Chemical investigations in drug discovery and drug delivery

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Content i Chemical Investigations in Drug Discovery and Drug Delivery Caitlin M. DeAngelo A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (CHEMISTRY) December 2018 Copyright 2018 Caitlin M. DeAngelo ii Dedication To Mom and Dad. iii Acknowledgements I would like to thank Professor Nicos Petasis for his confidence in me, constant encouragement, and unwavering support of my professional and personal goals. I will carry on your problem-solving strategies and excitement for new discoveries in the rest of my life and career. Additionally, I would like to thank the members of my committee: Professors G.K. Surya Prakash, Valery Fokin, Chao Zhang, Yong (Tiger) Zhang, and Stan Louie. Also, other chemistry faculty who have been particularly helpful in research and teaching include Professors Travis Williams, Stephen Bradforth, Smaranda Marinescu, Barry Thompson, Jennifer Moore, and especially Rebecca Broyer who has been a wonderful mentor. A very big thank you goes out to all Petasis lab members, past and present: Dr. Marcos A. Sainz for training me to be a thorough researcher and being a great friend; Caroline Black for being my teammate; Varun Jain and Yuki Kuromaru for being hardworking undergraduate researchers; as well as Dr. Kalyan Nagulapalli Venkata, Dr. Stephen J. Glynn, Dr. Kevin Gaffney, Dr. Rong Yang, Dr. Nikita Vlassenko, Robert Nshimiyimana, Ting Fung Lam, Kevin Kossick, Blake Houser, and Katelyn Fallows. I would like to also thank the LHI and USC Chemistry staff: Michele Dea, Magnolia Benitez, Dr. Robert Aniszfeld, Jessie May, and especially David Hunter for all the sweets and smiles and Carole Phillips for putting up with the lab’s shenanigans. Thank you to my parents for always believing in me, my sister Michele, my brother Michael, and the rest of my family and friends including Dr. Courtney A. Downes, Dr. Betsy L. Melenbrink, Amanda Baxter, Shannon Moore, Lacey Axemaker-O’Keefe, Megan, Anna, Isabella, Adrianna, Ellamena, and Layla. Finally, I would not be the chemist or person I am today without Ian Harvey’s love and encouragement. Thank you. iv Table of Contents Dedication ……………………………………………………………………………………………………………………………… ii Acknowledgements ………………………………………………………………………………………..……………………… iii Table of Contents …………………………………………………………………………………………………………………… iv List of Tables …………………………………………………………………………………………………..……………………… vii List of Figures …………………………………………………………………………………………………..………….………… viii List of Schemes …………………………………………………………………………………………………….………………… xi Abstract ………………………………………………………………………………………………………………………………… xiv Chapter 1. The N-Formyl Functional Group in Disease and Drug Discovery ……………………………. 1 1.1. Introduction ………………………………………………………………………………………………………… 1 1.2. FDA Approved Drugs Containing N-Formyl Functional Group ……………………….……… 3 1.2.1. Natural Products ……………………………………………………………………….…………… 3 1.2.2. Derivatives of Natural Products ……………………………………..……………….……… 5 1.2.3. Synthetic …………………………………………………………………………..…….……….…… 15 1.3. Investigational Non-FDA Approved Drug Candidates …………………..…….…………..…… 17 1.4. N-Formyl Peptide Receptors ………………………………………………………..…….……….….…… 22 1.5. Conclusions …………………………………………………………………..……………..…….………….…… 28 1.6. References …………………………………………………………………..……………..…….………..….…… 29 Chapter 2. Direct N-Formylation with N,N’-Dimethylformamide Facilitated by Water and Oxygen ……………………………………………………………………………………………. 37 2.1. Introduction …………………………………………………………………..……………..…….……..….…… 37 2.1.1. A Summary of Previously Reported N-Formylation Methods ………….…..… 37 v 2.1.2. N-Formylation Methods Using DMF ……..…….………………………………..……… 37 2.2. The Role of Water and Oxygen & Proposed Mechanism ……..…….…………..….…..…… 38 2.3. Scope and Limitations …………………………………………………………………..……….……….…… 41 2.3.1. N-Formylation in Air and O 2 ………………………………………………..…………….…… 41 2.3.2. N-Formylation in DMF with Catalytic Formic Acid ……..…….…………………… 43 2.4. Conclusions …………………………………………………………………..……………..……..………...…… 43 2.5. Experimental …………………………………………………………………..……………..……..……….…… 45 2.6. References …………………………………………………………………..……………..…….……….…….… 53 Chapter 3. Synthetic Methods for the Functionalization of Cyanine Dyes ……………………..………. 59 3.1. Introduction …………………………………………………………………..……………..……..…..………… 59 3.1.1. Heptamethine Cyanine Dyes ……………………………………………….…………..…… 59 3.1.2. Applications of heptamethine cyanine dyes in drug discovery and drug delivery …………………………………………………………..……………..…….………..…….… 64 3.2. Cyanine dye synthesis and relationship between structure and absorbance ….…... 68 3.3. BP114: A Proof of Concept Compound ……………………………………………………….….…… 70 3.4. The Role of the Linker ………………………………………………..……………..…….…………..……… 74 3.5. Functionalization with Carboxylate Salts ………………………………………………….…....…… 76 3.6. Conclusions …………………………………………………………………..……………..…….…...…….…… 80 3.7. Experimental …………………………………………………………………..……………..……..……….…… 81 3.8. References …………………………………………………………………..……………..…….……..….…… 101 Chapter 4. Light-Based Drug Delivery Applications for Traumatic Brain Injury ……………….……. 106 4.1. Introduction …………………………………………………………………..……………..…….……….…… 106 vi 4.1.1. Traumatic Brain Injury ………………………………………………..………..……….…… 106 4.1.2. Cyclosporin-A in TBI ………………………………………………………..…….……….…… 107 4.1.3. Gabapentin in TBI ………………………………………………………..…….………….…… 108 4.1.4 Near-IR Light and TBI ………………………………………………………..…….……….…… 109 4.2. Functionalization of a Cyanine Dye with GABA and Gabapentin ……..…….……….…… 109 4.3. Functionalization of a Cyanine Dye with Cyclosporin-A ……..…….………………..……… 117 4.4. Conclusions …………………………………………………………………..……………..…….………..…… 120 4.5. Experimental …………………………………………………………………..…………..…….………..…… 121 4.6. References …………………………………………………………………..……………..…….……………… 139 Chapter 5. Synthesis and Evaluation of Lead Compounds for the Inhibition of dUTPase ……… 141 5.1. Introduction …………………………………………………………………..……………..…….……….…… 141 5.2. Efficacy of designed dUTPase inhibitors ………………………………………………….…….…… 144 5.3. Scale-Up of Lead Compounds ………………………………………………………..…….……….…… 148 5.4. Thymine and 5-Fluorouracil Derivatives of Lead Compounds ……..…….………..…..… 151 5.5. Conclusions …………………………………………………………………..……………..…….…..………… 154 5.6. Experimental …………………………………………………………………..……………..……..….….…… 155 5.7. References …………………………………………………………………..……………..…….……….…..… 188 Bibliography …………………………………………………………………..……………..………………………………..…… 194 Appendix: Selected Spectra ………………………………………………………………..……………..…….…….…… 218 vii List of Tables Table 2.1 Conditions for N-formylation of benzylamine. 39 Table 5.1 EC50 values for cytotoxicity of designed dUTPase inhibitors against HCT116 colorectal carcinoma cell line. 146 Table 5.2 EC50 values for cytotoxicity of designed dUTPase inhibitors against HCT116 colorectal carcinoma cell line. 147 viii List of Figures Figure 1.1 FDA Approved Drugs Containing N-Formyl Functional Group. (A) Natural products. (B) Derivatives of natural products. (C) Synthetic 2 Figure 1.2 Vincristine and vinblastine 3 Figure 1.3 Gramicidin D is a mixture of gramicidins A, B, and C 5 Figure 1.4 (A) Leucovorin and the natural product it is derived from, Folate. (B) First anticancer drug, Aminopterin, and the natural product, Pterin. 6 Figure 1.5 Enantiopure levoleucovorin 7 Figure 1.6 Thiamine, also known as vitamin B1, and derivative benfotiamine 8 Figure 1.7 FDA-approved drug, Fursultiamine, and natural products allithiamine and prosultiamine. 11 Figure 1.8 Orlistat and the natural product it is derived from, Lipstatin. 13 Figure 1.9 Formoterol (racemic mixture) and enantiopure Arformoterol. 16 Figure 1.10 Macimorelin diagnostic compound. 16 Figure 1.11 Fosmidomycin antimalarial in Phase III. 18 Figure 1.12 FOXY-5 anticancer in Phase I. 19 Figure 1.13 Lanopepden antibacterial in Phase II. 20 Figure 1.14 Ioforminol contrast agent in Phase II. 21 Figure 1.15 N-formyl-methionyl-leucyl-phenylalanine (fMLF) 22 Figure 1.16 Formyl peptide receptors (FPRs) in disease. 23 Figure 1.17 (Left) Cyclosporins A and H; (Right) Plant phenol PP-6. 25 Figure 3.1 Indocyanine green (ICG) 59 ix Figure 3.2 Representation of the variety of cyanine dyes synthesized for covalent modification of biomolecules 60 Figure 3.3 Representation of the variety of cyanine dyes synthesized for non- covalent modification of biomolecules 61 Figure 3.4 (A) pH sensitive cyanine dyes. (B) & (C) Dimeric cyanine dyes with alkyl, aryl, and PEG type linkers 62 Figure 3.5 (A) Cyanine dye with a crown ether for detecting metal cations. (B) Cyanine dye with a Cu 2+ detecting lactam. (C) Cyanine dyes with hydroxy and carboxy groups for metal binding 63 Figure 3.6 (A) Cyanine dyes modified with polyamines for nucleus targeting and triphenylphosphine for mitochondria targeting. (B) A cyanine dye that has a larger Stokes shift and lysosomal targeting 64 Figure 3.7 (A) Cyanine dye that is encased in a nanocapsule for light therapy applications. (B) A cyanine dye that displays antitumor properties. 65 Figure 3.8 Drug-polymer-dye conjugate. 65 Figure 3.9 (A) Cyanine dye-drug conjugate that releases cargo upon irradiation with NIR light. (B) Cyanine dye-drug-antibody conjugates. 66 Figure 3.10 Cyanine dye structure examples and their maximum absorbances. 68 Figure 3.11 Upon irradiation with NIR light, the cyanine dye breaks down leading to a decrease in absorbance at 735nm and release of the coumarin compound gives an increase in fluorescence specific to that coumarin. 74 x Figure 4.1 Representation of attempted IR-820 coupling reactions with Cyclosporine 118 Figure 5.1 dUTPase catalyzes the reaction to make dUMP, the native substrate for TS. 142 Figure 5.2 dUTPase 143 Figure 5.3 (A) Native substrate, dUTP, broken down to into uracil (U), sugar (S), and phosphate (P) regions. (B) Literature dUTPase inhibitors do not mimic the native substrate. (C) Docked drug-like fragments. (D) Top fragments reconstituted and docked. 144 Figure 5.4 Comparison of cytotoxicity of thymine- and uracil-based inhibitors. 152 xi List of Schemes Scheme 2.1 N-formylation of piperidine moiety. 38 Scheme 2.2 Proposed mechanism for N-formylation 40 Scheme 2.3 Scope a of N-Formylation by DMF in air and O 2-rich b conditions 42 Scheme 2.4 Scope a of N-Formylation in Air with Formic Acid Catalyst at Room Temperature and 120°C 44 Scheme 3.1 Cyanine dye-drug conjugate with peroxide sensing boronic ester drug delivery trigger. 67 Scheme 3.2 Synthesis of IR-820 cyanine dye. 69 Scheme 3.3 Self-immolating drug or reporter release mechanism. 70 Scheme 3.4 Synthesis of BP-114 proof of concept compound from IR-820 cyanine dye. 71 Scheme 3.5a The mechanism of release of cargo begins with excitation of an electron in the cyanine dye conjugated system which upon energy transfer makes the more reactive singlet oxygen. 72 Scheme 3.5b Oxidative cleavage leads to either aldehyde 3.8a or ketone 3.8b which upon hydrolysis liberates the cargo with the amine linker intact. 73 Scheme 3.5c Self immolation of the amine linker leads to release of the now fluorescent coumarin compound. 73 Scheme 3.6 Preparation of a variety of dye-linker compounds. 75 Scheme 3.7 Synthesis of dye-linker compounds activated for carboxylic acid attachment. 76 xii Scheme 3.8 Activated dye-linker compounds react with 4-phenylbutyric acid in the presence of cesium carbonate and sodium iodide in acetonitrile. 77 Scheme 3.9 Carboxylate salt with potassium counterion works well only with amine linker and methyl-chloride. 78 Scheme 3.10 Preparation of dansyl carboxylic acid compound. 79 Scheme 3.11 Preparation of cyanine dye-dansyl conjugate. 79 Scheme 4.1 (A) Cyanine dyes modified with different GABA receptor agonists. (B) Mechanism for the reversible and pH dependent imine formation when amine is secondary and not tertiary. 110 Scheme 4.2 Synthesis of cyanine dye with extended linker and gabapentin. 112 Scheme 4.3 Proposed mechanism of release of extended gabapentin cyanine dye conjugate. 112 Scheme 4.4 Real-time monitoring of drug release using a coumarin linker. 113 Scheme 4.5 Synthesis of modified coumarin compound. 114 Scheme 4.6 Synthesis of real-time monitoring of drug release of Gabapentin cyanine dye conjugate. 115 Scheme 4.7 Synthesis of protected linker-coumarin-Gabapentin cargo. 115 Scheme 4.8 Literature precedence for the rapid deprotection and functionalization of the amine linker. 116 Scheme 4.9 Attempted coupling of linker-coumarin-Gabapentin 4.26 to IR-820 (4.1) 116 Scheme 4.10 Activation of CsA-diol with 4-nitrophenyl chloroformate. 118 xiii Scheme 4.11 Coupling CsA to the cyanine dye. 119 Scheme 5.1 The reactions that are catalyzed by dUTPase and TS. 143 Scheme 5.2 Synthesis of lead compounds with phenyl linker. 148 Scheme 5.3 Synthesis of lead compounds with thiophene linker. 149 Scheme 5.4 Synthesis of positive control dUTPase inhibitor that is in Phase I in Japan. 150 Scheme 5.5 Synthesis of TMS-protected uracil. 151 Scheme 5.6 Final step in synthesis of positive control dUTPase inhibitor. 151 Scheme 5.7 Synthesis of thymine and 5-fluorouracil conjugates of lead inhibitors. 152 Scheme 5.8 Synthesis of TMS-protected thymine and 5-fluorouracil. 153 Scheme 5.9 Synthesis of 5-fluorouracil conjugate of positive control dUTPase inhibitor. 154 Scheme 5.10 Synthesis of thymine conjugate of positive control dUTPase inhibitor. 154 xiv Abstract In Chapter 1, the relationship between the structure and function of therapeutics is explored with a focus on the N-formyl functional group. Drugs that contain N-formyl groups are involved in the treatment of chronic inflammatory diseases, obesity, cancer, and bacterial infections. Also, formyl peptide receptors are a major part of the pro-inflammatory process in the body. Information in this chapter serves as overarching bridge that connects the other chapters. In Chapter 2, a novel mechanism for N-formylation by N,N’-dimethylformamide (DMF) facilitated by water and oxygen is proposed to involve the formation of a catalytic N-oxide species that is formed when amines are heated in the presence of oxygen or an oxidant. Conditions were found to afford the N-formylation of a variety of benzyl, aromatic, and aliphatic amines. In Chapter 3, cyanine dyes are functionalized with a variety of cargo compounds that are released upon irradiation with near-IR light, effectively acting as a drug delivery system. The novel synthetic methods permit for the attachment of amine-, alcohol-, and carboxylate-containing molecules, which allows for easy diversification of this drug-delivery system in the future. In Chapter 4, two lead candidates for the treatment of traumatic brain injury (Gabapentin and Cyclosporin-A) are synthetically coupled to a cyanine dye carrier and are released in a NIR light- controlled fashion. Drug release can be acutely controlled by using either continuous 850nm light or intermittent light. In Chapter 5, the synthesis of lead dUTPase inhibitors for 5-fluorouracil-resistant cancers is discussed. 1 Chapter 1: The N-Formyl Functional Group in Disease and Drug Discovery 1.1 Introduction N-formylation is an important biological process and an interesting and vital synthetic strategy in organic chemistry and drug discovery. A novel mechanism for N-formylation by N,N’- dimethylformamide (DMF) facilitated by water and oxygen is proposed in Chapter 2. N- formylated peptides from bacteria are responsible for activating formyl peptide receptors (FPR) and consequently other pro-inflammatory processes that have been linked to Alzheimer’s disease, cancer, chronic inflammatory diseases, and many others. Cyclosporine, a cyclic peptide that is traditionally used as an immunosuppressant drug, is a potent and selective FPR1 inhibitor and also has demonstrated a protective effect on mitochondrial ultrastructure and function in cases of traumatic brain injury (TBI). A light-based drug delivery system for targeted and controlled delivery of cyclosporine in cases of TBI is discussed in Chapter 4 along with many synthetic methods for diversification to accommodate other drugs discussed in Chapter 3. Finally, a small number of FDA approved drugs exist that contain N-formyl functionality, with many of them being either natural products or derivatives of natural products. One such drug is Leucovorin, also called folinic acid, and it is used to stabilize the binding of 5-fluorouracil, an anticancer drug, to its target, thymidylate synthase. Novel dUTPase inhibitors for 5-fluorouracil- resistant cancers are discussed in Chapter 5. These N-formyl-containing FDA approved drugs have very interesting histories, intriguing mechanisms of action, and sometimes overlapping biological functions that afford a better understanding of why nature uses the N-formyl functional group and how scientists can use this information to design better drugs in the future. 2 H N O N H O H N O R 1 O N H O H N O N H O H N O N H NH O H N O N H R 2 O H N O N H NH O H N O N H NH O H N OH OH OAc MeO N N N H CO 2 Me CHO MeO 2 C N OH Gramicidin D Vincristine H O N N H O N H OH O OH O N H O HN H 2 N N Leucovorin/Levoleucovorin Fursultiamine HN N Me NH N O Me S S OH O Benfotiamine HN N Me NH N O Me S O O P OH O OH O O O O H N O Orlistat H 2 N O N H NH O H N NH H N O Macimorelin O N H OH N H O HO Formoterol/Arformoterol Figure 1.1. FDA Approved Drugs Containing N-Formyl Functional Group. (A) Natural products. (B) Derivatives of natural products. (C) Synthetic A B C 3 1.2 FDA Approved Drugs Containing N-Formyl Functional Group 1.2.1 Natural products Vincristine Vincristine is a naturally occurring vinca alkaloid that was first extracted from the leaves of the periwinkle plant Catharanthus roseus. 1 Vincristine is a large molecule at 923.1 kilodaltons with nine chiral centers, and is an asymmetrical dimer of catharanthine and vindoline, which are also natural products. 2 Because of its complexity, the biosynthesis of vincristine in periwinkle requires more than 30 enzymes and 35 chemical intermediates. 3 Extracts of the periwinkle plant have been used for medicinal applications for several hundred years, but the first successful clinical use of vincristine was published in 1962. Like other vinca alkaloids, vincristine inhibits cell proliferation by binding to tubulin and preventing its polymerization to microtubules. 4-7 This disrupts cell division during metaphase when the microtubules assist in aligning the original and duplicated chromosomes along the center of the cell. The binding site for these vinca alkaloids is at the inter-dimer interface and is Figure 1.2. Vincristine and vinblastine OH OAc MeO N N N H CO 2 Me R MeO 2 C N OH Vincristine R=CHO Vinblastine R=Me 4 unique from other tubulin-binding drugs like taxol or colchicine. When compared to vinblastine, vincristine has a higher overall affinity (K1K2) for tubulin and therefore the lowest maximum dosage. The dose-limiting event for vincristine is the occurrence of peripheral neuropathy in the patient. The first signs of neuropathy include limb weakness and numbness, abdominal pain, and constipation. 8,9 Because there is no antidote for these symptoms, vincristine treatment must be suspended to allow the symptoms to subside. If treatment is not suspended, severe neurotoxic symptoms including seizures, paralytic ileus, hypertension, and severe musculoskeletal pain may occur. Neuronal cell damage was also shown to include motor and memory performance in rats treated with vincristine. 10 This neurotoxicity is unique to vincristine as patients treated with vinblastine do not exhibit neurotoxicity but rather bone marrow toxicity and myelosuppression, symptoms not seen in vincristine treatment. This inconsistency may be attributed to the only functional group difference between vincristine and vinblastine, an N- formyl (N-CHO) versus N-methyl (N-Me) in the lower vindoline unit. Gramicidin D Gramicidin D was the first antibiotic to be successfully used in the clinic in 1939. 11 It is a linear pentadecapeptide antibiotic with alternating D and L amino acids that is a heterogeneous mixture of six unique compounds. 12 Gramicidins A, B, and C typically occur in a ratio of 7:1:2 in Bacillus brevis bacteria where their natural function is not known, but they may play a role in gene regulation during the shift from vegetative growth to sporulation. 11,13 These peptides are known to form ion channels in lipid membranes that are specific to monovalent cations with the 5 selectivity of Li + <Na + <K + <Rb + <Cs + . 11 The channel is essentially impermeable to anions and multivalent cations; divalent cations such as Ca 2+ block the channel. 11 The channel is composed of two single-stranded right  6.3 -helices that are joined by hydrogen bonds between the N-termini. The channel has a conductance on the order of ~10 7 ions per second and increases the permeability of the bacterial cell membrane to these cations. 11 This in turn disrupts the ion balance between the intracellular and the extracellular environments. When isolated from each other, the structure is different than the heterogeneous mixture. For example, gramicidin A when alone crystallizes as a left-handed double-stranded antiparallel  5-6 -helical dimer and cannot function as an ion channel in lipid bilayers. 14 1.2.2 Derivatives of Natural Products Leucovorin & Levoleucovorin Leucovorin, also known as folinic acid, was first made in 1945, and is a racemic mixture of both the levo and dextro isomers. 15 It is a derivative of folic acid, also called folate, that is found Figure 1.3. Gramicidin D is a mixture of gramicidins A, B, and C. H N O N H O H N O R 1 O N H O H N O N H O H N O N H NH O H N O N H R 2 O H N O N H NH O H N O N H NH O H N OH NH OH Trp Gramicidin A Phe Gramicidin B Tyr Gramicidin C Val Ile R 1 = R 2 = 6 in many foods like dark green leafy vegetables, liver, and lentils, and was first isolated from spinach leaves in 1941. 16 Research on folic acid led to the synthesis of antifolate compound aminopterin which was the first ever anticancer drug in 1948. 17 Aminopterin is a competitive inhibitor of the folate binding site of the enzyme dihydrofolate reductase. The inhibition of this enzyme leads to the depletion of nucleotide precursors and the prevention of DNA, RNA, and protein synthesis. The “pterin” part of the word comes from the compound that was first discovered in the pigments of butterfly wings and is now a part of many larger drug compounds. 18 Leucovorin is used to modulate the cytotoxicity of the cancer chemotherapy drug 5- fluorouracil. 15 After absorption, it is metabolized into 5,10-methylenetetrahydrofolate that stabilizes the binding of the active metabolite of 5-fluorouracil, fluorodeoxyuridylic acid, to its target thymidylate synthase, and therefore enhancing the inhibition. 15 This is the standard of Figure 1.4. (A) Leucovorin and the natural product it is derived from, Folate. (B) First anticancer drug, Aminopterin, and the natural product, Pterin. H O N N H O N H OH O OH O N H O HN H 2 N N Leucovorin N N H O N H OH O OH O N O HN H 2 N N Folate, folic acid, vitamin B9 N N H O N H OH O OH O N N H 2 N N NH 2 N N O HN H 2 N N Aminopterin Pterin B A 7 treatment for metastatic colon cancer. Colorectal cancer is the 4 th most frequently diagnosed cancer in the United States and the 2 nd leading cause of cancer deaths. 19 Leucovorin is used in combination with 5-FU as well as with 5-FU and a cocktail of other anticancer drugs like irinotecan and oxaliplatin, the full combination being called FOLFIRINOX. 20 This cocktail is used in advanced cases of pancreatic cancer, one of the most deadly of cancers that has a 5-year survival rate as low as 6% in the United States. 20 It was hypothesized that the active isomer, the levo or l isomer, must compete for transport into cells with the inactive isomer, the dextro or d isomer. 15 Therefore, the enantiomerically pure version of the drug was synthesized and called levoleucovorin. Levoleucovorin can be used in half the doses of leucovorin considering only half the doses of leucovorin are the isomerically active compound, but the enantiomerically pure compound shows no difference in efficacy or side effects and is economically less efficient to produce. 