound because TopoLI D has the lowest GB binding energy of -9.487 kcaVmol. It has a non-solvent (explicit solvation) binding energy of -107.6 kcaVmol; it has the lowest PBSA binding energy of 73.41 kcaVmol. Also, TopoLI D was selected because it has the highest number of pi-interactions, namely, three pi-cation interactions with magnesium and one pi-cation interaction with Arg98. This pose has the lowest electrostatic energy of 1.085 kcaVmol, and has 4.19 A coordination distances from the magnesium metal cation. 32

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Example 2 (TopoL28): This particular pose (Figure B-2) has a non-solvent (explicit solvation) binding energy of -179.3 kcaVmol. It also has the lowest GB binding energy of -2.316 kcaVmol. This pose has the lowest Poisson Boltzmann with a non polar Surface Area PBSA binding energy of 107.6 kcaVmol. It also has the lowest electrostatic energy of 9.764 kcaVmol. Pi-interactions were not considered for the poses as they all have the same pi-pi interaction with Phe 142. The distance between the ligand and the metal cation is a major issue for the poses of L2 because the metal cation is too close to the ligand molecule. However, L2B has the farthest coordination distance (0. 961 A) from the magnesium metal cation. 4.3 SYNTHESES. Ethyl-2-nitrobenzoylacetate (14). To a suspension of 41 mL of ethanol containing 23.3 g (0.2 mol) of magnesium ethoxide, 83 mL of toluene containing 31 g (0.2 mol) of diethylmalonate was added drop wise. After stirring at a bath temperature of 70 C for two hours; the prepared mixture was cooled to room temperature and then cooled to 0C, and 21 mL of toluene containing 35 g (0.18 mol) of 2-nitrobenzoyl chloride was added thereto. During the process ofthis addition process, the temperature must not go beyond 1 0 C. After stirring at room temperature for 3 hours, 83 mL of water containing 19.8 g of sulfuric acid, the thus prepared mixture was poured into 120 mL of water and separated out after the addition of 30 mL of ethyl acetate. The organic layer obtained was washed with 120 mL of a saturated aqueous solution of sodium 33

PAGE 48

chloride and dried over sodium sulfate. After filtration and concentration, the resulting residue was dried in vacuo to obtain 55.51 g (95%) of a crude product of diethyl 2-(2-nitrobenzoyl) malonate. TLC (CHCb) Rf = 0. 17. To 55.39 g ofthis crude product diethyl 2-(2-nitrobenzoyl) malonate, 73.85 ml of water and 77.4 mg (0.406 mmol) of p-toluene sulfonic acid monohydrate were added. The prepared mixture was refluxed for 2.5 hours and then cooled to room temperature; 74 mL ofCHCb was added to the mixture to separate it out. The organic layer obtained was washed with 74 mL of 7% aqueous NaHC03 and 74 mL of saturated aqueous solution of sodium chloride. After drying over sodium sulfate, the resulting residue was filtered, concentrated and dried in vacuo to obtain 37.71 g (88% yield) of crude oily product TLC (CHCh) Rr= 0.20. The crude product was purified through continuous extraction process that is similar to soxhlet extraction method using hexane as the solvent, to give 25 g (59% yield) 1H NMR (CDCb) a 8 18(d, J = 8.2Hz 1H), 7.77 (t, J = 8.0Hz, 1H), 7.53 (d J = 7.6Hz, lH), 4.17( q, J = 7.2Hz, 2H), 3.89( s 2H) 1.25 (t, J = 7.2Hz 3H). 7-acetamido-4-(2-acetamidophenyl)-5,8-dimethoxyquinoline (15). 50 mg (0 136 mrnol) of 9 was dissolved in 5.44 mL of absolute ethanol to which was added 108 mg of 10% Pd on Carbon, and 2.72 mL of cyclohexene. The reaction mixture was refluxed for approximately 2 hours. The product was recovered from the catalyst by filtering over a plug of celite while hot. The catalyst was washed with ethanol until 34

