Synthesis and Pharmacological Evaluation of Nitrogen Oxide Releasing Prodrugs

Item Type text; Electronic Dissertation

Authors Bharadwaj, Gaurav

Publisher The University of Arizona.

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Download date 04/10/2021 11:39:05

Link to Item http://hdl.handle.net/10150/301748

SYNTHESIS AND PHARMACOLOGICAL EVALUATION OF NITROGEN OXIDE RELEASING PRODRUGS

by

Gaurav Bharadwaj

______

A Dissertation Submitted to the Faculty of the

DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY

In Partial Fulfillment of the Requirements For the Degree of

DOCTOR OF PHILOSOPHY WITH A MAJOR IN CHEMISTRY

In the Graduate College

THE UNIVERSITY OF ARIZONA

2013

2

THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE

As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Gaurav Bharadwaj entitled " SYNTHESIS AND PHARMCOLOGICAL EVALUATION OF NITROGEN OXIDE RELEASING PRODRUGS " and recommend that it be accepted as fulfilling the dissertation requirement for the

Degree of Doctor of Philosophy

______Date: 03/18/2013 Dr. Katrina M. Miranda

______Date: 03/18/2013 Dr. F. Ann Walker

______Date: 03/18/2013 Dr. Richard Glass

______Date: 03/18/2013 Dr. Hamish Christie

Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.

______Date: 07/30/13 Dissertation Director: Dr. Katrina M. Miranda 3

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the author.

SIGNED: Gaurav Bharadwaj 4

ACKNOWLEDGEMENTS

It is my privilege to acknowledge the people who have been constant source of support and guidance though out my research work.

First of all, I would like to express my indebtedness to my advisor Dr. Katrina Miranda for her inspiring and scholarly guidance, valuable suggestions, and constant support throughout my time at The University of Arizona work. She gave me complete freedom to explore new area and was a constant source of support during ups and downs in accomplishing those undertakings.

I would also like to sincerely thank my committee members namely Dr. Walker, Dr. Glass and Dr. Christie for spending their valuable time and their constructive suggestions at various stages of the work that enabled me to improve upon the quality of the thesis. I would also thank faculty members of the Department of Chemistry and Biochemistry for their courses, which prepared me to understand the basics that were very useful during my research work.

I would like to thank Dr. Debashree Basudhar, who started the NONO-NSAID project and I had the opportunity to work with her and get to know her. Presently she is my lovely wife, who has always believed in me and is a constant source of support. I would also like to express my sincere gratitude to present and past members of the Miranda research group at University of Arizona. They were not only a source of inspiration but also made working in the lab fun. I would specially like to thank Patricia Benini, who started the cyclic amine NONOate project. Also I would like to thank all the undergrads (Cyf, Michelle, Yannon and Kavya) with whom I got the chance to work and without their contribution, this work would not have been possible.

Finally, I would like to thank my parents for motivating me to try for higher noble goals and render the best effort to achieve the same without being afraid of failure. Their sacrifice, patience and inspiration made me work harder. My brothers and my sister-in- law have always been a constant source of inspiration and support.

5

DEDICATION

To my parents, my wife, brothers and sister-in-law, who have been a great source of

inspiration and always supported me through good and bad times. 6

TABLE OF CONTENTS

LIST OF FIGURES ...... 9

LIST OF SCHEMES ...... 14

LIST OF TABLES…………………………………………………………………………16

ABSTRACT ...... 17

1 INTRODUCTION ...... 19 1.1. Nitric oxide ...... 19 1.2. Chemical biology of NO ...... 22 1.3. Donors of NO ...... 24 1.3.1. Organic nitrates ...... 24 1.3.2. Metal nitrosyl ...... 26 1.3.3. S-Nitrosothiols (RSNO) ...... 29 1.3.4. Diazeniumdiolates...... 30 1.4. Nitroxyl ...... 34 1.4.1. Donors of HNO ...... 34 1.4.1.1. Angeli’s salt ...... 35 1.4.1.2. Piloty’s acid ...... 35 1.4.1.3 Acyl nitroso compounds ...... 36 1.4.1.4 Acyloxy nitroso Compounds ...... 37 1.4.1.5 Primary amine based diazeniumdiolates ...... 38 1.5. Cancer and therapeutic potential of NO and HNO ...... 39 1.6. Breast cancer ...... 44 1.7. Tamoxifen ...... 46 1.8. Chlorambucil...... 47 1.9. NO-NSAIDs ...... 51 1.10. Conclusion ...... 52 Abbreviations ...... 54 7

2 NITROGEN OXIDE RELEASING DIAZEN-1-IUM-1,2-DIOLATE BASED ADDUCTS OF N-DESMETHYL-TAMOXIFEN ...... 55 2.1. Introduction ...... 55 2.2. Materials and methods ...... 60 2.3. Results and discussions ...... 66 2.3.1. Half-life of DEA/NO-AcOM and carbamate adducts ...... 67 2.3.2. NO release profile of DEA/NO-AcOM ...... 68 2.3.3. Chemiluminescence detection of NO/HNO from N-desmethytamoxifen adducts ...... 69 2.3.4. Intracellular release of NO/HNO ...... 70 2.3.5. Cytotoxicity...... 73 2.3.6. Attempted synthesis of new hybrid NO/HNO-N-desmethyltamoxifen adduct ...... 78 2.4. Conclusions ...... 82 Abbreviations ...... 82

3 COMPARISON OF HNO AND NO DONATING PROPERTIES OF CYCLIC AMINE DIAZENIUMDIOLATES ...... 84 3.1. Introduction ...... 84 3.2. Methods and materials ...... 87 3.3. Results and discussions ...... 95 3.3.1. Decomposition profile and half-life ...... 96 3.3.2. NO/HNO release profile ...... 99 3.3.3. Quantification of HNO release from ionic diazeniumdiolates ...... 101 3.3.4. Cell survival assay ...... 102 3.3.5. Synthesis of acetoxymethyl protected diazeniumdiolates ...... 103 3.3.6. Decomposition half-life of CPA/NO-AcOM ...... 104 3.3.7. Intracellular NO and HNO release ...... 109 3.3.8. Cytotoxicity...... 111 3.3.9. Effect of CPA/NO-AcOM on cytotoxicity of tamoxifen ...... 112 3.4. Conclusions ...... 114 Abbreviations ...... 115 8

4 CHLORAMBUCIL ANALOGUES OF PABA/NO ...... 117 4.1. Introduction ...... 117 4.2. Methods and materials ...... 120 4.3. Results and discussions ...... 123 4.3.1. Attempted synthesis ...... 123 4.3.2. Cytotoxicity...... 126 4.3.3. Intracellular NO release ...... 127 4.4. Conclusions ...... 128 Abbreviations ...... 128

5 SYNTHESIS AND CHARACTERIZATION OF NITROGEN OXIDE ADDUCTS WITH NON-STEROIDAL ANTI-INFLAMMATORY DRUGS ...... 130 5.1. Introduction ...... 130 5.2. Methods and materials ...... 132 5.3. Results and discussions ...... 140 5.3.1. Characterizing NO/HNO release ...... 141 5.3.2. Intracellular release ...... 146 5.3.3. Cytotoxicity...... 147 5.4. Conclusions ...... 150 Abbreviations ...... 150

6 FUTURE DIRECTIONS ...... 152

APPENDIX A: NMR DATA ...... 159

REFERENCES ...... 209

9

LIST OF FIGURES

Figure 1.1: Dimeric structure of nitric oxide synthase ...... 20

Figure 1.2: Direct and indirect effects of NO ...... 23

Figure 1.3: Examples of clinically used organic nitrates ...... 25

Figure 1.4: Suggested enzymatic or non enzymatic release of NO from organic nitrates ..26

Figure 1.5: Dinitrosyl iron complex ...... 27

Figure 1.6: Iron nitrosyl based NO donor...... 28

Figure 1.7: Examples of stable S-nitrosothiols...... 30

Figure 1.8: O2-Substituted diazeniumdiolates designed to be activated for NO release

by specific enzymes ...... 32

Figure 1.9: Cellular response to different level of NO ...... 42

Figure 1.10: Effects of low vs high concentration of NO in cancer biology ...... 43

Figure 1.11: Three types of estrogen hormone ...... 45

Figure 1.12: Tamoxifen ...... 46

Figure 1.13: Key metabolites of tamoxifen ...... 47

Figure 1.14: Alkylating drug chlorambucil and its active metabolite ...... 48

Figure 1.15: Structure of GST inhibitor: ethacrynic acid ...... 50

Figure 1.16: Representative examples of NO-NSAIDs ...... 52

Figure 2.1: The pH-dependence of the first-order rate constants of decomposition of

DEA/NO-AcOM at 37°C in PBS containing 50 μM DTPA measured at 250

nm in presence of 2% guinea pig serum ...... 68 10

Figure 2.2: Detection of NO using 50 µM MbO2 reacted with 50 µM DEA/NO-AcOM

in the presence of 2% guinea pig serum at 37°C and aerated condition...... 69

Figure 2.3: Detection of NO using chemiluminescence from head space of reaction of

DEA/NO-N-des-tam (100 µM) in the presence of 2% guinea pig serum in

pH 7.4 buffer at room temperature...... 70

Figure 2.4: (A) NO release measured under physiological conditions in MB-231 (n=6,

2trial) cells from reaction of 10 µM DAF-2DA with 10 µM of DEA/NO-

AcOM in DMSO (<0.1%) or DEA/NO 100 µM in 10 mM NaOH (B) NO

or HNO release measured after 12 h under physiological conditions in

MCF-7, MB-468 and MB-231 cells from reaction of 10 µM DAF-2DA

with 10 µM of DEA/NO-N-des-tam and IPA/NO-N-des-tam in DMSO

(<0.1%)...... 72

Figure 2.5: The effect of varied concentrations of DEA/NO-AcOM on cell survival of

(A) MCF-7, and (B) MB-231cell lines...... 74

Figure 2.6: The effect of different concentrations of DEA/NO-AcOM on cell survival of

(A) MCF-7 along with 0.1μM tamoxifen, and (B) MB-231cell lines along

with 10 μM. Cells were treated with different concentrations of DEA/NO-

AcOM (0-100 µM) and cell survival was determined using MTT assay after

48 h...... 76

Figure 2.7: The effect of different concentrations of DEA/NO-N-des-tam, IPA/NO-N-

des-tam and tamoxifen on cell survival in MCF-7 cells. Cell survival was

determined using MTT assay after 48 h ...... 77 11

Figure 2.8: New proposed NO/HNO donor of N-desmethyltamoxifen...... 78

Figure 3.1: Spontaneous decomposition of CPA/NO at 37°C in assay buffer at pH

7.4…...... 97

Figure 3.2: The pH-dependence of the first-order rate constants of decomposition of

CPA/NO, CHA/NO, CHPA/NO and COA/NO at 37°C in PBS containing

50μM DTPA measured at 250 nm...... 98

Figure 3.3: The maximum current intensity from an NO-specific electrode during

decomposition of 50 µM CPA/NO, CHA/NO, CHPA/NO and COA/NO at

pH 7.4 assay buffer ± 1 mM ferricyanide (green bars, NO + HNO) at room

temperature...... 99

Figure 3.4: The pH dependence of maximum current intensity from an NO-specific

electrode during decomposition of 5 µM CPA/NO in PBS containing 50

μM DTPA (blue bars, NO alone) ± 1 mM ferricyanide (green bars, NO +

HNO) at room temperature...... 101

Figure 3.5: Toxicity towards MCF-7 cells of cyclic diazeniumdiolates...... 103

Figure 3.6: Hydrolysis of CPA/NO-AcOM in the presence of 2% guinea pig serum in

assay buffer at pH 7.4 and 37°C...... 105

Figure 3.7: The pH dependent maximum current intensity from an NO-specific

electrode during decomposition of CPA/NO-AcOM (100 μM) in presence

of 2% guinea pig serum in assay buffer (blue bars, NO alone) ± 1 mM

ferricyanide (green bars, NO + HNO) at room temperature...... 106

Figure 3.8: Hydrolysis of CPA/NO-AcOM in assay buffer at pH 7.4 and 37°C ...... 107 12

Figure 3.9: Reductive nitrosylation of metMb (50 μM, blue spectra) with A) CPA/NO

or B) CPA/NO-AcOM (100 μM). The assay was performed in pH 7.4 assay

buffer at 37 °C (red spectra, compound alone) ± 1 mM GSH (green spectra)

under deaerated conditions ...... 109

Figure 3.10: NO and HNO release measured in MB-231 cells ...... 110

Figure 3.11: Toxicity of CPA/NO-AcOM towards MCF-7 and MDA-MB-231 cells

by MTT cell survival assay at 37°C...... 112

Figure 3.12: The effect of CPA/NO-AcOM (75 µM), tamoxifen (10 µM) or

combination (CPA/NO-AcOM 75 µM + tamoxifen 10 µM) on survival of

MDA-MB-231 cells...... 114

Figure 4.1: NO releasing JS-K ...... 119

Figure 4.2: PABA/NO...... 120

Figure 4.3: NO releasing chlorambucil analog of PABA/NO...... 120

Figure 4.4: The effect of DMA-DNB-chlorambucil, chlorambucil and DMSO control

(0-200 µM, respectively) on cell survival of MB-231 cells...... 127

Figure 4.5: NO or HNO release measured after 12 h under physiological conditions in

MB-231 cells from reaction of 10 µM DAF-2DA with 10 µM of

chlorambucil and 3 in DMSO...... 128

Figure 5.1: Naproxinod, a derivative of naproxen with a nitroxybutyl ester ...... 131

Figure 5.2: Representative spectral changes (n ≥ 3) indicating trapping of NO and

HNO by MbO2 (10 μM) during guinea pig serum induced decomposition at

37°C of DEA/NO-NSAIDs or IPA/NO-NSAIDs in assay buffer ± GSH (1 13

mM): (A) 10 µM DEA/NO-indomethacin, (B) 10 µM DEA/NO-niflumic

acid, (C) 10 µM IPA/NO-indoemthacin and (D) 10 µM IPA/NO-niflumic

acid...... 143

Figure 5.3: NO and HNO release measured using an NO-specific electrode from 100

µM of (A) IPA/NO-NSAIDs analogues or (B) DEA/NO-NSAIDs

analogues in assay buffer (pH 7.4) containing 2% guinea pig serum (± 1

mM ferricyanide in A). Niflumic derivative red color, indomethacin blue

color and aspirin derivative green colored...... 145

Figure 5.4: NO and HNO release measured in MB-231 cells ...... 146

Figure 5.5: The effect of NONO-NSAIDs prodrugs and appropriate controls (25-100

µM, respectively) on cell survival of MB-231 cells with (A) DEA/NO-

NSAIDs, (B) IPA/NO-NSAIDS and in MCF-7 cells with (C) DEA/NO-

NSAIDs and (D) IPA/NO-NSAIDs...... 149

Figure 6.1: Fluorescent tag attached derivatized primary amine based

diazeniumdiolate… ...... 155

Figure 6.2: Structure of alkylating agent melphelan and cyclophosphamide ...... 156

Figure 6.3: NSAIDs ibuprofen, naproxen and diclofenac ...... 157

Figure 6.4: Second generation NSAIDs ...... 158

14

LIST OF SCHEMES

Scheme 1.1 Biosynthetic pathway of NO production……………………………...... 19

Scheme 1.2 Preparative routes for organic nitrates…………………………………...... 24

Scheme 1.3 Decomposition of RSNO……………………………………………………... 29

Scheme 1.4 Dual decomposition mechanisms for primary amine based diazeniumdiolates…………………………………………………………….. 38

Scheme 1.5 General mechanism for reactivity of nitrogen mustard………………………. 49

Scheme 1.6 GST activated release of NO…………………………………………………. 50

Scheme 2.1 Partial depiction of CYP mediated metabolism of tamoxifen………………... 57

Scheme 2.2 Acetoxymethyl protected DEA/NO (DEA/NO-AcOM)…………………………... 59

Scheme 2.3 Synthesis of potentially NO/HNO releasing N-desmethyltamoxifen derivative……………………………………………………………………… 60

Scheme 2.4 Proposed in vivo release of NO/HNO from diazeniumdiolate-N- desmethyltamoxifen conjugate………………………………………………... 67

Scheme 2.5 Proposed esterase mediated hydrolysis………………………………………..78

Scheme 2.6 Attempted synthesis of hybrid ester based drug using succinic acid…………. 80

Scheme 2.7 Attempted synthesis of hybrid ester based drug using protected succinic acid. 81

Scheme 3.1 Common HNO donor structures……………………………………………… 85

Scheme 3.2 Mechanism of NO and HNO release from diazeniumdiolates……………….. 86

Scheme 3.3 Mechanism of decomposition of acetoxymethyl protected IPA/NO in PBS..... 87

Scheme 3.4 Synthesis of stable cyclic amine diazeniumdiolates………………………….. 96

Scheme 3.5 Synthesis of CPA/NO-AcOM………………………………………………… 104

Scheme 3.6 Decomposition of O2-(acetoxymethyl)-1-(cyclopentylamino) diazen-1-ium- 1,2-diolate at PBS pH 7.4 and 37°C in the presence of serum...... 105 15

Scheme 4.1 GST mediated NO release from JS-K…………………………………………119

Scheme 4.2 Attempted aromatic nucleophilic substitution to form compound 3…………. 123

Scheme 4.3 Attempted DCC coupling of intermediate 2 with chlorambucil……………… 124

Scheme 4.4 Attempted DMAP catalyzed DCC coupling………………………………….. 125

Scheme 4.5 Attempted synthesis of target compound via acid halide intermediate………. 125

Scheme 4.6 Synthesis of DMA-dinitrobenzene-chlorambucil adduct…………………….. 126

Scheme 5.1 Synthesis of NO/HNO releasing NSAIDs derivatives……………………….. 134

Scheme 6.1 Proposed synthetic route for NO releasing 4-hydroxytamoxifen conjugate………… 153

Scheme 6.2 Proposed synthetic routes for antibody coupled derivatized diazeniumdiolates……………………………………………………………... 154

16

LIST OF TABLES

Table 1.1: Diazeniumdiolates with their half-life ...... 31

Table 3.1: Decomposition data for cyclic amine diazeniumdiolates at pH 7.4 and 37°C..

...... 98

Table 3.2: Percent HNO from NONOates using 50 μM GSH at pH 7.0 and at 7.4...... 102

17

ABSTRACT

The main goals of this research were to synthesize nitrogen oxide releasing diazeniumdiolates and their prodrugs and to evaluate their pharmacological effects. The different projects and their results are described below. i. Comparison of HNO and NO donating properties of cyclic amine diazeniumdiolates

Diazeniumdiolates are an attractive class of donor compounds as they can be tuned to release NO or both NO and HNO depending upon the amine backbone. Isopropylamine

(IPA/NO) and cyclohexylamine (CHA/NO) diazeniumdiolates are currently the only examples of primary amine based diazeniumdiolates. A series of structurally related cyclic amine based diazeniumdiolates were synthesized and characterized. An acetoxymethyl derivative was also synthesized to facilitate cellular uptake and to achieve higher HNO levels in cells. ii. Nitrogen oxide releasing diazeiumdiolate based adducts of N-des-methyl-tamoxifen

Nitrogen oxide (NO/HNO) donating diazeniumdiolate adducts of N-desmethyltamoxifen

(a key metabolite of the breast cancer drug tamoxifen) were synthesized. DEA/NO-

AcOM, an NO donor was also synthesized to monitor the effect of NO on breast cancer cell survival. Derivatives of N-desmethyltamoxifen were found to be effective towards estrogen receptor positive (ER+) cells only. DEA/NO-AcOM was found to be cytotoxic towards estrogen-dependent and independent cell lines, in combination with tamoxifen, or by itself.

18

iii. Synthesis and characterization of nitrogen oxide adducts with non-steroidal anti- inflammatory drugs (NSAIDs)

Our group has shown HNO releasing diazeniumdiolate derivatized aspirin to be comparably effective in preventing gastric ulceration to NO-releasing diazeniumdiolate based aspirin analogues. Series of such NSAID adducts were further extended by synthesizing such derivatives of indomethacin and niflumic acid. NO/HNO releasing analogues of aspirin and indomethacin were cytotoxic towards two different breast cancer cell lines, irrespective of estrogen dependence. iv. Chlorambucil analogue of PABA/NO

Chlorambucil, an alkylating agent is used in leukemia treatment. Tumor cells resistant to alkylating agents often have increased glutathione levels and increased activity of glutathione-S-transferase (GST). PABA/NO is an NO donor with a promising anticancer profile. The chlorambucil analogue of PABA/NO was synthesized to utilize GST for releasing NO and to potentially overcome cellular resistance. 19

CHAPTER 1 INTRODUCTION

1.1 Nitric oxide

Nitric oxide (NO), an endogenous free radical messenger molecule, plays a vital

role in numerous physiological and pathological processes spanning from modulation of

blood pressure and neural communication to immune response.1-7 NO is biosynthesized

in an NADPH-dependent, five-electron oxidation of L-arginine to L-citrulline by NO

synthase (NOS; Scheme 1.1). N-Hydroxy-L-arginine (NOHA) is a key intermediate in

this pathway.8 NOS exists in three isoforms: endothelial NOS (eNOS),9 neuronal NOS

(nNOS)10 and inducible NOS (iNOS).11 These three isoforms share structural similarities

and have nearly identical catalytic mechanisms.12 nNOS and eNOS are constitutively

expressed whereas iNOS is inducible isoform that can be induced by cytokines and

lipopolysaccharides.13

H2N HO NH H2N NH NH O HN HN HN O2 H2O O2 H2O

+ NO NADPH NADPH

H3N H3N H3N O O O O O O

L-arginine NOHA L-citrulline

Scheme 1.1 Biosynthetic pathway of NO production

All three NOS isoforms are homodimers in their active form, with monomer

weight varying from 130 to 160 KDa.8,12,14,15 NOS consists of a heme or oxygenase 20

containing N-terminal domain and a reductase containing domain at its C-terminal.16 In

between the two domains lies the highly conserved calmodulin binding domain.17 The C- terminal domain is responsible for binding the reducing agents NADPH, FMN and

FAD.18 This domain functions to transfer electrons to the N-terminal domain, which

19 contains binding sites for heme, tetrahydrobiopterin (H4B) and L-arginine. The N-

terminal domain forms the active site where biosynthesis of NO occurs. Figure 1.1 shows

a schematic presentation of dimeric NOS.

Figure 1.1 Dimeric structure of nitric oxide synthase (adapted from Groves et al.).20

Each NOS isoform is associated with a particular physiological process. eNOS is a membrane-bound protein and is expressed in the vascular endothelium. NO generated by eNOS regulates smooth muscle relaxation and blood pressure through cyclic guanosine monophosphate (cGMP)-dependent pathway via activation of soluble gulanyl cyclase (sGC).21,22 eNOS also regulates platelet aggregation and leukocyte adherence.