21 Therefore, the standard of treatment has remained the racemic mixture, leucovorin. Fursultiamine & Benfotiamine Figure 1.5. Enantiopure levoleucovorin. H 2 N N O N H N H O N H OH O OH O N O N H Levoleucovorin 8 Thiamine, also called vitamin B1, is found naturally in legumes, whole grains, and some meats and fish. 22 It was first reported in 1911 by Dr. Umetaro Suzuki in Japan, and as isolated from rice bran. 23 Rice bran was being explored because of its effectiveness in treating beriberi, a disease later associated with lack of vitamin B1. 23 In “dry” beriberi, symptoms include partial paralysis, peripheral neuropathy, loss of tendon reflexes, mental confusion, speech difficulties, pain, involuntary eye movements, and vomiting. 24 The patient can also develop Wernicke’s encephalopathy which is a neuro-psychiatric disorder with paralysis of eye movements, abnormal stance and gait, and deranged mental function. “Wet” beriberi displays other symptoms such as heart failure, weakening of capillary walls, shortness of breath, peripheral edema, and dilated cardiomyopathy. 24 Indeed, beriberi is commonly observed in the developed world in individuals with alcohol dependence along with other severe nutritional and vitamin deficiencies. 25 Vitamin B1 deficiency erodes neurological pathways that may influence the ability to drink in moderation, see above the deranged mental function symptom. 25 Additionally, functional deficits in Figure 1.6. Thiamine, also known as vitamin B1, and derivative benfotiamine. Benfotiamine N N NH 2 Me N + S Me OH Thiamine HN N Me NH N O Me S O O P OH O OH 9 activation of thiamine-dependent enzymes have been reported in 35% of alcoholics. 25 Also, neuropathological brain lesions that are characteristic of thiamine deficiency have been reported in 12.5% of autopsied brain samples of alcoholics. 25 Rats exposed to dietary depletion of thiamine or treatment with thiamine antagonists have shown increased total alcohol consumption, which was then reversed upon thiamine rescue. In a double-blind, randomized and placebo-controlled study in 2013, individuals with alcohol dependency were treated with benfotiamine, a synthetic s-acyl derivative of thiamine, and alcohol consumption decreased from baseline levels in 9 out of 10 women. 25 Thiamine-dependent processes are also critical in glucose metabolism, and a reduction of glucose metabolism has been observed in the brain during Alzheimer’s disease. 26-28 It was proposed that cognitive function can be improved, and amyloid deposition reduced via thiamine- dependent mechanisms. However, treatment with thiamine itself exerts little beneficial effect. Benfotiamine, once absorbed, is dephosphorylated by ecto-alkaline phosphatase to lipid-soluble S-benzoyl thiamine. An 8-week treatment with benfotiamine dose-dependently enhanced the special memory of amyloid precursor protein/presenilin-1 mice in the Morris water maze test and also reduced amyloid plaque numbers and phosphorylated Tau levels. Interestingly, these results were not mimicked by other thiamine derivative, fursultiamine, even though they both increased free thiamine levels in the brain. Additionally, benfotiamine but not fursultiamine, elevated phosphorylation level of glycogen synthase kinase-3  and -3  and reduced their enzymatic activities in the mouse model brain. GSK-3 has recently been identified to play an important part of type II diabetes, Alzheimer’s disease, inflammation, cancer, and bipolar 10 disorder. GSK-3 has been shown to promote amyloid-beta production and the hyperphosphorylation of tau proteins. Benfotiamine increases the intracellular levels of thiamine diphosphate which is a cofactor necessary for transketolase leading to reduction of the tissue levels of advanced glycation end products (AGEs). 22 Elevated levels of AGEs have been implicated in diabetes complications because they accumulate rapidly in tissues and when they bind to their receptor (RAGE), it activates monocytes and endothelial cells leading to inflammatory events. AGEs additionally exaggerate oxidative stress in diabetes. The anti-AGE property of benfotiamine could make it a good treatment for diabetic neuropathy, nephropathy, and retinopathy. Indeed, clinical trials in Europe have shown significant improvement in symptoms of diabetic neuropathy with no adverse effects. AGEs are also formed from glucose flux through the polyol pathway, which also leads to accelerated generation of reactive oxygen species and activation of the diacylglycerol-protein kinase C pathway. 28 Reactive oxygen species can partially inhibit glyceraldehyde-phosphate dehydrogenase resulting in accumulation of glycerlahyde-3-phosphate which also promotes AGE formation, protein kinase C, and increased flux through the hexosamine pathway. Thiamine and benfotiamine have been shown to reduce intracellular glucose and increase expression and activity of transketolase which may shift excess glycolytic metabolites into the pentose phosphate cycle and accelerate glycolytic flux. 28 Both drugs are also shown to correct polyol pathway activation that was induced by high glucose in vascular cells. Thiamine could prevent cellular damage by removing excess glyceraldehyde-3-phosphate and facilitating utilization of acetyl-CoA derived from accelerated glycolysis. 11 Fursultiamine is a synthetic disulfide derivative of thiamine and is most closely structurally related to allithiamine, a natural compound found in garlic in Japan in 1951. 29,30 Disulfide derivatives of thiamine function by the disulfide bond fracturing and the prosthetic group remains outside of the cell. 30 This is particularly beneficial because it doesn’t need the rate limiting transport system that thiamine requires, leading to higher blood concentrations than if dosed with a thiamine salt instead. Once in the cell, thiamine is phosphorylated to thiamine pyrophosphate (TPP). 30 TPP is a vital component in the central nervous system; in the brain, it donates a phosphate to form ATP. Thiamine tetrahydrofurfuryl disulfide (TTFD), another name for fursultiamine, was first discovered during experimentation that started with thiamine propyl disulfide, prosultiamine, giving off a strong garlic odor. Prosultiamine was discovered in garlic along with allithiamine (thiamine allyl disulfide, TAD, vitamin B9) in garlic in Japan in the 1950s. Both have improved lipid solubilities and also do not depend on the rate-limiting transporters that thiamine uses. TTFD has been used in Japan for diabetic neuropathy in post-op paralytic ileus. 30 It has also been Figure 1.7. FDA-approved drug, Fursultiamine, and natural products allithiamine and prosultiamine. Fursultiamine N N NH 2 Me Allithiamine N O Me S S OH N N NH 2 Me Prosultiamine N O Me S S OH HN N Me NH N O Me S S OH O 12 shown to help in Alzheimer’s disease, where decreases in the activites of thiamine-dependent enzymes and thiamine diphosphatase observed in the Alzheimer’s brain. Inflammation plays a role in many diseases. Elevated hepcidin, a 25-amino acid peptide that functions as an iron regulatory hormone, has been detected in patients with chronic inflammatory diseases. 31 Anemia of inflammation (AI), a condition common in autoimmune diseases and cancer, is caused by excess of hepcidin. Fursultiamine has been shown to interfere with hepcidin binding to its receptor, ferroportin, by blocking C326 thiol residue that is essential for hepcidin binding. This in turn prevented hepcidin-induced ferroportin ubiquitination, endocytosis, and degradation in vitro and allowed for continuous cellular iron export despite the presence of hepcidin. Interestingly, thiamine alone or benfotiamine did not interfere with hepcidin effect on ferroportin, and other FDA-approved thiol-reactive compounds had a 1000- fold less potent effect when compared to fursultiamine. Unfortunately, these positive results were only observed in vitro because when rapid conversion in vivo to thiamine occurred and led to loss of effect. Ferroportin is the only conduit for delivery of cellular iron to plasma. It is expressed in enterocytes that absorb dietary iron, macrophages which recycle iron from senescent erythrocytes, and hepatocytes which are a major location for iron storage. Hepatocytes are triggered by interleukin-6 and other cytokines to synthesize more hepcidin. Orlistat Orlistat is a saturated derivative of a naturally occurring lipase inhibitor, lipstatin, which is produced by the bacteria Streptomyces toxytricini. 32 It is sold over the counter by 13 GlaxcoSmithKline by the brand name Alli for weight loss. Orlistat was first approved as a non- systemic treatment for obesity by inhibiting pancreatic lipase in the gastrointestinal tract without being absorbed and circulated throughout the body. 33 The way Orlistat helps with weight loss is because dietary fat that is consumed is not absorbed in the intestine unless subjected to the pancreatic lipase enzyme, which Orlistat inhibits irreversibly by means of a covalent bond to a serine in the lipase active site. The serine nucleophilically attacks the beta-lactone of orlistat. 34 In a 1999 published study, it has been shown to improve the lipid profiles of non-diabetic obese individuals as well as reduce total cholesterol and LDL cholesterol. 33 In diabetic patients treated with the drug, they had lost more weight, had improved serum lipid profiles, and significant improvements in glycaemic control overall when compared to those not treated with the drug. It was also discovered that orlistat could be tolerated over a long period of time, up to two years. Later in 2004, another 4-year double-blind, pacebo-controlled study confirmed that Orlistat reduced the incidence of type two diabetes and improved weight loss in obese patients. 35 Figure 1.8. Orlistat and the natural product it is derived from, Lipstatin. O O O O H N O Orlistat O O O O H N O Lipstatin 14 It was hypothesized that Orlistat could have other targets in the body and was discovered to be a novel inhibitor of the thioesterase domain of fatty acid synthase and have antitumor activity. 34 The inhibition of fatty acid synthase happens by the same mechanism of pancreatic lipase by means of nucleophilic attack of an active site serine on the beta-lactone functional group to form a covalent bond between orlistat and the enzyme. This discovery was made activity-based proteomics used to identify targets and screen inhibitors specifically for prostate cancer. The fatty acid synthase enzyme is strongly linked to tumor progression and is upregulated in prostate cancer. Orlistat was shown to inhibit the growth of prostate tumor cells in mice. 34 Then in 2010, 8 new covalent targets of orlistat were identified using a pull-down experiment that uses copper-catalyzed azide-alkyne cycloaddition chemistry to fluorescently label and identify orlistat targets. The targets labeled included fatty acid synthase and also Hsp90AB1 (involved in protein degradation), B-tubulin (a known anticancer target of vincristine, taxol, etc.), Annexin A2 (involved in cell proliferation and division), GAPDH, and three ribosomal proteins that are implicated in protein synthesis, cellular transformation, tumor growth, aggressiveness, and metastasis called RPL7A, RPL14, and RPS9. 36 Overexpression of the ribosomal proteins has been observed in colon, brain, liver, breast, and prostate cancers. Because of this, the antitumor activity of Orlistat may be related to targets other than fatty acid synthase. Orlistat has had a few reported cases of severe side effects including a case in 2000 when a 40-year-old woman was experiencing dizziness, peripheral edema, and a pulsating headache. 37 It was discovered that she had a regular heart rate but an extremely high blood pressure reading that lowered when the drug was halted. However, upon restarting the orlistat treatment, 15 her blood pressure skyrocketed once more, and treatment was stopped permanently. In another case in 2006, severe hepatic injury was observed. The patient was given vitamin K for 5 days to counteract the liver damage, but permanent improvement of liver function was only observed after completely halting the drug. 38 Because of this, recent efforts have been focused on finding new anti-obesity therapeutics, including scanning nature’s chemical toolbox for ideas. Many polyphenols, saponins, and flavonoids show strong anti-obesity activity and are being explored. 32 1.2.3. Synthetic Formoterol & Arformoterol Formoterol is a long-acting and highly selective bronchodilator that functions by being an agonist for B2-adrenoceptor. 39 It has minimal effect on B1-adrenoceptors and no effect on a- adernoceptors. 40 It was first synthesized and patented in 1972 in Japan. It consists of both enantiomers, the R,R and S,S forms of the molecule. Its prolonged duration of action may be a cause of the drug interacting with membrane lipid bilayers and then gradually leeching from the plasmalemma and continually activating the receptor. 40 Formoterol is generally inhaled as a powder and can cause increases in heart rate, lower blood pressures, increased plasma glucose levels, decreased serum potassium levels, and increased cAMP levels. 40 Pure (R,R)-formoterol, also called Arformoterol, is the active enantiomer of the drug and can produce effective broncodilation at lower doses than the racemate. 41 It has also shown that in addition to broncodilation, it can reduce IL-8 production from allergen-stimulated small airway epithelial cells. Arformoterol was recently introduced as the only first long-acting beta agonist 16 that was FDA approved for nebulized delivery, which can achieve similar plasma concentrations as the dry powder inhalation. 42 Macimorelin Macimorelin is the newest FDA approved compound in this list, and it is not a drug but rather a diagnostic tool. It is an orally active synthetic ghrelin mimetic that will stimulate growth hormone secretion by agonizing the growth hormone secretagogue receptor. 43 Ghrelin, a 28- amino acid peptide, is commonly called the “hunger hormone” as it is excreted when the stomach is empty. 44 Figure 1.9. Formoterol (racemic mixture) and enantiopure Arformoterol. O N H OH N H O HO Arformoterol O N H OH N H O HO Formoterol Figure 1.10. Macimorelin diagnostic compound. H 2 N O N H NH O H N NH H N O Macimorelin 17 Human growth hormone stimulation is needed to test for adult growth hormone deficiency (AGHD), which is traditionally diagnosed using an insulin tolerance test. Insulin tolerance tests, while the reference standard for AGHD diagnosis, are labor intensive, can cause sever hypoglycemia, and are not suitable for certain patients. 45 In an effort to overcome this, macimorelin was developed and received FDA approval in December of 2017 for use in AGHD diagnostic tests. 1.3 Investigational Non-FDA Approved Drug Candidates Fosmidomycin Fosmidomycin is a phosphonic acid derivative that is a natural antibacterial agent that was first isolated from Streptomyces lavendulae in the late 1970s. 46-48 It functions as a 1-deoxy- D-xylulose 5-phosphate (DOXP) reductoisomerase inhibitor. 49 DOXP is a key metabolite in the mevalonate-independent pathway of isoprenoid biosynthesis. 49 The DOXP/MEP pthway is present in algae, plants, bacteria and protozoans, but not in humans. 50 In 1999, it was discovered that fosmidomycin had antimalarial properties. Additionally, the DOXP/MEP pathway was a novel pathway for antimalarial drugs which is good because it can circumvent the mechanisms of resistance that are intrinsic or acquired to other antimalarial drugs. Considering that 3 million die every year from malaria, new treatments are necessary. The presence of two genes encoding the enzymes DOXP reductoisomerase and DOXP synthase suggested that the isoprenoid biosynthesis in Plasmodium falciparum depends on the DOXP pathway. Indeed, the drug treatment was able to suppress the growth of multidrug resistant P. falciparum strains of malaria and also cured mice that were infected with the rodent strain of malaria, P. vinckei. 49 18 By 2015, fosmidomycin had completed three phase II clinical trials with African and Asian adults and children as a monotherapy, six phase II clinical trials in combination with clindamycin in pediatric patients, and several other phase II clinical trials with a variety of other antimalarial drugs. 47 However, pharmacokinetic studies and drug delivery vehicle studies are still lacking, and fosmidomycin has not advanced further in the clinic. Additional research has been done in order to make the drug more lipophilic and increase its bioavailability, a variety of a-phenyl substituted fosmidomycin derivatives have been synthesized. 3,4-dichlorobenzyl-fosmidomycin was found to be twice as active as the original drug. 50 These results in addition to recently emerging mechanisms of resistance to the drug may lead the way for a synthetic analog of fosmidomycin to become a better candidate for further clinical studies. Fosmidomycin resistance has been predicted from a copy number variation event in the pfdxr gene that would enable the parasite to overcome the inhibition of isoprenoid biosynthesis. Foxy-5 There are 19 identified proteins in the Wnt family that have been linked to a variety of human cancers including breast, lung, colon, and skin cancers. 51 WNT-5A is a member of this Figure 1.11. Fosmidomycin antimalarial in Phase III. OH N P OH OH O O Fosmidomycin 19 family of cysteine-rich signaling proteins, and plays important roles in organ development, tissue orientation, and cell migration. It has been shown to act as both a suppressor and a promoter of tumor metastasis. 51 Some prostate cancer patients with high expression of WNT-5A in solid tumors have a more favorable outcome. It was proposed that the reconstitution of WNT-5A could help those patients with low expression achieve better prognoses. 52 FOXY-5 is a WNT-5A-derived peptide that was formulated to mimic the action of the protein. The added formyl group on the methionine at the 5’ end increased the hexapeptide’s effect on adhesion and could also protect the peptide from degradation at the site of inflammation and activate formyl peptide receptors which are GPCRs on the surfaces of leukocytes. When bacteria die in a host, they release N-formylated peptide fragments that come from bacterial protein synthesis being initiated by a formylated N-terminal methionine residue. The formyl group is later cleaved off by peptide deformylase enzymes. In a prostate cancer mouse model, FOXY-5 was able to inhibit the initial metastatic dissemination to regional and distal lymph nodes by 90% and 70% respectively. 52 Because of WNT-5A being an integral part of cell migration, FOXY-5 only targets the invasion of cells and not apoptosis or proliferation. This can still lead to better responses to prostate cancer chemotherapies. S N H O O H N OH O O N H O H N SH O N H OH O O H N OH O Foxy-5 Figure 1.12. FOXY-5 anticancer in Phase I. 20 Lanopepden Lanopepden, also called GSK1322322, is part of a new class of antibiotics that target peptide deformylase, an enzyme that is essential in bacteria for protein maturation. 53 Peptide deformylases (PDFs) are ubiquitous metalloenzymes that remove the formyl group that is carried by the initiator methionine in bacteria, mitochondria, and chloroblast proteins. Removal of the formyl group is imperative for final protein function. All PDFs are efficiently inhibited by the natural compound, actinonin, but this compound cannot be used as a therapeutic for several reasons: it targets other metalloenzymes, it induces apoptosis, it is rapidly exported by bacterial efflux pumps, and it induces bacterial resistance through several mechanisms. 54 Although PDFs are ubiquitous enzymes, the mitochondrial isoform in humans has several significant differences in the binding site that allow for bacterial-specific inhibitors to be explored. 54 There were three such inhibitors that entered clinical trials, but only lanopepden continued on to Phase II clinical trials for bacterial skin and skin structure infections. Right now, the only oral compound approved for treatment of complicated skin and skin structure infections due to MRSA is linezolid, a drug that has displayed many troubling side effects including myelosuppression, lactic acidosis, and serotonin syndrome. 53 Because lanopepden functions Figure 1.13. Lanopepden antibacterial in Phase II. N N H H N O N OH O F N N N O Lanopepden 21 through a novel mechanism, the potential is high for its clinical success in treating bacterial infections. Ioforminol MRI contrast agents are typically paramagnetic compounds such as stable free radicals or compounds with transition or lanthanide metals. 55 The most common contrast agent is GdGTPA- BMA which is a low molecular weight paramagnetic compound marketed under the brand name Omniscan . Occasionally, there is contrast-induced nephrotoxicity and ventricular fibrillation, so new contrast agents for MRIs as well as X-rays are needed. 56 Ioforminol, also called GE-145, is a new dimeric contrast agent that differs from Iodixanol by the presence of two N-formyl groups instead of N-acetyl groups. 57 Ioforminol is more hydrophilic than iodixanol and has a lower osmolality. 57,58 This allows for a more physiologic concentration of sodium and calcium ions to be added while still maintaining the desired isotonic properties. In a study using anesthetized pigs, ioforminol had a significantly lower propensity to cause ventricular fibrillation compared to other iodinated radiographic contrast medias. 58 Figure 1.14. Ioforminol contrast agent in Phase II. OH OH H N O I O NH OH OH I N OH N O I O HN HO HO I O H N OH HO I O I Ioforminol 22 1.4 N-Formyl Peptide Receptors N-formyl peptide receptors are a class of G-protein coupled receptors (GPCRs) that were first discovered to play a part in host defense against microbial infections. 59 It is a receptor for host-derived endogenous molecules that are known to be involved in proinflammatory responses. White blood cells, or leukocytes, are the cells of the immune system that are responsible for protecting the body against foreign invader organisms and infectious diseases. Recruitment of leukocytes to sites of inflammation is dependent on chemo attractants, or substances that have the ability to make or trigger other cells to move, in this case the white blood cells. N-formyl-methionyl-leucyl-phenylalanine (fMLF) is a chemo attractant that binds to N- formyl peptide receptor 1 (FPR1) and activates phagocytic leukocytes. 59,60 fMLF was first discovered in the supernatant of gram negative bacteria, and in 1976, a high affinity binding site for fMLF was found on the surface of neutrophils. 61 fMLF stimulates NFkB and production of inflammatory cytokines. 62 FPR1, a 350-amino acid protein, was the first GPCR to be described on the human neutrophil. There are three isoforms of the N-formyl peptide receptors: FPR1, H N O N H O H N O OH O S fMLF Figure 1.15. N-formyl-methionyl- leucyl-phenylalanine (fMLF) 23 FPR2, and FPR3. 63 For many years, only the first two were recognized and could be referred to in earlier publications as FPR and FPRL1 instead of FPR1 and FPR2 respectively. Each isoform plays entirely different roles and has a variety of unique ligands. FPR1 Because it is a GPCR, FPR1 has seven transmembrane domains that are connected by extracellular and intracellular loops. Arg84 and Lys85 in the second extracellular loop, Arg163 and Arg205 in the third extracellular loop, and Asp284 in the fourth extracellular loop are all found to be important for fMLF binding to FPR1. 61 The formyl group at the N-terminus of fMLF participates in an important hydrogen bond with FPR1 in the binding pocket using these charged Figure 1.16. Formyl peptide receptors (FPRs) in disease. 24 residues. fMLF is known to activate phospholipases C and A2, the release of intracellular Ca2+ store, and is an agonist for FPR1. 64 Classic studies suggested that the N-formyl group was crucial for binding to FPR1, but only bacterial and mitochondrial proteins are the sources of N-formyl peptides in nature. The mitochondria would passively release formylated peptides to trigger the start of the inflammatory process needed to clean up dead or dying cells. It was later discovered that the formyl group is not necessary for binding to the receptor. 60 Indeed, FPR1 agonists include fMLF but also Annexin-1 and HIV-1 envelope proteins. 