PAGE 49

the filtrate became clear. The filtrate (yellow fluorescing solution) was concentrated in vacuo to give a yellow residue. Without purification, the residue was dissolved in 8 mL of acetic acid and 8 mL of acetic anhydride (I: I). The color of the solution became orange after 15 minutes ofthe reaction. The reaction vessel was covered with drying tube (filled with drierite), and stirred at room temperature for four hours. The acetylated product was concentrated and dried in vacuo to afford a yellow solid (47.6mg, 92% yield). TLC (20% MeOH in EtOAc): Melting Point 243-245C. Rr = 0.26. 1H NMR (CDCh) 8.93 (d, J=5.2Hz, I H), 8.17(s, 2H), 8.11 (d, J=8Hz, 1 H), 7.41 (t, 1=8.0 IH), 7.19 (t, J=8.0Hz, 1H), 7.13 (s, 2H), 6.59(s, 1H), 4.17(s, 3H), 3.52(s, 3H), 2.32(s, 3H), 1.81 (s, 3H). 13C NMR (400 MHz, CDCI\ 168.8, 168.1, 162.2, 152.2, 149.9, 143.0, 136.1, 134.5, 133.6, 128.3, 128.0, 123.8, 122.3, 121.6, 116.0, 100.2, 62.16, 55. 95, 25.19, 24.29. lR: 3264.5, 2925.3, 1670.8, 1613.6, 1254.0, 1251.0. HRMS (m/z): [M +Ht calculated for C21H22N304, 380.1610; found, 380.1600. 7-acetamido-4-(2-acetamidophenyl)-5-methoxyquinoline-8-ol (1, AA-67). 23 mg (0.061 mmol) of the 15 starting material was dissolved in I mL of 2, 6-lutidine at room temperature under nitrogen; to this solution was added 17.6mg (2equivalent) of lithium iodide. The reaction was then reflux for 8 hours. The reaction mixture was allowed to cool to room temperature and poured into I 0 mL of water. The remaining product was transferred with 10 mL of water. The combined aqueous solution was 35

PAGE 50

neutralized to pH 7 with HCl on ice bath keeping the temperature between 0-5 C. The neutral solution was extracted with dichloromethane x 4 (35 mL). The organic layers were combined and washed with 40 ml of saturated sodium chloride. The aqueous layer was back extracted with DCM (35 mL) x 2. The organic layers were combined and dried over sodium sulfate. The solution was concentrated, and dried in vacuo. The crude product was purified by flash column chromatography and eluted with ethyl acetate I acetone (I: I) to give tan color solids ( 11.6 mg, 53 % yield). TLC (EtOAc/acetone (I: I)) Rr = 0.14 Melting point 238-240C 1 H NMR ( 400MHz CDCb) 8: 8.78(d, J= 4.7Hz, IH), 8.16(s, IH), 8.07(d, J= 7.7Hz, IH), 7.85(s, IH), 7.42 (t, J= 8.2Hz, I H), 7.23-7.14(3 H), 6.64(s, IH), 3.50(s, 3H), 2.31(s, 3H), 1.80(s, 3H), 13C NMR (400 MHz, Acetone d-6): 169.9, 168.6, 167.6, 164.2, 160.3, 156.3, 153.4, 152.7, 148.1, 146.9, 135.8, 134.1, 131.4, 128.6, 123.1, 116.8, 105.7, 55.3, 23.49, 22.39, IR: 3427, 3334, 2918,2849, 1674, 1627, 1584, 1445, HRMS (rnlz): [M +Hr calculated for C21H16N7, 366.1467; found, 366.1454. 7-acetamido-4(-2-acetamidophenyl)quinoline-5, 8-quinolinedione (16). 30 mg (0.079 mmol) of 15 was dissolved in 3 mL acetonitrile (ACN) and cooled to 0 C on ice bath. A solution of CAN (130 mg, 0.2374 moles) in 3 mL of water was added dropwise to the ice-cold CAN solution and stirred at 0C for 30m minutes. The solution was then allowed to warm to room temperature and stirred for another I hour at room temperature. The reaction solution was neutralized to pH 7 with saturated 36

PAGE 51

NaHC03. The neutral solution was extracted with CHCb x 2 (30 mL). The Chloroform extracts were combined, dried over sodium sulfate, concentrated and dried in vacuo. The solid obtained was purified by column chromatography using 60% acetone in EtOAc as the eluent, given orange color solids ( 11.6 mg, 43% yield). TLC (acetone/ EOAC (1:1)) Rr = 0.19. IR: 3333, 2917, 2847, 2361, 1679, 1654, 1654, 1585. rnlz: [M +Ht found 350.4 Iminoquinone derivative (17, AA-119). 5 mg (0.014 mmol) of 16 was dissolved in 5 mL of CHCh and to this solution was added 0.5 mL of 1 N NaOH. The reaction solution became cloudy, and was stirred at room temperature for 48 hours. The aqueous solution was extracted with CHCb 10 mL x3. The organic layers were combined dried over sodium sulfate and filtered. The filtrate was concentrated and dried in vacuo to give an orange solid (2.7 mg, 68% yield). TLC (CHCb/ MeOH (1:1)) Rr= 0.73 1H NMR (CDCI3) a 9.30(d, J= 6.4Hz, 1H), 8.86(s, 1H}, 8.64(d, J= 5.2Hz, 1H), 8.57(d, J= 8.4Hz, 1 H), 8.52(s, 1H), 8.29(d, J= 8.8 Hz, 1 H), 7.93(t, J= 8.2Hz, 1 H), 7. 79(t, J= 7.5Hz, 1 H), 2.35(s, 3H). IR: 3338, 2929, 2843, 2357, 2320, 2177, 1691, 1650, 1589, 1515, 1458, 1339, 1229, 1037. [M +Ht found 290.0 2,5-dimethoxy-3, 6-dinitroacetanilide (18). To a 4:1 solution of 70% nitric acid and acetic acid (25 mLI 6.25 mL) was added to 1g (4.16 mmol) of5 at room temperature. The solution was warmed between 30-40C for 15 minutes. The reaction solution was 37