Diminished NO production from eNOS has been implicated in the pathogenesis of 21

systemic and pulmonary hypertension, atherosclerosis and other vascular disorders.23,24

There is evidence that eNOS is also expressed in other cell lines such as airway epithelial cells.25,26 Epithelium-derived NO is responsible for smooth muscle relaxation,

bacteriostasis and modulation of ciliary motility among others.27-29 There is growing

evidence of possible epithelial eNOS expression attenuation during inflammatory

conditions, thereby contributing to airway dysfunction.30

nNOS is a cytosolic protein responsible for neurotransmission in the brain and peripheral nervous system by cGMP-dependent mechanisms.7 It also regulates neural

development,31 regeneration and the ability of two neurons to connect (synaptic

plasticity).32 Overproduction of NO resulting from deregulated nNOS activation can

initiate a neurotoxic cascade, following persistent stimulation of excitatory amino acid

receptors, mediating glutamate toxicity.33 Glutamate is the most abundant

neurotransmitter in the vertebrate nervous system and plays a key role in learning and

memory.34 Excessive release of glutamate results in neural cell damage and death.

Diseases like Alzheimers, autism, and intellectual disabilities are often attributed to

glutamate-induced toxicity.35-37 In vivo and in vitro studies with NO donors, NOS

inhibitors and glutamate receptor antagonists suggest a dual role of NO in glutamate

release.38 Low concentrations of NO decrease glutamate release, but when NO levels increase, the inhibitory effect on glutamate release is reversed.39

While both eNOS and nNOS are constitutive isoforms12 and require

calmodulin/calcium for NO production, iNOS11 is expressed as a result of immune

response against microorganisms, and iNOS is not dependent upon calcium/calmodulin 22

for its enzymatic action.40 The iNOS isoform expression can be induced by pro-

inflammatory cytokines such as interferons (IFN), interleukin(IL)-1, IL-2, tumor necrosis

factor (TNF)-α or bacterial lipopolysaccharide (LPS).41 Although iNOS is expressed in

essentially every cell type, macrophages represent the typical source of this enzyme and

can locally generate high quantities of NO.42-45 Generally, iNOS is assumed to produce

low micromolar levels of NO compared to picomolar-nanomolar concentrations by the constitutive isoforms. This is due to the high affinity of iNOS for calmodulin, which is tightly bound within physiological concentrations of calcium and thus produces sustained

NO in the absence of changes in calcium levels. In comparison, the constitutive isoforms require a rise in intracellular calcium level for calmodulin binding.42

Given the diverse roles of NO, an imbalance in regulation of NO synthesis (either

excessive or diminished concentration) may lead to pathological conditions. For instance

many autoimmune disorders arise from elevated NO concentrations.46 iNOS, for instance,

has been implicated as a marker of inflammation and cancer prognosis.47,48 Higher concentrations of NO produced by iNOS provide a chemical biology which is more rich and diverse than that which occurs from NO produced by constitutive isoforms. This difference can be attributed to direct or indirect effects of NO as described by Wink and

Mitchell.45

1.2 Chemical biology of NO

The biological importance of NO was firmly established in the early 1990’s as

NO was proven to be endothelium derived relaxing factor (EDRF), which controls blood 23

pressure. This discovery was acknowledged by the 1998 Nobel Prize in Medicine to

Ignarro, Furchgott, and Murad. NO can undergo various sets of reactions to exert effects

that are often physiologically categorized as direct and indirect effects (Figure 1.2).45

Direct effects occur through reactions in which NO interact directly with biological molecules, particularly metals and radicals. The most notable example of direct effect is binding of NO to sGC.49 This binding results in conversion of guanosine triphosphate

(GTP) to cyclic guanosine monophosphate (cGMP).50,51 In contrast, indirect effects are

mostly derived from the reaction of NO with either superoxide or O2, which results in

formation of reactive nitrogen species (RNS).52,53 Such species then mediate alterations

of macromolecules such as proteins and DNA.54-57 They can also cause cellular oxidation of small molecular species such as thiol glutathione.46

Figure 1.2 Direct and indirect effects of NO (figure adapted from Mancardi. et al.)58 24

NO is used clinically to treat persistent pulmonary hypertension in premature

59,60 61 neonates. However instability, autoxidation to form toxic NO2, site selectivity and

inconvenience in handling NO, necessitate design and synthesis of compounds that can

generate NO in situ.62

1.3 Donors of NO

Major progress over the past two decades in the field of chemical biology of NO

has been made possible by the availability and the extensive use of donor compounds,

which provide rich diversity in structure and mode of release of NO. Some of the major

NO donors such as organic nitrates, S-nitrosothiols, metal nitrosyl and diazeniumdiolates

with their properties are discussed here.

1.3.1 Organic nitrates

Organic nitrates (RONO2) are the oldest class of NO donor and have been used

clinically for over a century to relieve pain associated with angina,63 a medical state

arising from constriction of coronary arteries.64 Organic nitrates are nitric acid esters of

alcohols and can be readily prepared either by esterification (Scheme 1.2) of alcohols or by reacting alkyl halides with silver nitrate.65

Scheme 1.2 Preparative routes for organic nitrates 25

Common clinically used organic nitrates include glyceryl trinitrate (one of the best

studied organic nitrates; also known as nitroglycerin), isosorbide-5-mononitrate,

isosorbide dinitrate, pentaerythrityl and tetranitrate nicorandil (Figure 1.3).

Figure 1.3 Examples of clinically used organic nitrates

The clinical effects of nitrates are attributed to NO, which is released upon enzymatic or non-enzymatic three-electron reduction.66 The biochemical pathways

leading to release of NO are not fully understood, and it is likely that multiple

intracellular and extracellular pathways contribute to NO formation (Figure 1.4).67

Several studies suggest cellular thiols as a key factor in mediating release of NO68, although slower release of NO by thiols in the absence of enzymes suggests involvement of enzymes such as cytochrome P450, glutathione-S-transferase (GST) or xanthine oxidase (Figure 1.4).69-71 26

Figure 1.4 Suggested enzymatic or non enzymatic release of NO from organic nitrates

(adapted from Wang et al.)62

A limitation associated with use of organic nitrates is the attenuation of response on prolonged treatment, which is known as nitrate tolerance.72 The mechanism for

development of tolerance is unclear in part due to lack of understanding of the NO

release pathways.

1.3.2 Metal nitrosyl

Metal centers especially iron due to its abundance, are principal targets for NO under physiological conditions leading to metal nitrosyl complex formation. However metal nitrosyl complexes can also act as NO donors. Dinitrosyl iron complexes (DNICs,

Figure 1.5), are suggested to store and transport NO in vivo.73 Solutions of DNIC are

characterized by absorbance maxima near 320 nm.74 DNICs have been shown to inhibit

platelet aggregation much like NO, and to relax vascular vessels.75 They also enhance

cardiac resistance to ischemia and reperfusion.76 27

Figure 1.5 Dinitrosyl iron complex

Iron-sulfur clusters constitute another important class of iron nitrosyl complexes.77 Iron-sulfur cores exist in numerous enzymes78, and complexation with NO

gives rise to iron-sulfur cluster nitrosyls. Examples of such complexes (Figure 1.6) are

-1 synthetically prepared Roussin’s black salt (RBS, [Fe4S3(NO)7] ), Roussin’s red salt

2- 79 (RRS,[Fe2S2(NO)4] ) and Roussin’s red esters (Fe2(SR)2(NO)4, RRE, R = aliphatic

group).80 Such species are known to release NO upon photoactivation and have been

shown to inhibit platelet aggregation81 and to have bacteriostatic effects.82 The most well

studied iron nitrosyl complex is sodium nitroprusside (Na2[Fe(CN)5NO], SNP), which

has been used clinically for over 70 years to treat hypertensive emergencies. SNP is a

crystalline compound83 and if kept dry and protected from light, it can be stored for years.

Release of NO from SNP, although not completely understood, requires either light or

non enzymatic one-electron reduction.84,85 Although SNP inhibits platelet aggregation,86 a potential concern associated with the usage of SNP is cyanide release upon decomposition which can lead to cyanide toxicity.87 28

Figure 1.6 Iron nitrosyl based NO donor

Complexes of NO with ruthenium have also attracted considerable attention as an alternative NO donor to conventional iron-nitrosyls due to the high affinity of ruthenium for NO and better stability. Like SNP, they can also release NO upon one-electron reduction. The attractiveness arises from the tunability of the reduction potential of the

Ru-NO complexes by varying the π-bonding strength of other ligands and dissociation rates of the Ru-NO bond.88,89 Several studies have been documented which demonstrate

ruthenium nitrosyl complexes as effective antihypertensives.90,91 Some of the ruthenium-

nitrosyl release NO when exposed to light and thus has potential to be used in

photodynamic therapy.92

29

1.3.3 S-Nitrosothiols

Decomposition of S-nitrosothiols (RSNO) can proceed via homolytic or heterolytic cleavage (Scheme 1.3) of the S-NO bond to potentially release NO, NO+ or

NO-. RSNO can be potential vehicles for NO storage and delivery under physiological conditions.93 RSNO compounds exhibits antiplatelet and vasodilator properties, and their

antitumor properties have also been investigated.94,95

Scheme 1.3 Decomposition of RSNO

RSNOs can be synthesized by nitrosation of a thiol group (Eq 1)96, and are colored compounds with characteristic UV-Vis and NMR spectra.

Most S-nitrosothiols are unstable compounds with stability dependent upon R group, light, presence of other thiols, heat, or certain metal ions (Cu+ particularly).97 In

the presence of Cu+, heat or light, NO is released. On the other hand in the presence of

excess thiol, transnitrosation (transfer of NO+) or HNO production can occur.96,98,99

Examples of some stable RSNOs are shown in Figure 1.7. 30

Figure 1.7 Examples of stable S-nitrosothiols

A possible advantage of S-nitrosothiols over organic nitrates is the lack of tolerance development as they have far less strict metabolic activation requirements.

However non-specificity can lead to potential concern regarding effects at undesired sites.100 Other potential advantages include antiplatelet action at a dosage that does not

influence vascular tone.101,102

1.3.4 Diazeniumdiolates

Diazeniumdiolates, often called NONOates, have the general structure

X[N(O)NO]-, where X can be C, N, O or S-based nucleophilic center. N-Bound

diazeniumdiolates, first synthesized by Drago,103 were revived in 1991 by Maragos et al.

as controlled donors of NO under physiologically relevant conditions.104 NONOates are

widely used to study the effects of time and concentration-dependent exposure of NO.

Amine based diazeniumdiolates can be prepared by exposing amines to high pressure of NO (Eq 2).105,106

31

Diazeniumdiolates are typically stable as solids but dissociate spontaneously at rates that vary widely depending on their structure and the pH of the reaction medium.104

Secondary amine based diazeniumdiolates generate up to two moles of NO per donor molecule (Eq 3).

Keefer et al. have synthesized a variety of diazeniumdiolate based NO donors with half-

lives from 2 s to 20 h (Table 1.1).107

Table 1.1 Diazeniumdiolates and their half-lives under physiological condition107

Amine NONOate Half-life (pH 7.4, 37 ºC) L-proline PROLI/NO 2s

Pyrrolidine PYRRO/NO 2.8s

Diethylamine DEA/NO 2 min

L-spermine SPER/NO 37 min

Diethylenetriamine DETA/NO 20 h

Pyrrolidine (O2-vinyl) V-PYRRO/NO 6d

PROLI/NO with a 2 s half-life can be used for localized NO generation while

DETA/NO maintains a constant NO flux in cells for hours. The ability to produce NO

over a broad timescale has led to extensive use of diazeniumdiolates in understanding the

chemical biology of NO. The list of available NONOates has been further extended by 32

derivatization of the terminal oxygen of the ionic diazeniumdiolate moiety.

Derivatization of diazeniumdiolates significantly increases the ease of purification and

also the half-lives of these compounds (see Table 1.1, PYRRO/NO vs V-PYRRO/NO).

Moreover, site-specific NO delivery can be achieved by attaching groups cleaved by

specific enzymes or activating agents. A few examples of such compounds are shown in

Figure 1.8.

Figure 1.8 O2-Substituted diazeniumdiolates designed to be activated for NO release by

specific enzymes.108

V-PYRRO/NO was synthesized to selectively deliver NO to hepatocytes. NO

release is activated by cytochrome P450s that are present in liver. V-PYRRO/NO showed

significant protection of liver cells from apoptotic cell death induced by exposure to toxic

levels of TNF- in an in vivo model. Thus V-PYRRO/NO has potential for treatment of

hepatic disorders such as fulminant liver failure.109 PYRRO/NO has also been coupled to 33

a small peptide that is a substrate for prostate-specific antigen, and thus has the ability to

specifically target cancer cells.110 -Glc-PYRRO/NO is hydrolyzed by -D-glucosidase

to release NO and was found to have promising antileishmanial properties in targeting the

parasite Leishmania (a parasite that thrives in macrophages).111 The acetoxymethyl

derivatives of DEA/NO and PYRRO/NO undergo esterase-induced hydrolysis, and have

been investigated in leukemia cells.112 These acetoxymethyl protected diazeniumdiolates

showed significant in vitro antileukemic activity. JS-K, which is activated by glutathione

transferase, has emerged as a NO donor with promising anticancer properties. JS-K has

been shown to be effective against various cancer cell lines with IC50 values ranging from

0.2-1.2 µM, compared to much higher micromolar concentrations for other derivatized diazeniumdiolates.112,113 JS-K was further modified using computer modeling to an NO

donor that was specific toward the π isoform of glutathione transferase (an enzyme

involved in detoxification of xenobitics), an isoform that is over-expressed in cancer

114 cells. This led to the development of the NO donor PABA/NO, which was found to

release NO selectively by π isoform and showed promising activity against human

ovarian cancer xenografts in mice.

Other than showing potent anti-cancer property, diazeniumdiolates have also been

shown to be excellent antimicrobial agents.115 N-Diazeniumdiolate modified silica

nanoparticles caused three log reductions in planktonic Pseudomonas aeruginosa

cultures.116 In combination with the antibiotic silver sulfadiazine (AgSD), PROLI/NO was shown to synergize with AgSD against a range of pathogens.117 Thus, the wide range 34

of half-life and facile derivatization at the O2-position may lead to use of

diazeniumdiolate for treatment of a range of medical problems.

1.4 Nitroxyl

Nitroxyl (HNO), one electron more reduced than NO, has recently emerged as a

promising pharmacological agent with important cardiovascular118-120 and tumorocidal

properties.121 HNO has also been used clinically in the treatment of alcoholism in the

form of cyanamide, which is metabolized to HNO.122 It has also been shown in many

cases that HNO elicit pharmacological responses that are different from its redox

congener NO.123-126 Despite suggestions of possible endogenous production pathways to

HNO127-129, to date there is no definitive evidence of HNO biosynthesis in mammalian

systems. This may be in part due to spontaneous, irreversible dimerization (Eq 4), thereby

complicating detection.

1.4.1 Donors of HNO

Due to irreversible dimerization130-132 HNO cannot be stored, and donor compounds are necessary for in situ production of HNO. Donors that are well documented in the literature are Angeli’s salt, Piloty acid and derivatives, acyl nitroso compounds, acyloxy nitroso compounds and primary amine based diazeniumdiolates.

35

1.4.1.1 Angeli’s salt

To date the most prevalent donor used for understanding the chemistry and

pharmacological effects of HNO is Angeli’s salt (Na2N2O3, sodium trioxodinitrate).

Synthesized in 1896,133 widespread use of Angeli’s salt arose from the ability to

spontaneously release HNO in aqueous solution with a pH-independent rate from pH 4-8,

(Eq 5).134-136 Moreover, Angeli’s salt is commercially available, stable as a solid and in

- basic solution. Decomposition of Angeli’s salt also produces nitrite (NO2 ) which has its

own biological activity. Thus, care must be taken to conduct experiments with

decomposed Angeli’s salt as a control. In spite of its widespread usage, Angeli’s salt is

not suitable for analysis of chronic exposure of HNO due to its short half life of ~ 2

min.104

1.4.1.2 Piloty’s acid

Another class of commonly used and well studied HNO donors are N-

hydroxysulfonamide derivatives such as N-hydroxybenzenesulfonamide (Piloty’s acid;

137 C6H5SO2NHOH). Piloty’s acid is also commercially available and can be synthesized

readily by condensation of hydroxylamine with benzenesulfonyl chloride to give a solid

product (Eq 6).138

36

Piloty’s acid decomposes (Eq 7) through a base-catalyzed deprotonation

mechanism followed by S-N bond heterolysis.139 Decomposition follows first-order

kinetics with a rate constant at pH 13 of 1.8 × 10-3 s-1 at 37 ºC, which is comparable to that of Angeli’s salt at neutral pH.140

At neutral pH, however, production of HNO from Piloty’s acid decreases significantly, and under aerobic conditions Piloty’s acid undergoes oxidation to form NO instead of HNO (Eq 8).141

The slower rate of HNO release and NO production hampers the use of Piloty’s

acid as an HNO donor under neutral aerobic conditions. A number of Piloty’s acid

derivatives have been synthesized recently that show enhanced HNO production near

neutral pH.142

1.4.1.3 Acyl Nitroso compounds

Acyl nitroso compounds [RC(=O)NO] are organic based HNO donors. They are

highly reactive and thus stable compounds have not been reported. Evidence of their

existence was first determined by time resolved infrared spectroscopic measurements.143

Acyl nitroso compounds can be prepared by oxidation of N-acylhydroxylamine 37

derivatives such as N-hyroxyurea using agents such as sodium periodate. Acyl nitroso

generate HNO on reaction with nucleophile presumably via nucleophilic acyl substitution

(Eq 9).

Generation of nitrous oxide (N2O) in the above reaction provides strong evidence

for HNO production (eq 3).144 Since high instability of acyl niroso compounds requires in

situ generation, care must be taken to consider the chemistry/toxicity of precursor

molecules and the byproducts while elucidating the chemical targets of HNO under

biological conditions.

1.4.1.4 Acyloxy Nitroso Compounds

Acyloxy nitroso compounds are bright blue in color and are relatively new HNO

donor compounds. They can be prepared by oxidation of oximes145-147 with lead tetra-

acetate, and the acyl group can be varied by adding an excess amount of the appropriate

acid. Hydrolysis of the ester bond gives an unstable intermediate and the corresponding

acid. This intermediate then decomposes to release the corresponding ketone and HNO

(Eq 10).

Gas chromatographic headspace analysis shows production of N2O from these

compounds thereby providing strong evidence for formation of HNO (eq 3).148 Acyloxy 38

nitroso compounds are relatively stable under neutral conditions (t½ > 2 h for nitrosocyclohexyl acetate), but decomposition is accelerated under basic conditions (t½ =

0.8 min in 0.1 N NaOH).148 The rate of hydrolysis and the HNO yield varies with

structure.148 Preliminary studies have shown this class of donor compounds to inhibit

platelet aggregation as well as thrombus formation.145,146

1.4.1.5 Primary amine based diazeniumdiolates

Diazeniumdiolates are an attractive class of donor compounds as they can be tuned to release NO or both NO and HNO, depending upon the amine backbone.149,150

Secondary amine based diazeniumdiolates are firmly established as NO donors151 at all

pH values. On the other hand, the primary amine based diazeniumdiolates derived from

isopropylamine (IPA/NO) is primarily an HNO donor at pH > 8, an NO donor at pH < 5

and between pH range 5-8, it exhibits dual donor properties (Scheme 1.4).136,152

Scheme 1.4 Dual decomposition pathways for the primary amine based diazeniumdiolates. 39

The decomposition mechanism of IPA/NO has been studied theoretically as well as

experimentally.136,152 Tautomerization followed by N-N by heterolytic bond cleavage provides access to an HNO-donating pathway. On the other hand, protonation at the nitroso oxygen followed by tautomerization and subsequent N-N bond cleavage leads to production of NO. This mechanism is applicable to secondary amine diazeniumdiolates as well.153

IPA/NO has been shown to mimic the chemical and biological properties of

Angeli’s salt,123,150 however unlike Angeli’s salt, diazeniumdiolates can be readily derivatized at the O2-position,109,154,155 which enhances stability, purification and

controlled release106 enzymatically,112 photolytically,156 or by spontaneous

hydrolysis.154,157

NO has been used clinically to treat cardiovascular disease158 whereas HNO has

been used to treat alcoholism in form of drug cyanamide.122 Evidence is emerging for the

role of NO and HNO as anticancer agents13,121 and more recently for NO to overcome

cancer cell resistance.159,160 Their chemotherapeutic potential is discussed in the next

section.

1.5 Cancer and therapeutic potential of NO and HNO

Cancer is a complex disease that is marked by unregulated cell growth. Cancer can develop in almost any organ, and in many cases malignant tumors metastasize to other organs. In spite of significant success in treatment of cancer, early detection and treatment of late stage cancer remains significant challenge. Moreover, development of 40

resistance toward conventional chemotherapeutics remains a major hurdle in eradication

of cancer. Determining what causes normal cells to become cancerous is complicated, but

many agents are known to increase risk of cancer, such as tobacco use, exposure to

chemicals such as benzene, obesity, some viruses, prolonged exposure to sunlight, etc.

Since the discovery of NO as the EDRF,1 the specific role of NO in cancer

biology has been difficult to classify. This is in part due to conflicting results that have

been published over the past two decades. It is now recognized that NO mediates

angiogenesis (formation of new blood vessels),161-164 a key process on which cancer cell

survival depends. On the other hand, some reports suggest high concentrations of NO to

be anti-angiogenic.165,166 NO also affects apoptosis,167,168 proliferation, migration,

adhesion, DNA damage, and reports are often contradictory.89,90,13,169 It is now becoming

clear that whether NO promotes or inhibits tumor growth is generally dependent on the

level and duration of NO synthesis, its interaction with other free radicals, metal ions and

proteins, and the cell type.168,170-172 For example Jones et al. recently reported a

concentration/response curve of NO on tube formation (capillary like structures formed

by endothelial cells) using gastric endothelial cells.173 At concentrations up to 18.5 µM,

tube formation was enhanced, but then declined progressively with no apparent effect

observed at 36 µM. At 280 µM, tube formation was inhibited four-fold.

Aberrant expression of iNOS has been observed in many cancer patients,

including breast, colon and ovarian cancer.174,175 In a pilot study performed on breast

cancer tissues, iNOS expression was correlated with patient survival using multivariable

survival analysis. Increased expression predicted poor survival of patients who were 41

diagnosed with estrogen receptor negative (ER-) tumors.176 The constitutive isoform

eNOS is not only expressed in normal tissues, but is also extensively expressed in tumor

tissues.170,177,178 Lastly, nNOS has also been detected in some oligodendroglioma and

neuroblastoma cell lines.179 These findings, particularly in terms of iNOS expression,

have led several investigators to consider NO as a potential mediator of tumor

development. On the other hand, others have reported that major source of NO-mediating

tumor killing is macrophage iNOS.6 Macrophages can destroy a broad range of tumor

types via an NO-dependent pathway,180 both in vitro and in vivo. This dichotomy in

effect may be due to variation in the levels of NO, expression of pro- and anti-apoptotic genes and the presence of other reactive molecules.