63 Many bacteria-derived peptides that are released into the host during an infection bind to FPR1 and recruit leukocytes to the area to clear up the infection. Because the chronic propagation of inflammation plays an important role in many diseases, it was proposed that blocking the binding of fMLF to FPR1 was a potentially therapeutic pathway. However, the intrinsic functional redundancy of the chemoattractant/chemokine system may make blocking a single receptor difficult as a therapeutic approach. 64 Nevertheless, many FPR1 antagonists are under investigation. Cyclosporins A and H are among the most potent and receptor specific FPR1 antagonists and may be binding to an allosteric site that then blocks the binding site for fMLF. 59,64-66 They inhibit the binding of fMLF to leukocytes and abolish FPR1- mediated cell response to fMLF including chemotaxis, Ca 2+ mobilization, GT phase activation, and release of proinflammatory mediators. The cyclosporins are both isolated from the fungus Tolypocladium inflatum. 67 They were shown to dose-dependently displace [ 3 H]-fMLF from FPR1 selectively without effecting the other FPR variants. 64 Interestingly, an extensive structure- activity relationship study was performed using cyclosporin and led to the discovery of various 25 non-immunosuppressive cyclophilin inhibitors for Hepatitis C and other diseases, but their effects on the FPR proteins is currently unknown. 64 There are many other FPR1 antagonists that have been discovered using the natural product library. This includes the chemotaxis inhibitory protein (CHIPS) of Staphylococus aureus, a 121-residue protein, and modified versions of fMLF. 64 Upon substitution of the formyl group on fMLF with either t-Boc, i-Boc, or a carbobenzoxy group yielded peptides that displayed antagonistic properties. A lignan, or plant phenol, called PP-6 was shown to block fMLF binding to FPR1 and also inhibited fMLF-induced intracellular Ca 2+ mobilization, the production of reactive oxygen species, and phosphorylation of ERK 1 and 2 along with Akt and p38. This suggests that PP-6 has direct antagonistic activity, whereas there are many other plant phenols, coumarins, diterpenes and triterpenes that inhibit fMLF-induced responses like the reactive oxygen species production, HNE production by neutrophils, Ca 2+ flux in fMLF-stimulated human neutrophils, but they do not have the ability to displace fMLF binding from FPR1. 64 They may be functioning as ROS scavengers or have a mechanism that is not yet understood. The lipids OH O N O N O NH O N O N H O H N O N O N O N O N O HN * * (S) = CsA * (R) = CsH HO HO O O H H MeO OMe PP-6 Figure 1.17. (Left) Cyclosporins A and H; (Right) Plant phenol PP-6. 26 deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) antagonize FPR1, but oleanolic acid only inhibits the fMLF-induced responses. 59,64 In summary, FPR1 is a promiscuous receptor involved in inflammation that has the ability to bind peptides, large proteins, and lipids which do not share significant primary or tertiary structural similarities. FPR2 FPR2 reacts to more host-derived chemotactic agonists compared to FPR1 including acute phase protein serum amyloid A, the 42-amino acid form of amyloid beta, the prion protein fragment PrP106-126, mitochonidrial peptide fragment MYFINILTL that is derived from NADH dehydrogenase subunit 1, and LL-37 which is an enzymatic cleavage fragment of the neutrophil granule derived cathelicidin. 59 FPR2 recognizes both peptide agonists and also the lipid mediator lipoxin A4. It was observed that N-glycosylation was essential for peptide recognition and binding to FPR2 but not for binding of LXA4. 59 FPR2 can produce proinflammatory and anti-inflammatory responses. Serum amyloid A and leucine-37 yield pro-inflammatory results including neutrophil NF-kB activation and cytokine release. Annexin A1 and pro-resolving lipids LXA4 and resolvin D1 (RvD1) yield anti-inflammatory results including different dimerization states after agonist binding that alter receptor conformation and intracellular signaling. 61 RvD1 activates FPR2 and prevents accumulation of macrophages in adipose tissue in mice. Additionally, accumulation of inflammatory adipose tissue macrophages correlates with insulin resistance in humans, and FPR2 is expressed in adipose tissue. 61 One of the native ligands for FPR2 is amyloid-beta which plays an important role in Alzheimer’s disease (AD). 59,62,68 AD brains exhibit chronic inflammatory response 27 associated with amyloid-beta deposits. In fact, it was observed that chronic oral administration of ibuprofen, a well known anti-inflammatory drug, in amyloid-beta producing mice significantly reduced AB deposition, cerebral plaque load, plaque-associated microglial activation and over- production of IL-1. Activation of FPR2 by AB may be responsible for accumulation and activation of mononuclear phagocytes (monocytes and microglia). Microglial cells are essential in development, inflammation, and immunological responses in the central nervous system. They may be involved in the conversion of nonfibrillar AB into amyloid fibrils, a result previously ascribed to peripheral macrophages. Upon binding, the FPR2/AB complexes are rapidly internalized into the cytoplasmic compartment, and consistent exposure of FPR2-expressing cells to AB resulted in intracellular retention of the complex and the formation of Congo-red-positive fibrils in mononuclear phagocytes. 68 This means that FPR2 mediates pro-inflammatory response and participates in AB uptake and resultant fibrillar formation. Interaction of AB with FPR2 is then clearly associated with cell activation and release of proinflammatory and neurotoxic mediators. FPR2 has not been identified as a target with therapeutic potential considering the duality of the receptor in both pro-inflammatory and anti-inflammatory events. FPR3 FPR3 is the least characterized of the formyl-peptide receptors and has no orthology in mice. 63 It is proposed to play a role in the pathogenesis of allergic disease. It is not expressed by neutrophils but is found on eosinophils, monocytes, macrophages, and dendritic cells. It does not respond to formylated peptides in any way, and has only one high affinity ligand, peptide F2L, 28 which is derived from the heme-binding protein. 63 FL2 is an endogenous 21-amino acid peptide that binds to FPR3 and causes monocyte intracellular calcium flux, ERK1 and 2 phosphorylation, and chemotaxis while also augmenting LPS-mediated IL-12 production in dendritic cells, inhibiting their maturation. Humanin, a neuroprotective peptide that binds to FPR2, is the only other ligand of FPR3 identified. 62 1.5 Conclusions In conclusion, N-formylated FDA-approved drugs and diagnostic tools are involved in the treatment of chronic inflammatory diseases, obesity, cancer, and bacterial infections. Formyl peptide receptors in the human body are also linked to these same conditions and are a major part of the pro-inflammatory process in the body. Some drugs, like FOXY-5, have the N-formyl group added in order to target activation of the pro-inflammatory process in the body. Others have the N-formyl group added to alter solubility profiles, like Ioforminol, or to target formyl- detecting enzymes like peptide deformylase, like Lanopepden. Most drugs are natural products or derivatives of natural products, and the exact purpose of the N-formyl functional group is not known. It is obvious, though, that its presence is important and occasionally transformative, like in the case of Vincristine versus Vinblastine; these compounds have the same molecular formulas and same structure apart from Vincristine containing an N-formyl group and Vinblastine containing a methyl, but they have completely different dose-limiting side effects in humans. Ideally in the future, a more defined relationship between the structures of therapeutics and their functions in the body will be common knowledge among drug discovery scientists and will allow for easier drug design and better treatment outcomes for all diseases and ailments. 29 1.6 References 1. Gidding, C. E. 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Journal of Leukocyte Biology 72, 628–635 (2002). 37 Chapter 2: Direct N-Formylation with N,N’-Dimethylformamide Facilitated by Water and Oxygen 2.1 Introduction 2.1.1 Summary of Previously Reported N-Formylation Methods Formamide functional groups appear as intermediates and as final products in the syntheses of potentially therapeutic compounds in drug discovery. 1 They also are used in the formation of isocyanate, formamidine, and nitrile functional groups, and other reactions including functional group conversions, the Vilsmeier formylation, allylation, and hydrosilation of carbonyl compounds. 2 There have been many synthetic approaches discovered for forming formamides through the formylation of amine compounds. 1 The formyl derivatives of aromatic amines were first reported in 1895 by using formamide as a transformylating agent, a reagent that was not improved upon until 2013 with the addition of sodium methoxide. 3 Formic acid has been widely used as a formylating agent without any additive and also with a variety of organic and inorganic additives in a variety of conditions. 4 2.1.2 N-Formylation Methods Using DMF DMF is one of the most widely used solvents but is also a reagent in a variety of transformations. 5 It was first reported as an N-formylating reagent in 1959 by Pettit in addition to sodium methoxide for aromatic amines at reflux. 6 Soon after, aliphatic amines were heated in DMF under a stream of CO 2 to afford formylated products. 7 It was first reported in 1973 that heating benzylamine in DMF without any other additive could yield an N-formylated product. 8 Interestingly, it was noted the reaction did not proceed any better under argon but the addition of sulfuric acid improved reactions times and yields. 38 NH R N R O Cl R 2 DMF, 120°C N R R 2 The scope of DMF as a formylating agent can be expanded using a variety of conditions and additives. N-alkylphthalimides can be transformed into N-alkylformamides in DMF using hydrazine hydrate. 9 2,3-Dihydro-1,4-phthalazinedione can promote the N-formylation of primary amines in DMF. 10 DMF can be mixed with silyl chlorides to afford the N-formylated products of a variety of secondary aliphatic amines and benzylamines. 11 The same scope of N-formylated products can be achieved using microwave irradiation at 200°C of amines in DMF with methyl benzoate. 12 It can also be used with inorganic catalysts such as cerium oxide and hydrous zirconium oxide. 13 Finally, amino acid esters can be N-formylated using imidazole in DMF, but these conditions were sluggish for benzylamine and did not afford any product for aniline or bulkier secondary amines apart from amino acid esters. 14 2.2 The Role of Water and Oxygen & Proposed Mechanism Although it is known that DMF can act as an N-formylating reagent at higher temperatures without the need for any catalyst or promoter, the mechanism of this reaction has never been explored. In our lab, we observed N-formylation of a piperidine moiety when running an alkylation reaction at high temperature in DMF (Scheme 2.1). Upon further exploration, we determined that the yield of the N-formylated product is improved when the reaction is allowed to be open to air and when the DMF used is “wet” or contains a small amount of water. Scheme 2.1. N-formylation of piperidine moiety. 39 It was hypothesized that the air was acting as an oxygen source, so it was replaced with several alternative oxygen sources to confirm. 10 mol% of N-methylmorpholine-N-oxide (NMO) provided modest yields of N-benzylformamide from benzylamine when paired with 1 molar equivalent of water (Table 2.1). Running the reaction in 100% oxygen atmosphere provided excellent conversion to the formylated product both with and without added water seen in entries 1h and 1i in Table 1. Similar yields were also achieved in the absence of oxygen with 10 mol% of mCPBA oxidant instead. entry atmosphere oxidant H 2O/Anh. yield a (%) 1a Air none none 81 1b Argon none Anh. 15 1c Air none H 2O 63 1d Air none Anh. 25 1e Argon none H 2O 13 1f Argon NMO H 2 O 55 1g Argon NMO Anh. 13 1h O 2 O 2 H 2O 86 1i O 2 O 2 Anh. 90 1j Argon mCPBA H 2O 86 1k Argon mCPBA Anh. 84 Table 2.1. Conditions for N-formylation of benzylamine. a Isolated yield. 40 Based on these results, we propose a novel mechanism for this transformation that starts with the formation of a catalytic N-oxide species that is generated when heating an amine in the presence of oxygen (Scheme 2.2). The oxygen of the N-oxide then attacks the carbonyl carbon of DMF. The nitrogen of DMF is protonated to form a dimethylamine leaving group that is released upon reformation of the carbonyl. An additional molecule of starting material amine attacks the carbonyl, and then, after a proton transfer and carbonyl reformation, the final formylated product is liberated and the catalytic N-oxide species is reconstituted. H N O R 2 R 1 N Me Me O H N O R 2 R 1 N Me Me HO H N Me Me HO H N O R 2 R 1 N Me Me HO H N O R 2 R 1 H OH H N O R 2 R 1 O H N O R 2 R 1 N H R 2 R 1 N H R 2 R 1 H N O R 2 R 1 N H R 2 R 1 O H N O R 2 R 1 N H R 2 R 1 O H N O R 2 R 1 H N R 2 R 1 O + Scheme 2.2. Proposed mechanism for N-formylation. 41 2.3 Scope and Limitations 2.3.1 N-Formylation in Air and O 2 Scheme 2.3 examines a variety of benzyl, aromatic, and aliphatic amines when heated in DMF in both air and 100% oxygen atmospheres. Benzylamines 1, 2, 4, 5, and 7 were efficiently converted to formylated products when heating in DMF in air. Dibenzylamine (3) and N- isopropylbenzylamine (6) appear to be too sterically hindered, and 2-picolylamine (8) too electron poor to afford decent yields of formylated product in either air or oxygen-rich atmospheres. The same trend can be seen when looking at aromatic anilines as well. Aniline (9) and 2,5-dimethoxyaniline (13) can be formylated with excellent conversion in air, but the percent conversion is not improved upon with the addition of an oxygen-rich environment. Slightly electron poor anilines 10 and 11 as well as very electron poor aniline 12 were not formylated in decent yields in either condition as was 2,6-diethylaniline possibly due to steric hindrance from the ethyl groups. The addition of oxygen atmosphere was able to improve the yields of aliphatic amines 15, 16, and 17, although air was sufficient for conversion of 15 and 17 to their formylated products. 42 N H Me O H N O H N O F H N I O N O N H O N O O N O H N O NO 2 F H N O OMe MeO H N O Et Et N H O MeO OMe N O H N OMe MeO O N O N H N O Cl N H O 1.1a,b (81%, 100%) 1.3a,b (13%, 19%) 1.2a,b (71%, 42%) 1.4a,b (80%, ?%) 1.5a,b (83%, 70%) 1.6a,b (0%, 6%) 1.7a,b (79%, 81%) 1.8a,b (0%, 0%) 1.9a,b (94%, 3%) 1.10a,b (1%, 6%) 1.11a,b (1%, 6%) 1.12a,b (4%, 5%) 1.13a,b (100%, 21%) 1.14a,b (0%, 3%) 1.15a,b (81%, 93%) 1.16a,b (21%, 74%) 1.17a,b (89%, 100%) R 1 NH R 1 N O DMF 120°C 24h air/O 2 R 2 R 2 Scheme 2.3. Scope a of N-Formylation by DMF in air and O 2-rich b conditions a All percentages are percent conversion from the starting material as calculated by 1 H NMR analysis. b O 2 atmosphere achieved with balloon of O 2. 43 2.3.2 N-Formylation in DMF with Catalytic Formic Acid The rate of reaction as well as the percent conversion to formylated product can both be increased by the addition of a catalytic amount (10 mol%) of formic acid (Scheme 2.4). In fact, most benzyl and aliphatic amines can be converted to formylated product upon stirring at room temperature in DMF with formic acid. The formyl group still comes from DMF as can be seen when using DMF-d7 as the solvent in the reaction. Electron poor 2-picolylamine (8c,d) and 5- fluoro-2-nitroaniline (12c,d) still would not react even upon heading with formic acid. 2.4 Conclusions In conclusion, the mechanism for the formylation of amines in DMF was found to involve the formation of a catalytic N-oxide that is formed when amines are heated in the presence of oxygen or an oxidant. Conditions were found to afford the N-formylation of a variety of benzyl, aromatic, and aliphatic amines. 44 N H Me O H N O H N O F H N I O N O N H O N O O N O H N O NO 2 F H N O OMe MeO H N O Et Et N H O MeO OMe N O H N OMe MeO O N O N H N O Cl N H O 1.1c,d (94%, 100%) 1.3c,d (100%, 100%) 1.2c,d (74%, 76%) 1.4c,d (100%, 100%) 1.5c,d (81%, 95%) 1.6c,d (69%, 99%) 1.7c,d (98%, 96%) 1.8c,d (0%, 0%) 1.9c,d (90%, 99%) 1.10c,d (16%, 83%) 1.11c,d (19%, 99%) 1.12c,d (4%, 6%) 1.13c,d (58%, 100%) 1.14c,d (6%, 70%) 1.15c,d (86%, 100%) 1.16c,d (80%, 85%) 1.17c,d (87%, 92%) R 1 NH R 1 N O DMF HCOOH (cat.) 24h, air rt/120°C R 2 R 2 Scheme 2.4. Scope a of N-Formylation in Air with Formic Acid Catalyst at Room Temperature and 120°C a All percentages are percent conversion from the starting material as calculated by 1 H NMR analysis. b 10 mol% of formic acid used in all reactions. 45 2.5 Experimental Chemicals purchased from Sigma Aldrich and Combi-Blocks. TLC plates, screw-cap vials, and microwave vials and caps were purchased from VWR International. TLC plates were visualized with UV light and cerium ammonium molybdate stain. Flash column chromatography was performed on a Biotage Isolera One autocolumn system with Biotage KP-Sil Snap Cartitridges. NMR spectra taken on either Varian Mercury 400 or Varian 400MR instruments. Chemical shifts reported as from CDCl 3. LCMS data received from Agilent Technologies 1260 Infinity Liquid Chromotograph and Agilent Technologies 6120 Quadrupole LC/MS. NMR and LC/MS data was analyzed in MestReNova. N-benzylformamide (1.1) (Table 1). A dry microwave vial with a stir bar was charged with any solid reagent and capped and placed under the respective atmosphere by evacuating and backfilling with desired gas (argon, oxygen), or just capped with air atmosphere. N,N’- dimethylformamide (DMF, 2mL) and benzylamine ( mL, 0.96mmol) were added to vial through the top septum. If the reaction conditions called for the addition of water, it was added last through the top septum. Balloon atmosphere was removed and the top cap was parafilmed. Reactions were then immersed in an oil bath at 125°C and heated for 24 hours. The reaction mixture was diluted with ethyl acetate (10mL) and water (10mL), and the organic layer was removed. The aqueous layer was extracted with 2 x 10mL ethyl acetate, and the combined organic extracts were washed with H 2 O (10mL), brine (10mL), dried over Na 2 SO 4 , and 46 concentrated under reduced pressure to yield a white solid. 1 H NMR (400 MHz, Chloroform-d) δ 8.20 (s, 1H), 7.41 – 7.20 (m, 5H), 6.62 – 6.36 (m, 1H), 4.43 (d, J = 5.8 Hz, 2H). 13 C NMR (400 MHz, CDCl 3, Including Rotamers) δ 164.80, 161.33, 137.45, 128.89, 128.73, 127.94, 127.74, 127.64, 126.98, 77.39, 77.07, 76.75, 45.72, 42.21. 1a: Air. Reagent-grade DMF. NMR ratio of product to starting material = 1:0. Total yield = 0.1053g, Adjusted yield = 0.1053g, 81%. 1b: Argon. Reagent-grade DMF. NMR ratio of product to starting material = 1:1.18. Total yield = 0.0422g, Adjusted yield = 0.019g, 15%. 1c: Air. Drisolv® DMF. Water (0.018mL, 0.96mmol). NMR ratio of product to starting material 1:0.21. Total yield = 0.0992g, Adjusted yield = 0.082g, 63%. 1d: Air. Drisolv® DMF. NMR ratio of product to starting material = 1:2.13. Total yield = 0.1019g, Adjusted yield = 0.033g, 25%. 1e: Argon. Drisolv® DMF. Water (0.018mL, 0.96mmol). NMR ratio of product to starting material = 1:2.27. Total yield = 0.0544g, Adjusted yield = 0.017g, 13%. 1f: Argon. Drisolv® DMF. Water (0.018mL, 0.96mmol). N-methylmorpholine-N-oxide (g, 0.096mmol). NMR ratio of product to starting material = 1:0. Total yield = 0.0714g, Adjusted yield = 0.0714g, 55%. 1g: Argon. Drisolv® DMF. N-methylmorpholine-N-oxide (g, 0.096mmol). NMR ratio of product to starting material = 1:1.96. Total yield = 0.0495g, Adjusted yield = 0.017g, 13%. 1h: Oxygen. Drisolv® DMF. Water (0.018mL, 0.96mmol). NMR ratio of product to starting material = 1:0. Total yield = 0.1112g, Adjusted yield = 0.1112g, 86%. 47 1i: Oxygen. Drisolv® DMF. NMR ratio of product to starting material = 1:0. Total yield = 0.1175g, Adjusted yield = 0.1175g, 90%. 1j: Argon. Drisolv® DMF. Water (0.018mL, 0.96mmol). meta-Chloroperoxybenzoic acid (g, 0.096mmol). NMR ratio of product to starting material = 1:0. Total yield = 0.1119g, Adjusted yield = 0.1119g, 86%. 1k: Argon. Drisolv® DMF. meta-Chloroperoxybenzoic acid (g, 0.096mmol). NMR ratio of product to starting material = 1:0. Total yield = 0.1097g, Adjusted yield = 0.1097g, 84%. General procedure for products 1.2-1.14, 1.17: An 8-dram vial with a stir bar was charged with amine of interest and DMF (2mL) and subjected to 4 conditions: A. Room temperature, 24h B. Room temperature, formic acid (0.006mL, 0.096mmol), 24h C. 125°C, 24h D. 125°C, formic acid (0.006mL, 0.096mmol), 24h After completion of reaction, the reaction mixture was diluted with ethyl acetate (10mL) and water (10mL), and the organic layer was removed. The aqueous layer was extracted with 2 x 10mL ethyl acetate, and the combined organic extracts were washed with H 2O (10mL), brine (10mL), dried over Na 2 SO 4 , and concentrated under reduced pressure 48 (S)-N-formyl-1-phenylethylamine (1.2). Clear oil. 1 H NMR (400 MHz, Chloroform-d) δ 8.07 – 7.99 (m, 1H), 7.39 – 7.17 (m, 5H), 6.97 – 6.73 (m, 1H), 5.13 (p, J = 7.2 Hz, 1H), 1.45 (dd, J = 7.0, 1.3 Hz, 3H). 13 C NMR (400 MHz, CDCl 3, Including Rotamers) δ 164.53, 160.66, 142.73, 128.73, 127.33, 126.21, 126.01, 51.98, 47.58, 21.79. N,N-dibenzylformamide (1.3). Clear oil. 1 H NMR (400 MHz, Chloroform-d) δ 8.41 (s, 1H), 7.42 – 7.24 (m, 6H), 7.18 (ddd, J = 10.9, 7.9, 1.6 Hz, 4H), 4.41 (s, 2H), 4.26 (s, 2H). 13 C NMR (400 MHz, CDCl 3, Including Rotamers) δ 162.85, 136.02, 135.65, 128.92, 128.70, 128.51, 128.14, 127.72, 127.66, 50.25, 44.65. N-(3-chlorobenzyl)formamide (1.4). White solid. 1 H NMR (400 MHz, Chloroform-d) δ 8.31 – 8.22 (m, 1H), 7.33 – 7.22 (m, 3H), 7.16 (dddd, J = 6.2, 3.0, 1.4, 0.7 Hz, 1H), 4.45 (dt, J = 6.1, 0.8 Hz, 2H). 13 C NMR (400 MHz, CDCl 3, Including Rotamers) δ N-(2,4-dimethoxybenzyl)formamide (1.5). Fluffy white solid. 1 H NMR (400 MHz, Chloroform-d) δ 8.11 (s, 1H), 7.13 (dd, J = 8.1, 2.4 Hz, 1H), 6.56 – 6.31 (m, 3H), 4.35 (dd, J = 5.9, 2.4 Hz, 2H), 3.77 49 (dd, J = 9.6, 2.6 Hz, 6H). 13 C NMR (400 MHz, CDCl 3, Including Rotamers) δ 164.82, 161.03, 160.34, 158.30, 129.94, 129.28, 118.31, 103.89, 98.39, 98.30, 55.25, 55.22, 41.21, 36.98. N-benzyl-N-isopropylformamide (1.6). Clear oil. 1 H NMR (400 MHz, Chloroform-d) δ 8.37 (d, J = 1.2 Hz, 1H), 7.38 – 7.18 (m, 5H), 4.51 (d, J = 1.2 Hz, 2H), 4.35 (s, 1H), 1.17 (dd, J = 6.8, 1.3 Hz, 6H). 13 C NMR (400 MHz, CDCl 3 , Including Rotamers) δ 163.45, 162.50, 138.00, 128.68, 128.44, 127.73, 127.54, 127.14, 127.11, 50.05, 48.39, 45.05, 44.10, 22.22, 20.13. 2-Formyl-1,2,3,4-tetrahydroisoquinoline (1.7). Clear oil. 1 H NMR (400 MHz, Chloroform-d) δ 8.21 (d, J = 20.2 Hz, 1H), 7.24 – 7.04 (m, 4H), 4.66 (s, 1H), 4.52 (s, 1H), 3.83 – 3.71 (m, 1H), 3.69 – 3.57 (m, 1H), 2.87 (dt, J = 13.9, 6.1 Hz, 2H). 13 C NMR (400 MHz, CDCl 3, Including Rotamers) δ 162.34, 161.76, 161.25, 134.37, 134.06, 133.53, 131.70, 129.18, 129.14, 128.90, 127.76, 127.58, 127.08, 126.70, 126.63, 126.59, 126.47, 125.87, 47.31, 43.25, 42.30, 38.61, 38.02, 29.68, 27.90, 27.32. Formanilide (1.9). Pale yellow oil. 1 H NMR (400 MHz, Chloroform-d) δ 8.38 (d, J = 1.8 Hz, 1H), 7.55 (dt, J = 7.9, 1.1 Hz, 1H), 7.40 – 7.28 (m, 2H), 7.23 – 7.07 (m, 2H). 13 C NMR (400 MHz, CDCl 3, 50 Including Rotamers) δ 162.88, 159.39, 136.78, 136.66, 129.74, 129.09, 125.34, 124.92, 120.10, 118.84, 77.35, 77.03, 76.71. N-(2-fluorophenyl)formamide (1.10). Clear oil. 1 H NMR (400 MHz, Chloroform-d) δ 8.47 (d, J = 1.6 Hz, 1H), 7.53 (s, 1H), 7.25 (dd, J = 6.7, 1.7 Hz, 1H), 7.20 – 7.02 (m, 3H). 13 C NMR (400 MHz, CDCl 3, Including Rotamers) δ 158.82, 153.39, 150.96, 125.40, 125.30, 122.15, 122.09. N-(4-iodo-phenyl)-formamide (1.11). Fluffy white solid. 1 H NMR (400 MHz, Chloroform-d) δ 8.30 (s, 1H), 7.56 (ddd, J = 14.3, 8.8, 1.9 Hz, 2H), 7.35 – 7.24 (m, 1H), 6.90 – 6.77 (m, 1H). 13 C NMR (400 MHz, CDCl 3, Including Rotamers) δ 162.57, 146.21, 137.84, 124.55, 123.42, 117.31. N-(5-fluoro-2-nitrophenyl)formamide (1.12). Bright yellow solid. 1 H NMR (400 MHz, Chloroform-d) δ 7.92 (dd, J = 9.7, 1.5 Hz, 1H), 6.57 – 6.13 (m, 1H), 6.09 (ddd, J = 9.7, 2.7, 1.3 Hz, 1H), 5.80 (dd, J = 2.8, 1.3 Hz, 1H). 13 C NMR (400 MHz, CDCl 3, Including Rotamers) δ 168.16, 165.62, 162.73, 147.16, 147.03, 129.26, 129.14, 105.69, 105.44, 103.83, 103.57. 51 N-(2,5-dimethoxyphenyl)formamide (1.13). White needles. 