PAGE 52

poured into ice-cold water and allowed to precipitate overnight in the fridge. The precipitate was filtered off and washed with ice-cold water. The solid was dried in vacuo to afford 1.068 g (90% yield). TLC ( 10% MeOH in CHCb) Rr = 0.52. 1 H NMR (400 MHz CDCb) a: 8.57(s, 1 H), 7.87 (s, 1 H), 3.99(s, 3H), 3.93(s, 3H), 2.32(s, 3H). 13C(400MHzCDCb)a: 168.1, 148.9, 138.3, 136.0, 132.8, 127.0, 105.3,62.52, 56.65, and 24.32. IR: 3354.4, 3129.6, 3076.5, 2533.0, 2353.2, 1703.5, 1613.6, 1552.3, 1041.5 4, 7-dimethoxy-2-methyl-6-nitroimidazoquinoline (19). A solution consisting of 18 (50 mg, 0.175 mmol), 20 mg of 10% Pd on Carbon and cyclohexene (35 mg, 0.426mmol) in 1.25mL of absolute ethanol was refluxed for 15 minutes. The reaction was filtered hot through a pad of celite and the orange solution was concentrated in vacuo giving red prisms. The red prisms were dissolved in 2 mL of acetic acid containing 0.2 mL of acetic anhydride. The reaction solution was refluxed for 4 hours, removed from the heating source, and cooled to room temperature. The crude product was concentrated, dried in vacuo to give 37.8 mg (91% yield). The product was purified by column chromatography using EtOAc as the eluent to give 24.8 mg (60% yield). TLC (EtOAc), Rr = 0.33. 1H NMR (400MHz CDCb) a: 8.55(s, I H), 7.82(s, 1 H), 3.98 (s, 3H), 3.93 (s, 3H), 2.32 (s, 3H). HRMS (m/z): [M +Ht; found 38

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6-acetamido-4, 7-dimethoxy-2-methy( -2-nitrobenzoyl)benzimidazole (20). To a solution of 19 (14lmg, 0.68 mmol) in 40 mL of methanol, was added 50mg of 10% Pd on carbon. The mixture was purged under nitrogen for 5 minutes; the reaction mixture was hydrogenated at 50 psi for 18 hours under hydrogenator. The product was recovered from the catalyst by filtering over a pad of celite. The filtrate was concentrated in vacuo to the aniline product. Without purification, the aniline product was added to 178 mg (0.745 mmol) of ethyl-2-nitrobenzoylacetate 14, which were dissolved in 1.5 mL of anhydrous DMF. The reaction solution was placed on a preheated sand bath ( 160-180C) for 7 hours. The reaction was stooped and allowed to cool to room temperature and poured on ice cold ether which was triturated to obtain some solids. The product was purified by column chromatography using EtOAc as the eluent to afford 45 mg (20% yield). TLC (EtOAc) RF 0.24 1H NMR (400 MHz CDCb) a: 8.14 (d, J= 6.0Hz, 2H), 7.98 (s, 1H), 7.89 (s, 1H), 7.71 (t, J= 6.8Hz, 1H), 7.63 (t, J= 8.0Hz, 1H), 7.58 ( d, J= 7.6Hz, 1H), 3.94 (s, 3H), 3.81 (s, 3 H), 3.55(s, 2H), 2.25 (s, 3H). 13C (400 MHz CDCb) a: 168.6, 165.7, 147.3, 145.1, 136.6, 135.3, 133.9, 132.5, 130.4, 129.8, 128.0, 124.6, 114.9, 100.7, 62.37, 56.15, 44.75, and 25.08. IR: 3333.9, 3121.5, 3011.1, 2925.3, 1691.3, 1593.2, 1519.6, 1405. HRMS (m/z): [M +Hf; found for C,9HI906N4, 399.1289, and [M-Hr m/z, found for C,9HI706N4, 397.1154. 39