It is now accepted that low levels such as nanomolar to picomolar levels of NO172 induce activation of cancer-promoting pathways while higher levels (micromolar) promote tumor suppressing pathways.181 NO or RNS derived from NO can post-

translationally modify several key proteins involved in cellular process. In a study carried

out in MCF-7 and endothelial cells using the SPER/NO diazeniumdiolate as the NO donor (Figure 1.9), sustained low levels of NO (10-30 nM), resulted in cGMP dependent phosphorylation of extracellular signal-regulated kinase (ERK).182 It plays an important

role in cell growth, differentiation and survival and has been the subject of intense

research in developing pharmacologic inhibitors for the treatment of cancer.183

42

Figure 1.9 Cellular response to different levels of NO (copied with permission from

David Wink).184

A slightly higher level of NO (30-60 nM) resulted in Akt phosphorylation.182,185

Akt is a protein kinase that plays an important role in several cellular processes including angiogenesis and is implicated with tumor cell survival, proliferation, and invasiveness.186,187 Akt activation triggers a cascade of responses, from cell growth and proliferation to survival and motility that drive tumor progression.188

At an NO concentration of 100-300 nM, hypoxia-inducible factor (HIF-1α) stabilization occurs. HIF-1 α is a transcription factor that mediates the effects of hypoxia.

It is often over-expressed in cancer cells.189 At 400-800 nM NO, p53 phosphorylation occurs, a protein that plays an important role in the cellular response to DNA damage and

act as a tumor suppressor protein.190 In 50% of human malignancies, the p53 gene is

mutated, thus implying the importance of this protein in tumor biology as accumulation 43

of p53 can induce growth arrest and apoptosis.191,192 At concentrations above 1 µM nitrostrative stress occurs which results in nitrosation of several key proteins such as the

DNA repair protein poly(ADP-ribose) polymerase (PARP), caspase that are involved in initiating apoptosis.193

Various cellular processes that are crucial to cancer survival or suppression and are affected by NO, directly or indirectly are summarized in Figure 1.10.

Figure 1.10. Effects of low vs high concentration of NO in cancer biology (Adapted from

Nitric Oxide, Biology and Pathology, Ignarro, L. (2000))

Given that exogenous donors can generate high concentrations of NO and that NO has the ability to reverse the resistance shown by cancer cells towards chemotherapy, site 44

targeted delivery of NO donors is gaining popularity such as development of JS-K which undergoes glutathione-S-transferase mediated release of NO or NO donating non- steroidal anti-inflammatory adducts.108,113,159,160,194,195

Compared to NO, HNO has only recently generated interest as a promising

chemotherapeutic agent. Under aerobic conditions HNO can be cytotoxic,196 in part due

to formation of RNOS that mediate DNA double strand breaks.197,198 HNO has been

shown to inhibit breast121 and neuroblastoma199 cancer proliferation in mouse xenografts.

HNO donors such as Angeli’s salt, and NO donor such as DEA/NO are typically

cytotoxic in the millimolar range.121 HNO is also known to deplete the intracellular level of GSH196 and to irreversibly inhibit enzymes with critical thiols such as glyceraldehyde

phosphate dehydrogenase (GAPDH).200 This critical enzyme is involved mainly in

glycolysis and other cellular process such as apoptosis and response to oxidative

stress.201-204 Cancer cells have a higher rate of glycolysis than normal cells (Warburg

effect)205 and hence inhibition of GAPDH has chemotherapeutic potential.206

1.6 Breast cancer

Breast cancer is one of the most common types of cancer among women.207 80% of breast cancers are ER+, which means they require estrogen for growth and survival.

Estrogen dependency represents a unique feature that can be utilized to control growth and/or prevent tumor development. Estrogen is required for the proliferation and differentiation of healthy breast epithelium but has also been associated with breast 45

cancer.208,209 There are three major naturally occurring estrogens estrone (E1), estradiol

(E2) and estriol (E3) (Figure 1.11).

Figure 1.11 Three types of estrogen hormone

A common strategy for treatment of breast cancer is centered on blocking the action of estrogen. This strategy can involve blocking the production of estrogen using aromatase inhibitors,210,211 down-regulating the levels of protein receptor to which

estrogens binds using an antiestrogen such as fulvestrant,212,213 or inhibiting estrogen

from binding to estrogen receptors using a selective estrogen receptor modulator

(SERM)214 such as tamoxifen.215,216

SERMs are a class of therapeutic compounds that have been used clinically to

treat breast cancer and osteoporosis. Unlike estrogens, which are uniformly agonists, and

antiestrogens, which are fully antagonists, responses to SERM are tissue-selective for

example they are antagonist in breast tissue but agonists on bones. Two of the most well

known SERMs are raloxifene (used mainly in osteoporosis) 217 and tamoxifen (used in

treatment of breast cancer).216

46

1.7 Tamoxifen

Tamoxifen (Figure 1.12) is a pioneering non-steroidal SERM that has been used

clinically worldwide for treatment of all stages ER+ breast cancer. Originally developed

as a contraceptive (ICI 46474)218, it was the first targeted antiestrogenic therapy for breast

cancer.216

Figure 1.12 Tamoxifen

Tamoxifen is also prescribed prophylactically for women at high risk of

developing breast cancer.219 There is also evidence that tamoxifen preserves bone density

in postmenopausal patients.220 Unfortunately, long-term usage of tamoxifen has been

linked with increased incidence of endometrial cancer in breast cancer patients.221 Also in

a rat model, tamoxifen has been reported to be a potent hepatocarcinogen,222 but fortunately, hepatocarcinogenicity has not been observed in humans, likely due to sophisticated mechanisms of DNA repair inherent to humans cells.223,224 Other than

endometrial cancer, tamoxifen may induce serious side effects including blood clots and

stroke.219,225

Tamoxifen is absorbed readily after oral administration with the usual dosage

being 20 mg per day.226,227 Upon oral administration tamoxifen is extensively 47

metabolized in the liver by cytochrome P450s (CYPs) and flavin-containing

monooxygenases to give various hydroxylated and demethylated metabolites (Figure

1.13, important metabolites shown), each metabolite exhibits unique estrogenic or

antiestrogenic activity. 228-231 It is now well accepted that overall action of tamoxifen is a combined effect, arising from the activity of tamoxifen and its metabolites.232

Figure 1.13 Key metabolites of tamoxifen233

1.8 Chlorambucil

Chlorambucil (Figure 1.14, sold by GlaxoSmithKline as Leukeran) is an aromatic

nitrogen mustard (alkylating agent) that has been used for decades as the standard first

line treatment for chronic lymphocytic leukemia,234 as it is well tolerated by patients. It is also used in treatment of ovarian and breast carcinomas and Hodgkin’s disease.235-238 It is

readily taken up by various tumor cells, where it exerts its cytotoxic effects by interacting

with DNA.239 The major side effect associated with chlorambucil is bone marrow

suppression. Although other nitrogen mustards such as bendamustine240 or the purine

analogue fludarabine,241 are now more commonly prescribed, chlorambucil still remains

in use for treatment of elderly patients with chronic lymphocytic leukemia.242 48

Chlorambucil is extensively metabolized in the liver to {4-[bis(2-

chloroethyl)amino]phenylacetic acid (Figure 1.14), which is also an active alkylating

agent. 243,244 Upon oral administration of chlorambucil, the plasma half-life of

chlorambucil was found to be 92 min and that of its active metabolite phenylacetic acid

mustard was 145 min. 245,246

Figure 1.14 Alkylating drug chlorambucil and its active metabolite

Chlorambucil, and in general other nitrogen mustards, exert their cyctotoxicity by binding with DNA, forming inter-stand crosslinks that eventually lead to apoptosis.247

This reaction is initiated by intramolecular nucleophilic substitution to form highly reactive aziridinium ion (Scheme 1.5) that reacts with nucleophilic species such as DNA bases or with detoxifying agents such as GSH and water to form mono adducts. This mono adduct can then undergo another intramolecular substitution to form another aziridinium ion that can react with another DNA base to form cross linked DNA.248-250 49

Scheme 1.5 General mechanism for reactivity of nitrogen mustard

60-80% of patients initially respond to chlorambucil administration, often for years, however resistance to this agent is also common.251 Several mechanisms have been put

forward for resistance development: elevated DNA topoisomerase II activity, 252over- expression of metallothionein protein,253 detoxification via GST ( mainly π isoform)

mediated coupling of GSH with chloroethyl alkylating moiety,254,255 multidrug resistance

protein 1 (MRP1) working in synergy with GST to remove GSH-chlorambucil

conjugate.256 Of all the possible mechanisms behind the development of resistance, over-

expression of GSTs, is a significant one, as several tumor cells are known to over-express

GSTs as a way of detoxifying cancer drugs.257-260

GSTs have emerged as a promising anticancer target, with approaches including

design of inhibitors and GST activated prodrugs. 113,261-266 One such inhibitor ethacryrnic

acid (figure 1.15) was used as co-treatment with chlorambucil and patients achieved

further benefits.267 However, phase II trials were abandoned due to severe side effects.

50

Figure 1.15 Structure of GST inhibitor: ethacrynic acid

GST activated prodrug approach has been used extensively by the Keefer group at

NCI Fredrick, and two of the promising molecules that have shown promising anti-cancer

activity are JS-K and PABA/NO (Figure 1.8).113,268 On activation by GST, they release

diazeniumdiolates, which then decompose to release NO (Scheme 1.6).

Scheme 1.6 GST activated release of NO

Chlorambucil can be converted to GST activated donor of both NO and the parent

drug by replacing the ester linker with chlorambucil, and is discussed in Chapter 4.

Several studies have shown synergistic effects between NO and anticancer agents

such as doxorubicin, cisplatin and alkylating agent melphalan.160,195,269,270 Most of the

studies that were done, involve co-treatment of anticancer drugs with an NO donor. Few

studies have been reported where NO donor were covalently attached to cancer 51

drugs.271,272 Of all the drugs that have been studied in conjunction with NO, non-steroidal

anti-inflammatory drugs (NSAIDs) represent the most extensively studied class. NSAID

are primarily used to treat pain, fever and inflammation, also possess anti-cancer

property.273-275 Several NO donors have been covalently attached to various NSAID and

are collectively called as NO-NSAID.

1.9 NO-NSAID

NSAIDs such as aspirin, ibuprofen are among the most widely used of all

therapeutic agents. There are currently dozens of NSAIDs available.273,276 However, long

term usage of NSAIDs can lead to serious side effects including gastric ulceration and

renal failure.274,277,278 NSAIDs act by inhibiting cyclooxygenases (COX), which are

involved in production of prostaglandins.274,279-282 The discovery that there are two

isoforms of COX, namely COX-1 and COX-2, with varied roles, sparked interest in

designing selective inhibitors, as targeting COX-2 isoform can play a key role in

relieving pain and inflammation, while inhibition of COX-1 leads to associated side

effects.283 This led to production of second generation NSAID such as celecoxibs and

rofecoxib,284-286 which were selective inhibitors of COX-2. However, this new generation

of NSAIDs suffered from new side effects such as increased incidence of heart attack and

stroke,287,288 which led to the withdrawal of rofecoxib from the market.

Given the side effects associated with first and second generation NSAIDs, there

is a need to develop new anti-inflammatory agents showing high activity in combination

with an improved degree of safety. This led to the development of NO donating NSAIDs 52

(NO-NSAID).289,290 This third generation is still in development, but has gained wide spread attention due to the safety profile in various animal models.194,291-294 Several NO-

NSAIDs (Figure 1.16) have been developed by coupling NSAIDs such as aspirin,

ibuprofen, indoemethacin with organic nitrates291,295 and diazeniumdiolates.296-298 These molecules were shown to retain the anti-inflammatory properties of the parent NSAID and at the same time overcome the side effects of parent NSAIDs.299 They were also

shown to have efficacy as cancer therapeutics.194

Figure 1.16 Representative examples of NO-NSAIDs

1.10 Conclusion

Diazeniumdiolate-based NO and HNO donors are important tools for unveiling the chemical biology of NO and HNO. Since the discovery of the biphasic nature of NO in cancer biology, several studies have been done to unfold the exact outcome of NO exposure. Inhibiting NOS is one viable alternative as several cancer cells express NOS.300

On the other end of the spectrum is the utility of NO donors that can produce a sustained 53

level of NO and thus act as potential anticancer agents. NO has also been shown to revert

the resistance to conventional chemotherapeutic agents such as cisplatin and doxorubicin

in cancer cells.160 HNO, which is another important nitrogen oxide, has also been shown

to have promising anti-cancer potential and requires further exploration. Contrary to the

large number of secondary amine diazeniumdiolates based NO donors there are only two

known primary amine based diazeniumdiolates of which only one (IPA/NO) has been

extensively examined.

To further establish diazeniumdiolates as HNO donors and to generalize the

properties of IPA/NO to be applicable to other diazeniumdiolates in general, there is need

to synthesize primary amine based diazeniumdiolates and to study their properties.

Synthesizing and characterizing such primary amine based diazeniumdiolates is

discussed in Chapter 3. Also given the well documented anticancer profile of nitrate ester

based NO-NSAIDs and promising results on HNO (diazeniumdiolates based) releasing

NSAIDs from our lab, there is a need to further functionalize different NSAIDs with

diazeniumdiolates (NO or HNO releasing, Chapter 5) and establish them as viable

alternatives to nitrate ester analogues. As the current literature on NO/HNO based

anticancer drugs has very few reported cases where NO or HNO donors were directly

attached to known anti cancer drugs, synthesis and evaluation of chemical and biological

properties of the diazeniumdiolate based chemotherapeutic drugs is discussed in Chapters

2 and 4.

54

Abbreviations

Angeli's salt, sodium trioxodinitrate; cGMP, cyclic guanosine monophosphate; COX,

cyclooxygenase; COXIB, selective inhibitors of COX-2; CINOD, COX inhibiting nitric

oxide donors; COX-1, cyclooxygenase-1; COX-2, cyclooxygenase-2; CYP, cytochrome

P450; DNIC, dinitrosyl iron complexes, eNOS, endothelial nitric oxide synthase; EDRF, endothelium derived relaxing factor; ER+, estrogen receptor positive; ER(−), estrogen

receptor negative; ERK, extracellular signal-regulated kinase; FAD, flavin adenine

dinucleotide; FMN, flavin mononucleotide; GAPDH, glyceraldehyde-3-phosphate

dehydrogenase; GST, glutathione-S-transferase; GTP, guanosine triphosphate; HIF-1α,

hypoxia-inducible factor; HNO, nitroxyl; iNOS, inducible nitric oxide synthase; IFN,

interferons; IL, interleukin; JS-K, O2-(2, 4-dinitrophenyl)-1-[(4-ethoxycarbonyl)

piperazin-1-yl] diazeniumdiolate); MRP1, multidrug resistance protein 1; NADPH,

nicotinamide adenine dinucleotide phosphate; NOHA, N-Hydroxy-L-arginine; NO, nitric

oxide; NSAID, non-steroidal anti-inflammatory drugs; NO-NSAIDs, nitric oxide

donating NSAIDs; NOS, nitric oxide synthase; nNOS, neuronal nitric oxide synthase;

PARP, poly(ADP-ribose) polymerase; PYRRO/NO, sodium 1-(pyrrolidin-1-yl)diazen-1-

ium-1,2-diolate; Piloty’s salt, N-hydroxy benzene sulfonamide; ROS, reactive oxygen

species; RNS, reactive nitrogen species; AgSD, sulfadiazine;sGC, soluble gulanyl cyclase; SNP, sodium nitroprusside; RSNO, S-nitrosothiols; SERM, selective estrogen

receptor modulator; AgSD, silver sulfadiazine; SNP, Sodium nitroprusside, N2O, nitrous

oxide; H4B, tetrahydrobiopterin; TNF, tumor necrosis factor.

55

CHAPTER 2: NITROGEN OXIDE RELEASING DIAZEN-1-IUM-1,2-DIOLATE

BASED ADDUCTS OF N-DESMETHYL-TAMOXIFEN

2.1 Introduction

Estrogens are a class of steroid based hormones that are essential to the function

of the female reproductive system. They exert their action by binding to the estrogen

receptor. Even though estrogen is essential for the growth and development of breast

tissue, it has also been associated with breast cancer.1 It is one of the most common types

of cancer among women, with more than 190,000 new cases reported in 2009 in the USA

alone.2 The dependence on estrogen has been used to broadly classify breast cancer. In

70-80% of breast cancer cases, estrogen receptors are over-expressed and cells are

classified as estrogen receptor positive (ER+).1,3 Such cancer cells require estrogen for

survival and are managed by blocking receptor function. ER negative (ER-) cancers,

which do not require estrogen hormone for growth, are more aggressive, and are further

classified into triple negative [ER-, human epidermal growth factor receptor 2 negative

(HER 2-) and progesterone receptor negative (PR-)] and ER-/HER2 positive cancers.3, 4

Patients with both early and metastatic ER+ breast cancer have the option to receive hormonal based therapies including tamoxifen5 and aromatase inhibitors6, which decrease mortality. Tamoxifen, a nonsteroidal selective estrogen receptor modulator

(SERM), is a pioneering medicine used worldwide in treatment of all stages of ER+ 56

breast cancer. Tamoxifen is also prescribed prophylactically for women at high risk of

developing breast cancer.

Upon oral administration, tamoxifen is extensively metabolized7-11 by

cytochrome P450s (CYPs) and flavin containing monooxygenases (Scheme 2.1). Many

of the metabolites of tamoxifen may contribute to the anti-estrogenic action in vivo.10,12,13

For instance, N-desmethyltamoxifen is the major circulating metabolite, but low concentration species such as 4-hydroxytamoxifen and endoxifen are 30-100 fold more effective than the parent drug.12,13 Despite being highly effective against breast cancer,

tamoxifen may induce serious side effects including blood clots, stroke and endometrial

cancer.14-16 Also, as in common with chemotherapy, in many cases tamoxifen therapy

eventually fails as cancer cells develop resistance.17 This is often attributed to altered

expression of several growth factor receptors such as insulin-like growth factor-1

receptor and downstream signaling molecules.4,18,19 57

Scheme 2.1. Partial depiction of CYP mediated metabolism of tamoxifen20

Over the last two decades, nitric oxide (NO) has emerged as biological mediator

of diverse physiological functions, ranging from neurological functions, vascular

homeostasis, immune regulation and inhibition of platelet aggregation.21-27 NO is endogenously synthesized by aerobic oxidation of L-arginine by NO synthases (NOS).28

The role of NO in cancer is complicated. NO production can be mutagenic, and NO can also affect apoptosis, proliferation, migration, adhesion, angiogenesis and vascular permeability.29 Whether NO promotes or inhibits tumor growth and metastasis is

generally dependent on the level and duration of NO synthesis.30-32 For instance,

expression of endothelial NOS, which produces sustained low levels of NO, is associated

with a higher incidence of breast cancer.31,33 Low to intermediate steady-state 58

concentrations of NO (50–100 nM)34 induce activation of cancer-promoting pathways,

whereas considerably higher levels (>400 nM) cause p53 phosphorylation,35 leading to

apoptosis in malignant cells. NO also has the potential to revert the resistance to

conventional chemotherapeutic agents such as cisplatin and doxorubicin in cancer cells.

Hence, site directed delivery of NO donors in combination with known cancer drugs is

being explored.36-39

Recently tamoxifen has been shown to induce apoptosis in MCF-7 (ER+ breast cancer) cells in an NO-dependent pathway.40 Tamoxifen elevates mitochondrial lipid

peroxidation, and releases cytochrome c by enhancing NO production from NOS. Given

that NO can sensitize cancer cells towards chemotherapy,36,38 adducts of tamoxifen with

an NO donor can be a viable alternative to tamoxifen.

Among the class of hybrid NO-drug compounds (NO donor attached covalently to

a known drug), adducts with non-steroidal anti-inflammatory drugs (NSAIDs) have been

extensively studied,41-43 with organic nitrates as the major NO donor used.44,45 Their development was based on the assumption that gastrointestinal side effects associated with NSAIDs would be reduced by release of NO. Moreover, a potential application of such hybrids is in the area of chemotherapy41,46 as NSAIDs are also known to be

chemopreventive agents.47,48 More recently, NO releasing NSAIDs have been produced

by derivatization of secondary amine diazeniumdiolates, which are enzymatically cleaved

to induce spontaneous release of NO from the diazeniumdiolate moiety without requiring

metabolic pathways associated with organic nitrates.49 Among the advantages that

diazeniumdiolates offer over other classes of NO donors is the spontaneous release of NO 59

at controlled, tunable rates.50-52 Derivatization of diazeniumdiolates has been known to

modify half life, intracellular release and cytotoxicity.53-55 For example, the acetoxy

methyl ester of diethylamine diazeniumdiolates (DEA/NO-AcOM, Scheme 2.2) has been reported to be significantly more cytotoxic in cancer cell lines and to have a longer half-

life than the parent compound DEA/NO.53

Scheme 2.2 Acetoxymethyl protected DEA/NO (DEA/NO-AcOM)

Nitroxyl (HNO) has also emerged as a biologically relevant nitrogen oxide, with

promising results in treatment of cardiovascular diseases such as heart failure and

ischemic reperfusion.56,57 The cytotoxicity of HNO, particularly in aerobic environments,

is substantially higher than that of NO.58 Although HNO has yet to be demonstrated to be

endogenously produced, a role in inhibition of tumor development including breast cancer, by HNO donors is emerging.59 HNO is known to deplete intracellular levels of glutathione58, a tripeptide involved in maintaining cellular redox status.60 HNO can also inhibit key enzymes that have thiols at their active sites such as glyceraldehyde phosphate

dehydrogenase (GAPDH).61 Even though HNO has shown promise for being an anti-

cancer agent, studies have been mainly done with ionic diazeniumdiolates that have

shorter half-lives of decomposition. Adducts of HNO donors with longer half-lives would

be an ideal step in further exploring the potential of HNO in chemotherapy. 60

Given the initial promise of NO-releasing NSAIDs, synthesis of nitrogen oxide

(NO/HNO) donating adducts of N-desmethyltamoxifen (Scheme 2.3), and a comparison

of their chemical and biological properties is reported here. The potential of converting

major and long- lived metabolites of tamoxifen into prodrugs is projected to reduce side

effects by requiring a lower dose of drug, particularly for chronic use.

Scheme 2.3 Synthesis of NO/HNO releasing N-desmethyltamoxifen derivatives

2.2 Materials and methods

Reagents

Tamoxifen, diethylamine, isopropylamine, bromomethylacetate were purchased

from Sigma-Aldrich. Chloromethylchloroformate was purschsed from Alfa-Aesar. Thin

layer chromatography (TLC) was carried out using Analtech silica gel GF (250 micron)

glass-backed plates. Flash chromatography was performed using the indicated solvent

system on Silica Gel 60 (230-450 mesh size; purchased from Alfa Aesar). IPA/NO and

DEA/NO, DEA/NO-AcOM were synthesized according to previously published

procedures.50,51,53 Concentrations of NONOate stock solutions (>10 mM), prepared in 10

mM NaOH and stored at –20°C, were determined directly prior to use from the extinction 61

coefficients at 250 nm (ε of 8,000 M-1cm-1).52 Stock solutions other than of nitrogen

oxide donors were prepared fresh daily at 100 in MilliQ or Barnstead Nanopure

Diamond filtered H2O, unless specified. Typically, the assay buffer consisted of the metal

chelator diethylenetriaminepentaacetic acid (DTPA, 50 μM) in calcium- and magnesium-

free Dulbecco’s phosphate-buffered saline (PBS, pH 7.4). By sequestering contaminating

metals, addition of DTPA quenched the oxidation of HNO to NO.62 All reactions were

performed at 37 °C.