1 H NMR (400 MHz, Chloroform-d) δ 8.04 (d, J = 3.1 Hz, 1H), 6.83 – 6.71 (m, 2H), 6.54 (dd, J = 8.9, 3.0 Hz, 1H), 3.78 (s, 3H), 3.72 (s, 3H). 13 C NMR (400 MHz, CDCl 3, Including Rotamers) δ 162.52, 154.38, 141.84, 137.30, 111.33, 102.00, 101.84, 56.08, 55.46. 2,6-Diethylphenylformamide (1.14). White needles. 1 H NMR (400 MHz, Chloroform-d) δ 8.44 (d, J = 1.9 Hz, 1H), 7.32 – 7.20 (m, 1H), 7.20 – 7.05 (m, 2H), 6.87 (s, 1H), 2.75 – 2.55 (m, 4H), 1.26 – 1.10 (m, 6H). 13 C NMR (400 MHz, CDCl 3, Including Rotamers) δ 141.66, 127.70, 126.07, 118.34, 24.37, 13.13. N-[2-(3,4-dimethoxyphenyl)ethyl]formamide (1.17). Clear oil. 1 H NMR (400 MHz, Chloroform- d) δ 8.10 (s, 1H), 6.78 (dd, J = 7.9, 1.6 Hz, 1H), 6.74 – 6.63 (m, 2H), 6.05 (s, 1H), 3.83 (dd, J = 5.3, 1.6 Hz, 6H), 3.57 – 3.39 (m, 2H), 2.76 (td, J = 7.1, 1.6 Hz, 2H). 13 C NMR (400 MHz, CDCl 3, Including 52 Rotamers) δ 164.92, 161.44, 148.99, 147.69, 131.00, 130.14, 120.88, 120.64, 112.05, 111.90, 111.50, 111.40, 55.89, 55.84, 43.48, 39.33, 37.18, 35.03, 29.64. General procedure for products 1.15-1.16: An 8-dram vial with a stir bar was charged with amine of interest and DMF (2mL) and subjected to 4 conditions: A. Room temperature, 24h B. Room temperature, formic acid (0.006mL, 0.096mmol), 24h C. 125°C, 24h D. 125°C, formic acid (0.006mL, 0.096mmol), 24h After completion of reaction, the reaction mixture was transferred to a 50mL round-bottom flask and concentrated under reduced pressure. The residue was charged with heptane (5mL) to aid in the removal of residual DMF and concentrated under reduced pressure. 1-Formyl-4-methylpiperidine (1.15). Pale yellow oil. 1 H NMR (400 MHz, Chloroform-d) δ 7.99 (s, 1H), 4.34 (ddd, J = 13.3, 4.4, 2.2 Hz, 1H), 3.66 – 3.47 (m, 1H), 3.04 (td, J = 12.8, 2.8 Hz, 1H), 2.69 – 2.51 (m, 1H), 1.78 – 1.54 (m, 3H), 1.17 – 0.98 (m, 3H), 0.95 (dd, J = 6.5, 1.6 Hz, 3H). 13 C NMR (400 MHz, CDCl 3, Including Rotamers) δ 160.79, 46.18, 39.95, 34.68, 33.24, 31.35, 21.71. 53 N-Formylmorpholine (1.16). Clear oil. 1 H NMR (400 MHz, Chloroform-d) δ 8.04 (s, 1H), 3.72 – 3.60 (m, 4H), 3.59 – 3.50 (m, 2H), 3.41 – 3.34 (m, 2H). 13 C NMR (400 MHz, CDCl 3, Including Rotamers) δ 163.30, 160.91, 67.19, 66.40, 63.77, 45.82, 43.29, 40.62. 2.6 References (1) For formamide functional group occurrence in drug discovery see: (a) Gerack, C.J., McElwee-White, L. Formylation of Amines. Molecules 2014, 19, 7689–7713. (b) Grant, H.G.; Summers, L.A. Synthesis of N-methyl-N-(2,2,2-trichloro-1- arylaminoethyl)tormamides and related-compounds as potential fungicides. Aust. J. Chem. 1980, 33, 613–617. (c) Kobayashi, K.; Nagato, S.; Kawakita, M.; Morikawa, O.; Konishi, H. Synthesis of 1-formyl-1,2- dihydroquinoline derivatives by a lewis acid-catalyzed cyclization of o-(1-hydroxy-2- alkenyl)phenyl isocyanides. Chem. Lett. 1995, 24, 575–576. (d) Jackson, A.; Meth-Cohn, O. A new short and efficient strategy for the synthesis of quinolone antibiotics. J. Chem. Soc. Chem. Commun. 1995, 1319–1319. (e) Pettit, G.; Kalnins, M.; Liu, T.; Thomas, E.; Parent, K. Notes- potential cancerocidal agents. III. Formanilides. J. Org. Chem. 1961, 26, 2563–2566. (2) For formamide use in functional group conversion see: 54 (a) Faraj, M.K. Synthesis of Isocyanate Precursors from Primary Formamides. U.S. Patent 5,686,645, 1997. (b) Han, Y.; Cai, L. An efficient and convenient synthesis of formamidines. Tetrahedron Lett. 1997, 38, 5423–5426. (c) Arlt, D.; Klein, G. Preparation of Nitriles from Formamides. U.S. Patent 4,419,297, 1983. (d) Muzart, J. N,N-Dimethylformamide: much more than a solvent. Tetrahedron 2009, 65, 8313- 8323. (e) Downie, I.M.; Earle, M.J.; Heaney, H.; Shuhaibar, K.F. Vilsmeier formylation and glyoxylation reactions of nucleophilic aromatic compounds using pyrophosphoryl chloride. Tetrahedron 1993, 49, 4015–4034. (f) Kobayashi, S.; Nishio, K. Facile and highly stereoselective synthesis of homoallylic alcohols using organosilicon intermediates. J. Org. Chem. 1994, 59, 6620–6628. (g) Kobayashi, S.; Yasuda, M.; Hachiya, I. Trichlorosilane-dimethylformamide (Cl 3SiH-DMF) as an efficient reducing agent. Reduction of aldehydes and imines and reductive amination of aldehydes under mild conditions using hypervalent hydridosilicates. Chem. Lett. 1996, 25, 407– 408. (3) (a) Hirst, H. R. & Cohen, J. B. LXXXVI.—A method for preparing the formyl derivatives of the aromatic amines. J. Chem. Soc. 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An efficient, catalyst- and solvent- free N-formylation of aromatic and aliphatic amines. Green Chem. Lett. Rev. 2011, 4, 151–157. (f) Bose, A. K., Ganguly, S. N., Manhas, M. S., Guha, A. & Pombo-Villars, E. Microwave promoted energy-efficient N-formylation with aqueous formic acid. Tet. Lett. 2006, 47, 4605–4607. (g) Wei, D., Cui, C., Qu, Z., Zhu, Y. & Tang, M. A computational study on the reaction mechanisms of N-formylation of amines under a Lewis acid catalysis. J. Mol. Struct. (Theochem) 2010, 951, 89– 92. (h) Shekhar, A.C., Kumar, A.R., Sathaiah, G., Paul, V.L., Sridhar, M., Rao, P.S. Facile N-formylation of amines using Lewis acids as novel catalysts. Tet. Lett. 2009, 50, 7099-7101. (i) Krishnakumar, B. & Swaminathan, M. A convenient method for the N-formylation of amines at room temperature using TiO2-P25 or sulfated titania. J. Mol. Cat. 2011, 334, 98–102. (j) Pathare, S. P., Sawant, R. V. & Akamanchi, K. G. Sulfated tungstate catalyzed highly accelerated N-formylation. Tet. Lett. 2012, 53, 3259–3263. 56 (k) Hong, M. & Xiao, G. Hafnium(IV) bis(perfluorooctanesulfonyl)imide complex supported on fluorous silica gel catalyzed N-formylation of amines using aqueous formic acid. J. Fluor. Chem. 2013, 146, 11–14. (l) Patil, U. B., Singh, A. S. & Nagarkar, J. M. Nanoceria-catalyzed Highly Efficient Procedure for N- Formylation of Amines at Room Temperature under Solvent-free Conditions. Chem. Lett. 2013, 42, 524–526. (m) Kim, J.-G. & Jang, D. Facile and Highly Efficient N-Formylation of Amines Using a Catalytic Amount of Iodine under Solvent-Free Conditions. Synlett 2010, 2093–2096. (n) Hosseini-Sarvari, M. & Sharghi, H. ZnO as a New Catalyst for N-Formylation of Amines under Solvent-Free Conditions. J. Org. Chem. 2006, 71, 6652–6654. (o) Habibi, D., Heydari, S. & Afsharfarnia, M. A capable cobalt nano-catalyst for the N-formylation of various amines and its biological activity studies. Appl. Organometal. Chem. 2017, 31, 3874. (p) Aleiwi, B.A., Mitachi, K., Kurosu, M. Mild and convenient N-formylation protocol in water- containing solvents. Tet. Lett. 2013, 54, 2077-2081. (5) For reviews on DMF see: (a) Muzart, J. N,N-Dimethylformamide: much more than a solvent. Tetrahedron 2009, 65, 8313- 8323. (b) Ding, S. & Jiao, N. N,N-Dimethylformamide: A Multipurpose Building Block. Angew. Chem. Int. Ed. 2012, 51, 9226–9237. (6) Pettit, G. R. & Thomas, E. G. Formylation of Aromatic Amines with Dimethylformamide. Communications 24, 895–896 (1959). 57 (7) Otsuji, Y., Matsumura, N. & Imoto, E. Transacylation from Acid Amides to Amines Catalyzed by Carbon Dioxide. Short Comm. 1968, 1485. (8) Kraus, M. A. The Formylation of Aliphatic Amines by Dimethylformamide. Synthesis-Stuttgart 1973, 361–362. (9) Iwata, M. & Kuzuhara, H. A New Transformation Method of N-Alkylphthalimides to N- Alkylformamides with N,N-Dimethylformamide and Hydrazine Hydrate. Chemistry Letters 1986, 951–952. (10) Iwata, M. & Kuzuhara, H. N-Formylation of Aliphatic Primary Amines with N,N- Dimethylformamide Promoted by 2,3-Dihydro-1,4-phthalazinedione. Chemistry Letters 1989, 2029–2030. (11) (a) Berry, M. B., Blagg, J., Craig, D. & Willis, M. C. An Improved Procedure for N-Formylation of Secondary Amines Using Chlorotrimethylsilane-Imidazole-N,N-Dimethylformamide. Synlett 1992, 659-660. (b) Djuric, S. W. A mild and convenient procedure for the N-formylation of secondary amines using organosilicon chemistry. J. Org. Chem. 1984, 49, 1311–1312. (12) Yang, D.-S. & Jeon, H.-B. Convenient N-Formylation of Amines in Dimethylformamide with Methyl Benzoate under Microwave Irradiation. Bull. Korean Chem. Soc. 2010, 31, 1424–1426. (13) (a) Wang, Y., Wang, F., Zhang, C., Zhang, J., Li, M., Xu, J. Transformylating amine with DMF to formamide over CeO 2 catalyst. Chem. Commun. 2014, 50, 2438–2441. (b) Takahashi, K., Shibagaki, M. & Matsushita, H. Formylation of Amines by Dimethylformamide in the Presence of Hydrous Zirconium Oxide. Agric. Biol. Chem. 1988, 52, 853-854. 58 (14) Suchý , M., Elmehriki, A. A. H. & Hudson, R. H. E. A Remarkably Simple Protocol for the N- Formylation of Amino Acid Esters and Primary Amines. Org. Lett. 2011, 13, 3952–3955. 59 Chapter 3. Synthetic Methods for the Functionalization of Cyanine Dyes 3.1. Introduction Ever since the first cyanine dye was synthesized in 1856, these colorful organic compounds have been used for imaging and reporting purposes because of their high extinction coefficients and high quantum yields of fluorescence. Cyanine dyes can be classified according to the length of the internal carbon chain (monomethine, trimethine, etc.), with the addition of each vinylene (HC=CH) increasing the maximum absorbance of the compound about 100nm. 1 This shift of either absorbance or fluorescence from a lower wavelength to a higher wavelength is called a bathochromic shift. One of the most commonly used heptamethine cyanine dyes is indocyanine green (ICG), an FDA approved diagnostic tool for imaging purposes (Figure 3.1). The focus of this introduction will be on heptamethine cyanine dyes and their uses and syntheses. 3.1.1. Heptamethine cyanine dyes One of the first uses of heptamethine cyanine dyes was for the imaging of different biomolecules. Because the quantum yield of fluorescence increases when the dye compound is more rigid, the fluorescence signal increases when the compound is bound to a large N + N - O 3 S SO 3 - Na + ICG 1 2 3 4 5 6 7 Figure 3.1. Indocyanine green (ICG) 60 N + Me Me R N R Me Me Z O NH R = Et, (CH 2 ) 4 SO 3 - X = O, S Y = ClO4 - , I - Z = H, OH N + Me Me R N R Me Me X O O N O O N C S O O N C S N O O Y - N R Figure 3.2. Representation of the variety of cyanine dyes synthesized for covalent modification of biomolecules biomolecule. This allows the cyanine dye to report when it is bound to protein, DNA, or RNA and also allows for fluorescence-based imaging of whole cell extracts to determine the cellular location of biomolecules of interest. Initially, the cyanine dyes were convalently tethered to the biomolecule using two different reactive coupling moieties: the isothiocyanato group and a n- hydroxysuccinimide group. These groups are known to react with amino groups of proteins to make either a stable thiourea linkage or amide linkage. They were synthetically introduced to the center of several heptamethine cyanine dyes on either the top or bottom side as can be seen in Figure 3.2. 2-8 Additionally, a Stokes shift of around 20nm allows for the discrimination between scattered excitation light and fluorescence when using standard interference filters. 9 61 Non-covalent methods based on dispersion and hydrogen bond interactions 10 were also utilized for labeling antibodies, 11 human serum albumin, 12,13 bovine serum albumin, 14 and DNA (Figure 3.3). 4,11,15-18 Non-covalent labeling was faster than covalent labeling and took place in physiological pH and showed preference for hydrophobic sites. Covalent labeling uniformly labeled all amino groups at hydrophobic and hydrophilic sites and was also more stable and robust. 19 A pH-sensitive cyanine dye system can be created by hydration of the dye (Figure 3.4A). This compound is in equilibrium with the keto-form. In acidic solutions, the dye-OH dominates and the maximum absorbance is in the NIR range. In neutral or basic solutions, the keto form dominates and the maximum absorbance is much lower, around 500nm. 1,6,11 Two cyanine dyes can be covalently linked together either through a central alkyl or aryl linker or a indolium alkyl or PEG linker (Figure 3.4A and 3.4B). These bis-cyanine dyes reduced background fluorescence that was caused by intramolecular aggregation, but they tend to have lower extinction coefficients and low fluorescence quantum yields. 20-22 N + X R N X R Z N R R’ Y - X = C(CH 3 ) 2 , S, Se Y = ClO 4 - , BF 4 - , I - Z = Cl, Me, Ph, CO 2 HPh, MeO, MeNH, PhO, PhS, CO 2 HCH 2 S, P(Ph) 2 , 4-H 2 NPhS, 4-H 2 NPhNH, R = n-Bu, Et, (CH 2 ) 4 SO 3 - R’ = H, Cl N O Figure 3.3. Representation of the variety of cyanine dyes synthesized for non-covalent modification of biomolecules 62 Heptamethine cyanine dyes have also been used for the ratiometric determination of certain metal ions including calcium, lithium, copper, and palladium 23 using ortho-hydroxy- carboxy functionality for metal chelation (Figure 3.5B), 24 benzo-15-crown-5 moiety for measuring intracellular calcium concentrations and other metal ion contaminants in water sources (Figure 3.5A), 25,26 and a spirocyclic-opening reaction induced by the binding of Cu 2+ followed by accompanied photo-induced transfer (PET) process of the cyanine dye to metal-ligand centers (Figure 3.5C). 27 Heptamethine cyanine dyes have been shown to accumulate preferentially in cancer cells. 28 The dyes tend to concentrate in mitochondria and lysosomes. The cancer targeting effect N + Me Me R N R Me Me OH N Me Me R N R Me Me O N R R’ R = n-Bu, Et, (CH 2 ) 4 SO 3 - R’ = H, Cl N + Me Me N n-Bu Me Me Cl Linker N Me Me N + Me Me Cl n-Bu Linker = (CH 2 ) n , n = 4, 6, 8 PEG n , n = 1, 3 N + Me Me N n-Bu Me Me X N Me Me N + Me Me X n-Bu n-Bu n-Bu Linker X = NH, O, S Linker = Ph, (CH 2 ) n , n = 4, 8, 12 Figure 3.4. (A) pH sensitive cyanine dyes. (B) & (C) Dimeric cyanine dyes with alkyl, aryl, and PEG type linkers A B C 63 may be due to tumor cells having higher mitochondria membrane potentials. Cyanine dyes with targeting specific for lysosomes, mitochondria, and nuclei separately have all been synthesized. Polyamines have been shown to target the nucleus of cells, and so cyanine dyes modified with at least two polyamine chains showed selective labeling (Figure 3.6A). 29 Dyes modified with alkyl- triphenylphosphine have been shown to specifically target the mitochondria of cancer cells and display mild anticancer properties (Figure 3.6A). 30 Finally, specific lysosomal targeting can be achieved with cyanine dye that was synthesized in a dual effort to also increase the Stokes shift (Figure 3.6B). 31 This dye has an unprecedented Stokes shift of up to 165nm leading to almost no background fluorescence or light scattering in the range of measurement. N + Me Me Me N Me Me Me NH BF 4 - O O O O O N + N I - HN N O N H HN N + Me Me Me N Me Me Me CF 3 COO - HO COOH HOOC OH Cl N + Me Me N Me Me O ClO 4 - R 1 R 2 R 3 R 1 R 2 R 3 OH COOH H H OH COOH H COOH OH Figure 3.5. (A) Cyanine dye with a crown ether for detecting metal cations. (B) Cyanine dye with a Cu 2+ detecting lactam. (C) Cyanine dyes with hydroxy and carboxy groups for metal binding A B C 64 3.1.2. Applications of heptamethine cyanine dyes in drug discovery and drug delivery Some heptamethine cyanine dyes have anticancer properties as well. Photodynamic light therapy with cyanine dye seen in Figure 3.7 has been shown to be effective when using nanocapsules to increase the cellular uptake of the dye. 32 Additionally, dyes with pyridines on the ends have been shown to be antitumor against HELA cells. 33 Also, dyes with triphenyl phosphine have anticancer properties (Figure 3.6A). 30 N + Me Me N Me Me O O P + Ph Ph Ph N + Me Me N Me Me - O 3 S SO 3 - Na + O R O R H N H N N H NH 2 R = HN N + N N O O N O Figure 3.6. (A) Cyanine dyes modified with polyamines for nucleus targeting and triphenylphosphine for mitochondria targeting. (B) A cyanine dye that has a larger Stokes shift and lysosomal targeting B A 65 N N + HN Me Me Me Me OC 4 H 9 O OC 4 H 9 O Br- O NH CbzHN O HN NH O H 2 N NH O HN O O H 3 CO HN O N Doxorubicin 44 10 9 Figure 3.8. Drug-polymer-dye conjugate. Recently, cyanine dyes have been used to aid or monitor drug delivery methods. First, Yan and coworkers used chemical conjugation to connect Doxorubicin to a hydrophobic aminocyanine dye and a triblock copolypeptide via hydrazine and amide bonds (Figure 3.8). The pH sensitive hydrazone bond is hydrolyzed under acidic conditions and leads to the activation of the originally dormant drug delivery systems. The cyanine dye allows for the drug-polymer separation process to be conveniently and clearly observed during the incubation period. 34 After, Schnermann and coworkers use a cyanine dye as a non-phototoxic light-triggered cargo delivery molecule itself (Figure 3.9A). 35 Upon irradiation at or near the maximum N + N SO 3 - SO 3 - Cl N + Me Me N Me Me O ClO 4 - Figure 3.7. (A) Cyanine dye that is encased in a nanocapsule for light therapy applications. (B) A cyanine dye that displays antitumor properties. B A 66 absorbance of the cyanine dye-drug conjugate, the dye breaks down in the presence of oxygen and releases whatever is attached to it at the center point. The cargo spontaneously self immolates to release a reporter molecule that fluoresces, signaling the successful degradation of the dye carrier and release of active compound. The mechanism is explained in section 3.3. Additionally, Schnermann and coworkers designed a dual release cyanine dye carrier by attaching an antibody and a drug to it (Figure 3.9B). 36,37 N + Me Me N Me Me - O 3 S SO 3 - Na + N N Me O O Drug N + Me Me N Me Me - O 3 S SO 3 - Na + N N Me O O Drug N N N mAb N N + - O 3 S SO 3 - Na + Y LINKER CARGO Photoactivation w/ Near-IR Light CARGO Release of Active Drug LINKER DYE Figure 3.9. (A) Cyanine dye-drug conjugate that releases cargo upon irradiation with NIR light. (B) Cyanine dye-drug-antibody conjugates. B A 67 Scheme 3.1. Cyanine dye-drug conjugate with peroxide sensing boronic ester drug delivery trigger. N + N + - O 3 S - O 3 S O B O O O O O O N N O O H 2 O 2 N + N + - O 3 S - O 3 S O - O O O O N N O O HO O N N O O N + N + - O 3 S - O 3 S O H 2 O N N + - O 3 S - O 3 S O OH Then, Shabat and coworkers used a cyanine dye complexed with a boronic ester trigger and the drug camptothecin (Scheme 3.1). 38 The boronic ester trigger is cleaved when it comes in contact with a reactive oxygen species such as hydrogen peroxide that is overproduced in various cancer cell types. Once cleaved, the dye-phenol triggers both the release of the active camptothecin drug and the creation of QCy7, a fluorescent cyanine dye that can signal that the drug has been released. There is no innate fluorescence before the drug is released and before exposure to hydrogen peroxide. The percent drug released measured by HPLC corresponded linearly with measured fluorescence and was used to treat a glioblastoma cell line effectively. 68 Figure 3.10. Cyanine dye structure examples and their maximum absorbances. N + N SO 3 - SO 3 Na Cl N + N SO 3 - SO 3 Na Cl Cl N + N SO 3 - SO 3 Na IR-1010 N + N SO 3 - SO 3 - Cl N + N SO 3 - SO 3 Na Cl Cl N + N SO 3 - SO 3 Na IR-1030 IR-783 IR-806 IR-820 IR-840 3.2. Cyanine dye synthesis and relationship between structure and absorbance Interestingly, small changes to the structure of this cyanine dye can affect the maximum absorbance of the compound. As can be seen in Figure 3.10, the dyes with a central five- membered ring have consistently higher maximum absorbances compared to those with six- membered rings. Additionally, extending the pi-conjugated system by adding another aromatic ring on either end of the dye increases the maximum absorbance 30-40nm (IR-820 and IR-840). 69 Scheme 3.2. Synthesis of IR-820 cyanine dye. N + Me - O 3 S Me Me N SO 3 - Me Me Cl N + Me Me Me SO 3 - N Me Me O S O O N N H Cl O NH 2 N H O + + + + 140°C, neat 87% 1. DMF, POCl 3 , 0°C, 30min 2. Cyclohexanone, reflux 3. Aniline/EtOH (1:1), rt, 30min 4. H 2 O, HCl, 73% (AcO) 2 , EtOH, NaOAc reflux, 55% 3.1 3.2 3.3 The location of this extra benzene ring matters as can be seen when comparing IR-820 with IR-1010 and IR-840 with IR-1030. All of these fluctuations can allow the dye to be applied for many systems that may require different wavelengths of light to release attached cargo, making the system diverse and versatile. An example of one parent near-infrared (NIR) dye compound can be synthesized in three steps as illustrated in Scheme 3.2 with the first being the N-alkylation of 1,1,2- 70 Scheme 3.3. Self-immolating drug or reporter release mechanism. O N O N O O O NH O O NH Trigger Reporter Reporter O N O NH O O NH O O NH Reporter Reporter OH O O NH O O NH Reporter Reporter N N O O O O NH Reporter CO 2 NH 2 Reporter H 2 O OH O O NH Reporter HO CO 2 NH 2 Reporter O HO H 2 O OH HO HO trimethylbenz[e]indole with 1,4-butane sultone to yield zwitter ion 3.1. Hydrochloride salt 3.2 is synthesized using a Vilsmeier-Haak reaction starting with DMF and POCl 3. Cyclohexanone is added and the reaction is heated to reflux and then quenched with an aniline/ethanol mixture. Acidifcation of the solution will yield hydrochloride salt 3.2 as a dark purple solid. Indole 3.1 and intermediate 2 are then combined and heated to reflux in ethanol to yield the symmetrical dye compound IR-820 (3.3). 3.3. BP114: A Proof of Concept Compound The idea that using a compound that can be selectively triggered to release an active drug compound is an attractive theory for the field of drug discovery. The ability to have complete control over where and when the drug is released in the body would be very attractive for any drug therapy. Shabat and coworkers in 2003 reported such a system that uses a trigger to initiate a sequence of self-immolative reactions that lead to spontaneous release of reporter molecules 71 Scheme 3.4. Synthesis of BP-114 proof of concept compound from IR-820 cyanine dye. MeN MeN O Cl N + Me Me N - O 3 S SO 3 - Me Me MeN O O O Me O N + Me Me N - O 3 S SO 3 - Me Me MeN N + Me Me N - O 3 S SO 3 - Me Me MeN MeHN N + Me Me N - O 3 S SO 3 - Me Me Cl DIPEA, DMF, rt, 80min 91% MeHN NHMe Cl O Cl DIPEA, ACN, rt, 1.5h O O HO Me DIPEA, DMAP, ACN, rt, 24h 33% 3.3 3.4 3.5 3.6 (Scheme 3.3). 39 This cause and effect concept can allow for deeper understanding of cellular functions if a single specific event causes the release of a reporter molecule. It can also allow for targeted drug release in the presence of certain specific traits, whether it be enzymatic, pH, or any other trigger. A proof of concept compound was synthesized by adapting a published procedure. 35 Parent NIR-dye compound 3.3 was substituted using N,N’-dimethylethylenediamine in DMF at room temperature to yield dye-amine 3.4 (Scheme 3.4). Upon treatment with triphosgene, amino-acyl chloride 3.5 is formed and used directly without purification in the following reaction with 4-methylumbelliferone to yield dye-fluorophore 3.6. 72 Scheme 3.5a. The mechanism of release of cargo begins with excitation of an electron in the cyanine dye conjugated system which upon energy transfer makes the more reactive singlet oxygen. N 3.6 N + Me Me N - O 3 S SO 3 - Me Me MeN MeN O O O Me O hn 800nm N + Me Me SO 3 - SO 3 - Me Me MeN MeN O O O Me O 3 O 2 1 O 2 N + Me Me N SO 3 - SO 3 - Me Me MeN MeN O O O Me O + O O O O 3.6* 3.6 3.7a 3.7b The theoretical mechanism of release is illustrated in Scheme 3.5. Upon irradiation with near-infrared light, energy is transferred from the excited state of dye compound 3.6 to triplet oxygen, forming the more reactive singlet oxygen (Scheme 3.5a). Singlet oxygen can then form dioxetane intermediates 3.7a and 3.7b that thermally decompose to ketones 3.8a and 3.8b (Scheme 3.5b). Subsequent hydrolysis to alcohols 3.9a and 3.9b and rapid intramolecular cyclization of 3.10 provide byproduct 3.11 and the fluorescent coumarin (3.12, Scheme 3.5c). 73 N + Me Me SO 3 - MeN MeN O O O Me O O N + Me Me O SO 3 - MeN MeN O O O Me O 3.8a 3.8b N + Me Me SO 3 - O N + Me Me O SO 3 - OH OH 3.9a 3.9b MeHN MeN O O O Me O 3.10 Scheme 3.5b. Oxidative cleavage leads to either aldehyde 3.8a or ketone 3.8b which upon hydrolysis liberates the cargo with the amine linker intact. MeN NMe O + OH O Me O MeHN MeN O O O Me O 3.10 3.11 3.12 Scheme 3.5c. Self immolation of the amine linker leads to release of the now fluorescent coumarin compound. 