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3, 6-dimethoxy-1, 2, 4-triactamidobenzene (21). To 200 mg (0.770I mmol) of 18 was dissolved in 40mL of acetic acid and the solution was purged under nitrogen for 5 minutes. To this solution was added 50mg of I 0% Pd on carbon and the mixture was purged for another 5 minutes, to which was added 5 mL of acetic anhydride. The mixture was hydrogenated under 50 psi of H2 at room temperature for 6 hours. The catalyst was filtered off over a pad of celite and washed with acetic acid. The filtrate was concentrated and dried in vacuo to afford 209mg (96% yield). TLC (I 0% MeOH in CHCI3) RF 0.14. 1H NMR (400MHz CDCI3) a: 8.15 (s, lH), 8.10 (s, lH), 7.85 (s, IH), 7.40 (s, lH), 3.87 (s, 3H), 3.77 (s, 3H), 2.25(s, 3H), 2.21 (s, 3H), 2.I7(s, 3H). 13C (400 MHz CDCb) a: 169.4, 168.7, 150.5, 140.4, 130.7, 1I9.9, II4.8, 1I2.9, 1 04.5, 60.80, 56.I8, 24.34, and 23.29. IR: 3329.9, 3I99.1, 2941.7, 2839.5, 1658.6, 1601.4, 1540. HRMS (m/z): [M +Ht; found for C14H2oOsNJ. 310.I3, 4.4 DNA Decatenation Assay with neoamphimedine, and with AA-67 DNA Decatenation Assay with neoamphimedine, and with AA-67 (Ponder et al.) Topolla (TopoGen) was added to 70 ng of kinetoplast DNA (kDNA, TopoGen) comprising small interlocked supercoiled circular DNA in reaction buffer (50 mM Tris-HCl, pH 8.0, 120 mM KCl, 10 mM MgCI2, 0.5 mM OTT, 0.5 mM ATP) in the presence various concentrations of neoamphimedine and AA-67 ( I-50f..1M). Reaction mixtures were incubated at 37 oc for an optimized time of30 min. The reactions were 40

PAGE 55

then separated on a I% agarose gel containing 0.5 Jlg/mL ethidium bromide with decatenated and catenated DNA markers (TopoGen). Fluorescence intensity quantitative analysis was carried out using Quantity One software by calculating the percent decatenation by comparing the pixel intensities of both decatenated bands relative to the catenated band. The percent inhibition of decatenation was then determined using the following formula as described in ponder et al. o...-:: Inh'b' D 100 ( 1 %Decatenation with neo ) ,o 1 ttlon ecatenatton = -------------o/oDecatenation without neo 41

PAGE 56

APPENDIX A: SUPLEMENTARY ANALYTICAL DATA a:ICD--.1-......,..,....,...,....,. .... .. ......... .... .jOO.O"(Jo. ........ iii! !:it ..... o. .... ti Ll.!.., "\/ J 0 0 NO, 14 I -'------y y y y 0 ---i :: 8 Q '34 8 (I 7 6 7 2 6, 6 4 6 1) 56 52 48 44 40 3 6 32 28 24 2 ('1 I 2 08 FIGURE A-1 1 H NMR SPECTRUM OF ETHYL-2-NITROBENZOYLACET ATE 42

PAGE 57

I I a 10 0 0 UNO, 8 00 7 90 I I I 7 80 I I I I I 7 70 7 60 7 so 7 -10 7 30 7 20 FIGURE A-2 AROMATIC REGIONS OF ETHYL-2-NITROBENZOYLACET ATE ----------. i= ; ---"'" 'i '--\j '-Jy I I "' 0 "o h N)l____ H 0 I "' "' __jl_N h N H /0 15 I ( 1 v w w y v -:; = -g -PPM 9.0 8.0 7.0 6.0 5.0 4.0 J.O FIGURE A-3 1H NMR SPECTRUM OF 7-ACETAMI00-4(-2ACET AMIOOPHENYL)-5,8-DIMETHOXYQUINOLINE 43 ul v --i 2.0 1.0 0.0

PAGE 58

::!: mi \I \I l!r',J __ L----:" y y l r"lJ >PM --. 9.4 Q.2 9.0 8.8 8.6 8.4 8 2 8 0 78 76 7.4 7 2 FIGURE A-4 AROMATIC REGIONS OF 7-ACETAMID0-4(-2ACETAMIDOPHENYL)-5,8DIMETHOXYQUINOLINE .. o: _ !; _ -,1--' I I "' 0 'o _.-:. H 0 I "' h N / H /0 II l JL Ju.l Ji .I J 170 160 150 140 110 120 110 100 90 80 70 60 FIGURE A-5 13C NMR SPECTRUM OF 7-ACETAMID0-4(-2ACET AM I DOPHENYL)-5,8Dl M ETHOXYQUINOLINE 44 50 i y -7.0 68 6.6 -'-1, .l 40 10 20

PAGE 59

5 32; I SIJ: Ill; I as: a I II<; I !12; "'; 0 18; Njl____ H ,.: 0 _.)l.N 7A; H /0 n: ro: i / 6B;
PAGE 60