Instrumentation

UV-visible spectroscopy was performed with an Agilent Hewlett-Packard 8453

diode-array spectrophotometer equipped with an Agilent 89090A thermostat.

Electrochemical detection was accomplished with a World Precision Instruments Apollo

4000 system with NO, O2 and H2O2 sensitive electrodes. A Sievers NO analyzer (NOA

280i) was used for chemiluminescence measurements. A PerkinElmer HTS 7000 or

BioTek Synergy 2 microplate readersplate reader wereas utilized for absorbance and

fluorescence measurements. Solution pH was determined by use of a ThermoElectron

Orion 420A+ pH meter. 1H and 13C NMR spectra were acquired using a Bruker DRX-

500 spectrometer (500 MHz). Low resolution mass spectra were obtained with a JEOL

HX110A spectrometer

62

Synthesis

(Z)-Chloromethyl 2-(4-(1,2-diphenylbut-1-enyl)phenoxy)ethyl(methyl)carbamate

(1). Tamoxifen (371 mg, 1.00 mmol) was dissolved in 15 mL of anhydrous DCE.

Chloromethylchloroformate (625 μL, 7.00 mmol) was added to the reaction mixture and was allowed to stir for 10 min. Thereafter, it was refluxed for 24 h. On completion of

reflux, solvent was removed under vacuum to obtain a crude product, which was further

purified by column chromatography using hexane/dichloromethane (30% → 70%) to

1 obtain a viscous light yellow oil (420 mg, 93.5%). H NMR (CDCl3): δ 0.905 [t, J = 7.5

Hz, 3H], 2.439 [q, 2H], 3.006-3.028 [m, 3H], 3.571-3.615 [m, 2H], 3.936-4.002 [m, 2H],

5.732-5.748 [m, 2H], 6.502-6.530 [m, 2H, aromatic], 6.765 [d, J = 9Hz, 2H, aromatic],

13 7.108-7.353 [m, 10H, aromatic]. C NMR (CDCl3): δ 13.559, 29.009, 36.069, 36.342,

48.235, 49.208, 65.816, 66.018, 70.776, 113.16, 126.021, 126.515, 127.843, 128.080,

129.404, 129.645, 131.908, 135.870, 135.984, 138.056, 138.083, 141.459, 141.510,

142.321, 143.675, 153.468, 153.761, 156.161, 156.298. MS (LRMS) calculated M-Na+

472.2, found 472.2.

O2-(Z)-(((2-(4-(1,2-diphenylbut-1-en-1-

yl)phenoxy)ethyl)(methyl)carbamoyl)oxymethyl)-1-( N,N-Diethylamino)-diazen-1-ium-

1,2-diolate (2). A solution of DEA/NO (202.3 mg, 1.304 mmol) and 1 (586 mg, 1.302 mmol) in anhydrous DMSO (12 mL) was stirred at room temperature overnight.

Ethylacetate was added, and the organic layer was washed with NaCl solution (50 × 3 mL) dried over anhydrous Na2SO4 and evaporated to dryness. The crude product was purified on silica gel using 15% hexane/acetone as the eluting solvent to yield colorless 63

1 oil (342 mg, 48% yield). H NMR (CDCl3): δ 0.919 [d, J = 7.5 Hz, 3H], 1.006-1.074 [m,

6H], 2.429 [q, J = 7.5Hz, 2H], 2.98-3.007 [m, 3H], 3.114-3.164[m, 4H], 3.547-3.589 [m,

2H], 3.904-3.990 [m, 2H] 5.814-5.824 [m, 2H], 6.471-6.517 [m, 2H, aromatic], 6.752[d,

J= 9 Hz, 2H, aromatic], 7.104-7.351 [m, 10H, aromatic]. 13C NMR (CDCl3): δ 11.40,

11.43, 13.58, 29.02, 36.03, 36.37, 48.20, 48.29, 48.97, 66.09, 66.15, 88.53, 113.16,

113.20, 126.00, 126.50, 127.83, 128.06, 129.40, 129.64, 131.88, 135.78, 135.90, 138.08,

141.40, 141.46, 142.33, 143.71, 153.94, 154.24, 156.20, 156.34 MS (LRMS) calculated

+ M-NH4 564.3, found 564.1.

O2-(Z)-(((2-(4-(1,2-diphenylbut-1-en-1-

yl)phenoxy)ethyl)(methyl)carbamoyl)oxymethyl) -1-(N-isopropylamino)-diazen-1-ium-

1,2-diolate (3). A solution of IPA/NO (122.5 mg, 0.866 mmol) and 1 (390.0 mg, 0.866

mmol) in anhydrous DMSO (12 mL) was stirred at room temperature overnight.

Ethylacetate was added, and the organic layer was washed with NaCl solution (50 × 3

mL), dried over anhydrous Na2SO4 and evaporated to dryness. The product was purified

on silica gel using 15% hexane/acetone as the eluting solvent to yield light yellow color

1 oil (119 mg, 25.6% yield). H NMR (CDCl3): δ 0.919 [d, J = 7.5 Hz, 3H], 1.095-1.151

[m, 6H], 2.427 [q, J = 7.5Hz, 2H], 2.997-3.009 [m, 3H], 3.549-3.594 [m, 2H], 3.908-

3.998 [m, 3H] 5.748-5.758 [m, 2H], 6.061 [b, 1H], 6.487-6.525 [m, 2H, aromatic], 6.758

13 [d, J= 8.5 Hz, 2H, aromatic], 7.106-7.352 [m, 10H, aromatic]. C NMR (CDCl3): δ

13.56, 20.33, 29.01, 36.05, 36.30, 48.27, 48.98, 49.09, 49.17, 66.00, 66.10, 88.34, 88.38,

113.15, 113.20, 126.00, 127.83, 128.06, 129.38, 129.63, 131.88, 135.80, 135.89, 138.07, 64

141.41, 141.47, 142.30, 143.68, 154.08, 154.36, 156.20, 156.32 MS (LRMS) calculated

M-Na+ 555.2, found 555.1.

Cell culture

For cell culture, estrogen receptor α negative (ER-; MDA-MB-231), or estrogen

receptor α positive (ER+; MCF-7) human breast cancer cells (American Type Culture

Collection, Manassas, VA) were grown as a monolayer in RPMI 1640 media

supplemented with 10% fetal bovine serum (FBS, Hyclone), penicillin (50 units/mL) and

streptomycin (50 units/mL; Life Technologies, Inc., Grand Island, NY) at 37 °C in 5%

CO2 and 80% relative humidity. Single cell suspensions were obtained by trypsinization

(0.05% trypsin/EDTA).

Reaction of NO with oxymyoglobin

The reaction buffer was purged with ultra-high purity argon at a rate of ≥1 mL

/min, and solutions were transferred using gas-tight Hamilton syringes to maintain deaerated conditions. The cuvette was maintained under an argon atmosphere for the

-1 -1 duration of the experiment. Oxymyoglobin (MbO2 50 µM) (ε542 = 13.9 mM cm ; ε580 =

-1 -1 63 14.4 mM cm ) was reacted with DEA/NO-AcOM (50 µM) to form metMb (ε502 = 10.2

-1 -1 -1 -1 mM cm ; ε630 = 3.9 mM cm ).

65

Chemiluminescence detection of NO and HNO

The release of NO from DEA/NO-N-des-tam and NO/HNO from IPA/NO-N-des- tam were examined via NO-selective chemiluminescence detection.64 The reaction buffer

(5 mL) was purged with ultra-high purity argon at a rate of ≥1 mL/min followed by

addition of 2% guinea pig serum using gas-tight Hamilton syringes to maintain deaerated

conditions. In the chemiluminescence assay, adducts dissolved in DMSO were added to a

final concentration of 100 µM at room temperature. The NO signal was measured by

injecting 500 µL of headspace of the reaction mixture using gas-tight Hamilton syringes

into the argon-purged, reaction vessel at various time intervals (1-10 h). For IPA/NO-N-

des-tam, the experiment was repeated in the presence of 1 mM potassium ferricyanide for

indirect measurement of HNO.

NO and HNO release in MCF-7, MB-231 and MB-468 cells

Cells were plated at 30,000 cells per well in a 96 well plate and grown for 12 h in

RPMI 1640 media at 37 °C in 5% CO2 and 80% relative humidity. A stock of 4-amino-5-

methylamino-2’,7’-difluorofluorescein diacetate (DAF-FM-2DA, Invitrogen, Carlsbad,

CA)65 in DMSO (100×) was diluted to a final concentration of 10 µM using DTPA free

assay buffer. The media was aspirated from each well and replaced by 100 µL of 10 µM

DAF-FM diacetate. The plate was incubated for 45 min at 37 ºC. Each well was then

washed three times with PBS pH 7.4 to remove excess dye. Diazeniumdiolate-N-

desmethyltamoxifen derivatives dissolved in DMSO (1000 were then added to achieve a 66

final concentration of 10 or 100 µM. The increase in fluorescence intensity was

measured at an excitation of 485 nm and emission of 535 nm

Effect of IPA/NO-N-des-tam, DEA/NO-N-des-tam and DEA/NO-AcOM on cell survival

Cells were plated at a concentration of 8,000-10,000 per well in a 96 well plate and grown overnight. The cells were then treated with different concentrations (0-100

µM) of the prodrugs for 48 h. After addition of a solution of 2 mg/mL 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to each well, the plate was incubated for 1 h. The media was then removed, 100 µL DMSO was added to each well, and the absorbance was recorded at 550 nm.66 For combination assay in which cells were

co-incubated with tamoxifen and DEA/NO-AcOM, 0.1 or 10 µM of tamoxifen was used

with variable concentrations (0-100 µM) of DEA/NO-AcOM for 48 h.

2.3 Results and discussion

NO has been shown to sensitize hypoxic cells to ionizing radiation67 and to enhance the cytotoxicity of several anti-cancer drugs such as cisplatin36 and melphalan.68

However, to our knowledge no studies on the effect of NO donors with tamoxifen in ER+ and ER- cell lines have been reported. Herein, we present the outcome of NO released from DEA/NO-AcOM on cell viability in the presence of tamoxifen. The cytotoxicity of two newly synthesized NO or HNO releasing diazeniumdiolate based carbamate derivatives of N-desmethyltamoxifen is also reported. The carbamate based prodrug approach has been reported earlier for primary or secondary amine based drugs,69,70 and 67

the same approach was used for N-desmethyltamoxifen. Prepared derivatives were

expected to undergo carboxylesterase-mediated hydrolysis to release NO/HNO as shown

in Scheme 2.4.

Scheme 2.4 Proposed in vivo release of NO/HNO from diazeniumdiolate-N-

desmethyltamoxifen conjugates

2.3.1 Half-life of DEA/NO-AcOM and carbamate adducts

O2-Derivatization is known to stabilize NONOates.71 The half-life of

decomposition for DEA/NO-AcOM in assay buffer in the presence or absence of serum

was determined to be 2.8 min and 9.7 h, respectively. The pH-dependence of

decomposition in the presence of 2% guinea pig serum is shown in Figure 2.1. For both

IPA/NO- and DEA/NO-N-des-tam, half-life measurement was not feasible due to the

aromatic portion masking the peak around 240 nm that has been previously reported for

other derivatized NONOates.72 68

600

500

400

300

half life (s) life half 200

100

0 45677.489

pH

Figure 2.1 The pH-dependence of the first-order rate constants of decomposition of

DEA/NO-AcOM at 37°C in PBS containing 50 μM DTPA measured at 250 nm in

presence of 2% guinea pig serum (mean ± SD, n ≥ 3)

2.3.2 NO release profile of DEA/NO-AcOM

The NO release profile of DEA/NO-AcOM was studied using oxymyoglobin

(MbO2) (Eq. 1) due to its ability to react with NO. This method was used earlier to

suggest that DEA/NO is a donor of only NO under physiological conditions (signal of

oxymyoglobin conversion was not affected by using glutathione, a known scavenger of

HNO).73 As shown in Figure 2.2, oxymyoglobin was converted to metMb upon treatment

with DEA/NO-AcOM, indicating that production of NO from DEA/NO was not affected

by derivatization. 69

 7 -1 -1 74 MbO2    metMb   (4 × 10 M s ) (Eq 1)

MbO (50M) 0.8 2 + DEA/NO-AcOM (50M)

0.6

0.4 Absorbance 0.2

0 450 500 550 600 650 700 Wavelength (nm)

Figure 2.2 Detection of NO using 50 µM MbO2 reacted with 50 µM DEA/NO-AcOM in

the presence of 2% guinea pig serum at 37°C and aerated condition (n ≥ 2).

2.3.3 Chemiluminescence detection of NO/HNO from N-desmethytamoxifen

adducts

For N-desmethyltamoxifen-diazeniumdiolates adducts, significant NO or HNO

signal was not observed using methods such as trapping by oxymyoglobin or detection

with an NO electrode. Therefore adducts were evaluated for their release profile using

chemiluminescence. While NO release can be directly detected, ferricyanide was used to

detect HNO release from IPA/NO-N-des-tam (Eq. 2), as feriicyanide oxidizes HNO to

NO. 70

3- 4- + HNO + [Fe(CN)6]  NO + [Fe(CN)6] + H (Eq 2)

In the presence of 2% guinea pig serum, a small amount of NO release was

observed from the DEA/NO-analogue (Figure 2.3), whereas no appreciable signal was

observed for the IPA/NO analogue with or without ferricyanide (data not shown).

Insignificant release of NO or HNO from these conjugates suggests negligible hydrolysis

of the carbamate bond.

Figure 2.3 Detection of NO using chemiluminescence from head space of reaction of

DEA/NO-N-des-tam (100 µM) in the presence of 2% guinea pig serum in pH 7.4 buffer

at room temperature.

2.3.4 Intracellular release of NO/HNO

To determine whether hydrolysis of DEA/NO-AcOM or the N-

desmethyltamoxifen-diazeniumdiolate adduct will occur intracellularly, DAF-FM-2DA 71

was used as a reporter molecule in breast cancer cells.75 Both NO and HNO have been

previously reported to increase the fluorescence of DAF.76 The autoxidation product of

HNO with DAF-2DA produces a higher relative fluorescence compared to NO.

Figure 2.4A shows signal intensity produced by DEA/NO-AcOM in MB-231

cells. The fluorescence generated by DEA/NO-AcOM is a function of direct uptake by

the cell as well as esterase mediated decomposition kinetics. The higher fluorescence of

DEA/NO-AcOM (10 μM) compared to DEA/NO (10 μM) is likely due to facilitation of

cellular uptake and decomposition in close proximity to DAF, thus reducing cellular

scavenging of NO. The ionic compound, DEA/NO is expected to decompose outside the

cell, requiring diffusion of NO across the cellular membrane.

A 1e+5 DEA/NO-AcOM (10 M) DEA/NO (10 M) 8e+4 Vehicle

6e+4 RFU 4e+4

2e+4

0 0 50 100 150 200 250 Time (min)

72

B 2.5e+4 Control DEA/NO-N-des-tam 2e+4 IPA/NO-N-des-tam

1.5e+4

RFU 1e+4

5000

0 MB-231 MB-468 MCF-7

Figure 2.4 (A) NO release measured under physiological conditions in MB-231 (N=6 per

plate, two trial) cells from reaction of 10 µM DAF-2DA with 10 µM of DEA/NO-AcOM

in DMSO (<0.1%) or DEA/NO 100 µM in 10 mM NaOH (B) NO or HNO release

measured after 12 h under physiological conditions in MCF-7, MB-468 and MB-231 cells from reaction of 10 µM DAF-2DA with 10 µM of DEA/NO-N-des-tam and

IPA/NO-N-des-tam in DMSO (<0.1%).

For N-demethyltamoxifen based diazeniumdiolate analogues, intracellular release results were varied (Figure 2.4B). In MB-468 cells, as compared to the DMSO control, there was a slight time-dependent increase in signal from both DEA/NO-N-des-tam and

IPA/NO-N-des-tam with the signal being higher for IPA/NO adduct. In MCF-7 cells there was a slight increase in signal only from the IPA/NO-N-des-tam adduct. For MB-

231 cells, both adducts did not show any difference in signal as compared to the control.

The slight increase in some of the cases could be due to low yield of NO and HNO from these compounds (presumably due to slower enzymatic or non-enzymatic hydrolysis). 73

Compared to DEA/NO-AcOM (Figure 2.4A) and other known ester based derivatized

O2-diazeniumdiolates53,77 where significant NO release was observed, carbamate based

N-desmethyltamoxifen-diazeniumdiolates hydrolysis was quite low. Lack of significant

NO/HNO release can limit the usefulness of these compounds as beneficial effects of

NO/HNO in overcoming limitation of tamoxifen may be lacking.

2.3.5 Cytotoxicity

DEA/NO-AcOM and the N-desmethyltamoxifen adducts were evaluated for their cytotoxicity in both ER+ and ER- breast cancer cell lines. DEA/NO-AcOM has been shown previously to be cytotoxic towards leukemia cell lines (HL-60 and U937 with IC50 values of 8.3 and 53 µM respectively).53 When evaluated at 48 h, DEA/NO-AcOM

showed a concentration- dependent inhibition of cell survival in both types of breast

cancer cells with IC50 values of ~70 µM for MCF-7 (Figure 2.5A) and ~100 µM for MB-

231 (Figure 2.5B) cells. 74

A 120

100

80

60

% Survival 40

20

0 0 10 25 50 100 Concentration (M)

B 120

100

80

60

% Survival 40

20

0 0 10255075100 Concentration (M)

Figure 2.5 The effect of varied concentrations of DEA/NO-AcOM on cell survival of (A)

MCF-7, and (B) MB-231cell lines. Cells were treated with different concentrations of 75

prodrugs and controls, and cell survival was determined using the MTT assay after 48 h

(n ≥ 4)

To determine whether growth inhibitory potency of DEA/NO-AcOM can be used

in conjunction with non-cytotoxic levels of tamoxifen, cells were treated with varied

concentrations of DEA/NO-AcOM with a co-treatment of fixed concentration of

tamoxifen (0.1 µM or 10 µM for MCF-7 or MB-231 cells respectively). For MCF-7 cells

(Figure 2.6A), there was no apparent change in the IC50 value as compared to DEA/NO-

AcOM treatment (Figure 2.5A). However in MB-231 cells, the IC50 value was determined

to be ~50 µM (Figure 2.6B) as compared to 100 µM observed for DEA/NO-AcOM

(Figure 2.5A).

Tamoxifen is approved for the treatment of ER+ breast cancer worldwide.5 The

present study demonstrate that DEA/NO-AcOM was cytotoxic to breast cancer cell line

regardless of the dependence on estrogen, presumably due to sustained release of NO in

the cells. It was also apparent that DEA/NO-AcOM was able to overcome the dose

requirement for tamoxifen in ER- cells as a combination alternative. The exact nature of

this enhanced cytotoxicity requires further exploration. 76

A 120

100

80

60

% Survival 40

20

0 0.0 tam (0.1 M) 10 25 50 100 Concentration (M)

B 120

100

80

60

% Survival 40

20

0 0 tam (10 M) 10 25 50 75 100

Concentration (M)

Figure 2.6 The effect of different concentrations of DEA/NO-AcOM on cell survival of

(A) MCF-7 along with 0.1μM tamoxifen, and (B) MB-231cell lines along with 10 μM.

Cells were treated with different concentrations of DEA/NO-AcOM (0-100 µM) and cell

survival was determined using MTT assay after 48 h (n ≥ 4) 77

Contrary to the combination results, for the carbamate based adducts DEA/NO-N- des-tam and IPA/NO-N-des-tam, less cytotoxicity was observed towards ER- cells. For

MB-468 cells, IC50 values were closer to 100 μM (data not shown), much higer then

tamoxifen (~20 μM, data not shown). In MB-231 cells, they were not cytotoxic even at

100 μM (data not shown). This reduced cytotoxicity may be explained in part due to negligible hydrolysis of these adducts. However, for the ER+ MCF-7 cells, these compounds do show cytotoxicity comparable to that of parent drug tamoxifen (Figure

2.7). Reasons for their similar cytotoxicity are not known and it can be possible that the whole compound by itself is competing with estrogen binding.

120

tamoxifen 100 DEA/NO-N-des-tam IPA/NO-N-des-tam 80

60

% Survival 40

20

0 0 0.01 1 10 100 Concentration (M)

Figure 2.7 The effect of different concentrations of DEA/NO-N-des-tam, IPA/NO-N-des- tam and tamoxifen on cell survival in MCF-7 cells. Cell survival was determined using

MTT assay after 48 h (n ≥ 3)

78

2.3.6 Attempted synthesis of new hybrid NO/HNO-N-desmethyltamoxifen adduct

Carbamate based hybrid drugs failed to hydrolyze and thereby release NO/HNO.

Therefore, synthesis of a new donor based on ester derivatization was explored (Figure

2.8). Succinic acid was incorporated as a linker connecting N-desmethyltamoxifen with

diazeniumdiolates.

N O O O R O O N N O O N+ R' O O-

Figure 2.8. New proposed NO/HNO donor of N-desmethyltamoxifen

It is postulated that such prodrugs will not require carbamate hydrolysis in order to

release the parent drug (Scheme 2.5).

R N O N O -O O O O N+ R' O OH + HO O O N OH O esterase O R O O O N N + HO O N R' O-

HCHO HCH + O CO2 R N-desmethyltamoxifen N N -O N+ R' O- Scheme 2.5. Proposed esterase mediated hydrolysis

The first attempted routes for synthesizing succinic acid based diazeniumdiolate-N-

desmethyltamoxifen are summarized in Scheme 2.6A-C 79

80

Scheme 2.6 Attempted synthesis of hybrid ester based drug using succinic acid

Both Schemes 2.6A-B failed when the substitution of the chloro group [O2-

(Chloromethyl-diazeniumdiolate or 1] with carboxylate was attempted. Coupling was attempted by changing the solvent (DMSO, CH3CN) or the base (Et3N, imidazole)

without success. In contrast to Scheme 2.6A-B, substitution of chlorine in

chloromethylchlorosulfide with the carboxylate of succinic acid was successful and the

mono thiomethyl protected succinic acid was isolated. This was then coupled with 1, to

afford the intermediate which has succinic acid linked to N-desmethyltamoxifen.