74 0 25 50 75 100 125 150 175 200 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 Time (minutes) % Change Absorbance (735 nm) from BL % Absorbance (738 nm) % Fluorescence (460/510nm) % Change Fluorescence (460/510 nm) from BL Figure 3.11. Upon irradiation with NIR light, the cyanine dye breaks down leading to a decrease in absorbance at 735nm and release of the coumarin compound gives an increase in fluorescence specific to that coumarin. The fluorescence of the coumarin is dependent on the ability of the phenol hydroxyl group to have a negative charge, which is not possible in the bound formation with the NIR dye. Once it is released, it can now fluoresce, giving quantitative feedback on the effectiveness of cargo release upon irradiation (Figure 3.11). As the NIR dye breaks down into smaller decomposition products, the NIR absorbance disappears. At the same time, the fluorescence of the coumarin goes up, indicating effective cargo release. 3.4. The Role of the Linker Substitution of the dye with different linker compounds can affect the maximum absorbance and the reactivity of the entire molecule. The maximum absorbance of the compound is important because the higher the wavelength, the lower the energy of light required to release any attached cargo, making it safer. Additionally, the higher the wavelength, the better tissue and bone penetration of the light allowing for more accurate control over cargo 75 Scheme 3.6. Preparation of a variety of dye-linker compounds. N N + Me Me Me Me - O 3 S SO 3 - Cl l max = 820nm 3.3 DIPEA, DMF, rt 91% MeHN X 3.4, n = 1, X = NHMe, 80min, 91%, lmax = 778nm 3.13, n = 2, X = NHMe, 80min, 92%, lmax = 734nm 3.14, n = 1, X = OH, 7h, 88%, lmax = 710nm 3.15, n = 2, X = OH, 16h, 86%, lmax = 715nm N N + MeN Me Me Me Me - O 3 S SO 3 - X n n release. With different chain lengths and the addition of heteroatoms comes the possibility to change the rate of the self-immolative drug release due to the formation of a different heterocyclic byproduct in each case. Because of the importance of maximum absorbance and the potential for derivatization of dye-drug conjugates, investigating the role of the linker is of merit. Using N,N’-dimethylethylenediamine as a starting point, the most simple deviations are lengthening the carbon chain from 2-3 carbons and changing one of the secondary amines to an alcohol (Scheme 3.6). All four dye-linkers can be made in great yield using the same conditions with varying reaction times. For diamine linkers, the longer the carbon chain, the lower the maximum absorbance of the entire compound. However, this does not seem to apply to the oxygen-containing linkers, which are overall lower. The oxygen-containing linkers are less reactive in the upcoming synthetic steps used for drug-cargo attachment to the dye, but once the cargo is attached, the maximum absorbance goes up, the opposite trend when compared to amine linkers. This can be seen in Scheme 3.8 when attaching carboxylic acid-containing molecules. The method was first explored using 4-phenylbutyric acid but can easily be adapted to any carboxylic acid-containing drug. 76 Scheme 3.7. Synthesis of dye-linker compounds activated for carboxylic acid attachment. N N + MeN Me Me Me Me - O 3 S SO 3 - X O O R Cl N N + MeN Me Me Me Me - O 3 S SO 3 - X Cl O O Cl Me DIPEA, ACN, 40°C, 16h 3.4, X = NHMe, lmax = 778nm 3.16, X = OH, lmax = 710nm 3.17, X = NMe, R = Me, lmax = 750nm 3.18, X = NMe, R = iPr, lmax = 747nm 3.19, X = O, R = Me, lmax = 795nm 3.20, X = O, R = iPr, lmax = 802nm 3.5. Functionalization with Carboxylate Salts The proof of concept compound demonstrates the ability of the dye to breakdown and release phenol-containing compounds upon irradiation with NIR light. In order to begin to apply this delivery method for actual drug compounds, it is important for there to be a variety of synthetic methods to choose from that allow for the synthetic attachment of a variety of drugs. In addition to phenols, it would be useful to be able to conjugate the cyanine dye with carboxylic acid or carboxylate salt containing compounds such as anti-inflammatory lipids. Dye-linker compounds 3.4 and 3.16 were synthesized as previously described and treated with either 1- chloroethyl chloroformate or 1-chloro-2-methylpropyl chloroformate to yield intermediates 3.17-3.20 that were used directly in the following step without isolation or purification. Interestingly, the 2-(methylamino)ethanol linker lowered the maximum absorbance of the dye compound over 100nm when going from dye-Cl to dye-linker, a much larger shift than with N,N- dimethylethylenediamine linker. However, when activated with the chloroformate compounds, the maximum absorbances goes back up to well above those of the amine linker counterparts. 77 Scheme 3.8. Activated dye-linker compounds react with 4-phenylbutyric acid in the presence of cesium carbonate and sodium iodide in acetonitrile. N N + MeN Me Me Me Me - O 3 S SO 3 - X O O R O O N N + MeN Me Me Me Me - O 3 S SO 3 - X O O R Cl OH O Cs 2 CO 3 , NaI, ACN, 40°C, 24h 3.17, X = NMe, R = Me, lmax = 750nm 3.18, X = NMe, R = iPr, lmax = 747nm 3.19, X = O, R = Me, lmax = 795nm 3.20, X = O, R = iPr, lmax = 802nm 3.21, X = NMe, R = Me, lmax = 745nm, 23% 3.22, X = NMe, R = iPr, lmax = 742nm, 19% 3.23, X = O, R = Me, lmax = 801nm, 14% 3.24, X = O, R = iPr, lmax = 800nm, 8% After the dye-linker is activated with the desired chloroformate compound, the desired carboxylate salt can be made from the acid by mixing the carboxylic acid with cesium carbonate in acetonitrile at room temperature for five minutes (Scheme 3.9). The activated dye is charged with sodium iodide and then the carboxylate salt mixture and heated to 40°C for 24 hours. The corresponding dye conjugates were isolated and purified by reverse phase chromatography in relatively low yields. The diamine linker being more reactive afforded better conversion of product with both chloroformates, the more sterically hindered chloroformate being consistently lower for both diamine linker and ethanol linker. The OH of the other linker appears to be rather unreactive and only afforded very low yields of the desired products after purification, but the maximum absorbance of the oxygen-containing linkers were consistently 50-60nm higher than those of the amine linker. The cause of this is unknown. A marked correlation between the number of byproducts and the equivalents of cesium carbonate led us to find a way to make the carboxylate salt beforehand in order to eliminate the use of cesium carbonate. Stirring 4-phenylbutyric acid in a mixture of water and ethanol in the 78 Scheme 3.9. Carboxylate salt with potassium counterion works well only with amine linker and methyl-chloride. N O O N N + MeN Me Me Me Me - O 3 S SO 3 - MeN O O Me O O N + MeN Me Me Me Me - O 3 S SO 3 - MeN Me Cl O O - K + NaI, ACN, 40°C, 16h 63% 3.17 3.21 presence of KOH led to the formation of a potassium carboxylate salt compound that was isolated and dried thoroughly before use. After activation of the dye-linker compound with 1- chloromethyl chloroformate, the reaction mixture was charged with sodium iodide and the potassium salt compound and heated to 40°C overnight (Scheme 3.10). Remarkably, the isolated and purified yield of the desired product was 63% when the cesium carbonate reaction only yielded 23%. In an unprecedented and surprising twist, these conditions did not result in any conversion to the desired product when activating the dye with the more sterically hindered 1- chloro-2-methylpropyl chloroformate. With suitable reaction conditions in hand, focus was shifted to making a proof of concept compound to validate that this type of linkage to the dye has the potential to self-immolate and release the cargo upon irradiation with NIR light. In an attempt to model the 4-phenylbutyric acid used for method development, a derivative of dansyl chloride was synthesized in one step with the addition of 4-(Methylamino)butyric acid to dansyl chloride in the presence of Hunig’s base and a catalytic amount of DMAP in acetonitrile (Scheme 3.11). Completion of reaction was 79 Scheme 3.11. Preparation of cyanine dye-dansyl conjugate. MeN Me Me O MeN N N + Me Me - O 3 S SO 3 - MeN O O O Me O N Me S O O NMe 2 N N + MeN Me Me Me Me - O 3 S SO 3 - MeN O O Me Cl NMe 2 S O O OH Cs 2 CO 3 , NaI, ACN, 40°C, 24h 10% 3.17 3.26 Scheme 3.10. Preparation of dansyl carboxylic acid compound. MeHN O NMe 2 S O O MeN O OH NMe 2 S O O Cl OH DIPEA, DMAP(cat.), ACN rt, 1h 3.24 monitored by TLC with the carboxylic acid as the limiting reagent so that the final reaction mixture can be added directly to the following reaction. Once the reaction was complete, dansyl conjugate 3.25 was charged with cesium carbonate and added directly to a mixture of cyanine dye 3.17 and sodium iodide in acetonitrile (Scheme 3.12). After 24 hours of heating at 40°C, the product was isolated and purified to yield 10% of the desired compound 80 3.6. Conclusions In conclusion, cyanine dyes have been used extensively as reporter molecules and diagnostic tools in the past but have recently been adapted to serve as light-based drug carriers that can release attached molecules in a selective and targeted manner using near-IR light. IR- 820, a symmetrical cyanine dye that has a maximum absorbance at 820nm and is structurally- based on the FDA-approved diagnostic indocyanine green, was functionalized with a variety of cargo compounds using newly-developed synthetic methodologies. The cargo is delivered when the dye decomposes after it is irradiated with near-IR light, releasing a linker-cargo compound that self-immolates and releases the intact cargo. The novel synthetic methods permit for the synthetic attachment of amine-, alcohol-, and carboxylate-containing molecules, allowing for easy diversification of this drug-delivery system to accommodate many other drugs in the future. 81 3.7. Experimental 4-(1,1,2-trimethyl-1H-benzo[e]indol-3-ium-3-yl)butane-1-sulfonate (3.1) 1,1,2-Trimethylbenz[e]indole (4.123g, 19.7mmol) and 1,4-butane sultone (2.015mL, 19.7mmol) were combined in an oven-dried 50mL round bottom flask and heated to 120°C. After two hours, the reaction mixture had solidified and was cooled to room temperature. The reaction mixture was charged with 30mL of ethyl ether, and the solid was broken up with a spatula. The solid was collected by vacuum filtration and was washed with copious amounts of ethyl ether (200mL). The product was dried overnight in the vacuum oven to yield a light blue solid (6.4365g, 95%). 1 H NMR (400 MHz, DMSO-d 6) δ 10.96 (s, 1H), 10.02 (d, J = 13.3 Hz, 0H), 8.20 (s, 1H), 7.57 (d, J = 8.2 Hz, 1H), 7.51 – 7.36 (m, 2H), 7.37 (d, J = 5.7 Hz, 1H), 7.38 – 7.28 (m, 2H), 7.28 (t, J = 8.4 Hz, 2H), 7.21 – 7.05 (m, 1H), 4.58 (s, 1H), 4.15 (s, 2H), 2.97 (s, 1H), 2.91 (s, 1H). N-[(3-(anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline hydrochloride (3.2) N,N’-Dimethylformamide (3.7mL, 48mmol) was placed in an oven dried 100mL round bottom flask, placed under Argon, and cooled to 0°C. The DMF was charged dropwise with phosphorus 82 oxychloride (3.22mL, 34.5mmol) and stirred for 30 minutes at 0°C. Cyclohexanone (1.56mL, 15mmol) was added and the reaction mixture was refluxed for 1 hour. After cooling to room temperature, the flask was charged dropwise with 50mL of a 1:1 mixture of aniline and ethanol and stirred at room temperature for 30 minutes. The red solution was then poured into a flask with cold 2M HCl(aq) and was refrigerated for several hours. A purple solid was collected by vacuum filtration and washed with ice cold water. The solid was dried for several hours in a vacuum oven to yield a dark purple powdery solid (4.322g, 89%). 1 H NMR (400 MHz, DMSO-d 6) δ 11.52 (s, 3H), 10.38 (d, J = 13.8 Hz, 1H), 10.13 (s, 0H), 8.49 (s, 3H), 8.08 (d, J = 13.1 Hz, 1H), 7.63 (d, J = 7.9 Hz, 5H), 7.42 (t, J = 8.2 Hz, 8H), 7.23 (tt, J = 20.7, 10.2 Hz, 9H), 7.02 (dt, J = 14.1, 7.2 Hz, 2H), 2.74 (d, J = 6.6 Hz, 6H), 2.54 (t, J = 6.0 Hz, 2H), 2.34 – 2.27 (m, 1H), 1.83 – 1.76 (m, 4H), 1.62 (s, 1H), 1.18 (dt, J = 14.0, 7.0 Hz, 1H). IR-820 (3.3) Indole 3.1 (0.259g 0.802mmol), dianiline 3.2 (1.108g, 3.206mmol), and sodium acetate (0.1604g, 1.96mmol) were combined in an oven-dried 50mL flask and placed under Argon. The reagents were dissolved in 200 proof ethanol (10mL) and heated to reflux for 5 hours. The now blue-green solution was cooled to room temperature and concentrated under reduced pressure. The residue was dry loaded onto celite and purified by reverse phase column chromatography on a 83 C18 column with the product eluting in 60% acetonitrile in water. The relevant fractions were combined and concentrated under reduced pressure and transferred to an 8-dram vial with a minimal amount of DMF. The vial was filled with ethyl ether (25mL) and cooled in a refrigerator overnight. The vial was rewarmed to room temperature, and the top ether layer was removed with a pipette while leaving the crystallized solid at the bottom of the container. More fresh ether was added to the container with the green solid, and the solid was allowed to settle to the bottom. The ether layer was removed again once the solid settled to the bottom of the container. Subsequent washing with ether and removal of the ether was performed until the ether layer is clear. The solid was dried under a constant stream of dry nitrogen to afford IR-820 as a dark green powdery solid (0.396g, 60%). Drying the solid was only performed directly before usage of the compound. The product was stored in an 8-dram vial filled with ether to separate the solid at the bottom from the moisture and oxygen in the air. LC/MS analysis: λ max (820 nm), observed [M+(-H)] = 827 [M-(-H)] = 825. 1 H NMR (600 MHz, Methanol-d 4) δ 8.44 (d, J = 14.0 Hz, 2H), 7.51 (dd, J = 7.5, 1.1 Hz, 2H), 7.47 (dd, J = 7.5, 1.1 Hz, 2H), 7.43 (ddd, J = 8.4, 7.3, 1.2 Hz, 2H), 7.40 – 7.35 (m, 4H), 7.28 (td, J = 7.4, 1.0 Hz, 2H), 6.34 (d, J = 14.0 Hz, 2H), 4.22 (t, J = 7.4 Hz, 4H), 2.89 (t, J = 7.2 Hz, 4H), 2.76 (t, J = 6.2 Hz, 4H), 2.06 – 1.99 (m, 4H), 1.95 (tdd, J = 14.2, 6.5, 1.9 Hz, 4H), 1.74 (s, 12H). 84 IR-820-linker (3.4) To a solution of IR-820 (3.3, 0.0473 g, 0.0560 mmol) in DMF (4 mL) in an 8-dram vial was added N,N’-Diisopropylethylamine (0.030 mL, 0.167 mmol) and N,N’-dimethylethylenediamine (0.018 mL, 0.167 mmol). The reaction mixture was stirred at room temperature for 80 minutes, and the completion of reaction was confirmed by LC/MS. The reaction mixture was charged with approximately 25mL of ethyl ether, filling the 8-dram vial, and was cooled to 4°C for several hours. The vial was rewarmed to room temperature, and the top ether layer was removed with a pipette while leaving the crystallized solid at the bottom of the container. More fresh ether was added to the container with the blue solid, and the solid was allowed to settle to the bottom. The ether layer was removed again once the blue solid settled to the bottom of the container. Subsequent washing with ether and removal of the ether was performed until the ether layer is clear. The solid was dried under a constant stream of dry nitrogen to afford IR-820-linker as a dark blue solid (0.039 g, 91%, correcting for IR-820 because it contains 80% dye). Drying the solid was only performed directly before usage of the compound. The product was stored in an 8- dram vial filled with ether to separate the blue solid at the bottom from the moisture and oxygen in the air. LC/MS analysis: λ max (770 nm), observed [M+(-H)] = 879 [M-(-H)] = 877. 1 H NMR (600 MHz, Methanol-d 4) δ 8.44 (d, J = 14.0 Hz, 2H), 7.52-7.45 (m, 4H), 7.40 – 7.35 (m, 4H), 7.28 (td, J = 7.4, 1.0 Hz, 2H), 6.34 (d, J = 14.0 Hz, 2H), 4.22 (t, J = 7.4 Hz, 4H), 3.12 (m, 4H), 2.89 (t, J = 7.2 Hz, 4H), 2.76 (t, J = 6.2 Hz, 4H), 2.44 (d, J = 1.6 Hz, 3H), 2.06 – 1.99 (m, 4H), 1.95 (tdd, J = 14.2, 6.5, 1.9 Hz, 4H), 1.74 (s, 12H). 85 IR-820-linker-OCl (3.5) To a solution of 3.4 (0.0246 g, 0.028 mmol) in acetonitrile (12mL) was added N,N’- diisopropylethylamine (0.0146 mL, 0.084 mmol). Triphosgene in acetonitrile (0.0033 g, 0.0112 mmol) was added dropwise over several minutes, and reaction stirred at room temperature for 1.5 hours. LC/MS analysis confirmed the complete conversion to 3.5, observed [M - ] 939/940 (MW of 3.5: 963.62). 3.5 was used in the next step without purification. IR-820-linker-coumarin (BP-114) (3.6) To a solution of 3.5 (0.027 g, 0.028 mmol) in acetonitrile (12 mL) was added N,N’- diisopropylethylamine (0.0146 mL, 0.084 mmol) and 4-methylumbelliferone (0.0148 g, 0.084 mmol). Reaction was stirred at room temperature until LC/MS analysis confirmed the disappearance of 3.5 (12h). Mixture was added to ether and cooled to afford crystallization of 86 the product. Subsequent washing with ether afforded 3.6 as a blue solid (0.010 g, 33%). LC/MS analysis: λ abs (738 nm), observed [M - ] 1079 (MW of 3.6: 1103.33). IR-820-LL-NHMe (3.13) To a solution of IR-820 (3.3, 0.0403 g, 0.0474 mmol) in DMF (4 mL) in an 8-dram vial was added N,N’-Diisopropylethylamine (0.025 mL, 0.142 mmol) and N,N’-dimethyl-1,3-propanediamine (0.018 mL, 0.142 mmol). The reaction mixture was stirred at room temperature for 80 minutes, and the completion of reaction was confirmed by LC/MS. The reaction mixture was charged with approximately 25mL of ethyl ether, filling the 8-dram vial, and was cooled to 4°C for several hours. The vial was rewarmed to room temperature, and the top ether layer was removed with a pipette while leaving the crystallized solid at the bottom of the container. More fresh ether was added to the container with the blue solid, and the solid was allowed to settle to the bottom. The ether layer was removed again once the blue solid settled to the bottom of the container. Subsequent washing with ether and removal of the ether was performed until the ether layer is clear. The solid was dried under a constant stream of dry nitrogen to afford IR-820-LL-NHMe as a dark blue solid (0.039 g, 92%). Drying the solid was only performed directly before usage of the compound. The product was stored in an 8-dram vial filled with ether to separate the blue solid 87 at the bottom from the moisture and oxygen in the air. LC/MS analysis: λ max (734 nm), observed [M+(-H)] = 893 [M-(-H)] = 891. IR-820-linker-OH (3.14) To a solution of IR-820 (0.1133 g, 0.133 mmol) in DMF (7 mL) in an 8-dram vial was added N,N’- Diisopropylethylamine (0.070 mL, 0.4 mmol) and 2-(Methylamino)ethanol (0.032 mL, 0.4 mmol). The reaction mixture was stirred at room temperature for 7 hours, and the completion of reaction was confirmed by LC/MS. The reaction mixture was charged with approximately 25mL of ethyl ether, filling the 8-dram vial, and was cooled to 4°C for several hours. The vial was rewarmed to room temperature, and the top ether layer was removed with a pipette while leaving the crystallized solid at the bottom of the container. More fresh ether was added to the container with the blue solid, and the solid was allowed to settle to the bottom. The ether layer was removed again once the blue solid settled to the bottom of the container. Subsequent washing with ether and removal of the ether was performed until the ether layer is clear. The solid was dried under a constant stream of dry nitrogen to afford IR-820-linker-OH as a dark blue solid (0.0813 g, 88%, correcting for IR-820 because it contains 80% dye). Drying the solid was only performed directly before usage of the compound. The product was stored in an 8-dram vial 88 filled with ether to separate the blue solid at the bottom from the moisture and oxygen in the air. LC/MS analysis: λ max (710 nm), observed [M+(-H)] = 866 [M-(-H)] = 864. IR-820-LL-OH (3.15) To a solution of IR-820 (0.0364g, 0.0428mmol) in DMF (4 mL) in an 8-dram vial was added N,N’- Diisopropylethylamine (0.023mL, 0.128mmol) and 3-methylamino-1-propanol (0.013mL, 0.128mmol). The reaction mixture was stirred at room temperature for 16 hours, and the completion of reaction was confirmed by LC/MS. The reaction mixture was charged with approximately 25mL of ethyl ether, filling the 8-dram vial, and was cooled to 4°C for several hours. The vial was rewarmed to room temperature, and the top ether layer was removed with a pipette while leaving the crystallized solid at the bottom of the container. More fresh ether was added to the container with the blue solid, and the solid was allowed to settle to the bottom. The ether layer was removed again once the blue solid settled to the bottom of the container. Subsequent washing with ether and removal of the ether was performed until the ether layer is clear. The solid was dried under a constant stream of dry nitrogen to afford IR-820-LL-OH as a dark blue solid (0.0323g g, 86%). Drying the solid was only performed directly before usage of the compound. The product was stored in an 8-dram vial filled with ether to separate the blue 89 solid at the bottom from the moisture and oxygen in the air. LC/MS analysis: λ max (715 nm), observed [M+(-H)] = 880 [M-(-H)] = 878. IR-820-linker-ECl (3.16) IR-820-linker solid (0.0165g, 0.0188mmol) in an 8-dram vial was charged with Drisolv acetonitrile (2mL) and N,N’-Diisopropylethylamine (13.1 L, 0.0752 mmol) and sonicated until all solid was dissolved. The resulting solution was transferred to an oven-dried 10mL round-bottom flask and placed under Argon. To the blue solution was added a solution of 1-Chloroethyl chloroformate (4.1 L, 0.0376 mmol) in acetonitrile (1mL) in dropwise fashion. The reaction was stirred at room temperature overnight (12h), and the completion of reaction was confirmed by LC/MS. LC/MS analysis: λ max (750/810 nm), observed [M+(-H)] = 985 [M-(-H)] = 983. The reaction mixture was used directly in the next step. IR-820-linker-iPrCl (3.17) 90 IR-820-linker solid (0.0213g, 0.0242mmol) in an 8-dram vial was charged with Drisolv acetonitrile (2mL) and N,N’-Diisopropylethylamine (17 L, 0.0969 mmol) and sonicated until all solid was dissolved. The resulting solution was transferred to an oven-dried 10mL round-bottom flask and placed under Argon. To the blue solution was added a solution of 1-Chloro-2-methylpropyl chloroformate (8 L, 0.0485 mmol) in acetonitrile (1mL) in dropwise fashion. The reaction was stirred at room temperature overnight (12h), and the completion of reaction was confirmed by LC/MS. LC/MS analysis: λ max (747/810 nm), observed [M+(-H)] = 1013 [M-(-H)] = 1011. The reaction mixture was used directly in the next step. IR-820-linker-O-ECl (3.18) IR-820-linker-OH solid (0.0184g, 0.0213mmol) in an 8-dram vial was charged with Drisolv acetonitrile (2mL) and N,N’-Diisopropylethylamine (23 L, 0.128 mmol) and sonicated until all solid was dissolved. The resulting solution was transferred to an oven-dried 10mL round-bottom flask and placed under Argon. To the blue solution was added a solution of 1-Chloroethyl chloroformate (7 L, 0.0638 mmol) in acetonitrile (1mL) in dropwise fashion. The reaction was stirred at 40°C overnight (12h), and the completion of reaction was confirmed by LC/MS. LC/MS analysis: λ max (795 nm), observed [M+(-H)] = 972 [M-(-H)] = 970. The reaction mixture was used directly in the next step. 91 IR-820-linker-O-iPrCl (3.19) IR-820-linker-OH solid (0.0739g, 0.0854mmol) in an 8-dram vial was charged with Drisolv acetonitrile (6mL) and N,N’-Diisopropylethylamine (89 L, 0.513 mmol) and sonicated until all solid was dissolved. The resulting solution was transferred to an oven-dried 25mL round-bottom flask and placed under Argon. To the blue solution was added a solution of 1-Chloro-2- methylpropyl chloroformate (37 L, 0.256 mmol) in acetonitrile (3mL) in dropwise fashion. The reaction was stirred at 40°C overnight (12h), and the completion of reaction was confirmed by LC/MS. LC/MS analysis: λ max (802 nm), observed [M+(-H)] = 1000 [M-(-H)] = 998. The reaction mixture was used directly in the next step. 4-Phenylbutyric acid potassium salt 4-Phenylbutyric acid (0.7688g, 4.68mmol) was dissolved in 5mL of ethanol. Potassium hydroxide (0.2627g, 4.68mmol) was dissolved in 5mL deionized water. The KOH solution was slowly added to the acid solution in an 8 dram vial. The reaction was stirred at room temperature overnight. The mixture was transferred to a 50mL RBF and was concentrated under reduced pressure. The 92 resulting white solid was dried further under reduced pressure in a vacuum oven at 55°C to yield white crystalline product (0.889g, 94%). IR-820-linker-E-PhBA (3.20) The reaction mixture containing IR-820-linker-ECl (~0.0196g, 0.02mmol) in acetonitrile (3mL) was charged with NaI (0.0045g, 0.03mmol) and 4-Phenylbutyric acid, potassium salt (0.0122g, 0.06mmol) in acetonitrile (2mL). The reaction mixture was heated under Argon to 40°C overnight. After 16 hours, completion of reaction was confirmed