.. ........ .... .............. -gn ! "l "-."l I l.J-W I I 'o N)l____ 0 "' )l_N H OH 1 l 1 I lL .A y w y y v v -. --= !: i PPM 9.0 8.0 1.0 6.0 4.0 ].0 2.0 1.0 0.0 FIGURE A-8 1 H NMR SPECTRUM FOR AA-67 .. ---E!:: e .... #t. \1 I \1 I \(,) I 0 o '':(N)l 0 H )l.N D,t .) H OH i j{ I L I y Y\.,-Y y yYJ 'y' . . . i = PPM 9.4 9.2 90 8.8 8.6 8.4 8.2 8.0 7.8 1.6 7.4 1 2 1.0 68 64 6.2 FIGURE A-9 AROMATIC REGIONS FOR AA-67 46

PAGE 61

--;;;: = i; --i \( I II I Ill j) 0 J! 0 N H 0 .U N H OH ........_[ .d ..... ... 170 160 ISO 140 IJO 120 110 100 90 80 70 FIGURE A-10: 1JC NMR SPECTRUM FOR AA-67 OS2511E {0 028) Is C1 00. 1.00) C20H19N304 100 l66 ,.,. I 387 1085 0 )lN H : I .l 60 'o "" h OH ----05251 IE 441 {8.) AM {
PAGE 62

j # 96 !M 92 i CJ)J 82j IJJ: !i re: 761 72: mj 68: 66i 6-11 62: em l!DJ DD FIGURE A-12 IR SPECTRUM FOR AA-67 1(1) gg; .... ...-... ... 98; i -. ii i a 5 .. i 1 9:1) I(Dl 5IXJ "' 91; 0 :;; Njl___ H 0 .. _)l_N e H 0 16 ;: i! l!DJ DD 29XI liiiJ 19:1) I (Ill FIGURE A-13 IR SPECTRUM FOR 7-ACETAMID0-4(-2-ACETAMIDOPHENYL) QUINOLINE-5,8-QUINOLINEDIONE 48 $ 5IXJ i

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'''" "" "" I 0 j L 0 I N '"" H 0 j 1] I ll N N H 0 16 :.o..-; ::o..-; "" 1:]' 10-'!' DL. ,.r '"" '_l, 1217, 1Ne ,,.. :::ro" "'' ., 1g1 '"' "" ,.., "" "'' 200 "" ,.., "" ,.., "" "" ,.., FIGURE A-14 [M+Hf SPECTRUM FOR 7 -ACET AMID0-4( -2ACET AMIDOPHENYL) QUINOLINE-5,8-QUINOLINEDIONE _j_ l111o H I I 'M I YY :i iE ---lpM 9 0 80 7 0 J: // I 0 11 : / N. -/. N H n --r (, 0 s (I 0 17 4 0 3 (I FIGURE A-15 1H NMR SPECTRUM FOR AA-119 49 ,, t I ;: 0 l "" -T I 0 ""' ,.. ,,, 4X 1 -00

PAGE 64

CI)IJI lliCI) Cl) 0011' IJI!JI IJI OG om II II II I II I J I.J l J I I II I J y I r r r I --0 m 0 G 0 92 90 '3 3 86 FIGURE A-16 AROMATIC REGIONS OF AA-119 '"' '"" ... ... ... ... .. """ ,,. ,,., .... ''"" 3 "' ., ., N J jl [ .. N "" '"' T eu l T .l: >OO ,., ... ., ;po .... FIGURE A-17 fM +Hr SPECTRUM FOR AA-119 50 ''' GGG m" <.Owo Ill Ill l I 'r) '( w : 3 2 80 1 a ,., ""' "'' ""' ,., ""' ..,

PAGE 65

960; .,,. 9.& 0 J 9) 5: 9) 0: !II 9] 5' 0 9]0: )l_N 91 5: H 0 91 0: -.J 5: Dl )5(1) )]]) 29JJ FIGURE A-18 IR SPECTRUM FOR AA-119 I I 'o o, 02N NH /oo)...___ 18 y y -! I -9.0 8.0 7.0 6.0 5.0 JD) w ::: 4.0 '.! I I .J' ._ l' -. 1000 y -].0 2.0 1.0 FIGURE A-19: 1 H NMR SPECTRUM FOR 2, 5-DIMETHOXY -3, 6-DINITRO ACETANILIDE 51 0.0

PAGE 66

i ; i I I I I 'o NH I I I I I I I I I I I I I I I -170 160 ISO 140 I)() 120 llO 100 90 80 70 60 so 40 )() FIGURE A-20: nc NMR SPECTRUM FOR 2, 5-DIMETHOXY-3, 6-DINITRO 9&; \ !I I 83; IE>; !U; lll; 18; 1'6; '0 J / .: E i I l, I I J .;-!! ,,.; 7]; O,N 1 NH ro: .0 0).' 611: .. 66; 500 l(DJ !'lll W....,...._'\(cm-1) FIGURE A-21: IR SPECTRUM FOR 2, 5-DIMETHOXY -3, 6-DINITRO ACETANILIDE 52 HDl i c : : I I 20 / I 'lll