However, attempts to convert -SCH3 to -Cl with SO2Cl2 failed. On the basis of failure to

couple succinic acid (scheme 2.6A-B), mono protected succinic acids was explored

(Scheme 2.7A-B). 81

R N O NN O Cl O Pd/C R' R O OH N OBn H2,EtOH OH NN O O DMSO, Et3N R' O O O 14

Cl O N O O R O N OH 1 NN O O Prodrug 1 DMSO, Et N R' O 15 O 3

B

Scheme 2.7. Attempted synthesis of hybrid ester based drug using protected succinic acid

TBDMS protected succinic acid is not commercially available. Hence, synthesis

of silyl protected succinic acid was explored (Scheme 2.7A) under various conditions

(solvent and base combination). Despite several attempts, TBDMS protected succinic

acid could not be isolated. Attempts to go on to next step (coupling with NONO-CH2Cl)

without isolating TBDMS protected succinic acid was also not successful. Succinc acid

monobenzylester, which is commercially available from Astatech Inc., was subsequently

explored. Coupling with DEA/NO-CH2Cl worked (Scheme 2.7 B) to isolate 14. Attempts 82

to remove the benzyl protection by hydrogenolysis was however not successful. Attempts

are still underway to make the desired target compound.

2.4 Conclusions

With a goal of enhancing the current drug tamoxifen, adducts of N-

desmethyltamoxifen that can potentially release NO/HNO on hydrolysis of carbamate

bond were synthesized. Also DEA/NO-AcOM, an NO donor, was examined for its

cytotoxicity towards estrogen dependent and independent breast cancer cells and as a

combination with nontoxic level of tamoxifen for treating breast cancer. While the

derivatized compound of N-desmethyltamoxifen were found to be effective towards ER+

cells only, DEA/NO-AcOM was found to be cytotoxic towards both breast cancer types.

Also, non-toxic levels of DEA/NO-AcOM can be used in combination with a

concentration of tamoxifen that will not compromise the tamoxifen therapeutic index.

Thus, the work shown here with DEA/NO-AcOM can open up the possibility of

extending the usage of tamoxifen towards treatment of more aggressive, ER (-) breast

cancer.

Abbreviations

CYP, cytochrome P450; DAF-FM-2DA, 4-amino-5-methylamino- 2’,7’-

difluorofluorescein diacetate; DEA/NO, sodium 1-(N,N-diethylamino)diazen-1-ium-1,2-

diolate NONOate; DEA/NO-AcOM, O2-(acetoxymethyl) 1-(dimethylamino)diazen-1-

ium-1,2-diolate; DEA/NO-N-des-tam, O2-(Z)-(((2-(4-(1,2-diphenylbut-1-en-1- 83

yl)phenoxy)ethyl)(methyl)carbamoyl)oxymethyl)-1-(N,N-Diethylamino)-diazen-1-ium-

1,2-diolate; DMSO, dimethyl sulfoxide; DTPA, diethylenetriaminepentaacetic acid; ER, estrogen receptor; HNO, nitroxyl; IPA/NO, sodium 1-(N-isopropylamino)diazen-1-ium-

1,2-diolate; IPA/NO-N-des-tam, O2-(Z)-(((2-(4-(1,2-diphenylbut-1-en-1-

yl)phenoxy)ethyl)(methyl)carbamoyl)oxymethyl)-1-(N-isopropylamino)-diazen-1-ium-

1,2-diolate; MCF-7, Michigan Cancer Foundation-7 estrogen receptor positive breast

cancer cell line; MDA-MB-231: estrogen receptor negative breast cancer cell line; MDA-

MB-468, estrogen receptor negative breast cancer cell line metMb, ferric myoglobin;

MbNO, nitrosyl myoglobin; MbO2, oxymyoglobin; MTT, (3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide; N-des-tam, (Z)-chloromethyl 2-(4-(1,2-diphenylbut-1-

enyl)phenoxy)ethyl(methyl)carbamate; NO, nitric oxide; NSAID, non-steroidal anti-

inflammatory drug; PBS, phosphate buffered saline; RPMI, Roswell Park Memorial

Institute; SERM, selective estrogen receptor modulator; TLC, thin layer chromatography;

TBDMS, tert-butyldimethylsilyl ethers

84

CHAPTER 3 COMPARISON OF HNO AND NO DONATING PROPERTIES OF

CYCLIC AMINE DIAZENIUMDIOLATES

3.1 Introduction

Nitric oxide (NO) is a well studied physiological signaling molecule with effects

ranging from regulation of blood pressure, cellular defense against invading pathogens,

and neurotransmission to prevention of platelet aggregation.1-6 Nitroxyl (HNO), one

electron more reduced than NO, has recently emerged as a promising pharmacological

agent with important cardiovascular7,8 and tumorocidal properties.9 Cyanamide, has been

successfully used clinically in treatment of alcoholism through metabolism to HNO.10

Due to irreversible dimerization11,12 HNO cannot be stored, and donor compounds are

necessary for in situ production of HNO. Continuous progress in the pharmacology of

HNO requires development of a versatile platform for systematically generating reliable, tunable, controlled fluxes of HNO in physiological media.

The HNO donor that has been extensively used to study the chemical biology of

HNO is Angeli’s salt13 (Scheme 3.1), synthesized by Angeli in 1896. Angeli’s salt spontaneously releases HNO in aqueous solution with a pH independent rate between pH

4-8.14,15,16 The short half-life of ~2 min under physiological conditions inhibits analysis of the effect of chronic exposure.17 Derivatization of Angeli’s salt to form a more stable

HNO donor has been unsuccessful to date. Moreover, decomposition of Angeli’s salt

produces nitrite, which also has biological activity.18 85

Organic donors offer the ability to tune the HNO release rate. Piloty’s acid

19 20 (C6H5SO2NHOH) and derivatives are base sensitive HNO donors. Although the rate of

HNO release is tunable, these compounds often tend to generate NO under aerated conditions.21 Acyl nitroso compounds generate HNO on reaction with nucleophiles,22 but are unstable intermediates.23 The most general route of synthesizing such compounds is

via oxidation of N-acyl hydroxylamine derivatives.24 Acyloxy nitroso compounds are

relatively new HNO donors that are synthesized by oxidation of oximes.25 HNO release

can be mediated either by enzymatic or spontaneous hydrolysis of the ester bond.26

Scheme 3.1 Common HNO donor structures

Diazeniumdiolates, also known as NONOates, are adducts of an NO dimer with

nitrogen, carbon, oxygen or sulfur based nucleophiles.27 Angeli’s salt is formally a

diazeniumdiolate with the nucleophile being O2-. Secondary amine based

diazeniumdiolates generate NO upon spontaneous decomposition, with reliable half-lives

of decomposition ranging from 2 s to 20 h depending on the amine backbone.28-30

Importantly, diazeniumdiolates can be readily O2-derivatized,31,32 which facilitates

purification, increases stability and half life. Moreover, derivatization opens the door to

convert diazeniumdiolates to prodrugs that can be bioactivated, thus providing rational 86

design for targeted delivery of NO.33 In contrast to decomposition of secondary amine

based diazeniumdiolates, primary amine analogues can generate HNO via a

tautomerization pathway (Scheme 3.2).34

O- O- R N+ O- R N+ O- N N N N RNNO- + HNO H H H+

O- O- + H R N OH R N+ O- RNH +2NO N N N+ N 2 H H

Scheme 3.2 Mechanism of NO and HNO release from diazeniumdiolates

Unlike the large number of secondary amine based diazeniumdiolates, isopropylamine (IPA/NO) and cyclohexylamine diazeniumdiolates (CHA/NO) are

currently the only examples of primary amine analogues in the literature.35 IPA/NO has

been shown to be an effective HNO donor under physiological conditions, and like

Angeli’s salt has a short half-life of 6.7 min.16 O2-derivatization of IPA/NO increases

HNO production over the parent due to access to a unique decomposition pathway

(Scheme 3.3).36 87

Scheme 3.3 Mechanism of decomposition of acetoxymethyl protected IPA/NO in PBS.

To date, the HNO donating ability of CHA/NO has not been examined. To establish diazeniumdiolates as an alternative to currently existing donors, herein, we report the synthesis and characterization of a series of structurally related primary amine based cyclic diazeniumdiolates.

3.2 Materials and methods

Reagents

Unless otherwise noted, chemicals were purchased from Sigma-Aldrich and used without further purification. NO was purchased from Matheson or Air liquide.

Concentrations of diazeniumdiolate stock solutions (>10 mM), prepared in 10 mM NaOH and stored at –20 °C, were determined directly prior to use from the molar extinction coefficients at 250 nm (ε of 8,000 M-1cm-1).17 Stock solutions of O2-protected diazeniumdiolates (1000×) were prepared in EtOH and stored at –20 °C. Typically, the 88

assay buffer consisted of the metal chelator diethylenetriaminepentaacetic acid (DTPA,

50 µM) in calcium- and magnesium-free Dulbecco’s phosphate-buffered saline (PBS, pH

7.4). By sequestering contaminating metals, addition of DTPA quenched the oxidation of

HNO to NO. Stock solutions of glutathione (GSH; > 10 mM) were made in assay buffer and stored at 4 °C. All reactions were performed at 37 °C except those measured with the NO-specific electrode (room temperature). Thin layer chromatography (TLC) was

carried out using Analtech silica gel GF (250 micron) glass-backed plates. Flash

chromatography was performed using the indicated solvent system on Silica Gel 60 (230-

450 mesh size; Alfa Aesar). Figures are representative data sets, each from n ≥ 3 individual experiments.

Instrumentation

UV-visible spectroscopy was performed with an Agilent Hewlett-Packard 8453 diode-array spectrophotometer equipped with an Agilent 89090A thermostat. BioTek

Synergy 2 microplate readers were also utilized for absorbance and fluorescence measurements. Electrochemical detection was accomplished with a World Precision

Instruments Apollo 4000 system equipped with NO, O2 and H2O2 sensitive electrodes

(Sarasota, FL). Solution pH was determined by use of a ThermoElectron Orion 420A+

pH meter. 1H and 13C NMR was carried out on a Bruker DRX-500. Low resolution mass

spec was recorded on JEOL HX110A instrument. Elemental analysis was done at

Atlantic Microlab, Inc. (Norcross, GA).

89

Synthesis of cyclic amine diazeniumdiolates

Sodium 1-(N-cyclohexylamino)diazen-1-ium-1,2-diolate (CHA/NO) was synthesized according to a previously published procedure.35 For synthesis of other

diazeniumdiolates in general, a solution of the appropriate cyclic amine in diethyl ether

was placed in a 250 ml Parr vessel. The solution was degassed with argon, cooled in dry

ice, charged with 40 psi of NO, and allowed to stir. After 24-48 h, the solid precipitate

was collected by filtration and washed with diethyl ether. The ammonium salt was

converted to the sodium salt by dissolving in methanol and adding an equivalent amount

of sodium methoxide for cation exchange. Addition of diethylether precipitated out the sodium salt which was collected as a white solid upon vacuum filtration.

Sodium 1-(N-cyclopentylamino)diazen-1-ium-1,2-diolate (CPA/NO). A solution

of cyclopentylamine (15 mL, 0.15 mol) in 25 mL of diethylether gave 3.7 g (14% yield)

of desired sodium salt. 1H NMR (DMSO):  1.48-1.50 [m, 4H], 1.56-1.66 [m, 6H], 3.86-

13 3.88 [m, 1H], 6.00-6.01 [b, 1H] C NMR (DMSO): δ 24.67 [CH2], 31.27 [CH2], 58.19

-1 -1 [NCH]; UV: max 250 nm ( = 8,200 M cm ).

Sodium 1-(N-cycloheptylamino)diazen-1-ium-1,2-diolate (CHPA/NO). A solution

of cycloheptylamine (30 mL, 0.22 mol) in 60 mL of diethylether gave 4.2 g (19% yield)

of desired sodium salt. 1H NMR (DMSO):  1.43-1.66 [m, 13H], 3.31-3.42 [m, 1H],

13 5.89-5.90 [b, 1H]; C NMR (DMSO): δ 23.79, 28.00, 31.63, 56.00; UV: max 250 nm (

= 8,700 M-1cm-1).

Sodium 1-(N-cyclooctylamino)diazen-1-ium-1,2-diolate (COA/NO). A solution of

cyclooctylamine (15 mL, 0.11 mol) in 25 ml of diethylether gave 2.9 g (17% yield) of 90

desired sodium salt. 1H NMR(DMSO):  1.46-1.65 [14H, m], 3.31-3.46 [1H, m], 5.86-

13 5.87 [b, 1H] ; C NMR (DMSO): δ 23.57, 25.32, 26.82, 29.43, 55.14 UV: max 250 nm

( = 8,300 M-1cm-1)

Attempted synthesis of sodium 1-(N-cyclobutylamino)diazen-1-ium-1,2-diolate

(CBA/NO). A solution of cyclobutylamine (1.2 mL, 0.014 mol) in 8 mL of diethyl

ether:THF (1:1) was used in order to synthesize the corresponding diazeniumdiolate salt.

Product formation was suggested by observation of a 250 nm peak, but the final product

could not be isolated due to its instability.

Attempted synthesis of sodium 1-(N-cyclopropylamino)diazen-1-ium-1,2-diolate

CPPA/NO. A solution of cyclopropylamine (8.9 mL, 0.12 mol) in 20 mL diethylether:THF (1:1) was used in order to synthesize corresponding diazeniumdiolate.

Cyclopropylammonium 1-(N-cyclopropylamino)diazen-1-ium-1,2-diolate was isolated

-1 -1 λmax 250 nm (8,200 M cm ), but upon treatment with NaOMe, the final product was not

stable.

General synthesis of acetoxymethylated diazeniumdiolates

A solution of bromomethyl acetate in THF was added to a slurry of the relevant

sodium salt in DMSO at room temperature. The reaction mixture was stirred overnight,

whereupon 15 mL of water was added and stirring was continued for another 10 min. The

residue was extracted with dichloromethane, washed with NaHCO3 (5%), dried over

anhydrous sodium sulfate, filtered, and evaporated in vacuo. Column chromatography

was performed using hexane: acetone (4:1) to give the desired product 91

O2-(Acetoxymethyl)-1-(cyclopentylamino)diazen-1-ium-1,2-diolate (CPA/NO-

AcOM). A solution of bromomethyl acetate (183 mg, 1.19 mmol) in 3 mL of THF was added to a slurry of CPA/NO (200 mg, 1.19 mmol in 10 mL of DMSO) according to

above general procedure to give the desired product CPA/NO-AcOM (187 mg, 72%):

-1 -1 1 UV (ethanol) λ max (ε) 239 nm (10.5 mM cm ); H NMR (CDCl3) δ 1.47-1.55 (m, 2H),

1.57-1.75 (m, 4H), 1.92-2.01 (m, 2H), 2.12 (s, 3H), 4.12-4.20 (m, 1H), 5.75 (s, 2H), 6.56

13 (d, J = 7.4 Hz, 1H); C NMR (CDCl3) δ 20.67, 23.77, 30.93, 58.49, 86.95, 169.34. MS

+ (LCQ, ESI ionization method): 240.1 (MNa peak).Anal. Calcd for C8H15N3O4: C, 44.23;

H, 6.96; N, 19.34. Found: C, 44.44; H, 6.95; N, 19.40.

Cell culture

For cell culture, estrogen receptor α negative (ER-; MDA-MB-231), or estrogen

receptor α positive (ER+; MCF-7) human breast cancer cells (American Type Culture

Collection, Manassas, VA) were grown as monolayers in RPMI 1640 media

supplemented with 10% fetal bovine serum (FBS, Hyclone), penicillin (50 units/mL), and

streptomycin (50 units/mL; Life Technologies, Inc., Grand Island, NY) at 37 °C in 5%

CO2 and 80% relative humidity. Single cell suspensions were obtained by trypsinization

(0.05% trypsin/EDTA), and cells were counted using a Beckman cell counter or bright

line hemacytometer.

Decomposition profile and half-life

The rate constants of decomposition for ionic diazeniumdiolates were measured

spectrophotometrically by monitoring the decrease in absorbance near 250 nm in assay 92

buffer of the desired pH (adjusted prior to use by adding NaOH or HCl as necessary) and

37°C. The spectrophotometer was blanked after warming the cuvette containing PBS in

the instrument heat block for 5 min (37°C). The diazeniumdiolate (<10 μL of stock) was

added to buffer solution of desired pH, mixed, and spectra were collected at 2-60 s

intervals for time periods of 2-120 min, with constant stirring. The rate of CPA/NO-

AcOM decomposition in the presence and absence of 2% guinea pig serum (by volume)

was measured spectrophotometrically at 238 nm at various pH and 37°C. A solution of

CPA/NO-AcOM in EtOH (≤ 0.1%) was added to initiate the reaction, and spectra were

collected at 5-300 s intervals for 3-600 min. Kinetic analysis was performed by fitting the

-kt data to an exponential decay (A = Ae + A∞).

Reaction of HNO with myoglobin

The reaction buffer was purged with ultra-high purity argon for ≥1 mL/min, and

solutions were transferred using gas-tight Hamilton syringes to maintain deaerated

conditions. The cuvette was maintained under an argon atmosphere for the duration of the

experiment. Reductive nitrosylation of ferric myoglobin (metMb, 50 µM); (ε502 = 10.2

-1 -1 -1 -1 37 -1 -1 mM cm ; ε630 = 3.9 mM cm ) to nitrosyl myoglobin (MbNO) (ε543 = 11.6 mM cm ;

-1 -1 37 ε575 = 10.5 mM cm ) by HNO generated from the diazeniumdiolates, was monitored in

assay buffer at pH 7.4 and 37oC under deaerated conditions.

93

Electrochemical detection of NO and HNO

The NO electrode was stabilized in assay buffer of the desired pH, and in case of

CPA/NO-AcOM contains 2% serum, at room temperature. Ionic diazeniumdiolates (5 or

50 µM) or CPA/NO-AcOM (100 µM, EtOH ≤ 0.1%) were added, and the maximum signal was recorded. After the signal returned to baseline, the process was repeated to obtain triplicate measurements. Addition of 1 mM sodium ferricyanide, which oxidizes

HNO to NO, allows for indirect measurement of HNO with this system.38

HNO quantitation39

NaN3 stock solutions were prepared by weight in assay buffer. Borate buffer (20 mM) was made by weight and adjusting the pH to 9.2 with 1 M NaOH. NDA (Invitrogen molecular probes) stock solutions (80 mM) were prepared by weight in DMSO and stored at 4 °C protected from light and used for several weeks. GSH (500 μL) was added and either 5 μL of 10 mM NaN3 or PBS/DTPA (50 μM) buffer, pH 7.4 was added to an

Eppendorf tube. Then either 5 μL of NONOate or 10 mM NaOH (blank) was added.

The samples were vortexed, and the reaction was incubated at 37°C for 1 h. After incubation, 390 μL of 20 mM borate buffer, pH 9.2 and 100 μL of 1 M NaOH were added. NDA (80 mM, 5 μL) was added to the reaction, and the samples were reacted in the dark at 4 °C for 30 min before reading the plates. Fluorescence measurements were made on a Thermo Spectronic Aminco Bowman Series 2 Luminescence Spectrometer

(excitation wavelength 485 nm), or a BioTek Synergy 2 Microplate Reader with 485/20 nm excitation filter, a 528/20 nm emission filter, and a dichroic mirror (510 nm) using 94

costar 3915 assay plates (96-well, no lid, flat bottom, non-treated, non-sterile, black

polystyrene, total volume of 200 μL

Intracellular release of NO and HNO

MDA-MB-231 cells were plated at 60,000 cells per well in a 96 cell plate and

grown for 4 h in RPMI 1640 media at 37°C in 5% CO2 and 80% relative humidity. A

stock (100×) of 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF-FM-

2DA, Molecular Probes)40 in DMSO was diluted to a final concentration of 10 µM using

DTPA free assay buffer. The media was aspirated from each well, and the plate was

washed once with PBS. The cells were then exposed to 100 µL of 10 µM DAF-FM-2DA

for 75 min at 37°C and washed three times with PBS to remove excess dye. CPA/NO-

AcOM dissolved in EtOH or ionic diazeniumdiolates dissolved in 10 mM NaOH were

then added to achieve a final concentration of 10 µM. The increase in fluorescence

intensity as a function of time was then measured at an excitation of 485 nm and emission

of 535 nm.

Clonogenic cell survival assay

Cells were plated at 400,000 cells per 60 mm dish and grown for 48 h. The cells

were treated in growth media containing different concentrations (0-24 mM) of

diazeniumdiolates for 24 h. After treatment, the cells were washed twice with PBS, trypsinized, counted and plated at a density of 100, 1000 or 10,000 per 60 mm plate. For each dose determination, cells were plated in triplicate, and each experiment was repeated 95

at least twice. After 10-12 d, the colonies were stained with crystal violet (0.5% w/v) and counted using a stemi microscope.

MTT assay

The cytotoxicity of CPA/NO-AcOM was measured using 3-(4,5-dimethylthiazol-

2-yl)-2,5-diphenyltetrazolium bromide (MTT). Cells were plated at a concentration of

8,000-10,000 cells per well in a 96 well plate and grown overnight. The cells were treated with different concentrations (0-100 µM) of CPA/NO-AcOM and or control (ethanol <

1% by volume) for 48 h. A solution of 5 mg/mL MTT was added to each well, and the plate was incubated for 1 h. After removal of the media, 100 µL DMSO was added to each well, and the absorbance was recorded at 550 nm. Inhibition of growth is reported as percentage of the corresponding control. Figures are representative data sets, each from n

≥ 3 individual experiments. For combination drugs assay, cells were treated with 10 µM

tamoxifen, 75 µM of CPA/NO-AcOM or combination for 48 h.

3.3 Results and Discussion

The utility of NO-donating diazeniumdiolates is extensive due to ease of use as

well as controllable release time. We strove to expand the available number of HNO

releasing diazeniumdiolates. To begin, we selected analogues based on cyclic primary

amines as HNO donation profile of CHA/NO has never been determined previously.

The cyclic diazeniumdiolates were synthesized by exposing solutions of amine in

diethyl ether to high pressure of NO (Scheme 3.4). Evidence of diazeniumdiolate 96

formation was verified by the presence of a 250 nm peak, characteristic of the [N(O)NO]- moiety. The molar extinction coefficients for these compounds (7,900-8700 M-1cm-1) were in good agreement with other diazeniumdiolates (6,000-10,000M-1cm-1).17

Diazeniumdiolates based on cyclobutylamine and cyclopropylamine could not be isolated as the sodium salts due to low stability.

Scheme 3.4 Synthesis of stable cyclic amine diazeniumdiolates

3.3.1 Decomposition profile and half-life

Diazeniumdiolates are typically stable as solids and in highly basic solution and

can be stored at -20oC for long periods. Decomposition is accelerated with decreasing pH

and follows first order kinetics.17 The newly prepared cyclic amine diazeniumdiolates

decayed as expected as shown in Figure 3.1 with CPA/NO as the representative example. 97

1.5

1

Absorbance 0.5

0 200 220 240 260 280 300 320

Wavelength (nm)

Figure 3.1 Spontaneous decomposition of CPA/NO at 37°C in assay buffer at pH 7.4.

Spectra are shown at 0, 1, 2, 4, 6, 10 and 18 min.