by LC/MS. The reaction mixture was transferred to an 8-dram vial and charged with approximately 25mL of ethyl ether, filling the 8-dram vial, and was cooled to 4°C for several hours. The vial was rewarmed to room temperature, and the top ether layer was removed with a pipette while leaving the crystallized solid at the bottom of the container. More fresh ether was added to the container with the blue solid, and the solid was allowed to settle to the bottom. The ether layer was removed again once the blue solid settled to the bottom of the container. Subsequent washing with ether and removal of the ether was performed until the ether layer is clear. The solid was then dissolved in methanol, dry-loaded onto celite, and purified by reverse phase column chromatography (30- 100% acetonitrile/water). The product-containing fractions were identified by LC/MS, combined, 93 and concentrated under reduced pressure. The resulting blue residue was dissolved in DMF (4mL), transferred to an 8-dram vial, and charged with approximately 25mL of ethyl ether, filling the 8-dram vial, and was cooled to 4°C for several hours. The vial was rewarmed to room temperature, and the top ether layer was removed with a pipette while leaving the crystallized solid at the bottom of the container. More fresh ether was added to the container with the blue solid, and the solid was allowed to settle to the bottom. The ether layer was removed again once the blue solid settled to the bottom of the container. Subsequent washing with ether and removal of the ether was performed until the ether layer is clear. The solid was dried under a constant stream of dry nitrogen to afford IR-820-linker-E-PhBA as a dark blue solid (0.014 g, 63%). Drying the solid was only performed directly before usage of the compound. The product was stored in an 8-dram vial filled with ether to separate the blue solid at the bottom from the moisture and oxygen in the air. LC/MS analysis: λ max (745 nm), observed [M+(-H)] = 1113 [M-(- H)] = 1111. IR-820-linker-iPr-PhBA (3.21) The reaction mixture containing IR-820-linker-ECl (~0.0906g, 0.0895mmol) in acetonitrile (8mL) was charged with NaI (0.0201g, 0.134mmol) and a solution of Cs 2CO 3 (0.0292g, 0.0895mmol) and 94 4-Phenylbutyric acid (0.0735g, 0.448mmol) in acetonitrile (3mL). The reaction mixture was heated under Argon to 40°C overnight. After 24 hours, completion of reaction was confirmed by LC/MS. The reaction mixture was transferred to an 8-dram vial and charged with approximately 20mL of ethyl ether, filling the 8-dram vial, and was cooled to 4°C for several hours. The vial was rewarmed to room temperature, and the top ether layer was removed with a pipette while leaving the crystallized solid at the bottom of the container. More fresh ether was added to the container with the blue solid, and the solid was allowed to settle to the bottom. The ether layer was removed again once the blue solid settled to the bottom of the container. Subsequent washing with ether and removal of the ether was performed until the ether layer is clear. The solid was then dissolved in methanol, dry-loaded onto celite, and purified by reverse phase column chromatography (30-100% acetonitrile/water). The product-containing fractions were identified by LC/MS, combined, and concentrated under reduced pressure. The resulting blue residue was dissolved in DMF (4mL), transferred to an 8-dram vial, and charged with approximately 25mL of ethyl ether, filling the 8-dram vial, and was cooled to 4°C for several hours. The vial was rewarmed to room temperature, and the top ether layer was removed with a pipette while leaving the crystallized solid at the bottom of the container. More fresh ether was added to the container with the blue solid, and the solid was allowed to settle to the bottom. The ether layer was removed again once the blue solid settled to the bottom of the container. Subsequent washing with ether and removal of the ether was performed until the ether layer is clear. The solid was dried under a constant stream of dry nitrogen to afford IR-820-linker-iPr- PhBA as a dark blue solid. Drying the solid was only performed directly before usage of the compound. The product was stored in an 8-dram vial filled with ether to separate the blue solid 95 at the bottom from the moisture and oxygen in the air. LC/MS analysis: λ max (742 nm), observed [M+(-H)] = 1141 [M-(-H)] = 1139. IR-820-linker-O-E-PhBA (3.22) The reaction mixture containing IR-820-linker-O-ECl (~0.0206g, 0.0213mmol) in acetonitrile (3mL) was charged with NaI (0.0048g, 0.0319mmol) and a solution of Cs 2CO 3 (0.0069g, 0.0213mmol) and 4-Phenylbutyric acid (0.014g, 0.0852mmol) in acetonitrile (1mL). The reaction mixture was heated under Argon to 40°C overnight. After 24 hours, completion of reaction was confirmed by LC/MS. The reaction mixture was transferred to an 8-dram vial and charged with approximately 25mL of ethyl ether, filling the 8-dram vial, and was cooled to 4°C for several hours. The vial was rewarmed to room temperature, and the top ether layer was removed with a pipette while leaving the crystallized solid at the bottom of the container. More fresh ether was added to the container with the blue solid, and the solid was allowed to settle to the bottom. The ether layer was removed again once the blue solid settled to the bottom of the container. Subsequent washing with ether and removal of the ether was performed until the ether layer is clear. The solid was then dissolved in methanol, dry-loaded onto celite, and purified by reverse phase column chromatography (30-100% acetonitrile/water). The product-containing fractions 96 were identified by LC/MS, combined, and concentrated under reduced pressure. The resulting blue residue was dissolved in DMF (4mL), transferred to an 8-dram vial, and charged with approximately 25mL of ethyl ether, filling the 8-dram vial, and was cooled to 4°C for several hours. The vial was rewarmed to room temperature, and the top ether layer was removed with a pipette while leaving the crystallized solid at the bottom of the container. More fresh ether was added to the container with the blue solid, and the solid was allowed to settle to the bottom. The ether layer was removed again once the blue solid settled to the bottom of the container. Subsequent washing with ether and removal of the ether was performed until the ether layer is clear. The solid was dried under a constant stream of dry nitrogen to afford IR-820-linker-O-E- PhBA as a dark blue solid (0.009 g, 38%). Drying the solid was only performed directly before usage of the compound. The product was stored in an 8-dram vial filled with ether to separate the blue solid at the bottom from the moisture and oxygen in the air. LC/MS analysis: λ max (800 nm), observed [M+(-H)] = 1100 [M-(-H)] = 1098. IR-820-linker-O-iPr-PhBA (3.23) The reaction mixture containing IR-820-linker-O-iPrCl (~0.0854g, 0.0854mmol) in acetonitrile (9mL) was charged with NaI (0.0192g, 0.128mmol) and a solution of Cs 2CO 3 (0.0278g, 0.0854mmol) and 4-Phenylbutyric acid (0.0701g, 0.427mmol) in acetonitrile (3mL). The reaction 97 mixture was heated under Argon to 40°C overnight. After 24 hours, completion of reaction was confirmed by LC/MS. The reaction mixture was transferred to an 8-dram vial and charged with approximately 20mL of ethyl ether, filling the 8-dram vial, and was cooled to 4°C for several hours. The vial was rewarmed to room temperature, and the top ether layer was removed with a pipette while leaving the crystallized solid at the bottom of the container. More fresh ether was added to the container with the blue solid, and the solid was allowed to settle to the bottom. The ether layer was removed again once the blue solid settled to the bottom of the container. Subsequent washing with ether and removal of the ether was performed until the ether layer is clear. The solid was then dissolved in methanol, dry-loaded onto celite, and purified by reverse phase column chromatography (30-100% acetonitrile/water). The product-containing fractions were identified by LC/MS, combined, and concentrated under reduced pressure. The resulting blue residue was dissolved in DMF (4mL), transferred to an 8-dram vial, and charged with approximately 25mL of ethyl ether, filling the 8-dram vial, and was cooled to 4°C for several hours. The vial was rewarmed to room temperature, and the top ether layer was removed with a pipette while leaving the crystallized solid at the bottom of the container. More fresh ether was added to the container with the blue solid, and the solid was allowed to settle to the bottom. The ether layer was removed again once the blue solid settled to the bottom of the container. Subsequent washing with ether and removal of the ether was performed until the ether layer is clear. The solid was dried under a constant stream of dry nitrogen to afford IR-820-linker-O-iPr- PhBA as a dark blue solid (0.015 g, 15%). Drying the solid was only performed directly before usage of the compound. The product was stored in an 8-dram vial filled with ether to separate 98 the blue solid at the bottom from the moisture and oxygen in the air. LC/MS analysis: λ max (801 nm), observed [M+(-H)] = 1128 [M-(-H)] = 1126. 4-[[5-(dimethylamino)naphthalen-1-yl]sulfonyl-methylamino]butanoic acid (3.24) Dansyl chloride (0.2918g, 1.08mmol), N,N’-Diisopropylethylamine (0.56mL, 3.21mmol), DMAP (0.0065g, 0.054mmol), and 4-(methylamino)butyric acid HCl (0.1645, 1.07mmol) were combined in an oven-dried 25mL round bottom flask with acetonitrile (8mL) and stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure to yield a yellow waxy solid (0.337g, 90%). IR-820-linker-E-Dansyl (3.23) The reaction mixture containing IR-820-linker-ECl (~0.1103g, 0.112mmol) in acetonitrile (8mL) was charged with NaI (0.0252g, 0.168mmol) and a solution of Cs 2CO 3 (0.0366g, 0.112mmol) and Dansyl-acid (0.118g, 0.337mmol) in acetonitrile (3mL). The reaction mixture was heated under Argon to 40°C overnight. After 24 hours, completion of reaction was confirmed by LC/MS. The 99 reaction mixture was transferred to an 8-dram vial and charged with approximately 25mL of ethyl ether, filling the 8-dram vial, and was cooled to 4°C for several hours. The vial was rewarmed to room temperature, and the top ether layer was removed with a pipette while leaving the crystallized solid at the bottom of the container. More fresh ether was added to the container with the blue solid, and the solid was allowed to settle to the bottom. The ether layer was removed again once the blue solid settled to the bottom of the container. Subsequent washing with ether and removal of the ether was performed until the ether layer is clear. The solid was then dissolved in methanol, dry-loaded onto celite, and purified by reverse phase column chromatography (30-100% acetonitrile/water). The product-containing fractions were identified by LC/MS, combined, and concentrated under reduced pressure. The resulting blue residue was dissolved in DMF (4mL), transferred to an 8-dram vial, and charged with approximately 25mL of ethyl ether, filling the 8-dram vial, and was cooled to 4°C for several hours. The vial was rewarmed to room temperature, and the top ether layer was removed with a pipette while leaving the crystallized solid at the bottom of the container. More fresh ether was added to the container with the blue solid, and the solid was allowed to settle to the bottom. The ether layer was removed again once the blue solid settled to the bottom of the container. Subsequent washing with ether and removal of the ether was performed until the ether layer is clear. The solid was dried under a constant stream of dry nitrogen to afford IR-820-linker-E-Dansyl as a dark blue solid. Drying the solid was only performed directly before usage of the compound. The product was stored in an 8-dram vial filled with ether to separate the blue solid at the bottom from the moisture and oxygen in the air. LC/MS analysis: λ max (666/728nm), observed [M+(-H)] = 1299/1300, [(M+(-H))/2] = 650, [M-(-H)] = 1297/1298. 100 Photolysis Release Activity of BP-114 (3.6) Goals: 1. Determine the photolysis efficiency (dC/dt) of BP-114-6 at room temperature with 850nm light. 2. Determine the efficiency to liberate 4-methylumbelliferone (“coumarin”) cargo (dF/dt). Compounds max Time points (minutes) Evaluation BP-114-6 736nm 0, 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50 60, 75, 90, 120, 135, 150, 165, 180 max, abs/fluor. Steps for Photolysis: 1. BP-114-6 will be dissolved in sufficient MeOH to afford a 2mM stock. a. Mass in vial (mg) ÷ molecular weight × 500 = mL of MeOH 2. 0.25mL of 2mM stock is diluted to a final concentration of 50 µM in with 9.75mL of H 2O. 3. In a 20 mL glass beaker, 10 mL of 50 µM BP-114-6 is irradiated with an 850nm LED. 4. At the time points between 0-180 minutes, 0.5mL will remove to measure absorbance to determine the uncaging of cyanine dye and to measure fluorescence to determine coumarin release after light-triggered uncoupling. a. Time points: 0, 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 75, 90, 120, 135, 150, 165, & 180 minutes 5. From each time point, triplicates of 100µL will be aliquoted into a 96-well plate (Corning black opaque polypropylene for fluorescence and absorbance). The absorbance values of the caged compound ( max = 736nm) will be measured followed by fluorescence (460/510nm). 101 Analysis: After light activation, the time of degradation (dCT/dt) will be the photolysis efficiency. We will also determine the (dU/dt) to determine the cargo release. A correlation between the dC/dt will be compared with dU/dt to determine whether the rate of nanocaged complex correlate with the emergence of cargo release. 3.8. References (1) Henary, M., Mojzych, M., Say, M. & Strekowski, L. Functionalization of benzo[ c,d]indole system for the synthesis of visible and near-infrared dyes. J. Heterocyclic Chem. 2009, 46, 84–87. (2) Lipowska, M., Patonay, G. & Strekowski, L. New Near-Infrared Cyanine Dyes for Labelling of Proteins. Synthetic Communications 1993, 23, 3087–3094. (3) Strekowski, L., Mason, J. C., Lee, H. & Patonay, G. Synthesis of a Functionalized Cyanine Dye for Covalent Labeling of Biomolecules with a pH-Sensitive Chromophore. 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Synthesis, Chromatographic Separation, and Characterization of Near- Infrared Labeled DNA Oligomers for Use in DNA Sequencing. Analytical Chemistry 1995, 67, 247– 251. (10) Patonay, G., Salon, J., Sowell, J. & Strekowski, L. Noncovalent Labeling of Biomolecules with Red and Near- Infrared Dyes. Molecules 2004, 9, 40–49. (11) Mason, J. C., Patonay, G. & Strekowski, L. A New pH-Sensitive Near-Infrared Chromophore. Heterocyclic Communications 1997, 3, 409–411. (12) Sowell, J., Mason, J. C., Strekowski, L. & Patonay, G. Binding constant determination of drugs toward subdomain IIIA of human serum albumin by near-infrared dye-displacement capillary electrophoresis. Electrophoresis 2001, 22, 2512–2517. (13) Sowell, J. et al. Use of non-covalent labeling in illustrating ligand binding to human serum albumin via affinity capillary electrophoresis with near-infrared laser induced fluorescence detection. Journal of Chromatography 2001, 755, 91–99. (14) Kurutos, A. et al. Novel synthetic approach to near-infrared heptamethine cyanine dyes and spectroscopic characterization in presence of biological molecules. Journal of Photochemistry & Photobiology, A: Chemistry 2016, 328, 87–96. (15) Strekowski, L. et al. Further Studies on the Cyclization of Aromatic Azomethines Ortho- Substituted with a Trifluoromethyl Group: Synthesis of 2,4-Di- or 2,3,4-Trisubstituted Quinolines. J. Org. Chem. 1992, 196–201. 103 (16) Song, F. et al. Syntheses, spectral properties and photostabilities of novel water-soluble near-infrared cyanine dyes. Journal of Photochemistry & Photobiology, A: Chemistry 2004, 168, 53–57. (17) Salon, J., Wolinska, E., Raszkiewicz, A., Patonay, G. & Strekowski, L. Synthesis of Benz[e]indolium Heptamethine Cyanines Containing C-Substitutuents at the Central Portion of the Heptamethine Moiety. J. Heterocyclic Chem. 2005, 42, 959–961. (18) Lee, H., Berezin, M. Y., Henary, M., Strekowski, L. & Achilefu, S. Fluorescence lifetime properties of near-infrared cyanine dyes in relation to their structures. Journal of Photochemistry & Photobiology, A: Chemistry 2008, 200, 438–444. (19) Williams, R. J., Lipowska, M., Patonay, G. & Strekowski, L. Comparison of covalent and noncovalent labeling with near-infrared dyes for the high-performance liquid chromatographic determination of human serum albumin. Analytical Chemistry 1993, 65, 601–605. (20) KIM, J., KODAGAHALLY, R., STREKOWSKI, L. & PATONAY, G. A study of intramolecular H- complexes of novel bis(heptamethine cyanine) dyes. Talanta 2005, 67, 947–954. (21) Strekowski, L., Raszkiewicz, A. & Mojzych, M. Facile Synthesis of Dimeric Heptamethine Cyanine Dyes Containing a Linker at the Meso Positions. Heterocyclic Communications 2009, 15, 123–126. (22) Wolinska, E., Henary, M., Paliakov, E. & Strekowski, L. Near-infrared bis(indolium heptamethine cyanine) dyes with a spacer derived from oligo(ethylene glycol). J. Heterocyclic Chem. 2009, 46, 925–930. (23) Su, W. et al. A near-infrared and colorimetric fluorescent probe for palladium detection and bioimaging. Dyes and Pigments 2017, 137, 293–298. 104 (24) Gorecki, T., Patonay, G. & Strekowski, L. Synthesis of Novel Near-Infrared Cyanine Dyes for Metal Ion Determination. J. Heterocyclic Chem. 1996, 33, 1871–1876. (25) Ellis, A. L. et al. Design, synthesis, and characterization of a calcium-sensitive near infrared dye. Talanta 2002, 56, 1099–1107. (26) Tarazi, L. et al. Characterization of a novel crown ether-bearing near-infrared heptamethine cyanine dye. A study of fluorescence quenching by lithium. Microchemical Journal 2002, 72, 55– 62. (27) Xu, Z.-H. et al. A novel ratiometric colorimetric and NIR fluorescent probe for detecting Cu 2+ with high selectivity and sensitivity based on rhodamine-appended cyanine. Sensors & Actuators: B. Chemical 2014, 201, 469–474. (28) Luo, S., Zhang, E., Su, Y., Cheng, T. & Shi, C. A review of NIR dyes in cancer targeting and imaging. Biomaterials 2011, 32, 7127–7138. (29) König, S. G. & Krämer, R. Polyamine-modified near-infrared cyanine dyes for targeting the nuclei and nucleoli of cells. Dyes and Pigments 2017, 145, 80–94. (30) Ning, J.; Huang, B.; Wei, Z.; Li, W.; Zheng, H.; Ma, L.; Xing, Z.; Hiu, H.; Huang, W. Mitochondria targeting and near-infrared fluorescence imaging of a novel heptamethine cyanine anticancer agent. Molecular Medicine Reports, 2017, 15, 3761-3766. (31) Di Wu et al. Naphthalimide-modified near-infrared cyanine dye with a large stokes shift and its application in bioimaging. Chinese Chemical Letters 2017, 28, 1979–1982. (32) Pietkiewicz, J.; Zielinska, K.; Saczko, J.; Kulbacka, J.; Majkowski, M.; Wilk, K.A. New approach to hydrophobic cyanine-type photosensitizer delivery using polymeric oil-cored nanocarriers: 105 Hemolytic activity, in vitro cyclotoxicity and localization in caner cells. European Journal of Pharmaceutical Sciences, 2010, 39, 322-335. (33) Fadda, A. A. & El-Mekawy, R. E. Some studies in cyanine dyes incorporating pyridine rings endowed with pharmaceutical potency. Dyes and Pigments 2015, 118, 45–52. (34) Xing, Tao; Yang, Xianzhu; Wang, Feng; Lai, Bin; Yan, Lifeng. Synthesis of polypeptide conjugated with near-infrared fluorescence probe and doxorubicin for pH-responsive and image- guided drug delivery. J. Mater. Chem., 2012, 22, 22290-22300. (35) Gorka, A. P., Nani, R. R., Zhu, J., Mackem, S. & Schnermann, M. J. A Near-IR Uncaging Strategy Based on Cyanine Photochemistry. J. Am. Chem. Soc. 2014, 136, 14153–14159. (36) Nani, R. R., Gorka, A. P., Nagaya, T., Kobayashi, H. & Schnermann, M. J. Near-IR Light- Mediated Cleavage of Antibody-Drug Conjugates Using Cyanine Photocages. Angew. Chem. 2015, 127, 13839–13842. (37) Nani, R. R.; Gorka, A. P.; Nagaya, T.; Yamamoto, T.; Ivanic, J.; Kobayashi, H.; Schnermann, M. J. In Vivo Activation of Duocarmycin–Antibody Conjugates by Near-Infrared Light. ACS Cent. Sci. 2017, 3 (4), 329–337. (38) Redy-Keisar, Orit; Ferber, Shiran; Satchi-Fainaro, Ronit; Shabat, Doron. NIR Fluorogenic Dye as a Modular Platform for Prodrug Assembly: Real-Time in vivo Monitoring of Drug Release. ChemMedChem 2015, 10, 999-1007. (39) Amir, R. J., Pessah, N., Shamis, M. & Shabat, D. Self-Immolative Dendrimers. Angew. Chem. Int. Ed. 2003, 42, 4494–4499. 106 Chapter 4. Light-Based Drug Delivery Applications for Traumatic Brain Injury 4.1. Introduction 4.1.1. Traumatic Brain Injury Traumatic brain injury (TBI) is a leading cause of disability and death in young adults. There are approximately 1.5-2 million cases of TBI in the United States, and of those cases, 70,000 die before reaching medical care, 500,000 are hospitalized. 35% of those hospitalized have substantial impairment and 6-10% die. 1 There are a variety of accepted medical interventions to treat TBI. Head elevation to approximately 30° has been shown to help reduce swelling. Hyperosmolar therapy involves either intravenous Mannitol or hypertonic saline solutions to draw moisture out of the brain to reduce intracranial pressure. 2 A medically induced coma can help reduce cerebral blood flow and metabolic demand to balance oxygen supply and oxygen consumption in the brain. Cerebral metabolism can also be slowed down by therapeutic cooling or hypothermia. Surgical decompression may be needed in more severe cases or cases of TBI that have obvious open head injuries. Unfortunately, approximately 1/3 of patients who are awake and cooperative when they present for treatment end up dying because of complications from secondary injury. In TBI, primary injury is the immediate physical damage and secondary injury is a cascade of cellular and molecular processes that cause further damage. These can include ionic disturbance (elevated intracellular Ca 2+ levels), excitotoxicity (elevated extracellular glutamate levels), mitochondrial dysfunction, oxidative stress, neuroinflammation, blood brain barrier damage, and cell death. 3 Even though there have been several clinical trials for treatment of secondary injury, no suitable therapy currently exists. There are many therapeutic strategies that have been successful in 107 animal models such as calcium channel blockers (Nimodipine, failed phase III), NMDA receptor antagonists (Dexanabinol), glutamate receptor antagonists (topiramate, phencyclidine, and dextromethorphan), Erythropoetin (glycoprotein that promotes neuroprotection through binding to EpoR), S100B calcium-binding protein, free radical scavengers (Tirilazad, failed phase III), stem cell therapy, and Cyclosporin-A. 2,4-5 4.1.2. Cyclosporin-A in TBI Cyclosporin-A (CsA) is a cyclic peptide natural product that is FDA approved as an immunosuppressant in cases of organ transplant to prevent recipient rejection. It functions by inhibiting the immune responses of T-cell lymphocytes. 4 It has also demonstrated a protective effect on mitochondrial ultrastructure and function and blocks mitochondrial permeability transition pore (MPT) by inhibiting its activator cyclophilin D. Blocking MPT preserves mitochondrial integrity and prevents a bioenergetic crisis that is caused by ATP deficiency following a TBI. CsA normally would not cross the blood brain barrier, but in cases of TBI, the barrier is compromised and allows for the transport of CsA into the brain. 4 The protective effects of CsA in TBI was demonstrated in a documented case of a 14-year-old German girl who made a remarkable and unexpected recovery from a TBI when she was on chronic CsA treatment following a liver transplant. 6 There have been 16 animal-based studies that confirmed the positive effects between 1999 and 2007, and CsA then entered human clinical trials. However, the conclusions of multiple phase II trials and a phase III trial were contradictory, and the drug never received FDA approval for use in TBI. 4,6 108 4.1.3. Gabapentin in TBI Gabapentin (Neurontin ) is an FDA approved drug used as an anticonvulsant. 