PAGE 67

3Le 0492 100 I 95 '0 90 ;fl N02 85 I NH 80 75 /0 OJ 70 65 ., !! 60 E 55 50 286 0673 45 11 c; 40 a: 35 30 25 20 15 10 II 5 I II I 0 I 250 260 270 280 200 300 310 320 rrtz FIGURE A-22: [M +Hr SPECTRUM FOR 2, 5-DIMETHOXY -3, 6DINITRO ACETANILIDE ----00 I I \ 0 i H H I -o,N I H 0 19 j_f ---j J _._____ I II 0 0 ;: 'PM 8.0 7.0 6.0 5.0 40 ].0 2.0 1.0 0.0 FIGURE A -23: 1 H NMR SPECTRUM FOR 4, 7-DIMETHOXY -2METHYL-6-NITRO IMIDAZOQUINOLINE 53

PAGE 68

.................................. 'o 0 0 N I ,>-' N' / -,HOl /0 20 ll l II .11 V VY YW VY y 00 -: ::;:; : .! 8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 5.2 4.8 4.4 4.0 ).6 3.2 2.8 FIGURE A-24: 1 H NMR FOR 6-ACET AMID0-4, 7-DIMETHOXY -2-METHY( -2-NITROBENZOYL)IMIDAZOQUINOLINE PM 8.30 8.20 . y 8.10 y 0 8.00 Ill Ill II y y yy 0 'i 0 0 7.90 7.80 7.70 7.60 7.50 7.40 FIGURE A-25: AROMATIC REGIONS FOR 6-ACETAMID0-4,7DIMETHOXY -2-METHY( -2-NITROBENZOYL)IMIDAZOQUINOLINE 54 J y 2.4 7.3

PAGE 69

85 70 tf,-0 0 0 l a i 'I r N (l N H 0 NO, "''" :'fi' .c..,., 1; FIGURE A-26 IR SPECTRUM FOR 6-ACETAMID0-4,7-DIMETHOXY-2METHY( -2-NITROBENZOYL)IMIDAZOQUINOLINE ... -.................. ......................... : .,. ........... ............. i ) II \\\II((( I I I 0 0 0 N )I Jl i -NO, !I I II 1Jl11 l I I l PPM 160 140 120 100 80 60 40 20 FIGURE A-27 13C NMR FOR 6-ACETAMID0-4,7-DIMETHOXY-2-METHY(2-NITROBENZOYL)IMIDAZOQUINOLINE 55 I 0

PAGE 70

. 100 95 90 85 80 75 70 65 60 55 :i 50 "' 45 ;; 40 a: 35 30 25 20 15 10 4211105 399 1289 5 370 375 J80 385 390 395 400 405 410 415 420 425 430 rrlz FIGURE A-28 [M +H] FOR 6-ACETAMID0-4, 7-DIMETHOXY-2-METHY (-2-NITROBENZOYL) IMIDAZOQUINOLINE 100 95 90 85 80 75 7065 60 c 55 50J 1 ; .. : 40-i =J 25 20 ::j 0 L .._ Jl 150 200 250 300 350 rniZ 397 1154 400 459 2028 I .. l l. 450 500 550 FIGURE A-29 [M -H]' FOR 6-ACETAMID0-4,7-DIMETHOXY-2-METHY(2-NITROBENZOYL)IMIDAZOQUINOLINE 56

PAGE 71

-w w w lf, I I II yoo/ 0 )lN "' I N.Jl__ H H / 21 Jl { W-Y y w N l5 I I I I I I I I I I I I I 'PM 8.0 7.6 7.2 6.8 6.0 5.6 5.2 3.6 3.2 2.8 FIGURE A-30: 1 H NMR SPECTRUM FOR 3,6-DIMETHOXY -I ,2-4TRIACET AMIDOBENZENE N w w I I I yoo/ 0 )lN "'-N.Jl__ H _.....O H / / / A. y y 0 I I I I I I I I I I I I I I I PM 8.30 8.20 8.10 8.00 7.90 7.80 7.70 7.60 7.50 7.40 7.30 7.20 7.10 7.00 6.90 FIGURE A-31: AROMATIC REGIONS FOR 3, 6-DIMETHOXY -I ,2-4TRIACET AMIDO BENZENE 57 NNN lrfJ J WL -= I I 2.0 I I I 6.80 6.70 6.60

PAGE 72

.. 0 : v s 0 0 I I .. \I 'PM 160 ISO 140 IJO 120 110 100 90 80 70 60 50 40 JO 20 FIGURE A-32 13C NMR SPECTRUM FOR 3,6-DIMETHOXY-1,2-4TRIACET AMIDOBENZENE FIGURE A-33 IR SPECTRUM FOR 3,6-DIMETHOXY -I ,2-4TRIACET AMIDO BENZENE 58