The rate constants of decomposition and half-lives at pH 7.4 and 37oC are summarized in Table 3.1. The similar rates of decay rule out any ring effects on stability of isolated diazeniumdiolates. The acceleration of decomposition with decrease in pH and a 100-fold variance in rate constant (Figure 3.2) is also consistent with other known ionic diazeniumdiolates.41,16

98

Table 3.1 Decomposition data for cyclic amine diazeniumdiolates at pH 7.4 and 37°C

-3 -1 Compound k × 10 (s ) Half-life (min)

CPA/NO 3.2 ± 0.07 3.6 ± 0.08

CHA/NO 1.9 ± 0.06 6.1 ± 0.02

CHPA/NO 1.9 ± 0.01 5.9 ± 0.04

COA/NO 2.1 ± 0.02 5.4 ± 0.07

0.1 CPA/NO CHA/NO CHPA/NO 0.01 COA/NO obs k

0.001

0.0001 5677.48910

pH

Figure 3.2 The pH-dependence of the first-order rate constants of decomposition of

CPA/NO, CHA/NO, CHPA/NO and COA/NO at 37°C in PBS containing 50 μM DTPA measured at 250 nm (mean ± SD, n ≥ 3).

99

3.3.2 NO/HNO release profile

Numerous methods exist for detecting NO. Conversely direct detection of HNO is

complicated by irreversible dimerization42, and assessment of HNO release is often indirect. Our group has developed a protocol for HNO detection from donor

compounds.34,16 First, HNO production is assessed indirectly and qualitatively in the

presence of ferricyanide, which oxidizes HNO to NO.38 The current intensity from an

NO-specific electrode was elevated (2-8 fold) during decomposition of the ionic

diazeniumdiolates in the presence of ferricyanide (Figure 3.3, blue bars). This indicates

significant production of HNO at physiological pH from these new diazeniumdiolates.

7000

6000

5000

4000

3000

Current (pA) Current 2000

1000

0 CPA/NO CHA/NO CHPA/NO COA/NO

Figure 3.3 The maximum current intensity from an NO-specific electrode during

decomposition of 50 µM CPA/NO, CHA/NO, CHPA/NO and COA/NO at pH 7.4 assay 100

buffer ± 1 mM ferricyanide (green bars, NO + HNO) at room temperature (mean ± SD, n

≥ 3).

The difference in the NO signal without ferricyanide parallels the trend in decay rates (Table 3.1). A higher rate of product formation could lead to faster HNO consumption either by reacting with NO or by dimerization. In the presence of excess ferricyanide, the signal maxima indicate efficient trapping, leading to observation of similar product peaks. A difference in the ratio of HNO to NO production may also have an effect. As these results are purely qualitative, the extent of HNO/NO production requires further studies.

As previously shown for IPA/NO,34 the cyclic amine diazeniumdiolates also exhibit a pH dependent NO/HNO release profile. At physiological pH and above, these cyclic amine diazeniumdiolates primarily release HNO, whereas at lower pH, NO is the major decomposition product (Figure 3.4, CPA/NO as a representative example). Thus, our data establish release of HNO at physiological pH or above to be general property of primary amine based diazeniumdiolates.

101

5000

4000

3000

2000 Current (pA) Current

1000

0 345677.48

pH

Figure 3.4 The pH dependence of maximum current intensity from an NO-specific

electrode during decomposition of 5 µM CPA/NO in PBS containing 50 μM DTPA (blue

bars, NO alone) ± 1 mM ferricyanide (green bars, NO + HNO) at room temperature

(mean ± SD, n ≥ 3)

3.3.3 Quantification of HNO release from ionic diazeniumdiolates

The HNO yield from all four primary amine diazeniumdiolates at pH 7.0 and 7.4

was quantified (Table 3.2) using a newly available assay based on trapping with GSH.39

As expected, the percent of HNO donated is reduced at lower pH.

102

Table 3.2 Percent HNO from NONOates using 50 μM GSH at pH 7.0 and at 7.4.

[NONOate] Percent HNO, pH 7.0 Percent HNO, pH 7.4

CPA/NO 28 ± 1 54 ± 4

CHA/NO 25 ± 1 50 ± 4

CHPA/NO 24 ± 1 46 ± 4

COA/NO 31 ± 1 59 ± 2

3.3.4 Cell survival assay

HNO offers tremendous potential to be successfully applied as a preconditioning agent43 and as alcohol deterrent agent.44 To be able to use primary amine based diazeniumdiolates as pharmacological precursors of HNO for such treatment, low cytotoxicity is a requirement. Typically, simple ionic diazeniumdiolates become cytotoxic at millimolar concentrations.38,45,9 Similar cytotoxicity (Figure 3.5) was observed for the four new diazeniumdiolates as evaluated by colongenic assay in breast cancer cells. Cytotoxicity from the decomposed diazeniumdiolate products was observed at higher concentrations (≥ 20 mM, data not shown). 103

CPA/NO 100 CHA/NO CHPA/NO 80 COA/NO

60

40 Survival (%)

20

0 0 5 10 15 20

Concentration (mM)

Figure 3.5 Toxicity towards MCF-7 cells of cyclic diazeniumdiolates (0-20 mM, mean ±

SD, n ≥ 3).

HNO has also been shown to inhibit breast9 and neuroblastoma46 cancer proliferation in mouse xenografts as well as in culture through increased apoptosis. Due in part to short half-life and spontaneous decomposition outside cells, ionic diazeniumdiolates has limited potential from a chemotherapeutic point of view.

Conversion to neutral species that can release NO/HNO upon activation (enzymatic,

hydrolytic, photoactivation and others) has overcome these limitations.36

3.3.5 Synthesis of acetoxymethyl protected diazeniumdiolates

Given the lower cytotoxicity of CPA/NO, acetoxymethyl protected

diazeniumdiolates of CPA/NO, CPA/NO-AcOM was chosen for further detailed 104

characterization. Derivative was prepared (Scheme 3.5) by reacting bromomethylacetate

with CPA/NO under anhydrous condition. Acetoxymethyl protected derivatives of other cyclic NONOates were prepared similarly (data not shown) but were not included for further analysis. Presence of ester bond is expected to impart sensitivity towards esterase mediated hydrolysis to release parent diazeniumdiolates along with acetic acid and formaldehyde (Scheme 3.6).

Scheme 3.5 Synthesis of CPA/NO-AcOM

3.3.6 Decomposition half-life of CPA/NO-AcOM

In the presence of the esterases in serum, the half-life of decomposition at pH 7.4

and 37o C (5.8 min), was similar to the parent diazeniumdiolate (3.1 min). The initial peak at 238 nm shifted to 250 nm (Figure 3.6), indicating cleavage of the ester bond

(Scheme 3.6) and production of free diazeniumdiolate. 105

1.4

1.2

1

0.8

0.6

Absorbance 0.4

0.2

0 220 240 260 280 300 320

Wavelenghth (nm)

Figure 3.6 Hydrolysis of CPA/NO-AcOM in the presence of 2% guinea pig serum in

assay buffer at pH 7.4 and 37°C (n ≥ 3). Scans are plotted at 0, 1, 2, 3, 4, 6, 10, 14, 20

min.

Scheme 3.6 Decomposition of O2-(acetoxymethyl)-1-(cyclopentylamino) diazen-1-ium-

1,2-diolate at PBS pH 7.4 and 37°C in the presence of serum.

106

To further verify the esterase mediated decomposition profile of CPA/NO-AcOM, an NO electrode assay was performed in the presence of 2% guinea pig serum (Figure

3.7). The decomposition profile for CPA/NO-AcOM in the presence and absence of 1 mM ferricyanide at variable pH was similar to the parent diazeniumdiolates. This further suggests the production of free CPA/NO via scheme 3.6. The diminished signal at either end of the pH spectrum is likely due to esterase deactivation.

1.6 104 1.4 104 1.2 104 1 104 8000 6000 Current (pA) Current 4000 2000 0 345677.48910

pH

Figure 3.7 The pH dependent maximum current intensity from an NO-specific electrode during decomposition of CPA/NO-AcOM (100 μM) in presence of 2% guinea pig serum

in assay buffer (blue bars, NO alone) ± 1 mM ferricyanide (green bars, NO + HNO) at

room temperature (mean ± SD, n ≥ 3)

In the absence of serum, the half-life increases to 21 min at pH 7.4 and 37°C and

becomes less stable with increasing pH (rate constants 0.3, 1.3, 5.0 × 10-3 s-1 at pH 7, 8 107

and 9 respectively). The lack of an apparent peak at 250 nm (Figure 3.8) indicates that

free diazeniumdiolate is not produced during decomposition. All of these data are consistent with the previously determined mechanism for acetoxymethyl protected

IPA/NO (Scheme 3.3).36 Deprotonation of the amine proton would eventually lead to

formation of acylnitroso derivative, which produces HNO and acetic acid upon

hydrolysis.

1.4

1.2

1

0.8

0.6

Absorbance 0.4

0.2

0 200 220 240 260 280 300 320

Wavelength (nm)

Figure 3.8 Hydrolysis of CPA/NO-AcOM in assay buffer at pH 7.4 and 37°C (n≥3).

Scans are plotted at 0, 7, 13, 20, 27, 33, 40, 50, 60, 70, 100 min

Acetoxymethyl protected IPA/NO has been previously suggested to have

enhanced HNO production over the parent compound due to the unique decomposition

mechanism as shown above.36 To confirm further that CPA/NO-AcOM decomposes by 108

Scheme 3.3 in the absence of serum, the yield of HNO was assessed by trapping with metmyoglobin (metMb) (Eq. 1).37,47

metMb    MbNO + H+ (8  105 M-1 s-1) (Eq.1)

MetMb (50 µM) undergoes reductive nitrosylation with all the prepared cyclic diazeniumdiolates (Figure 3.9A by CPA/NO, a representative example). As with

IPA/NO-AcOM,36 a higher yield of MbNO (Figure 3.9B) was observed for CPA/NO-

AcOM (100 µM) suggesting increased production of HNO. HNO release was quenched

in the presence of 1 mM GSH (a known scavenger of HNO)48 further confirming HNO

production (Figure 3.9 A-B).

A 0.6

0.5

0.4

0.3

0.2 Absorbance

0.1

0 450 500 550 600 650 700

Wavelength (nm)

109

B 0.6

0.5

0.4

0.3

0.2 Absorbance

0.1

0 450 500 550 600 650 700

Wavelength (nm)

Figure 3.9 Reductive nitrosylation of metMb (50 μM, blue spectra) with A) CPA/NO or

B) CPA/NO-AcOM (100 μM). The assay was performed in pH 7.4 assay buffer at 37 °C

(red spectra, compound alone) ± 1 mM GSH (green spectra) under deaerated conditions until there were no further spectral changes (n ≥ 3).

3.3.7 Intracellular NO and HNO release

To determine whether hydrolysis of CPA/NO-AcOM will occur intracellularly,

DAF-FM-2DA was used as a reporter molecule in MB-231 cells. DAF reacts with the autoxidation product of both NO40 and HNO49, with higher fluorescence signal in latter

case. The diacetate (DAF-FM-2DA) is readily taken up by cells, where hydrolysis of the

ester bonds by intracellular esterase produces the non-permeable DAF. On treatment of

DAF-FM-2DA loaded cells, an increase in fluorescence intensity was observed in a time

dependent manner for all the five tested compounds compared to control. A relatively 110

similar signal was generated for the four ionic compounds. The signal from CPA/NO-

AcOM was ~3 fold higher than for CPA/NO, signifying enhanced HNO release within

the cells (Figure 3.10)

6 104 Control 5 104 CPA/NO CHA/NO CHPA/NO 4 4 10 COA/NO CPA/NO-AcOM 3 104 RFU

2 104

1 104

0 0 20406080100120 time (min)

Figure 3.10 NO and HNO release measured in MB-231 cells. The cells were exposed to

100 µL of 10 µM DAF-2DA in PBS pH 7.4 for 75 min at 37°C and washed three times with PBS to remove excess dye. Upon addition of 10 µM CPA/NO-AcOM (in

EtOH(<0.1%)), or CPA/NO, CHA/NO, CHPA/NO or COA/NO in 10 mM NaOH, the increase in fluorescence intensity at 535 nm was measured as a function of time at 37°C following excitation at 485 nm. The data are expressed as mean ± SD (n = 2, six replicates per plate).

111

3.3.8 Cytotoxicity

Under aerobic conditions HNO can be cytotoxic.38 This is in part due to

consumption of GSH and due to formation of reactive nitrogen oxide species formation that mediate DNA double strand breaks.50,51 HNO is also known to irreversibly inhibit

enzymes with critical thiols.52,53,54 Inhibition of one such enzyme glyceraldehyde phosphate dehydrogenase (GAPDH)55, a key player in glycolysis, has chemotherapeutic

potential given that cancer cells have a higher dependence on glycolysis than normal

cells.56,57

The cytotoxicity of CPA/NO-AcOM (Figure 3.11) was increased (IC50 ~ 100 µM)

in MCF7 and MDA-MB-231 cells compared to the parent. This enhanced cytotoxicity

can be explained in part by the increased uptake. Higher intracellular concentration of

HNO by CPA/NO-AcOM compared to CPA/NO could potentially deplete the glutathione

level38 thus changing cellular redox status. Enhanced HNO production also increases

DNA double strand break and inhibition of GAPDH to starve the cell. 112

100

80

60

40 Survival (%) Survival MDA-MB-231 20 MCF-7

0 0 20406080100

CPA/NO-AcOM concentration (M)

Figure 3.11 Toxicity of CPA/NO-AcOM towards MCF-7 and MDA-MB-231 cells by

MTT cell survival assay at 37°C (mean ± SD, n ≥ 3).

3.3.9 Effect of CPA/NO-AcOM on cytotoxicity of tamoxifen

Tamoxifen is a drug that has been clinically used worldwide in treatment of early and advanced stages of ER+ breast cancer and is prescribed as chemopreventive agent for women that are at high risk of developing it.58 Tamoxifen compets with binding of hormone estrogen to its receptor, which is crucial for breast cancer cell proliferation, thereby inducing growth arrest and apoptosis.59 However, successful application of tamoxifen to a broader population of breast cancer patients requires an urgent need for the development of novel and effective therapeutic approaches in targeting more aggressive hormone-insensitive breast cancer patients. Tamoxifen has been previously 113

shown to induce apoptosis in ER- cancer cells in an ER independent manner, albeit at a

quite high concentration.60

NO releasing donor compounds have been previously reported to increase the

cytotoxicity of cancer drugs such as cisplatin61 and doxorubicin towards cancer cells.62

Such analysis has not been reported for HNO donors. Here we evaluated the potential of using CPA/NO-AcOM in combination with tamoxifen for targeting ER- breast cancer.

To target the more aggressive form, MB-231 breast cells were used for evaluating combination potential. Figure 3.12 shows the surviving fractions of MB-231 cells treated with tamoxifen (10 µM) and/or CPA/NO-AcOM (75 µM) as evaluated by MTT assay after 48h treatment. The result showed that both tamoxifen (76 ± 13%) and CPA/NO-

AcOM (75 ± 6%) caused significant reduction in the surviving fraction. On the other hand co-administration of tamoxifen along with CPA/NO-AcOM demonstrates significantly higher cell killing compared with tamoxifen or CPA/NO-AcOM by itself

(47 ± 3%). It’s expected that the drug tamoxifen and HNO released from CPA/NO-

AcOM will have different molecular targets, thus reducing the chance of cell survival as a combination. Moreover long term combined usage will have less chance of resistance development compared to single treatment. Further investigation into these antitumor effects may lead to better understanding of the action of HNO in combination with other drugs and the intriguing possibility of using HNO in field of cancer treatment. 114

100

80

60

40 Survival (%)

20

0 control tamoxifen combination

CPA/NO-AcOM

Figure 3.12 The effect of CPA/NO-AcOM (75 µM), tamoxifen (10 µM) or combination

(CPA/NO-AcOM 75 µM + tamoxifen 10 µM) on survival of MDA-MB-231 cells. Effect was studied using MTT cell survival assay at 37°C (mean ± SD, n ≥ 3).

3.4 Conclusions

In conclusion we have shown diazeniumdiolates to be an alternative donor to other currently used HNO donors. Secondary amine based diazeniumdiolates are commercially available, and our data suggests facile synthesis and potential commercialization of primary amine based diazeniumdiolates. These diazeniumdiolates were shown to release NO/HNO in a way similar to that reported for IPA/NO, thus 115

establishing release profile to be common to primary amine based diazeniumdiolates.

Derivatization can dramatically change the production and release time of HNO, thus

making derivatized diazeniumdiolates usable for exploring HNO chronic exposure to

human cells. These features also hold promise in potential application for selectively

targeting cancer cells. Combination of less cytotoxic concentration of CPA/NO-AcOM

and tamoxifen resulted in significant reduction of breast cancer cell survival. In the future

additional compounds will be synthesized to generate a library of HNO donors with a

wide spectrum of half-lives of decomposition.

Abbreviations

Angeli's salt, sodium trioxodinitrate; CHA/NO, sodium 1-(N-

cyclohexylamino)diazen-1-ium-1,2-diolate; CHPA/NO, sodium 1-(N-

cycloheptylamino)diazen-1-ium-1,2-diolate; COA/NO, sodium 1-(N-

cyclooctylamino)diazen-1-ium-1,2-diolate; CPA/NO, sodium 1-(N-

cyclopentylamino)diazen-1-ium-1,2-diolate; CPA/NO-AcOM, O2-(acetoxymethyl)-1-

(cyclopentylamino)diazen-1-ium-1,2-diolate; DAF-2DA, 4-amino-5-methylamino- 2’,7’-

difluorofluorescein diacetate; DTPA, diethylenetriaminepentaacetic acid; ERα, estrogen

receptor alpha; ER(−), estrogen receptor negative; ER(+), estrogen receptor positive;

FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSH,

glutathione; HNO, nitroxyl; IPA/NO, sodium 1-(N-isopropylamino)diazen-1-ium-1,2-

diolate; MCF-7, estrogen receptor positive human breast cancer cell; MDA-MB-231,

estrogen receptor negative human breast cancer cell; MetMb, ferric myoglobin; MbNO, 116

nitrosyl myoglobin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;

NO, nitric oxide; PBS, phosphate-buffered saline; Piloty’s salt, N- hydroxylbenzenesulfonamide; RPMI, Roswell Park Memorial Institute.

117

CHAPTER 4: CHLORAMBUCIL ANALOGUES OF PABA/NO

4.1 Introduction

Nitric oxide (NO) is an important modulator of a large number of physiological

functions.1-8 Synthesis of NO in endothelial cells, in response to physical and chemical stimuli, has been found to play a crucial role in vasodilation and is essential for regulation of blood pressure.9,10 Moreover, NO inhibits aggregation and adhesion of platelets to

blood vessel walls,2,11 thereby significantly reducing the formation of blood clots. NO is also involved in the immune defense against pathogens.12 NO further plays an important

role in cell growth and in apoptosis.13,14 NO has a pivotal role in cancer biology with both

pro- and anti-cancer effects,15,16 and it has been shown to reverse multidrug resistance

(MDR) towards conventional chemotherapeutic drugs such as cisplatin and

doxorubicin.17-20 NO can deplete intracellular glutathione (GSH) levels,19 a tripeptide

which is often involved in the detoxification of drugs such as cisplatin and melphalan,

and can inhibit drug efflux by MDR-associated proteins (MRPs) such as P-

glycoprotein.18

Chlorambucil (sold by GlaxoSmithKline as Leukeran) is an aromatic nitrogen

mustard that has been used for the treatment of lymphocytic leukemia, ovarian and breast

carcinomas and Hodgkin’s disease.21-24 The cytotoxicity of chlorambucil as a bifunctional

alkylating agent is due to its ability to cross-link DNA.25 Chlorambucil hydrolyzes in

water and has a pKa of 5.8, implying significant ionization at physiological pH. 118

Chlorambucil is extensively metabolized in the liver to {4-[bis(2- chloroethyl)amino]phenylacetic acid, which has its own alkylating capabilities.26

The uptake of alkylating agents into cells is a critical factor in specificity towards cancer cells. The emergence of alkylating agent resistant tumor cells limits the clinical effectiveness of these drugs. Tumor cells resistant to alkylating agents often possess a higher ratio of protein-free to protein-bound thiols. GSH is the major intracellular nonprotein sulfhydryl compound and tumor cells commonly have increased GSH level.27

Many patients initially respond to chlorambucil administration but subsequent repeated dosing often eventually leads to resistance. Chlorambucil has been shown to be detoxified by glutathione transferase π (GST P1-1),28 an enzyme that uses GSH as a

substrate and is often over-expressed in cancer tissues.29,30 Over expression of GST π31 along with other factors is the major reason for development of resistance to chlorambucil. GSTs have emerged as promising anticancer targets with approaches varying from design of inhibitors to GST activated prodrugs.32-35 Considering the over-

expression of GSTs, Keefer et al. developed JS-K36 (Figure 4.1), which releases an NO producing diazeniumdiolate upon bioactivation.37 The diazeniumdiolate then undergoes

spontaneous decomposition to release NO (Scheme 4.1).

119

Figure 4.1 NO releasing JS-K

JS-K has been shown to have very effective anticancer activity in a variety of

cancer cell lines with IC50 values ranging from 0.2-1.2 µM, compared to millimolar

concentrations for simple NONOates.38,39 JS-K was effective in blocking angiogenesis,

thereby inhibiting cancer cell proliferation,40 and activated caspase-3 and-9, thereby up-

regulating apoptosis.41 JS-K substantially reduced the growth of lung cancer and multiple

myeloma in mice models.38,42 JS-K can also increase the accumulation of chemotherapeutic drugs like cisplatin in resistant cell lines.43

O NO2 O O N NO O N NO 2 2 N O 2 2 N O GSH N +N N N O N NO2 O N+ GST S O- S O- O O O O O O JS-K N OH N O H H H NH NH2 NH NH2 GS-2,4-DNP O OH O OH O O N O N O 2NO - N +N HN O N O-

EPC/NO sodium 1-[4-(methoxycarbonyl)piperazin-1-yl] diazen-1-ium-1,2-diolate

Scheme 4.1. GST mediated NO release from JS-K37 120

To take advantage of over-expression of the π isoform of GST, structural modifications

were made to increase GST π mediated diazeniumdiolate release while decreasing that by

a α isoform, which is expressed in normal cells. Computer modeling led to the synthesis

of PABA/NO (Figure 4.2)44 which was more π selective compared to JS-K.45,46

Figure 4.2. PABA/NO

The goal of this work was to utilize the resistance reverting property of NO in an

NO releasing chlorambucil analogue (Figure 4.3) based on the PABA/NO moiety and to evaluate its efficacy in chlorambucil resistant cancer cell lines.

Figure 4.3 NO releasing chlorambucil analog of PABA/NO

4.2 Materials and Methods

Reagents

Unless otherwise noted, chemicals were purchased from Sigma-Aldrich and were used without further purification. Thin layer chromatography (TLC) was carried out 121

using Analtech silica gel GF (250 micron) glass-backed plates. Eluted plates were

visualized using UV light. Flash chromatography was performed using the indicated

solvent system on Silica Gel 60 (230-450 mesh size; purchased from Alfa Aesar). Stock

solutions other than of nitrogen oxide donors were prepared fresh daily at 100 in MilliQ

or Barnstead Nanopure Diamond filtered H2O, unless specified. Typically, the assay buffer consisted of the metal chelator diethylenetriaminepentaacetic acid (DTPA, 50 μM)

in calcium- and magnesium-free Dulbecco’s phosphate-buffered saline (PBS, pH 7.4).