7 Although it was developed as a more lipophilic, non-hydrolyzable structural analog of -aminobutyric acid (GABA), its mechanism of action does not include alterations of ligand binding at GABA A or GABA B receptors like benzodiazepines or barbituates. Gabapentin does however increase GABA concentrations in the brain of rats and has been shown to increase the activity of partially- purified glutamic acid decarboxylase (GAD). This suggests that gabapentin might increase the synthesis of GABA from glutamate in brain tissues. As a major inhibitory neurotransmitter in the brain, low concentrations of GABA can lead to seizures from impaired synapses. Additionally, gabapentin has been shown to inhibit GABA transaminase, the enzyme that makes glutamate from a-ketoglutarate, and stimulates glutamate decarboxylase which makes GABA out of glutamate. 8 This is beneficial in cases of TBI because high extracellular L-glutamate concentration can be toxic to neurons and has been shown to happen post TBI. High glutamate concentrations lead to high intracellular Ca +2 concentrations. 4 Glutamate receptor antagonists are being explored as potential therapeutics for TBI. Experiments with 3 H-gabapentin showed that it had a single high-affinity binding target in the brain that is not a target of GABA. 8 Gabapentin binding protein (GBP) was purified from pig brain and discovered to be a  2  subunit of an L-type voltage-dependent Ca 2+ channel. Voltage- gated calcium channels are membrane proteins in excitable cells such as neurons and myocytes. They are heterotrimeric compounds with 1,  2 , and  subunits. A 1:1:1 ratio of subunits is required for function, and the  2  subunits are poorly understood. 8 Calcium channel blockers are 109 being explored as potential therapeutic strategies because increased intracellular calcium levels after TBI causes cellular damage. 2 4.1.4. Near-IR Light and TBI In order to adapt our cyanine dye light-based drug delivery system to treat cases of TBI, we must first be sure that NIR light can pass through the skull and brain to where is will be needed for dye breakdown and drug release. Using cadaver heads, the transcranial penetration of 850nm light was measured in the occipital, parietal, temporal and frontal regions by a collaborator at USC. 9 Additionally, finite element method (FEM) simulated measurements were calculated and compared to the experimental results for photon fluence rate into the different parts of the brain. It was ultimately concluded that a mutli-LED array would increase photon flux into the brain and yield a quasi-symmetrical light field while maintaining the temperature increase at 0.2°C or less. 9 4.2. Functionalization of a Cyanine Dye with GABA and Gabapentin Gabapentin can be attached to the NIR-dye carrier in one step by stirring gabapentin and IR-820 (4.1) in DMF at 40°C overnight (Scheme 4.1). Additionally, the neurotransmitter γ- aminobutyric acid (GABA) can be directly attached using the same reaction conditions. Interestingly, the max absorbance of the final dye-coupled conjugates is dependent on the amine substitution, with N-methyl-GABA yielding a product that has a maximum absorbance 50 nanometers higher. This is likely due to the formation of an imine that breaks the extended conjugation of the molecule, lowering the maximum absorbance (Scheme 4.1B). 110 - O 3 S SO 3 - Me Me N + Me Me O N + Me Me N - O 3 S SO 3 - Me Me HN N + Me Me N - O 3 S SO 3 - Me Me HN HO O N - O 3 S SO 3 - Me Me MeN HO O O HO N + Me Me N Cl HO O NH 2 HO O NHMe NH 2 HO O DIPEA, DMF, 40°C, 12h 21% DIPEA, DMF, 40°C, 12h 24% DIPEA, DMF, 40°C, 16h 18% l max = 688nm l max = 710nm l max = 660nm N + Me Me N - O 3 S SO 3 - Me Me HN HO O N Me Me N - O 3 S SO 3 - Me Me N HO 4.2 4.3 4.4 4.1 4.4 4. 4’ Scheme 4.1. (A) Cyanine dyes modified with different GABA receptor agonists. (B) Mechanism for the reversible and pH dependent imine formation when amine is secondary and not tertiary. A B 111 Gabapentin can also be synthetically attached to the NIR dye in an extended fashion. Previously synthesized intermediate 4.5 (see Chapter 3) can be stirred at room temperature with 4-hydroxybenzyl alcohol overnight in the presence of Hunig’s base and DMAP to yield dye-benzyl alcohol 4.6 (Scheme 4.2). The alcohol can be activated using 4-nitrophenyl chloroformate and then is used directly in a reaction with gabapentin to yield dye-gabapentin conjugate 4.8. The maximum absorbance of this gabapentin congjuate is now 733nm, which is a much higher and more desirable maximum absorbance than conjugate 4.2 at 688nm. The mechanism of release involves a few more steps, but it remains self immolative and based on the breakdown of the cyanine dye to begin (Scheme 4.3). 10,11 The use of fluorescence already described can be adapted to be used to monitor the release of gabapentin in real time by using a fluorophore as a spacer instead of 4-hydroxybenzyl alcohol. The mechanism of release begins similarly to others, but upon the liberation of phenolate, a spontaneous 1,8-elimination occurs to release carbamate gabapentin. This compound can then liberate CO 2 to afford the active drug. 12 This system was used by Shabat and coworkers in 2010 to deliver a cancer chemotherapy drug and was shown to provide a strong correlation between tumor cell growth inhibition and emitted fluorescence. 112 N N - O 3 S - O 3 S SO 3 - Me Me MeN MeN O O O O O NO 2 OH N + Me Me SO 3 - Me Me MeN MeN O O O NH O OH O N + Me Me N OH N + Me Me N - O 3 S SO 3 - Me Me MeN MeN O O N + Me Me - O 3 S SO 3 - Me Me MeN MeN O Cl O O NO 2 Cl HO DIPEA, DMAP, ACN, rt, 24h 79% DIPEA, DMAP, ACN, rt, 1h NH 2 HO O DIPEA, DMAP, ACN, rt, 12h 47% 4.5 4.6 4.7 4.8 Scheme 4.2. Synthesis of cyanine dye with extended linker and gabapentin. MeN MeN O O O NH O OH O O O NH O OH O MeN NMe O NH 2 HO O O CO 2 4.9 4.10 4.11 Scheme 4.3. Proposed mechanism of release of extended gabapentin cyanine dye conjugate 113 Drug Trigger O O O O Drug O - O O O Trigger Drug 1,8-elimination HO O O O H 2 O O HO O OH Scheme 4.4. Real-time monitoring of drug release using a coumarin linker. A modified coumarin can be synthesized to achieve this as can be seen below in Scheme 4.5. 2,4-Dihydroxybenzaldehyde (4.12) is first refluxed with diethylglutaconate overnight to give intermediate 4.13 as a yellow powder. Protection of the phenol alcohol followed by dihydroxylation of the double bond yield 1,2-diol 4.15. Sodium periodate can cleave the vicinal diol to give aldehyde 4.16. Finally, deprotection of the phenol followed by reduction of the aldehyde with sodium borohydride gives 7-hydroxy-3-(hydroxymethyl)-2H-chromen-2-one (4.18) in good yield. The dye conjugate that includes fluorophore 4.18 can be synthesized using analogous conditions as dye-gabapentin compound 4.8. Amine-acyl chloride 4.5 is first stirred with coumarin 4.18 at room temperature overnight (Scheme 4.6). Upon activation of the coumarin-alcohol, compound 4.20 can be substituted with gabapentin to yield final compound 4.21 (  max = 736nm). Upon irradiation with NIR light, the cyanine dye with break down and release both coumarin and gabapentin in equal molar quantities, allowing for the release to be 114 O HO OH H O HO O O O O O O O EtOH piperdine (cat) 80% O AcO O O O O AcO O O O AcO O O O OH OH Ac 2 O/pyridine 98% NMO/H 2 O, OsO 4 DCM 72% NaIO 4 , SiO 2 DCM/H 2 O 76% O HO O O NH 4 OH H 2 O/ACN 81% O HO O OH NaBH MeOH/H 2 O 85% 4.12 4.13 4.14 4.15 4.16 4.17 4.18 Scheme 4.5. Synthesis of modified coumarin compound. ratiometrically monitored by fluorescence (Figure 4.3). Quantifying this emitted fluorescence should allow for a prediction of release drug compound and its therapeutic effect. In an attempt to streamline the synthesis and derivitization of cyanine-reporter-drug conjugates, the synthetic preparation of cargo 4.26 was attempted (Scheme 4.7). N,N’- dimethylethylenediamine (4.22) can be mono-protected with Boc-anhydride in DCM at 0°C. The remaining secondary nitrogen can be activated using triphosgene and Hunig’s base in toluene. Reactive intermediate 4.24 is then treated with previously synthesized phenol-aldehyde compound 4.17, and the resulting coupled compound can be reduced to alcohol 4.25 using sodium borohydride in methanol. The alcohol is activated with 4-nitrophenylchloroformate and then treated with gabapentin in DMF to yield Boc-protected cargo 4.26. 115 Me Me N O O O O O N + Me Me N - O 3 S SO 3 - Me Me MeN MeN O O O O O NH O O OH N + - O 3 S SO 3 - Me Me MeN MeN O O O O OH HO O O OH N + Me Me N - O 3 S SO 3 - Me Me MeN MeN O O NO 2 N + Me Me N - O 3 S SO 3 - Me Me MeN MeN O Cl O O NO 2 Cl NH 2 HO O DIPEA, DMAP, ACN, rt, 12h 28% DIPEA, DMAP, ACN, rt, 1h DIPEA, DMAP, ACN, rt, 24h 82% 4.5 4.19 4.20 4.21 Scheme 4.6. Synthesis of real-time monitoring of drug release of Gabapentin cyanine dye conjugate. Scheme 4.7. Synthesis of protected linker-coumarin-Gabapentin cargo. O HO O O O N O H N O N O N Cl O HN H N O O O O O DCM, 0°C 21% Triphosgene DIPEA Toluene 50% O O O OH N O N O O 1. Pyridine, 16h 2. NaBH 4 , MeOH, 0°C, 2h, 78% 1. 4-Nitrphenylchloroformate DIPEA, pyridine (cat.) THF, 0°C, 1h 2. Gabapentin, DMF, TEA, rt, 2 days, 91% O O O O N O N O O O N H OH O 4.22 4.23 4.24 4.25 4.26 According to Shabat and coworkers, the Boc protecting group can be rapidly deprotected with TFA and reacted with an electrophilic compound as seen in Scheme 4.8. 11 Because of this 116 Scheme 4.8. Literature precedence for the rapid deprotection and functionalization of the amine linker O N O N O O O O O O O O Reporter Reporter Reporter Reporter Reporter Reporter 1. TFA 2. H N O O O O NO 2 N O N O O H N O literature precedence, Boc-protected cargo 4.26 was treated with TFA and exposed to a dropwise addition of IR-820 (4.1). Unfortunately, the self-immolation reaction appears to be quicker than the Cl-displacement reaction, and no product was isolated (Scheme 4.9). The self-immolation products included liberated coumarin compound, and significant fluorescence was observed as confirmation. It appears that the electrophilic compound must be very reactive in order for the desired reaction to occur instead of the self-immolation reaction. O O O O N O + H 2 N O N H OH O F 3 C O - O - O 3 S SO 3 - Me Me N + Me Me N Cl O O O O N O N O O O N H OH O 4.26 TFA 4.27 N + Me Me N - O 3 S SO 3 - Me Me MeN MeN O O O O O NH O O OH 4.21 4.1 DIPEA, DMF Scheme 4.9. Attempted coupling of linker-coumarin-Gabapentin 4.26 to IR-820 (4.1) 117 4.3. Functionalization of a Cyanine Dye with Cyclosporin-A Cyclosporin-A (CsA), an immunosuppressant FDA-approved drug, has shown promising results for the treatment of traumatic brain injury as described earlier. Because of the loss of activity that is observed when modifying the OH on CsA, it instantly was an attractive location to couple to the cyanine dye. 13 If the CsA was bound to the dye, it would not function, effectively making it a prodrug that will only function upon dye breakdown and release. Multiple synthetic methods were attempted to activate either the cyclosporine or the dye carrier to couple the two together (Figure 4.1). Coupling agents N,N’-disuccinimidyl carbonate (DSC), carbonyldiimidazole (CDI), and triphosgene would ideally activate either the CsA or the dye-amine compound 4.28, but the desired coupled product was not successfully synthesized using any combination in a variety of solvents (DMSO, DMF, ACN, H 2O). An alternative approach for connecting CsA to the dye was pursued using a hydroxylated cyclosporin derivative prepared in the Petasis lab by Dr. Rong Yang (4.29, Scheme 4.9) according to a published procedure. 14,15 The less sterically hindered alcohol on the CsA-diol can be activated using 4-nitrophenyl chloroformate to give activated CsA 4.30 (Scheme 4.10), which can react directly with the NIR-dye substituted with an amine linker that was previously synthesized to give NIR-dye-CsA conjugate 4.31 (Scheme 4.11). 118 OH O N O N O NH O N O N H O H N O N O N O N O N O HN O O NO 2 Cl O N O N O NH O N O N H O H N O N O N O N O N O HN OH DMAP, ACN, 3h 4.29 4.30 HO O O O O 2 N Scheme 4.10. Activation of CsA-diol with 4-nitrophenyl chloroformate Figure 4.1. Representation of attempted IR-820 coupling reactions with Cyclosporine CsA O N O N O NH O N O N H O H N O N O N O N O N O HN OH O N O N O NH O N O N H O H N O N O N O N O N O HN O Activating Group N N + Me Me - O 3 S SO 3 - Me Me MeN MeHN N N + Me Me - O 3 S SO 3 - Me Me MeN MeN Activating Group X O N O O O X N O N X O Cl 4.28 119 N + Me Me N - O 3 S SO 3 - Me Me MeN MeN O O O N O N O NH O N O N H O H N O N O N O N O N O HN OH ACN, 40°C, 16h 15% 4.28 4.31 O N O N O NH O N O N H O H N O N O N O N O N O HN OH 4.30 O O O O 2 N Scheme 4.11. Coupling CsA to the cyanine dye. It is of importance to study the control of light over the system of release: if you turn the light off, does the self-immolation cascade of release continue, or is it a function of the light being on and directed at the compound? It would be ideal to maintain as much control over the system as possible so that the ideal amount of drug can be delivered by using a specified amount/time of irradiation with NIR light. To test this, the IR-820-CsA conjugate 4.31 was irradiated with 850nm light continuously and then intermittently. Upon elimination of light, the release mechanism stops, the dye is not decomposing, and cyclosporine is no longer being released. These results indicate that we do indeed have the desired control over the system, and it will be of interest in the future to further our understanding of the mechanism of release. 120 4.4. Conclusions We have demonstrated that two lead candidates for the therapeutic treatment of traumatic brain injury can be synthetically coupled to a cyanine dye and released in a NIR light- controlled fashion. GABA and Gabapentin can be directly attached to the IR-820 cyanine dye, and gabapentin can also be attached in an extended fashion with or without a fluorophore that measures drug release in real time. Cyclosporin-A can be modified with an allylic bromination and hydroxylation to yield CsA-diol 4.29 that can be further activated and successfully coupled to dye-amine 4.28. The CsA can be released using continuous 850nm light or intermittent light, suggesting that when the light is turned off, the dye-drug conjugate remains intact and the release of the drug stops. 121 4.5. Experimental IR-820-Gabapentin (4.2) To a solution of IR-820 (0.062g, 0.073mmol) in DMF (5 mL) in an 8-dram vial was added N,N’- Diisopropylethylamine (0.038mL, 0.219mmol) and Gabapentin (0.075g, 0.438mmol). The reaction mixture was stirred at 40°C overnight, and the completion of reaction was confirmed by LC/MS. The reaction mixture was charged with approximately 25mL of ethyl ether, filling the 8- dram vial, and was cooled to 4°C for several hours. The vial was rewarmed to room temperature, and the top ether layer was removed with a pipette while leaving the crystallized solid at the bottom of the container. The solid was then dissolved in methanol, dry-loaded onto celite, and purified by reverse phase column chromatography (20-70% acetonitrile/water). The product- containing fractions were identified by LC/MS, combined, and concentrated under reduced pressure. The resulting blue residue was dissolved in DMF (4mL), transferred to an 8-dram vial, and charged with approximately 25mL of ethyl ether, filling the 8-dram vial, and was cooled to 4°C for several hours. More fresh ether was added to the container with the blue solid, and the solid was allowed to settle to the bottom. The ether layer was removed again once the blue solid settled to the bottom of the container. Subsequent washing with ether and removal of the ether was performed until the ether layer is clear. The solid was dried under a constant stream of dry 122 nitrogen to afford IR-820-Gabapentin (4.2) as a dark blue solid (0.015g, 21%). The product was stored in an 8-dram vial filled with ether to separate the blue solid at the bottom from the moisture and oxygen in the air. LC/MS analysis: λ max (688 nm), observed [M+(-H)] = 962 [M-(-H)] = 960. IR-820-GABA (4.3) To a solution of IR-820 (0.020g, 0.024mmol) in DMF (3mL) in an 8-dram vial was added N,N’- Diisopropylethylamine (0.013mL, 0.071mmol) and -aminobutyric acid (GABA, 0.0073g, 0.071mmol). The reaction mixture was stirred at 40°C for 3 days, and the completion of reaction was confirmed by LC/MS. The reaction mixture was charged with approximately 25mL of ethyl ether, filling the 8-dram vial, and was cooled to 4°C for several hours. The vial was rewarmed to room temperature, and the top ether layer was removed with a pipette while leaving the crystallized solid at the bottom of the container. The solid was then dissolved in methanol, dry- loaded onto celite, and purified by reverse phase column chromatography (20-70% acetonitrile/water). The product-containing fractions were identified by LC/MS, combined, and concentrated under reduced pressure. The resulting blue residue was dissolved in DMF (4mL), transferred to an 8-dram vial, and charged with approximately 25mL of ethyl ether, filling the 8- dram vial, and was cooled to 4°C for several hours. More fresh ether was added to the container 123 with the blue solid, and the solid was allowed to settle to the bottom. The ether layer was removed again once the blue solid settled to the bottom of the container. Subsequent washing with ether and removal of the ether was performed until the ether layer is clear. The solid was dried under a constant stream of dry nitrogen to afford IR-820-GABA (4.3) as a dark blue solid (0.0052g, 24%). The product was stored in an 8-dram vial filled with ether to separate the blue solid at the bottom from the moisture and oxygen in the air. LC/MS analysis: λ max (660 nm), observed [M+(-H)] = 894 [M-(-H)] = 892. IR-820-NMe-GABA (4.4) To a solution of IR-820 (0.1038g, 0.122mmol) in DMF (8mL) in an 8-dram vial was added N,N’- Diisopropylethylamine (0.53mL, 3.05mmol) and 4-(methylamino)butyric acid hydrochloride (0.375g, 2.44mmol). The reaction mixture was stirred at 40°C overnight, and the completion of reaction was confirmed by LC/MS. The reaction mixture was charged with approximately 20mL of ethyl ether, filling the 8-dram vial, and was cooled to 4°C for several hours. The vial was rewarmed to room temperature, and the top ether layer was removed with a pipette while leaving the crystallized solid at the bottom of the container. The solid was then dissolved in methanol, dry-loaded onto celite, and purified by reverse phase column chromatography (20- 70% acetonitrile/water). The product-containing fractions were identified by LC/MS, combined, 124 and concentrated under reduced pressure. The resulting blue residue was dissolved in DMF (4mL), transferred to an 8-dram vial, and charged with approximately 25mL of ethyl ether, filling the 8-dram vial, and was cooled to 4°C for several hours. More fresh ether was added to the container with the blue solid, and the solid was allowed to settle to the bottom. The ether layer was removed again once the blue solid settled to the bottom of the container. Subsequent washing with ether and removal of the ether was performed until the ether layer is clear. The solid was dried under a constant stream of dry nitrogen to afford IR-820-NMe-GABA (4.4) as a dark blue solid (0.020g, 18%). The product was stored in an 8-dram vial filled with ether to separate the blue solid at the bottom from the moisture and oxygen in the air. LC/MS analysis: λ max (710 nm), observed [M+(-H)] = 908 [M-(-H)] = 906. IR-820-linker-HBA (4.6) IR-820-linker-OCl (4.5) was synthesized according to previously described procedure. To a solution of 4.5 (0.107 g, 0.114 mmol) in acetonitrile (5 mL) was added N,N’-diisopropylethylamine (0.060 mL, 0.342 mmol), 4-dimethylaminopyridine (0.007 g, 0.057 mmol) and 4-hydroxybenzyl alcohol (0.042 g, 0.342 mmol). Reaction was stirred at room temperature until LC/MS analysis confirmed the disappearance of 4.5 (12h). Mixture was added to ether and cooled to afford 125 crystallization of the product. Subsequent washing with ether afforded 4.6 as a blue solid (0.093 g, 79%). LC/MS analysis: observed [M - ] 1027 (MW of 4.6: 1028.31). IR-820-linker-HBA-4NO (4.7) To a solution of 4.6 (0.093 g, 0.090 mmol) in acetonitrile (7 mL) was added N,N’- diisopropylethylamine (0.047 mL, 0.271 mmol) and 4-dimethylaminopyridine (0.006 g, 0.045 mmol). 4-nitrophenyl chloroformate (0.055 g, 0.271 mmol) in acetonitrile (1 mL) was added dropwise to reaction mixture. Reaction mixture was stirred for 1 hour, and LC/MS analysis confirmed the disappearance of 4.6, observed [M - ] 1192 (MW of 4.7: 1193.42). 4.7 was used in the next step without purification. IR-820-linker-HBA-Gabapentin (4.8) 126 To a solution of 4.7 (0.107 g, 0.090 mmol) in acetonitrile (11 mL) was added gabapentin (0.077 g, 0.452 mmol) in acetonitrile (2 mL). Reaction was stirred at room temperature until LC/MS analysis confirmed the complete conversion of 4.7 to 4.8 (12h). Mixture was filtered through a pipet to remove the excess gabapentin, transferred to an 8-dram vial, charged with 20mL of ether and cooled to 4°C for several hours. The vial was rewarmed to room temperature, and the top ether layer was removed with a pipette while leaving the crystallized solid at the bottom of the container. The solid was then dissolved in methanol, dry-loaded onto celite, and purified by reverse phase column chromatography (30-80% acetonitrile/water). The product-containing fractions were identified by LC/MS, combined, and concentrated under reduced pressure. The resulting blue residue was dissolved in DMF (4mL), transferred to an 8-dram vial, and charged with approximately 25mL of ethyl ether, filling the 8-dram vial, and was cooled to 4°C for several hours. More fresh ether was added to the container with the blue solid, and the solid was allowed to settle to the bottom. The ether layer was removed again once the blue solid settled to the bottom of the container. Subsequent washing with ether and removal of the ether was performed until the ether layer is clear. The solid was dried under a constant stream of dry nitrogen to afford 4.8 as a dark blue solid (0.052g, 47%). The product was stored in an 8-dram vial filled with ether to separate the blue solid at the bottom from the moisture and oxygen in the air. LC/MS analysis: λ max (733 nm), observed [M-(-H)] = 1224. 1 H NMR (600 MHz, DMSO-d 6 ) δ 8.17 (d, J = 9.8 Hz, 1H), 8.10 (d, J = 9.2 Hz, 2H), 8.04 (d, J = 8.6 Hz, 3H), 7.99 – 7.95 (m, 5H), 7.92 (s, 1H), 7.79 (dd, J = 17.1, 8.5 Hz, 6H), 7.59 (d, J = 13.7 Hz, 2H), 7.54 (d, J = 8.8 Hz, 4H), 7.46 (t, J = 7.6 Hz, 3H), 7.42 – 7.37 (m, 2H), 7.32 (t, J = 7.5 Hz, 3H), 7.21 (s, 1H), 7.05 (s, 1H), 6.97 (s, 1H), 6.02 (s, 2H), 5.94 (d, J = 13.0 Hz, 3H), 4.45 (s, 1H), 4.34 (s, 1H), 4.14 (s, 5H), 4.08 (s, 6H), 3.93 (s, 2H), 127 3.77 (s, 1H), 3.71 (s, 4H), 3.39 (s, 9H), 3.35 (d, J = 7.0 Hz, 1H), 3.35 (s, 2H), 3.03 (s, 1H), 2.95 (s, 1H), 2.86 (s, 2H), 2.80 – 2.74 (m, 1H), 2.70 (d, J = 5.7 Hz, 6H), 2.53 (s, 5H), 2.49 (d, J = 2.6 Hz, 2H), 2.48 (s, 7H), 1.92 (d, J = 8.0 Hz, 1H), 1.90 – 1.81 (m, 3H), 1.83 (s, 22H), 1.75 (ddt, J = 22.4, 17.1, 10.5 Hz, 16H), 1.71 (s, 22H), 1.06 (t, J = 7.0 Hz, 2H). 3-(hydroxy-2-oxo-2H-chromen-3-yl)acrylic acid ethyl ester (4.13) To a solution of 2,4-dihydroxybenzaldehyde (1.413g, 10.3mmol) in ethanol (30mL) in an oven- dried 100mL round bottom flask was added diethylglutaconate (1.9mL, 10.7mmol) and catalytic piperidine (3 drops). The reaction mixture was refluxed under Argon atmosphere for 24 hours. The now red solution was cooled to room temperature and then cooled further to -20°C for one hour. The resulting yellow precipitate was isolated by vacuum filtration and dried to yield a powdery yellow solid (2.1342g, 80%). (E)-ethyl 3-(7-acetoxy-2-oxo-2H-chromen-3-yl)acrylate (4.14) Phenol 4.13 (2.1178g, 8.2mmol) was dissolved in pyridine (15mL) in a 100mL round bottom flask and then charged dropwise with acetic anhydride (15mL) and stirred at room temperature for 30 minutes. The reaction mixture was then poured into an ice water mixture and stirred for 10 128 minutes. The resulting precipitate was collected by vacuum filtration and dried overnight in a vacuum oven to yield a white solid (2.42g, 98%). ethyl 3-(7-acetoxy-2-oxo-2H-chromen-3-yl)-2,3-dihydroxypropanoate (4.15)   -unsaturated ester 4.14 (0.930g, 3.08mmol) was dissolved in dichloromethane (15mL) and charged with a 1:1 w/w solution of N-methylmorpholine-N-oxide (0.721g, 6.16mmol) in water (0.8mL) and 3.0mL of 2.5wt% OsO 4 in t-BuOH. The solution was stirred at room temperature for 10 hours until completion was confirmed by TLC. The reaction was then quenched with water (50mL) and extracted with ethyl acetate (3 x 30mL). The combined organic layers were washed with brine (30mL), dried over Na 2SO 4, filtered, and concentrated under reduced pressure to yield a white solid (0.7486g, 72%). 