PAGE 73

332.1210 100 95 90 85 eo yoo_._. 75 0 70 )lN I N__J(_ 65 H O H 60 / i 55 50 ., i 45 "ii 0:: 40 35 30 25 20 15 10 I 5 I 0 J .I 290 300 310 320 330 340 350 mtz FIGURE A-34 [M +H]' SPECTRUM FOR 3,6-DIMETHOXY-1,2-4-TRIACETAMIDO BENZENE 59

PAGE 74

B: SUPPLEMAENTARY MOLECULAR MODELING DATA Asn95 FIGURE B-1: TOPOLID INTERACTIONS WITH THE ATPASE SITE OF TOPOIIa. Non solvent binding energy= -I 07.6kcal/mol. The key interactions are a network of hydrogen bonds (green dash lines) with Asnl50, Serl49, Lysl68, Tyrl65, Glyl66 a!ld Alal67. The Active site amino acid residues are shown as white carbon atoms and Mg2 as a blue sphere. Coordination interactions are shown as green lines while the orange line denotes pi-cation attractions. 60

PAGE 75

FIGURE B-2: TOPOL2B INTERACTIONS WITH THE ATPASE SITE OF TOPOIIa. Non-solvent binding energy= -107 .6kcal/mol. The key interactions are a network of hydrogen bond (green dash lines) with Asn91, Ser149. The Active site amino acid residues are shown as white carbon atoms and Mg2 as a blue sphere. Coordination interaction is shown as light blue line while the orange line denotes pi-pi attraction. 61

PAGE 76

REFERENCES Azarova, A.M., Lyu, Y. L., Lin, C. P., Tsai, Y. C., Lau, J. Y. N., Wang, J. C., and Liu, L. F. "From the Cover: Roles of DNA Topoisomerase II lsozymes in Chemotherapy and Secondary Malignancies." Proceedings of the National Academy ofSciences 104.26 (2007): 11014-19. Bates, A. D., Berger, J. M., and Maxwell, A. "The Ancestral Role of Atp Hydrolysis in Type II Topoisomerases: Prevention of DNA Double-Strand Breaks." Nucleic Acids Research 39.15 (2011): 6327-39. Blanco, M.d. M., Avendano, C., Cabezas, N., and Menendez, J. C. "A Friedlaender Approach to 3-Substituted 2,5,8-(lh)-Quinolinetriones." Heterocycles 36.6 (1993): 1387-98. Brahic, C., Darro, F., Belloir, M., Bastide, J., Kiss, R., and Delfoume, E. "Synthesis and Cytotoxic Evaluation of Analogues ofthe Marine Pyridoacridine Amphimedine." Bioorganic & Medicinal Chemistry 10.9 (2002): 2845-53. Chene, P., Rudloff, J., Schoepfer, J., Furet, P., Meier, P., Qian, Z., Schlaeppi, J.-M., Schmitz, R., and Radimerski, T. "Catalytic Inhibition ofTopoisomerase II by a Novel Rationally Designed Atp-Competitive Purine Analogue." BMC Chemical Biology 9.1 (2009): 1-16. Cherif, M., Cotelle, P., and Catteau, J. P. "General Synthesis of2,3-Substituted 5Membered Heterocyclic Quinones." Heterocycles 34.9 (1992): 1749-58. Corbett, K. D., and Berger, J. M. "Structure, Molecular Mechanisms, and Evolutionary Relationships in DNA Topoisomerases." Annual Review of Biophysics and Biomolecular Stmcture 33.1 (2004): 95-118. Echavaren, A.M., and Stille, J. K. "Total Synthesis of Amphimedine." Journal of the American Chemical Society 110 ( 1988): 4051-53. Faulkner, D. J. "Highlights of Marine Natural Products Chemistry (1972-1999)." Natural product reports 17 .I (2000): 1-6. Guo, W., Reigan, P., Siegel, D., and Ross, D. "Enzymatic Reduction and Glutathione Conjugation of Benzoquinone Ansamycin Heat Shock Protein 90 Inhibitors: 62