Instrumentation

UV-visible spectroscopy was performed with an Agilent Hewlett-Packard 8453

diode-array spectrophotometer equipped with an Agilent 89090A thermostat. BioTek

Synergy 2 microplate readers were utilized for absorbance and fluorescence

measurements. Solution pH was determined by use of a ThermoElectron Orion 420A+

pH meter. 1H and 13C NMR spectra were acquired using a Bruker DRX-500 spectrometer

(500 MHz). Mass spectra were obtained with a JEOL HX110A spectrometer.

Cell culture

Human breast cancer cells (MCF-7 and MB-231, American Type Culture

Collection, Manassas, VA) were grown as monolayers in RPMI 1640 media

supplemented with 10% FBS, penicillin and streptomycin (50 U/mL, Life Technologies,

Inc., Grand Island, NY) at 37°C in 5% CO2 and 80% relative humidity. Single cell 122

suspensions were obtained by trypsinization (0.05% trypsin/EDTA), and cells were

counted with a Bright line Hemacytometer.

Effect of chlorambucil-NO prodrugs on cell viability as determined with the standard

colorimetric MTT assay

Cells were plated at 8,000-10,000 cells per well in a 96-well plate and grown

overnight as above. The cells were then treated with varied concentrations (0-200 µM) of

the prodrugs for 48-72 h. After addition of 10 μl of 2 mg/mL of 3-(4,5-dimethylthiazol-2-

yl)-2,5-diphenyltetrazolium bromide (MTT) to each well, the plate was incubated for 4 h

at 37°C. The media was then removed, 100 µL DMSO was added to each well, and the

absorbance was recorded at 550 nm using a BioTek Synergy 2 microplate reader.

Synthesis

O2-(2,4-dinitro-5-fluorophenyl) 1-(N,N-dimethylamino)- diazen-1-ium-1,2-diolate (1) and O2-(2,4-dinitro-5- hydroxyphenyl) 1-(N,N-

dimethylamino)diazen-1-ium-1,2-diolate (2) were synthesized according to previously published procedures.44,47

O2-[2,4-dinitro-5-((4-(4-(bis(2-chloroethyl)amino)phenyl)butanoyl)oxy)phenyl]-

1-(N,N-Dimethylamino)diazen-1-ium-1,2-diolate (3) To a stirred solution of chlorambucil

(100.9 mg, 0.331 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.HCl (63.5 mg,

0.331mmol) and DMAP (~5 mg) in DCM under an inert atmosphere was added drop

wise to a solution of 2 (105 mg, 0.331 mmol) in dichloromethane. The reaction mixture 123

was stirred overnight, and then was filtered, and the filtrate was concentrated under

vacuum. Column purification with 1:1:8 (EtOAc/DCM/hexane) gave 20 mg (0.034

1 mmol, 10% yield) of a sticky, orange solid. H NMR (CDCl3): δ 2.05 [t, J = 7.5 Hz, 2H],

2.65- 2.71 [m, 4H], 3.30[s, 6H], 3.61-3.64[t, 4H, J = 7.5 Hz], 3.70-3.73[ [t, 4H, J = 7.5

Hz], 6.64 [d, J = 6 Hz, 2H], 7.10[d, J= 6Hz, 2H], 7.33 [s, 1H], 8.89 [s, 1H]. 13C NMR

(CDCl3): δ 26.03, 33.32, 33.68, 40.49, 41.75, 53.55, 112.19, 113.12, 124.81, 129.63,

129.69, 129.86, 133.87, 135.31, 144.49, 148.92, 154.08, 170.22. Mass spec (LRMS) calculated M-H+ 573.12, found 573.11

4.3 Results and discussion

4.3.1 Attempted synthesis

Synthesis of the target compound proved to be challenging. Several routes were

explored, some based on the method used by Keefer et al. in the synthesis of related

PABA/NO analogues.48 Scheme 4.2-4.5 shows the attempted synthesis of compound 3.

Scheme 4.2 Attempted aromatic nucleophilic substitution to form compound 3

Aromatic nucleophilic substitution (Scheme 4.2) was the first choice for synthesis

due to the low number of steps involved. The reaction was run overnight. On workup

and purification three different fractions were collected. None of the fractions were the 124

expected compound. The reaction was repeated in dichloromethane and dimethylformamide with no success. Next DCC coupling was explored, as DCC is

widely used for activating carboxylic acids toward amide and ester formation.

Scheme 4.3 Attempted DCC coupling of intermediate 2 with chlorambucil

DCC coupling was attempted under anhydrous condition for overnight reaction. A precipitate was visible as early as 1 h. The solution was filtered, and the filtrate was concentrated under vacuum. Attempts were made to purify the reaction mixture.

Different solvents systems for column were explored but it always ended up as a mixture.

In a modified procedure, DMAP was used as shown in Scheme 4.4, in a catalytic amount.

DMAP is used for alcohols, which are difficult to esterify. However, usage of DMAP also ended up in a mixture (identity unknown) that was difficult to purify. 125

Scheme 4.4 Attempted DMAP catalyzed DCC coupling

Due to the failure of DCC coupling and aromatic substitution, the acid halide route was used in an attempt to synthesize the target compound (Scheme 4.5).

Scheme 4.5 Attempted synthesis of target compound via acid halide intermediate

Chlorambucil was converted into the corresponding acid halide according to a previously published procedure.49 Nucleophilic acyl substitution was then attempted with

2 under an inert atmosphere. After 3 h of stirring, dil. HCl (50 mL) was added to the 126

reaction mixture and then extracted with EtOAc (50 × 3 mL). Column chromatography

was performed using 50% CHCl3/hexane as the eluting solvent to give an orange colored

liquid. NMR analysis of the liquid fraction showed that it was not the desired compound.

Carbodiimide based coupling was revisited but instead of using DCC, water soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was used for coupling

(Scheme 4.6). The reaction was successful, and target compound 3 was isolated as an orange solid.

Scheme 4.6 Synthesis of DMA-dinitrobenzene-chlorambucil adduct

4.3.2 Cytotoxicity

The efficacy of our DMA-DNB-chlorambucil (3) along with chlorambucil was studied on proliferation of MB-231 (an estrogen receptor negative cell line representing aggressive breast cancer) cells by MTT assay. On treating the MB-231 cells for 3 d with chlorambucil or the diazeniumdiolate adduct, significant reduction in cell survival was observed (Figure 4.4). At 50 µM, both the compounds showed nearly a log kill. 127

120 Chlorambucil DMA-DNB-Chlorambucil 100

80

60

% Survival 40

20

0 0 1 10 50 100 200 Concentration (M)

Figure 4.4 The effect of DMA-DNB-chlorambucil, chlorambucil and DMSO control (0-

200 µM, respectively) on cell survival of MB-231 cells. Cells were treated for 72 h at

370C and then analyzed by the specrophotometric MTT assay (n = 3 for MB-231 for at

least triplicate per plate).

4.3.3 Intracellular NO release

To determine intracellular release of NO from 3, DAF-FM-2DA50 was used as a

reporter molecule in breast cancer cells MB-231. Cells were treated with DMSO

(control), chlorambucil (10 µM) or 3, and the increase in fluorescence signal was

monitored over the time. As shown in Figure 4.5, 3 generated significant amount of

higher signal, compared to control and parent drug chlorambucil. These data suggest that

3 is able to permeate cell membrane and release NO inside. 128

2.5e+4 Control Chlorambucil 2e+4 DMA-DNB-Chlorambucil

1.5e+4

RFU 1e+4

5000

0

Figure 4.5 NO or HNO release measured after 12 h under physiological conditions in

MB-231 cells from reaction of 10 µM DAF-2DA with 10 µM of chlorambucil and 3 in

DMSO (<0.1%). (n=8 per plate, two trial)

4.4 Conclusion

In conclusion, PABA/NO analogues of alkylating drug chlorambucil can be prepared via EDC coupling. The cytotoxicity assay suggests that adducts as well as the parent compound to be quite cytotoxic at 50 µM. DAF-FM-2da assay suggest that compound 3 can release NO intracellularly and thus has the potential in enhancing the

cytotoxicity in chlorambucil resistant cancer cells.

Abbreviations

NO, nitric oxide; DCC, Dicyclohexylcarbodiimide; DMA/NO, sodium 1-(N,N-

dimethylamino)diazen-1-ium-1,2-diolate; DTPA, diethylenetriaminepentaacetic acid; 129

EDC, ethyl-3-(3-dimethylaminopropyl)carbodiimide; GSH, glutathione; GST P1-1,

glutathione transferase Pi; MDR, multidrug resistance; MRPs, MDR-associated proteins;

MDA-MB-231: estrogen receptor alpha negative breast cancer cell line; MTT, 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate buffered saline;

RPMI, Roswell Park Memorial Institute;

130

CHAPTER 5: SYNTHESIS AND CHARACTERIZATION OF NITROGEN

OXIDE ADDUCTS WITH NON-STEROIDAL ANTI-INFLAMMATORY

DRUGS

5.1 Introduction

Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, indomethacin, naproxen are widely used for treatment of pain, fever and inflammation.1 Their mode of

action relies on inhibition of cyclooxygenase (COX-1 and COX-2) derived

prostaglandins (PGs) synthesis, which are involved in various physiological as well as

pathological processes.2-11 NSAIDs, and in particular aspirin, have been suggested to be

chemopreventive agents against several cancers.12,13 However, long term usage of

NSAIDs can lead to serious side effects, particularly gastric ulceration and renal

failure.14,15 In a 199 study on the mortality rate in U.S. from seven different disorders,

NSAID toxicity related deaths were third highest in patients with rheumatoid arthritis or

osteoarthritis.16

Targeting the COX-2 isoform can play a key role not only in relieving pain and

inflammation17 but also in treatment of many pathological conditions while inhibition of

COX-1 leads to associated side effects. COX-2 is over-expressed in various types of

cancer18-21 and serves as an important biomarker in breast cancer.19,22,23 Inhibition of

COX-2 is an attractive target in treatment of cancer.24,25 Several studies have emerged

that show that daily intake of NSAIDs resulted in a significant risk reduction for cancer

occurrence,12 such as breast cancer (71 %), colon cancer (70) and lung cancer (79 %).26

131

As a family, NSAIDs show a broad range of relative COX-1 and COX-2 selectivity. NSAIDs such as indomethacin have high COX-1 selectivity, while niflumic acid is at the borderline and diclofenac is more selective towards COX-2.27 The quest for

NSAIDs with minimal side effects has led to development of selective inhibitors of

COX-2 (COXIBs) such as celecoxibs and rofecoxib.28 In spite of being safer to the

gastrointestinal tract, COXIB use leads to increased incidence of heart attack and stroke,

which ultimately resulted in withdrawal of rofecoxib from the market.29,30

A number of strategies have recently emerged to retain the NSAID moiety but to

improve the safety profile, attachment of a nitric oxide (NO) donor,31,32 or more recently, a hydrogen sulfide releasing agent has been reported.33 Development of NO-NSAIDs was

based on the well accepted role of NO in mediating mucosal defense,34 thus providing the

rationale to decrease NSAID-induced damage by delivery of NO at the affected site.

Several NO-NSIADs have been developed over the last decade, with organic nitrates35,36

and diazeniumdiolates37,38 being the NO donors of choice, and aspirin, ibuprofen,

indomethacin as the NSAID counterpart.39 One such NO-NSAID, naproxcinod,40 developed by NicOX has completed phase III trials (Figure 5.1) and is in late stage development.

Figure 5.1 Naproxinod, a derivative of naproxen with a nitroxybutyl ester

132

We have recently shown that nitroxyl (HNO) releasing diazeniumdiolate derivatized aspirin to be comparably effective in preventing gastric ulceration to NO releasing diazeniumdiolate derivatized aspirin.41 To further expand the list of these diazeniumdiolate-based NSAID derivatives, herein we report the synthesis, nitrogen oxide release properties, cytotoxicity studies of NO- and HNO-releasing derivatives of

niflumic acid, indomethacin, and compare their efficacy to that of the parent NSAID, as

well as to that of the aspirin analogues.

5.2 Materials and methods

Reagents

Unless otherwise noted, chemicals were purchased from Sigma-Aldrich and were

used without further purification. Thin layer chromatography (TLC) was carried out

using Analtech silica gel GF (250 micron) glass-backed plates. Eluted plates were

visualized using UV light. Flash chromatography was performed using the indicated

solvent system on Silica Gel 60 (230-450 mesh size, purchased from Alfa Aesar).

Concentrations of NONOate stock solutions (>10 mM), prepared in 10 mM NaOH and

stored at –20°C, were determined directly prior to use from the extinction coefficients at

250 nm (ε of 8,000 M-1cm-1).42 Stock solutions other than of nitrogen oxide donors were

prepared fresh daily at 100 in MilliQ or Barnstead Nanopure Diamond filtered H2O, unless specified. Typically, the assay buffer consisted of the metal chelator diethylenetriaminepentaacetic acid (DTPA, 50 μM) in calcium- and magnesium-free

Dulbecco’s phosphate-buffered saline (PBS, pH 7.4). By sequestering contaminating

133

metals, addition of DTPA quenched the oxidation of HNO to NO.43 All reactions were

performed at 37°C except those measured with the NO-specific electrode, which were

run at room temperature.

Instrumentation

UV-visible spectroscopy was performed with an Agilent Hewlett-Packard 8453

diode-array spectrophotometer equipped with an Agilent 89090A thermostat.

Electrochemical detection was accomplished with a World Precision Instruments Apollo

4000 system with NO, O2 and H2O2 sensitive electrodes. BioTek Synergy 2 microplate readers were utilized for absorbance and fluorescence measurements. Solution pH was determined by use of a ThermoElectron Orion 420A+ pH meter. 1H and 13C NMR spectra were acquired using a Bruker AM-500 spectrometer (500 MHz). Mass spectra were obtained with a JEOL HX110A spectrometer. CHN microanalyses were performed at

Columbia Analytical Services, Tucson.

Synthesis

IPA/NO (1) and DEA/NO (2) IPA/NO-SH3 (3), DEA/NO-SCH3 (4), IPA/NO-

CH2Cl (5), DEA/NO-CH2Cl (6), IPA/NO-aspirin (9) and DEA/NO aspirin (10) were

synthesized according to previously published procedures.41,44 Other NONO-NSAIDs

were synthesized as shown in Scheme 5.1

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Scheme 5.1. Synthesis of NO/HNO releasing NSAIDs derivatives

O2-(2-(3-(trifluoromethyl)phenylamino)nicotinate)-1-(N-isopropylamino)-diazen-

1-ium-1,2-diolate (7). Niflumic acid (3.34 g, 11.8 mmol) was dissolved in DMSO (30 mL). Triethylamine (1.66 mL, 11.8 mmol) was then added, and the reaction mixture was stirred for 50 min at room temperature. Then, a solution of 5 in DMSO (25 mL) was added drop wise to the reaction mixture, which was stirred for 15 h and then ethyl acetate

(80 mL) was added. The organic layer was washed with dilute HCl (5 × 60 mL), dried over sodium sulfate and then evaporated to obtain the crude product. Purification was performed by column chromatography, first using 22% ethyl acetate:hexane and then 1% ethyl acetate:dichloromethane to obtain 7 (1.10 g, 2.66 mmol, 37.8%) as a light yellow

135

1 solid. H NMR (CDCl3): δ 1.15 [d, J=6.5 Hz, 6H, (CH3)2], 3.98 [septet, J=6.5 Hz, 1H,

CH), 5.99 [s, 2H, OCH2O], 6.108 [b, 1H, NH] 6.785 [dd, J1= 5Hz, J2=8Hz, 1H arom]

7.28-7.44 [m, 2H arom], 7.82 [m, 1H arom], 8.07 [s, 1H arom] 8.27-8.30 [dd, J=2Hz,

8Hz, 1H, arom] 8.41-8.43 [dd, J1=5 Hz, 2 Hz, 1H, arom], 10.17 [s, 1H, NH]. 13C NMR

(CDCl3): δ 20.41, 49.27, 87.56, 106.20, 114.14, 117.38, 117.42, 119.37, 119.40, 123.75,

129.22, 139.99, 140.64, 153.94, 156.05, 166.01 Mass spec (HRMS) calculated M-H+

-1 -1 414.1384, found 414.1389, UV: ε (λ240nm) = 7.87 mM cm

O2-(2-(3-(trifluoromethyl)phenylamino)nicotinate)-1-(N,N-Diethylamino)-diazen-

1-ium-1,2-diolate (8). Niflumic acid (6.21 g, 22.0 mmol) was dissolved in DMSO (150 mL). Triethylamine (3.07 mL, 22.0 mmol) was then added and the reaction mixture was stirred for 30 min at room temperature. Then, a solution of 6 (3.99 g, 22.0 mmol) in

DMSO (50 mL) was added drop wise to the reaction mixture, which was stirred for 24 h and after completion of the reaction ethyl acetate (150 mL) was added. The organic layer was washed with saturated NaHCO3 solution (5 × 100 mL), dried over sodium sulfate

and then evaporated to obtain the crude product. Further purification was performed by

column chromatography (30% ethyl acetate-hexane) to obtain 6.22 g (66.2%) of pure

1 product. H NMR (CDCl3): δ 1.09 [t, J=3.5 Hz, 6H, (CH3)2], 3.18 [q, J=3.5 Hz, 4H, CH2),

6.08 (s, 2H, OCH2O), 6.78 [dd, J1= 5 Hz, J2=8 Hz, 1H arom] 7.29-7.45 [m, 2H arom],

7.82 [d, J=8 Hz, 1H arom], 8.10 [s, 1H arom] 8.27-8.29[dd, J=2 Hz, 8 Hz, 1H, arom]

13 8.42-8.43 [dd, J1=4.5 Hz, J2=2 Hz, 1H, arom], 10.19 [s, 1H, NH] C NMR (CDCl3): δ

11.37, 48.14 87.68, 106.14, 114.07, 117.32, 117.35, 119.28. 119.32, 123.70, 129.18,

139.98, 140.50, 153.89, 156.01, 165.91 Elemental analysis (C13H17N3O6): C=50.59;

136

H=4.72; N=16.39 (theoretical), C=50.82; H=4.55; N=16.16 (experimental), MS (LCQ,

ESI): 450.1 (MNa+ peak).

O2-(methyl2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetate)-1-

(N-isopropylamino)-diazen-1-ium-1,2-diolate (11). Indomethacin (1.53 g, 4.29 mmol) was dissolved in DMSO (15 mL). Triethylamine (0.598 mL, 4.29 mmol) was then added and the reaction mixture was stirred for 50 min at room temperature. Then an equimolar solution of 5 in DMSO (15 ml) was added drop wise to the reaction mixture, which was stirred for 15 h and after completion of the reaction; it was quenched with ethyl acetate

(50 mL). The organic layer was washed with dil. HCl (5 × 40 mL), dried over sodium sulfate and then evaporated to obtain the crude product. Purification was performed by column chromatography, first using 22% ethyl Acetate: hexane and then with 1% ethyl acetate: dichloromethane to obtain 11 (46 mg, 0.094 mmol, 2.2%) as light yellow liquid.

1 H NMR (CDCl3): δ 1.13 [d, J=6.5 Hz, 6H, (CH3)2], 2.35 [s, 3H, ArCH3], 3.70 [s, 2H,

ArCH], 3.81 [s, 3H, OCH3], 3.88 [sep, J=6.5 Hz, 1H, CH] 5.75 [s,2H, OCH2O], 6.03 [b,

1H, NH], 6.66 [dd, J=9 Hz, 2.5 Hz, 1H, Indonyl ring], 6.86 [d, J= 9 Hz, 1H, Indonyl],

6.93 [d, J= 2.5Hz, 1H indonyl] 7.44-7.46 [dd, J= 2 Hz, 7 Hz, 2H, benzoyl], 7.62-7.64 [dd,

13 J= 2 Hz, 7 Hz, 2H, benzoyl] C NMR (CDCl3): δ 13.32, 20.30, 30.25, 49.07, 55.74,

87.40, 101.33, 111.59, 111.67, 114.92, 129.12, 130.37, 130.78, 131.17, 133.75, 136.15,

139.32, 156.00, 168.25, 169.26. Mass spec (HRMS) calculated M-H+ 489.1535, found

489.153

O2-(methyl2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetate)-1-

(N,N-Diethylamino)-diazen-1-ium-1,2-diolate (12). Indomethacin (12.4 g, 34.6 mmol)

137

was dissolved in DMSO (150 mL). Triethylamine (4.84 mL, 34.6 mmol) was then added and the reaction mixture was stirred for 30 min at room temperature. Then a solution of 6

(6.26 g, 34.6 mmol) in DMSO (50 ml) was added drop wise to the reaction mixture, which was stirred for 24 h and after completion of the reaction; it was quenched with ethyl acetate (150 mL). The organic layer was washed with saturated NaHCO3 (5 × 100 mL), dried over sodium sulfate and then evaporated to obtain the crude product. Further purification was performed by gradient column chromatography (30% ethyl acetate-

1 hexane) to obtain 6.67 g (38.2%) of pure product. H NMR (CDCl3): δ 1.04 [t, J=7 Hz,

6H, (CH3)2], 2.35 [s, 3H, ArCH3], 3.16 [q, J= 7Hz, 4H (CH2)2] 3.70 [s, 2H, ArCH2], 3.83

[s, 3H, OCH3], 5.83 [s,2H, OCH2O], 6.67[dd, J=9Hz, 2.5 Hz, 1H, Indonyl], 6.86 [d, J= 9

Hz, 1H, Indonyl], 6.93[d, J= 2.5 Hz, 1H Indonyl] 7.46-7.48 [dd, J= 2 Hz, 7 Hz, benzoyl],

13 7.64-7.66 [dd, J= 2H, 7Hz, benzoyl] C NMR (CDCl3): δ 11.29, 13.32, 30.19, 47.85,

55.70, 87.75, 101.12, 111.60, 111.85, 114.93, 129.13, 130.40, 130.79, 131.16, 133.85,

136.10, 139.31, 156.14, 168.23, 169.16. HRMS calculated (MH+ peak) 503.1692, found

503.1690.

Reaction of NO and HNO with oxymyoglobin

The spectrophotometer was blanked with assay buffer (2 or 3 mL) ± 40 or 60 µL

- of guinea pig serum at pH 7.4 and 37°C. Oxymyoglobin (MbO2; 10 µM) (ε415 = 131 mM

1 -1 45,46 -1 - cm ) was exposed to NONO-NSAIDs (10µM) to form metMb (ε408 = 188 mM cm

1). Formation of HNO was further verified by inclusion of GSH, which quenches HNO47

138

without interacting directly with low concentrations of NO48. Spectra were collected at

30-60 s intervals for 1500-2200 s.

Electrochemical detection of NO and HNO

The NO electrode was stabilized in assay buffer of the desired pH (adjusted prior

to use by adding 1 M NaOH or HCl as necessary) containing 50 µM DTPA and 2%

serum at room temperature. NONO-NSAID were added to a final concentration of 100

µM (total DMSO volume ≤ 0.1%), and the maximum NO signal was recorded. After the

signal returned to baseline, the process was repeated to obtain triplicate measurements.

Addition of 1 mM sodium ferricyanide, which oxidizes HNO to NO, allows for indirect

measurement of HNO with this system.49

Cell culture

Human breast cancer cells (MCF-7 and MB-231; American Type Culture

Collection, Manassas, VA) were grown as monolayers in RPMI 1640 media

supplemented with 10% FBS, penicillin and streptomycin (50 U/mL; Life Technologies,

Inc., Grand Island, NY) at 37°C in 5% CO2 and 80% relative humidity. Single cell

suspensions were obtained by trypsinization (0.05% trypsin/EDTA), and cells were

counted with a Bright line Hemacytometer.

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Intracellular release of NO and HNO

MB-231 cells were plated at 60,000 cells per well in a 96-well plate and grown for 4 h. A stock (100×) of 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate50

(DAF-FM-2DA, Molecular Probes) in DMSO was diluted to a final concentration of 10

µM in PBS. The media was aspirated from each well, and the plate was washed once with PBS. The cells were then exposed to 100 µL of 10 µM DAF-FM-2DA for 75 min at

37°C and washed three times with PBS to remove excess dye. NONO-NSAID derivatives dissolved in DMSO (1000×) and ionic NONOates dissolved in 10 mM NaOH (1000×) were then added to achieve a final concentration of 10 µM. The increase in fluorescence intensity at 535 nm as a function of time was then measured following excitation at 485.

MTT assay

Cells were plated at 8,000-10,000 cells per well in a 96-well plate and grown overnight as above. The cells were then treated with different concentrations (0-100 µM) of the prodrugs for 48 h. After addition of 10 μL of a solution of 2 mg/mL of 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to each well, the plate was incubated for 2 h at 37°C. The media was then removed, 100 µL DMSO was added to each well, and the absorbance was recorded at 550 nm using a BioTek Synergy 2 microplate reader.

140

5.3 Results and Discussion

NO is a critical mediator of mucosal defense51 much like prostaglandins in the

gastrointestinal tract.2 These properties of NO led to development of NO-NSAIDs, which

have an improved safety profiles compared to traditional NSAIDs.37,40 Our recent work

with an HNO releasing aspirin prodrug has shown such derivatives to have significantly reduced ulcerogenecity and better cardioprotetctive profile with enhanced cytotoxicity against cancer cells than NO-aspirin.41

As part of our ongoing research targeted toward establishing NO/HNO donors as

drugs with multiple applications, here, we report new NO or HNO releasing

diazeniumdiolate based analogues of the NSAIDs indomethacin and niflumic acid in an effort to compare the effect of variable degree of COX-1 and COX-2 inhibition to better understand their mechanism of action and to investigate their effectiveness in cancer treatment.

O2-Derivatized diazeniumdiolates have been previously shown to be stabilized to

hydrolysis at physiological pH52,53 compared to the parent diazeniumdiolates.

Derivatization not only facilitates purification by column chromatography but also

provides a platform for selective delivery of NO/HNO payloads.54 O2-Protection also

leads to a shift in the diazeniumdiolate absorption maximum from 250 to ~240 nm,55,41,53 and this peak has been previously used to determine half-life of spontaneous or esterase induced hydrolysis. For the indomethacin and niflumic analogues, this peak was masked by the aromatic functionalities of the NSAIDs.

141

5.3.1 Characterizing NO/HNO release.

The prodrugs undergo slow hydrolysis at physiological pH and temperature. Slow

NO or HNO release leads to low local concentrations, thus making detection difficult.

Thus, the prodrugs were subjected to enzymatic hydrolysis using guinea pig serum to assess the release of NO or HNO. Both NO and HNO can be detected using a spectrophotometric method, with oxymyoglobin as the trapping agent.56,57,47 NO can also

be detected via electrochemical methods using an NO specific electrode.49 Indirect detection of HNO can similarly occur in presence of an oxidant such as ferricyanide.

These multistep protocols have been used previously58 and were used similarly for the

newly prepared drugs.

Although both NO (4 × 107 M-1s-1)57,56 and HNO (1 × 107 M-1s-1)47 react with

6 - MbO2 to produce metMb, addition of GSH (1 mM), which reacts with HNO (2  10 M

1s-1)47 but not appreciably with NO,48 provides specificity to the assay. Both IPA/NO and

DEA/NO derivatized NSAIDs converted MbO2 to metMb (Figure 5.2A-D). For IPA/NO-

NSAIDs complete quenching by GSH (Figure 5.2C-D) was not observed in the presence

of serum, indicating that NO production may be significant while DEA/NO-NSAIDs

(Figure 5.2A-B) were not affected by the presence of GSH.

142

A 1.6 1.4 1.2 1 0.8 0.6 Absorbance Absorbance 0.4 0.2 0 300 350 400 450 500 Wavelength (nm)

B 1.6

1.2

0.8 Absorbance 0.4

0 300 350 400 450 500 Wavelength (nm)

143

C 1.6 1.4 1.2 1 0.8 0.6 Absorbance 0.4 0.2 0 300 350 400 450 500 Wavelength (nm)

D 1.6

1.2

0.8 Absorbance 0.4

0 300 350 400 450 500 Wavelength (nm)

Figure 5.2 Representative spectral changes (n ≥ 3) indicating trapping of NO and HNO

by MbO2 (10 μM) during guinea pig serum induced decomposition at 37°C of DEA/NO-

NSAIDs or IPA/NO-NSAIDs in assay buffer ± GSH (1 mM): (A) 10 µM DEA/NO-

144

indomethacin, (B) 10 µM DEA/NO-niflumic acid, (C) 10 µM IPA/NO-indomethacin and

(D) 10 µM IPA/NO-niflumic acid. Initial scans are in red (MbO2), final scans without

GSH are in blue and final scans with GSH are in green.

In the presence of an oxidant such as ferricyanide, detection methods for NO can

be used to indirectly and indicate formation of HNO qualitatively.49 The slow rate of spontaneous hydrolysis from these derivatized diazeniumdiolates impedes such analysis.

The maximum current intensities observed with an NO-specific electrode during decomposition of the prodrugs in the presence of guinea pig serum at physiological pH is shown in Figure 5.3A-B. Even in the presence of serum, the maximum current signal for niflumic acid analogues was significantly lower, suggesting a relatively longer half-life of hydrolysis. For the IPA/NO analogue, regardless of the identity of the NSAID attached, the signal increased 2-4 folds in the presence of ferricyanide, implying significant production of HNO at physiological pH. For the DEA/NO analogues, the assay was not performed in the presence of ferricyanide, as it has been previously shown not to affect

NO signals from the parent diazeniumdiolate DEA/NO.59 Electrode data suggest niflumic

acid analogues might find use for conditions that require sustained, low levels of NO.

145

A 2500

2000

1500

1000 Current (pA) Current

500

0 without ferricyanide with ferricyanide

B 6000

5000

4000

3000

Current (pA) Current 2000

1000

0

Figure 5.3 NO and HNO release measured using an NO-specific electrode from 100 µM of (A) IPA/NO-NSAIDs analogues or (B) DEA/NO-NSAIDs analogues in assay buffer

(pH 7.4) containing 2% guinea pig serum (± 1 mM ferricyanide in A). Niflumic derivative red color, indomethacin blue color and aspirin derivative green colored. The data are expressed as mean ± SD (n ≥ 3).

146

5.3.2 Intracellular release

To confirm intracellular release of NO/HNO and cell permeability of these new compounds, the cell membrane permeable dye DAF-FM diacetate was used, which has been extensively employed in the literature to detect intracellular NO50 and recently for

HNO.60 Although typically non-specific toward HNO and NO, a higher relative signal is

produced by HNO compared to NO.60 Cells were pre-loaded with dye DAF-FM,

followed by treatment with DMSO solutions of prodrugs 7, 8, 11 and 12 or the parent

NSAID. As shown in Figure 5.4, the fluorescence intensity was considerably higher in

cells treated with the prodrugs (except DEA/NO-niflumic acid) as compared to vehicle

treated control cells or the parent NSAID. The higher signal intensities for the prodrugs

thus indicate significant cellular uptake, allowing HNO and NO to be produced in close

proximity to the reporter molecule. Higher signal for HNO releasing derivatives was as

per previously reported results.41 Aspirin analogues were not included as they have already been shown to release NO/HNO intracellularly.41

2x104

1.5x104 IPA/NO-Niflumic acid DEA/NO-Niflumic acid IPA/NO-indomethacin 4 RFU 1x10 DEA/NO-indomethacin niflumic acid indomethacin 3 5x10 DMSO

0 024681012 Time (h) Figure 5.4 NO and HNO release measured in MB-231 cells. The cells were exposed to

147

100 µL of 10 µM DAF-2DA in PBS pH 7.4 for 75 min at 37°C and washed three times with PBS pH 7.4 to remove excess dye. Upon addition of 10 µM of DEA/NO- indomethacin, DEA/NO-niflumic acid, IPA/NO-indomethacin, IPA/NO-niflumic acid, indomethacin or niflumic acid in DMSO (<0.1%), (n = 2, six replicates per plate), the increase in fluorescence intensity at 535 nm was measured at 37°C following excitation at 485 nm. The data are expressed as mean ± SD.

5.3.3 Cytotoxicity

NSAIDs have been shown to improve the efficacy of chemotherapy for certain tumors and even to reduce the risk of developing a variety of cancers.61,12 The role of NO in

cancer has been investigated extensively,62,63,64 while HNO donors have very recently been shown to inhibit breast65 and neuroblastoma66 cancer proliferation both in vitro and

in vivo. The cytotoxicity of both aspirin and ionic diazeniumdiolates in various cancer

67,58 cells lines is low (IC50 > 1 mM). We have recently shown the cytotoxicity of our NO and HNO releasing aspirin to be significantly higher in lung cancer cell line (A549).41

The effect of our newly synthesized prodrugs NONO-NSAIDs along with NONO-aspirin on proliferation was studied (Figure 5.5) in two breast cancer cell line, MB-231 (estrogen receptor- negative, representing aggressive breast cancer) and MCF-7 (estrogen receptor positive cell line, more treatable breast cancer). The DEA/NO and IPA/NO-analogues of aspirin and indomethacin had IC50 value of ~100 µM in both MB-231 and MCF-7 cells.

The niflumic acid analogue, were comparatively less cytotoxic, presumably due to slower release of NO/HNO (see Figure 5.3).

148

120 A DEA/NO-aspirin 100 DEA/NO-indomethacin DEA/NO-niflumic

80

60

% Survival 40

20

0 0 10 25 50 100 Concentration (M)

120 B IPA/NO-aspirin IPA/NO-indomethacin 100 IPA/NO-niflumic

80

60

% Survival 40

20

0 0 102550100 Concentration (M)

149

120 C DEA/NO-aspirin 100 DEA/NO-indomethacin DEA/NO-niflumic acid 80

60

% Survival 40

20

0 0 102550100 Concentraion (M)

120 D IPA/NO-aspirin IPA/NO-indomethacin 100 IPA/NO-nifluimic acid 80

60

% Survival 40

20

0 0 102550100 Concentration (M)

Figure 5.5 The effect of NONO-NSAIDs prodrugs and appropriate controls (25-100 µM, respectively) on cell survival of MB-231 cells with (A) DEA/NO-NSAIDs, (B) IPA/NO-

NSAIDS and in MCF-7 cells with (C) DEA/NO-NSAIDs and (D) IPA/NO-NSAIDs.

150

Cells were treated for 48 h at 37°C and then analyzed by the specrophotometric MTT assay (n = 2 for MB-231 and n = 2 for MCF-7 for in at least triplicate per plate).

5.4 Conclusions

Four new diazeniumdiolates based NO/HNO releasing NSAIDs were synthesized and NO/HNO release was examined by electrochemical and spectrophotometric methods.

The IPA/NO-NSAID adducts were readily taken up by the cells while only DEA/NO- indomethacin showed significant cellular levels of NO. Of particular interest is the observation of NO/HNO releasing analogues of aspirin and indomethacin to be cytotoxic towards breast cancer cells, irrespective of estrogen dependence, suggesting a potential for use in cancer treatment or prevention. Niflumic acid analogues produces sustained low level of NO/HNO and were comparatively less cytotoxic.

Abbreviations

Angeli's salt, sodium trioxodinitrate; NSAID, non-steroidal anti-inflammatory drugs; HNO, nitroxyl; NO, nitric oxide; IPA/NO, sodium 1-(N-isopropylamino)diazen-1- ium-1,2-diolate; DEA/NO, sodium 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate

NONOate; NONO-NSAIDs, adducts of NSAIDs with diazeniumdiolates; metMb, ferric myoglobin; MbNO, nitrosyl myoglobin; MbO2, oxymyoglobin; DTPA,

diethylenetriaminepentaacetic acid; PBS, phosphate buffered saline; MDA-MB-231: estrogen receptor alpha negative breast cancer cell line; RPMI, Roswell Park Memorial

Institute; DAF-FM-2DA, 4-amino-5-methylamino- 2’,7’-difluorofluorescein diacetate;

151

IPA/NO-AcOM, O2-(acetoxymethyl) 1-(isopropylamino)diazen-1-ium-1,2-diolate;

IPA/NO-indomethacin, O2-(methyl-2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-

indol-3-yl)acetate)-1-(N-isopropylamino)-diazen-1-ium-1,2-diolate; DEA/NO-

indomethacin, O2-(methyl-2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-

yl)acetate)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate; IPA/NO-niflumic acid, O2-(2-

(3-(trifluoromethyl)phenylamino)nicotinate)-1-(N-isopropylamino)-diazen-1-ium-1,2- diolate; DEA/NO-niflumic acid, O2-(2-(3-(trifluoromethyl)phenylamino)nicotinate)-1-

(N,N-diethylamino)diazen-1-ium-1,2-diolate

152

CHAPTER 6: FUTURE DIRECTIONS

The discovery of NO as a signaling molecule and its involvement in various physiological and pathological processes has led to increasing interest towards

1-9 biologically active small molecules like H2S and HNO. Even though HNO has been speculated to be formed under physiological conditions, this has yet to be determined.5,10,11 However, HNO shows varied biological properties that have generated much interest in this small molecule.8,12,13

In Chapter 2, N-desmethyltamoxifen has been derivatized with IPA/NO and

DEA/NO. However, the presence of the carbamate linkage leads to stability towards decomposition and hence diminished release of NO/HNO. In spite of diminished release of NO/HNO, adducts were found to be cytotoxic towards MCF-7 cells. The present study also clearly demonstrates that the NO donor DEA/NO-AcOM by itself was cytotoxic towards breast cancer cells and cytotoxicity was independent of estrogen dependence. In combination, DEA/NO-AcOM and tamoxifen together caused significantly higher cell killing in MB-231 cells compared with tamoxifen or DEA/NO-AcOM treatment alone.

In the future we will evaluate protein levels of various markers of cell death such as caspase, BCl2, TNF family and cleaved PARP using Western blots to elucidate the mechanism of cell death caused by carbamate adducts in MCF-7 cells and for the combination treatment.14-16 Many of the physiological properties of NO are cGMP- dependent. Thus, the effect of the adducts on cGMP levels will also be measured.17 We further plan to analyze inhibition of the glycolytic enzyme GAPDH in MCF-7 cells as 153

HNO-generating IPA/NO-N-desmethyltamoxifen can target GAPDH.18 It is a crucial enzyme for cancer cells as cancer cells undergo glycolysis at a substantially accelerated rate compared to normal cells.19 4-Hydroxytamoxifen is a potent but short-lived metabolite and its derivatization with NO and HNO releasing moieties will also be explored (Scheme 6.1). This may not only lead to release of NO/HNO and 4- hydroxytamoxifen, both of which are potent agents but derivatization may increase the stability of 4-hydroxytamoxifen.

Scheme 6.1. Proposed synthetic route for NO releasing 4-hydroxytamoxifen conjugate

One of the major drawbacks in the investigation of the chemical biology of HNO is the limited number of donor compounds compared to NO.20-24 Thus there is a need to synthesize new donors and hybrid molecules that are able to release HNO in a controlled manner. Compared to secondary amine based diazeniumdiolates, only two primary amine based diazeniumdiolates are reported in the literature. To extend the list, Chapter 3 desribes three more new primary amine based diazeniumdiolates were synthesized based on cyclic amines. One of the derivatized diazeniumdioate CPA/NO-AcOM was effective in intracellular release of HNO and also enhanced cytotoxicity. To further explore the utility of derivatized CPA/NO, its cytotoxicity will be evaluated in other cancer cell lines 154

and in the in vivo model. Moreover, enzyme specific derivatization of CPA/NO will be attempted such as by enzyme -D-glucosidase to target the parasite Leishmania.25 To further diversify this group of HNO donors, synthesis of HNO-releasing diazeniumdiolates based on primary amines with alcohol side chains (Scheme 6.1) will be explored as they can be derivatized with antibodies to achieve site-specific delivery.

Scheme 6.2 Proposed synthetic route to an antibody coupled derivatized diazeniumdiolates

These ionic and derivatized compounds can be useful in understanding the molecular biology of HNO and evaluation for their role in treatment of alcoholism,6 cancer18 and as a preconditioning agent.26

We have demonstrated that HNO releasing drug conjugates can be useful to overcome various side effects associated with the parent drug.27 However, for a drug success, there are other aspects that need attention. First of all, there is a need to study the distribution of these drugs in different organs followed by study of metabolism and effects of metabolites. In spite of various biological utilities of HNO, there may be 155

associated side effects such as neurotoxicity.28 In a study done with the HNO donor

Angeli’s salt, a time and concentration-dependent increase in neural cell death was observed on exposure to Angeli’s salt.28 Thus site-specifc delivery and molecular imaging of HNO release (Figure 6.1) can be important for better understanding localization of HNO.

Figure 6.1. Fluorescent tag attached derivatized primary amine based diazeniumdiolate

In Chapter 4, the NO releasing derivative of chrorambucil was synthesized based on the known NO donor PABA/NO.29 Chlorambucil is a alkylating agent,30 which is mostly used for treatment of lymphocytic leukemia.31 As it is also known to be effective against breast cancer,32-34 the NO releasing chloambucil and its parent drug were tested on MB-231 cell lines and were found to inhibit the cell survival in dose dependent manner Since the trend in percent survival was quite similar for both of the compounds, cytotoxicity experiments will be conducted with concentration varying from 0-50 µM to see if there is a difference in cytotoxicity between the two compounds. Intracellular NO release assessed by DAF reporter molecule showed significant release from the NO releasing chlorambucil adduct, compared to the parent drug and control. In the future, the

MTT assay will be performed in other cell lines as well as in leukemia cells as these cell shows sensitivity to both NO and chlorambucil.35-37 Such NO releasing PABA/NO based 156

analogues will be explored for other alkylating agents such as melphalan and cyclophosphamide (Figure 6.2) as they are also the substrate of glutathione-S- transferase.38,39 Also HNO releasing chlorambucil synthesis will also be explored by substituting NO donor DMA/NO with primary amine based diazeniumdiolates.

Figure 6.2 Structure of alkylating agent melphelan and cyclophosphamide

In Chapter 5 we have shown the potential of NO/HNO releasing NSAIDs by derivatizing the NO/HNO-releasing diazeniumdiolate with NSAIDs such as aspirin, indomethacin and niflumic acid. Qualitative analysis suggests niflumic acid analogues to undergo slower hydrolysis as compared to aspirin and indomethacin analogues. Aspirin and indomethacin analogues were found to be cytotoxic in breast cancer cells, independent of estrogen dependence. The niflumic acid analogues were found to be comparatively less cytotoxic. The IPA/NO-indomethacin and niflumic adducts were readily taken up by the cells while only DEA/NO-indomethacin showed significant cellular levels of NO. In the future, aspirin and indomethacin analogues will be further evaluated in MCF-10A cells, which are non-tumorogenic immortalized breast epithelial cells. The utility of ERK, HIF-1α, AKT and p-53 has been well established as dosimeter 157

for variable response on NO in cells.40 We will examine the signaling of these compounds on the above mentioned pathways using western blot technique. Reactive oxygen species and reactive nitrogen species (denoted as RNOS) are critical mediators of programmed cell death.41 Elevation in RNOS levels potentially damage biomolecules and can cause activation of specific signaling pathways.42,43 Elevation in RNOS levels by the synthesized compounds within cells will be analyzed using fluorescence spectroscopy by loading with the cell permeable dyes 2′,7′-dichlorofluorescin diacetate (DCFH-DA).44

Aspirin and indomethacin analogues efficacy will be analyzed and compared in the in vivo model. Toxicity of the adducts will be determined in nude mice in order to determine the dosage for treatment of tumor growth and metastasis studies. Efficacy will be examined in luciferase or GFP transfected MCF-7 or MB231 models for reduction of tumor growth and metastasis in mice. Other than breast cancer cells, cytotoxicity from these compounds will be screened in other cancer cell lines such as colon, lung and leukemia cancer cells. Niflumic acid analogues on the other hand will be examined for their cardio-protective potential. NO/HNO releasing analogues of other NSAIDs such as ibuprofen, naproxen and diclofenac (Figure 6.3) will be synthesized to expand the list of diazeniumdiolates based NSAIDs adducts.

Figure 6.3 NSAIDs ibuprofen, naproxen and diclofenac 158

It will be interesting to see if similar adducts can be synthesized from selective inhibitor of cyclooxygenase-II such as celecoxib and rofecoxib (Figure 6.4). These were the second generation NSAIDs which lacked ulceration side effect but suffered setback due to increased risk of cardiovascular events.45 Once synthesized these NO/HNO releasing adducts of second generation NSAIDs will be analyzed for their cardiovascular risks.

Figure 6.4 Second generation NSAIDs

159

APPENDIX A: NMR DATA

Compound Structure 1H/Page 13C/Page number number - O- DEA/NO O Na+ 163 164 N N N

IPA/NO 165 166

DEA/NO-SCH3 167 168

IPA/NO-SCH3 169 170

DEA/NO-Cl 171 172

IPA/NO-Cl 173 174

DEA/NO- 175 176 Indomethacin 160

DEA/NO-Niflumic 177 178 acid

DEA/NO-aspirin 179 180

IPA/NO- 181 182 Indomethacin

IPA/NO-Niflumic 183 184 acid

IPA/NO-aspirin 185 186

COA/NO 187 188

CHPA/NO 189 190 161

CHA/NO 191 192

CPA/NO 193 194

CPA/NO-AcOM 195 196

N- 197 198 desmethyltamoxifen- carbamate

IPA/NO-N-des-tam 199 200

DEA/NO-N-des-tam 201 202

DMA-DNB-Fluoro 203 204

DMA-DNB-OH 205 206 162

Cl O DMA-DNB- O- 207 208 Chlorambucil O O N+ N N N Cl O2N NO2

163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209

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Chapter 2

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Chapter 4

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Chapter 5

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Chapter 6

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