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Relevance for Toxicity and Mechanism of Action." Drug metabolism and disposition: the biological fate of chemicals 36.10 (2008): 2050-57. Hande, K. "Topoisomerase II Inhibitors." Update on Cancer Therapeutics 1.1 (2006): 3-15. Kubo, A., and Nakahara, S. "Synthesis of Amphimedine, a New Fused Aromatic Alkaloid from a Pacific Sponge, Amphimedon Sp." Heterocycles 27.9 (1988): 209598. LaBarbera, D. V., Bugni, T. S., and Ireland, C. M. "The Total Synthesis of Neoamphimedine." Journal ofOrganic Chemistry 72.22 (2007): 8501-05. Linka, R. M., Porter, A. C. G., Volkov, A., Mielke, C., Boege, F., and Christensen, M. 0. "C-Terminal Regions ofTopoisomerase II Determine Isoform-Specific Functioning ofthe Enzymes in Vivo." Nucleic Acids Research 35.11 (2007): 381022. Marshall, K. "The Anti-Neoplastic and Novel Topoisomerase 11-Mediated Cytotoxicity ofNeoamphimedine, a Marine Pyridoacridine." Biochemical Pharmacology 66.3 (2003): 447-58. Marshall, K. M., Andjelic, C. D., Tasdemir, D., Concepcion, G. P., Ireland, C. M., and Barrows, L. R. "Deoxyamphimedine, a Pyridoacridine Alkaloid, Damages DNA Via the Production of Reactive Oxygen Species." Marine Drugs 7.2 (2009): 196-209. Matsumoto, S. S., Biggs, J., Copp, B. R., Holden, J. A., and Barrows, L. R. "Mechanism of Ascididemin-lnduced Cytotoxicity." Chemical research in toxicology 16.2 (2003): 113-22. Nakahara, S., Tanaka, Y., and Kubo, A. "Total Synthesis of Amphimedine." Heterocycles 43.10 ( 1996): 2113-23. Needham, E. R., and Perkin, W. H. "0-Nitrobenzoylacetic Acid." Journal of the Chemical Society 88 ( 1904 ): 148-54. Nitiss, J. L. "DNA Topoisomerase II and Its Growing Repertoire of Biological Functions." Nature Reviews Cancer 9.5 (2009): 327-37. Nitiss, J.L. "Targeting DNA Topoisomerase II in Cancer Chemotherapy." Nature Reviews Cancer 9.5 (2009): 338-50. 63

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Okazoe, T., and Morizawa, Y. 2-Arylquinolines and Process for Producing the Same. Asahi Glass Co. and the Green Cross Corporation assignee. 1999. Park, B. K., Kitteringham, N. R., Maggs, J. L., Pirmohamed, M., and Williams, D.P. "The Role ofMetabolic Activation in Drug-Induced Hepatotoxicity." Annual Review of Pharmacology and Toxicology 45 (2005): 177-202. Phillips, M.A. "The Fonnation of2-Substituted Benzimidazoles." J.Chem.Soc. ( 1928): 2393-99. Ponder, J., Yoo, B. H., Abraham, A. D., Li, Q., Ashley, A. K., Amerin, C. L., Zhou, Q., Reid, B. G., Reigan, P., Hromas, R., Nickoloff, J. A., and LaBarbera, D. V. "Neoamphimedine Circumvents Metnase-Enhanced DNA Topoisomerase lia Activity through Atp-Competitive Inhibition." Marine Drugs 9.11 (2011): 2397-408. Saltiel, E., and McGuire, W. "Doxorubicin (Adriamycin) Cardiomyopathy." Western Journal of Medicine 139.3 ( 1983): 332-41. Schmitz, F. J., Agarwal, S. K., Gunasedera, S. P., Schmidt, P. G., and Shoolery, J. N. "Amphimedine, New Aromatic Alkaloid from a Pacific Sponge, Amphimedon Sp. Carbon Connectivity Detennination from Natural Abundance Carbon-13-Carbon-13 Coupling." Journal of the American Chemical Society 105.14 (1983): 4835-36. Shinkre, B. A., Raisch, K. P., Fan, L., and Velu, S. E. "Analogs ofthe Marine Alkaloid Makaluvamines: Synthesis, Topoisomerase li Inhibition, and Anticancer Activity." Bioorganic and Medicinal Chemistry Letters 17.10 (2007): 2890-93. Sicker, D., and Mann, G. "Synthesis of Ethyl Ortho-Substituted Benzoylacetates and Tautomerism and Ms Fragmentaion Behavior." Collection Czechoslovak Chemical Communication 53 ( 1988): 839-50. Wang, J. C. "Cellular Roles of Dna Topoisomerases: A Molecular Perspective." Nature Reviews Molecular Cell Biology 3.6 (2002): 430-40. Williamson, E. A., Rasila, K. K., Corwin, L. K., Wray, J., Beck, B. D., Severns, V., Mobarak, C., Lee, S. H., Nickoloff, J. A., and Hromas, R. "The Set and Transposase Domain Protein Metnase Enhances Chromosome Decatenation: Regulation by Automethylation." Nucleic Acids Research 36.18 (2008): 5822-31. Wray, J., Williamson, E. A., Royce, M., Shaheen, M., Beck, B. D., Lee, S. H., Nickoloff, J. A., and Hromas, R. "Metnase Mediates Resistance to Topoisomerase li Inhibitors in Breast Cancer Cells." PLoS One 4.4 (2009): (e5323) 1-5. 64

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Wray, J., Williamson, E. A., Sheema, S., Lee, S. H., Libby, E., Willman, C. L., Nickoloff, J. A., and Hromas, R. "Metnase Mediates Chromosome Decatenation in Acute Leukemia Cells." Blood 114.9 (2009): 1852-58. 65