3-formyl-2-oxo-2H-chromen-7-yl acetate (4.16) Diol 4.15 (0.7486g 2.23mmol) was added to a slurry of silica gel (5g) in dichloromethane (10mL). While stirring vigorously, the reaction mixture was charged dropwise with a solution of sodium periodate (NaIO 4, 0.6333g, 2.96mmol) over 5 minutes and then stirred at room temperature for 2 hours. The reaction mixture was filtered through a cake of celite and washed with copious amounts of ethyl acetate. The ethyl acetate was washed with water (30mL) and brine (30mL), 129 dried over Na 2SO 4, filtered, and dry-loaded onto celite. The crude reaction mixture was purified by silica gel chromatography using a 5-50% ethyl acetate in hexane, and the product eluted in 45%. The relevant fractions were combined and concentrated under reduced pressure to yield a white solid (0.396g, 76%). 7-hydroxy-2-oxo-2H-chromene-3-carbaldehyde (4.17) Aldehyde 4.16 (0.395g, 1.7mmol) was dissolved in 30% w/w NH 4OH in water (5mL) and charged with acetonitrile (5mL) slowly. The solution was then stirred 10 minutes at room temperature before being quenched with ethyl acetate (30mL). The organic layer was then washed with 1M HCl (2 x 20mL), dried over Na 2SO 4, filtered, and concentrated under reduced pressure to yield a yellow solid (0.2605g, 81%). 7-hydroxy-3-(hydroxymethyl)-2H-chromen-2-one (4.18) Phenol 4.17 (0.0541g, 0.28mmol) was dissolved in methanol (5mL) and cooled to 0°C. Solid NaBH 4 (0.013g, 0.34mmol) was added slowly, and the reaction mixture was stirred at 0°C for 30 minutes. The mixture was then diluted with ethyl acetate (20mL) and washed with saturated NH 4Cl(aq) solution (20mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to yield a yellow solid (0.0465g, 85%). 130 IR-820-linker-coumarin-OH (4.19) 4.5 synthesized according to previously described procedure. To a solution of 4.5 (0.097 g, 0.103 mmol) in acetonitrile (11 mL) was added N,N’-diisopropylethylamine (0.054 mL, 0.309 mmol), 4- dimethylaminopyridine (0.006 g, 0.052 mmol) and 7-hydroxy-3-(hydroxymethyl)-2H-chromen-2- one (0.059 g, 0.309 mmol). Reaction was stirred at room temperature until LC/MS analysis confirmed the disappearance of 4.5 (16h). Mixture was added to ether and cooled to afford crystallization of the product. Subsequent washing with ether afforded 4.19 as a dark blue solid (0.0929 g, 82%) that was used in the next step without any further purification. LC/MS analysis: observed [M - ] 1081 (MW of 4.19: 1105.31). IR-820-linker-coumarin-4NO (4.20) 131 Compound 4.19 (0.1282g, 0.117mmol) was dissolved in acetonitrile (12mL) and placed in a 50mL round bottom flask under Argon. The flask was then charged with N,N’-diisopropylethylamine (0.061mL, 0.351 mmol), DMAP (0.022g, 0.176mmol), and dropwise with a solution of 4- nitrophenyl chloroformate (0.059g, 0.293 mmol) in acetonitrile (2 mL). The reaction mixture was stirred for 1 hour, and LC/MS analysis confirmed the disappearance of 4.19. The product was used directly in the next step without isolation or purification. LC/MS analysis: λ max (738 nm), observed [M-(-H)] = 1285, [M-(-H)] = 1283. IR-820-linker-coumarin-Gabapentin (4.21) To the reaction mixture containing product 4.20 was added gabapentin (0.100g, 0.585mmol) and the reaction was stirred at room temperature for 12 hours, until LC/MS analysis confirmed the complete conversion of to 4.21. The mixture was transferred to an 8-dram vial, charged with 20mL of ether and cooled to 4°C for several hours. The vial was rewarmed to room temperature, and the top ether layer was removed with a pipette while leaving the crystallized solid at the bottom of the container. The solid was then dissolved in methanol, dry-loaded onto celite, and purified by reverse phase column chromatography (30-80% acetonitrile/water). The product- containing fractions were identified by LC/MS, combined, and concentrated under reduced 132 pressure. The resulting blue residue was dissolved in DMF (4mL), transferred to an 8-dram vial, and charged with approximately 25mL of ethyl ether, filling the 8-dram vial, and was cooled to 4°C for several hours. More fresh ether was added to the container with the blue solid, and the solid was allowed to settle to the bottom. The ether layer was removed again once the blue solid settled to the bottom of the container. Subsequent washing with ether and removal of the ether was performed until the ether layer is clear. The solid was dried under a constant stream of dry nitrogen to afford 4.21 as a dark blue solid (0.042g, 28%). The product was stored in an 8-dram vial filled with ether to separate the blue solid at the bottom from the moisture and oxygen in the air. LC/MS analysis: λ max (736 nm), observed [M+(-H)+(Na)] = 1316, [M-(-H)+(Na)] = 1314. 1 H NMR (600 MHz, DMSO-d 6) δ 8.17 (d, J = 9.8 Hz, 1H), 8.10 (d, J = 9.2 Hz, 2H), 8.04 (d, J = 8.6 Hz, 3H), 7.99 – 7.95 (m, 5H), 7.92 (s, 1H), 7.79 (dd, J = 17.1, 8.5 Hz, 6H), 7.59 (d, J = 13.7 Hz, 2H), 7.54 (d, J = 8.8 Hz, 4H), 7.46 (t, J = 7.6 Hz, 3H), 7.42 – 7.37 (m, 2H), 7.32 (t, J = 7.5 Hz, 3H), 7.21 (s, 1H), 7.05 (s, 1H), 6.97 (s, 1H), 6.02 (s, 2H), 5.94 (d, J = 13.0 Hz, 3H), 4.45 (s, 1H), 4.34 (s, 1H), 4.14 (s, 5H), 4.08 (s, 6H), 3.93 (s, 2H), 3.77 (s, 1H), 3.71 (s, 4H), 3.39 (s, 9H), 3.35 (d, J = 7.0 Hz, 1H), 3.35 (s, 2H), 3.03 (s, 1H), 2.95 (s, 1H), 2.86 (s, 2H), 2.80 – 2.74 (m, 1H), 2.70 (d, J = 5.7 Hz, 6H), 2.53 (s, 5H), 2.49 (d, J = 2.6 Hz, 2H), 2.48 (s, 7H), 1.92 (d, J = 8.0 Hz, 1H), 1.90 – 1.81 (m, 3H), 1.83 (s, 22H), 1.75 (ddt, J = 22.4, 17.1, 10.5 Hz, 16H), 1.71 (s, 22H), 1.06 (t, J = 7.0 Hz, 2H). tert-butyl methyl(2-(methylamino)ethyl)carbamate (4.23) 133 N,N’-Dimethylethylenediamine (3.0mL, 27.9mmol) was dissolved in dichloromethane (30mL) and cooled to 0°C. The reaction mixture was charged dropwise with di-tert-butyl dicarboxylate (2.18mL, 9.5mmol) and allowed to warm to room temperature and stir overnight. The dichloromethane was removed under reduced pressure, and the residue was dissolved in ethyl acetate (30mL), washed with brine (20mL), dried over Na 2SO 4, filtered, and concentrated under reduced pressure to yield a clear oil (2.0495g, 39%). 1 H NMR (400 MHz, Chloroform-d) δ 3.37 (s, 1H), 2.88 (d, J = 0.8 Hz, 1H), 2.78 (s, 2H), 2.49 (s, 1H), 1.50 – 1.42 (m, 4H). 13 C NMR (400 MHz, CDCl 3) δ 152.14, 80.99, 77.32, 77.01, 76.69, 30.20, 27.98, 27.88, 27.86. tert-butyl (2-((chlorocarbonyl)(methyl)amino)ethyl)(methyl)carbamate (4.24) Triphosgene (1.292g, 4.35mmol) was weighed into an oven-dried 100mL round-bottom flask and was placed under argon atmosphere. 10 mL of dry toluene was added, and the solution was cooled to 0°C. Mono-boc-N,N-dimethylethylene diamine (4.23, 2.0495g, 10.9mmol) was weighed into an oven-dried 50mL pear-bottom flask and was placed under argon atmosphere. The diamine was charged with 20mL of dry toluene and N,N-diisopropylethylamine (DIPEA, 5.7mL, 32.7mmol). The diamine solution was then added dropwise to the triphosgene solution at 0°C over the course of 30 minutes. The reaction mixture was warmed to room temperature and stirred overnight. After 12 hours, the completion of the reaction was confirmed by TLC in a 50% ethyl acetate/hexane mixture (product rf=0.7). Solids were filtered off through cotton and rinsed with ethyl acetate. The filtrate was concentrated under reduced pressure and dry-loaded 134 onto celite. The crude reaction mixture was purified by silica gel chromatography using a 20- 100% ethyl acetate in hexane, and the product eluted in 40% (pale yellow oil, 0.5812g, 21%). 1 H NMR (400 MHz, Chloroform-d) δ 3.62 – 3.56 (m, 1H), 3.50 (d, J = 6.2 Hz, 1H), 3.42 (dd, J = 7.0, 5.7 Hz, 1H), 3.14 (s, 1H), 3.05 (s, 1H), 2.88 (d, J = 9.4 Hz, 2H), 1.44 (s, 6H). 13 C NMR (400 MHz, CDCl 3) δ 171.10, 148.98, 77.35, 77.03, 76.71, 60.35, 30.32, 28.35, 28.33, 21.00, 14.17. tert-butyl (3-formyl-2-oxo-2H-chromen-7-yl) ethane-1,2-diylbis(methylcarbamate) Phenol compound (0.1208g, 0.636mmol) was weighed into an oven-dried 10mL round bottom flask and was placed under argon atmosphere. 2mL of dry pyridine was added. Diamine compound (0.1912g, 0.763mmol) was added and the reaction was stirred at room temperature overnight. The completion of the reaction was confirmed after 15 hours by TLC in 50% ethyl acetate/hexane (product rf=0.4). The reaction mixture was diluted with ethyl acetate and washed with a pH 5 HCl aqueous solution and brine. The organic layer was dried over Na 2SO 4, filtered, concentrated under reduced pressure, and dry-loaded onto celite. The crude mixture was purified by silica gel chromatography using a 40-100% ethyl acetate in hexane, and the product eluted in 80% (off-white solid, 0.221g, 86%). 135 tert-butyl (3-(hydroxymethyl)-2-oxo-2H-chromen-7-yl) ethane-1,2-diylbis(methylcarbamate) (4.25) Starting material (0.0851g, 0.21mmol) was dissolved in methanol (1.5mL) and cooled to 0°C. Solid sodium borohydride (0.011g, 0.3mmol) was added slowly, and the reaction was stirred at 0°C for 30 minutes. The completion of the reaction was confirmed by TLC in 80% ethyl acetate/hexane (product rf=0.55). The reaction mixture was diluted with ethyl acetate, washed with saturated NH 4Cl solution, dried over Na 2SO 4, filtered, and concentrated under reduced pressure. The crude compound was used without further purification (white solid, 0.078g, 91%). tert-butyl (3-((((4-nitrophenoxy)carbonyl)oxy)methyl)-2-oxo-2H-chromen-7-yl) ethane-1,2- diylbis(methylcarbamate) Starting material (0.078g, 0.19mmol) was weighed into an oven-dried 10mL round-bottom flask and placed under argon atmosphere. Dry THF (2.5mL) was added, and the solution was cooled to 0°C. DIPEA (0.13mL, 0.77mmol) was added, followed by 4-nitrochloroformate (0.116g, 0.58mmol, added in 0.5mL dry THF), and pyridine (5 L, 0.06mmol). The reaction was stirred at 0°C for one hour and confirmed completion by TLC in 50% ethyl acetate in hexane (product rf=0.4). The reaction mixture was diluted with ethyl acetate, washed twice with saturated NH 4Cl 136 solution and once with brine. The organic phase was dried over Na 2SO 4, filtered, concentrated under reduced pressure, and dry-loaded onto celite. The crude mixture was purified using a 0- 100% ethyl acetate in hexane gradient, with the product eluting in 70% ethyl acetate (white solid, 0.0387g, 50%). 2-(1-(((((7-(((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-2-oxo-2H- chromen-3-yl)methoxy)carbonyl)amino)methyl)cyclohexyl)acetic acid (4.26) Para-nitro starting material (0.010g, 0.0175mmol) was added to an oven-dried 5mL round- bottom flask and placed under argon atmosphere. Dry DMF (1mL) was added, followed by triethylamine (6.1 L, 0.044mmol) and gabapentin (0.006g, 0.035mmol). The reaction mixture was stirred at room temperature for 36 hours. Completion of reaction was confirmed by TLC in 100% ethyl acetate (product rf=0.5). The reaction mixture was diluted with ethyl acetate and aqueous HCl solution at pH 5. The aqueous layer was extracted three times with ethyl acetate. The combined organic layers were dried over Na 2SO 4, filtered, concentrated under reduced pressure, and dry loaded onto celite. The crude mixture was purified using a 50-100% ethyl acetate in hexane gradient, with the product eluting in 100% ethyl acetate (white solid, 0.010g, 96%). 137 CsA-4NO (4.30) To an oven-dried 5mL round bottom flask under Argon was added CsA analog 4.29 (0.010g, 0.0082mmol), DMAP (0.001g, 0.0082mmol), and ACN (1mL). 4-Nitrophenyl chloroformate (0.005g, 0.025mmol) in 0.5mL ACN was added dropwise to the reaction, and the mixture was stirred at room temperature for 3 hours. 4.30 was confirmed by LC/MS analysis with some unreacted starting material leftover and subsequently used in the next step without purification (MW: 1383.74; observed M/Z= 1384). More 4-nitrophenyl chloroformate was not added to minimize impurities in the next step. 138 IR-820-linker-CsA (BP-121) (4.31) IR-820-linker (4.28, 0.014g, 0.016mmol) in 0.5mL ACN was added to the reaction mixture of 4.30 and the reaction was heated to 40°C and stirred for 16 hours. The completion of reaction was monitored by LCMS (m/z= (2122+2)/2=1062). Upon completion (completion was determined by the complete consumption of the activated cyclosporine starting material), the reaction mixture was diluted with 25mL of ethyl ether and cooled for several hours. The ether layer was separated from the resulting solid at the bottom of the flask, the solid was washed with 3x25mL more ethyl ether. After removing the final ether wash, the solid was then dried under a stream of dry nitrogen to yield a dark blue solid 4.31 (0.0052g, 30%) (MW: 2122.81; observed M/Z= 1062). 1 H NMR (600 MHz, DMSO-d 6) δ 8.40 (s, 1H), 8.14 (s, 3H), 8.04 (d, J = 8.7 Hz, 1H), 7.97 (d, J = 8.5 Hz, 7H), 7.92 (s, 1H), 7.93 – 7.82 (m, 2H), 7.64 (s, 7H), 7.55 (s, 4H), 7.52 – 7.41 (m, 2H), 7.39 (s, 4H), 7.37 – 7.26 (m, 2H), 7.16 (s, 1H), 6.53 (d, J = 7.7 Hz, 0H), 6.01 (s, 3H), 5.59 (d, J = 13.0 Hz, 1H), 5.51 (s, 4H), 5.34 (s, 1H), 5.20 (s, 1H), 5.02 (s, 1H), 4.94 (s, 1H), 4.78 (s, 1H), 4.46 (s, 6H), 4.31 (s, 1H), 4.23 – 4.18 (m, 1H), 4.14 (s, 7H), 3.91 (t, J = 7.2 Hz, 1H), 3.80 (s, 5H), 3.69 (s, 1H), 3.59 (s, 3H), 3.35 (d, J = 3.0 Hz, 1H), 3.28 (s, 3H), 3.21 (s, 1H), 3.04 – 2.99 (m, 1H), 2.96 (s, 1H), 2.94 – 2.87 (m, 3H), 2.86 (s, 4H), 2.84 (s, 8H), 2.81 (d, J = 3.3 Hz, 1H), 2.82 – 2.75 (m, 4H), 2.77 – 2.71 (m, 1H), 2.72 (s, 1H), 2.70 (s, 4H), 2.66 (d, J = 10.9 Hz, 1H), 2.64 – 2.58 (m, 1H), 2.53 (s, 3H), 2.47 – 2.42 (m, 4H), 2.17 (s, 2H), 1.87 (s, 18H), 1.83 – 1.64 (m, 15H), 1.55 (s, 1H), 1.54 – 1.49 (m, 1H), 1.48 (s, 1H), 1.28 (s, 3H), 1.20 – 1.07 (m, 9H), 1.06 (t, J = 7.0 Hz, 1H), 0.98 – 0.89 (m, 1H), 0.89 – 0.83 (m, 2H), 0.86 (s, 11H), 0.85 – 0.72 (m, 9H), 0.69 (dd, J = 10.9, 6.4 Hz, 2H), 0.60 (d, J = 6.3 Hz, 1H), 0.55 (s, 1H). 139 4.6. References (1) Laurer, H. L.; McIntosh, T. K. Pharmacologic Therapy in Traumatic Brain Injury: Update on Experimental Treatment Strategies. Current Pharmaceutical Design 2001, 7, 1505–1516. (2) Galgano, M.; Toshkezi, G.; Qiu, X.; Russell, T.; Chin, L.; Zhao, L.-R. Traumatic Brain Injury. Cell Transplant 2017, 26 (7), 1118–1130. (3) Tran, L. V. Understanding the Pathophysiology of Traumatic Brain Injury and the Mechanisms of Action of Neuroprotective Interventions. Journal of Trauma Nursing 2014, 21 (1), 30–35. (4) Clausen, T.; Bullock, R. Medical Treatment and Neuroprotection in Traumatic Brain Injury. Current Pharmaceutical Design 2001, No. 7, 1517–1532. (5) Bayir, H.; Clark, R. S. B.; Kochanek, P. M. Promising Strategies to Minimize Secondary Brain Injury After Head Trauma. Crit. Care. Med. 2003, 31, 112–117. (6) Lulic, D.; Burns, J.; Bae, E. C.; van Loveren, H.; Borlongan, C. V. A Review of Laboratory and Clinical Data Supporting the Safety and Efficacy of Cyclosporin a in Traumatic Brain Injury. Neurosurgery 2011, 68 (5), 1172–1186. (7) Taylor, C. P.; Gee, N. S.; Su, T.-Z.; Kocsis, J. D.; Welty, D. F.; Brown, J. P.; Dooley, D. J.; Boden, P.; Singh, L. A Summary of Mechanistic Hypotheses of Gabapentin Pharmacology. Epilepsy Research 1998, 29, 233–249. (8) Offord, J.; Isom, L. L. Drugging the Undruggable: Gabapentin, Pregabalin and the Calcium Channel Α 2δ Subunit. Critical Reviews in Biochemistry and Molecular Biology 2016, 51 (4), 246– 256. 140 (9) Yue, L.; Monge, M.; Ozgur, M. H.; Murphy, K.; Louie, S.; Miller, C. A.; Emami, A.; Humayun, M. S. Simulation and Measurement of Transcranial Near Infrared Light Penetration; Jansen, E. D., Ed.; SPIE BiOS, 2015; 9321, 93210S–6. (10) Amir, R. J.; Pessah, N.; Shamis, M.; Shabat, D. Self-Immolative Dendrimers. Angew. Chem. Int. Ed. 2003, 42 (37), 4494–4499. (11) Shamis, M.; Shabat, D. Single-Triggered AB6 Self-Immolative Dendritic Amplifiers. Chem. Eur. J. 2007, 13 (16), 4523–4528. (12) Weinstein, R.; Segal, E.; Satchi-Fainaro, R.; Shabat, D. Real-time monitoring of drug release. Chem. Commun. 2010, 46, 553-555. (13) Hamel, A. R.; Hubler, F.; Carrupt, A.; Wenger, R. M.; Mutter, M. Cyclosporin a Prodrugs: Design, Synthesis and Biophysical Properties. J. Peptide Res. 2004, 63, 147–154. (14) Eberle, M. K.; Nuninger, F. Synthesis of the Main Metabolite (OL-17) of Cyclosporin-A. J. Org. Chem. 1992, 57, 2689–2691. (15) Prell, E.; Kahlert, V.; Rücknagel, K. P.; Malešević, M.; Fischer, G. Fine Tuning the Inhibition Profile of Cyclosporine a by Derivatization of the MeBmt Residue. ChemBioChem 2013, 14 (1), 63–65. 141 Chapter 5. Synthesis and Evaluation of Lead Compounds for the Inhibition of dUTPase 5.1. Introduction In a 1954 publication, it was reported that liver tumor cells absorbed more radioactive uracil than normal cells. 1 Shortly after, 5-fluorouracil was first synthesized and shown to have anticancer properties in solid tumors in rats and mice that were resultant from interference in uracil and thymine metabolism. 2,3 5-fluorouracil (5-FU) functions as an antimetabolite; it inhibits essential biosynthetic pathways and incorporates itself into macromolecules like DNA and RNA and inhibits their natural functions. 3,4 5-FU is metabolized in the body into fluorodeoxyuridine monophosphate (FdUMP) which functions as a competitive inhibitor for an enzyme called thymidylate synthase (TS). 5 TS naturally converts deoxyuridine monophosphate (dUMP) into deoxythymidine monophosphate (dTMP), feeding into thymine production for DNA replication and synthesis (Figure 5.1 and Scheme 5.1). 6 This process is essential in any cell, but it is of particular importance for cells that are rapidly dividing, necessitating copious amounts of DNA replication, like cancer cells. 7,8 5-FU works so well because the only difference between the active metabolite, FdUMP, and the native substrate, dUMP, is the presence of a fluorine atom instead of a hydrogen atom. Fluorine and hydrogen have approximately the same atomic radius, allowing the FdUMP to fit into the enzyme active site the same way as the native substrate. 9 However, the electronics of a carbon-fluorine bond differ from those of a carbon-hydrogen bond which prevents the enzyme from breaking said bond and installing a methyl group to change it to thymine. This causes a depletion of available thymine, mis incorporation of uracil into DNA, and “thymineless death” of the cancer cells. 10 5-fluorouracil has been used as an anticancer drug 142 effectively for a variety of cancers including colon, breast, cervical, pancreatic, stomach, and esophageal cancers since 1962. 6 Unfortunately, there is an intrinsic mechanism of resistance that can overcome 5- fluorouracil treatment. Because 5-fluorouracil functions as a competitive inhibitor of TS, its efficacy is dependent on the concentration difference between FdUMP and dUMP; FdUMP needs to be at a higher concentration in order to prevent any native substrate from binding. 11 If enough of dUMP binds to the enzyme, a suitable concentration of thymine derivatives will be synthesized and incorporated into DNA, allowing the cancer cells to survive. The cancer cells achieve this upset of the balance of concentrations by overexpressing an enzyme called deoxyuridine triphosphatase (dUTPase). 10 DNA AP Sites Repair CELL DEATH dUTP dUMP dUDP dUTPase dTMP dTDP dTTP TS UDP dCMP DNA Figure 5.1. dUTPase catalyzes the reaction to make dUMP, the native substrate for TS. 143 dUTPase is an essential, ubiquitous, homotrimeric enzyme that catalyzes the transformation of deoxyuridine triphosphate (dUTP) to dUMP (Figures 5.1, 5.2 and Scheme 5.1). 12-14 By overexpressing this enzyme, cancer cells can increase the concentration of dUMP and upset the concentration balance between dUMP and FdUMP. Indeed, dUTPase overexpression has been observed in the clinic particularly in colon cancers. 10,15-20 Because of this, dUTPase has emerged as a novel target for anticancer chemotherapy for use in 5-fluorouracil-resistant cancers. 21-25 Figure 5.2. dUTPase (PDB:2HQU) Scheme 5.1. The reactions that are catalyzed by dUTPase and TS. 144 5.2. Efficacy of designed dUTPase Inhibitors First generation literature inhibitors of dUTPase closely resembled the chemical structure of the native substrate, dUTP, while incorporating a variety of phosphate isosteres. Unfortunately, these compounds were plagued by poor bioavailability and difficult syntheses. Because of these problems, a novel inhibitor scaffold was pursued. To better understand the binding of the native substrate, it was docked computationally using Schrödinger’s Glide (Figure 5.3A) by Petasis lab members Dr. Marcos Sainz and Dr. Kevin Gaffney. 26,27 The native substrate binding is best understood by breaking the compound up into uracil, sugar and phosphate regions. The uracil region would need to be conserved when designing an inhibitor because that area of the binding pocket is extremely specific to uracil. Figure 5.3. (A) Native substrate, dUTP, broken down to into uracil (U), sugar (S), and phosphate (P) regions. (B) Literature dUTPase inhibitors do not mimic the native substrate. (C) Docked drug-like fragments. (D) Top fragments reconstituted and docked. (PDB:2HQU) A B C D 145 Interestingly, when a number of published dUTPase inhibitor compounds were docked, they appeared to not fit in the binding pocket the same way that the native substrate does (Figure 5.3B). These inhibitors take advantage of an upper, allosteric area of the binding pocket that is not conserved among all species and not used for the enzymes catalytic function. In order to better target the phosphate region of the binding pocket, the uracil region was blocked off and 350,000 drug-like fragments from the ZINC database (MW ≤ 250 daltons, logP ≤ 3.5, # rotatable bonds ≤ 5) were docked into the active site (Figure 5.3C). 28 From these top fragments, an inhibitor scaffold was pieced together and docked again (Figure 5.3D). The inhibitor design was broken down into uracil, linker, and hydrogen bond donor regions. 4-carbon and 5-carbon alkyl linkers along with meta and para phenyl linkers were synthesized with amide, sulfonamide, and benzimidazole hydrogen bond donors (Table 5.1). The cyclopropylmethoxy-substituted benzimidazoles displayed the lowest EC50 values for cytotoxicity against HCT116, a colorectal carcinoma cell line. This led to the synthesis of alternative polar functionality on the benzimidazole, namely cyano-, methylester- trifluoromethoxy- and trifluoromethyl-benzimidazoles with both phenyl and thiophenyl linkers (Table 5.2). The unsubstituted benzimidazole with any linker did not have remarkable cytotoxicity, and therefore the polar functionality must be necessary. Indeed, the trifluoromethoxy- and trifluoromethyl-benzimidazoles with both the phenyl and thiophenyl linkers achieved the lowest EC50 concentration, making them the lead candidates for dUTPase inhibition. The MTT cell viability assay was performed previously in the Prof. Nicos Petasis lab and Prof. Stan Louie lab by Dr. Marcos Sainz. 146 EC50 ( M) EC50 ( M) 160 135 >160 >160 >160 108 103 75 53 19 75 Table 5.1. EC50 values for cytotoxicity of designed dUTPase inhibitors against HCT116 colorectal carcinoma cell line. 147 EC50 ( M) EC50 ( M) 35 >100 22 45 80 20 140 Table 5.2. EC50 values for cytotoxicity of designed dUTPase inhibitors against HCT116 colorectal carcinoma cell line. 148 5.3. Scale-Up of Lead Compounds It is beneficial when completing biological studies that the compound or inhibitor of interest be from the same batch to prevent the possible batch variability from affecting the reproducibility of the results. Because of this, the lead compounds were synthesized with a multi- gram scaled-up procedure (Scheme 5.2). To begin, 4-(bromomethyl)benzoate was treated with uracil in the presence of potassium carbonate in DMSO to yield uracil intermediate 5.1, which upon saponification yield benzoic acid 5.2. An amide coupling with either trifluoromethyl- or trifluoromethoxy-substituted dianilines yielded 5.3 and 5.4 respectively. Finally, heating overnight in acetic acid provided efficient condensation to provide the substituted benzimidazole final products. Scheme 5.2. Synthesis of lead compounds with phenyl linker. Br OMe O HN N O O O OMe HN N O O HN N R 1 HN N O O O OH HN N O O H N O NH 2 R 1 Uracil K 2 CO 3 DMSO rt, 19h 59% 2M NaOH MeOH/H 2 O rt, 14h 95% Diamine HATU DIPEA DMF rt, 20h 5.3, R 1 = CF 3 , 87% 5.4, R 1 = OCF 3 , 84% 5.5, R 1 = CF 3 , 88% 5.6, R 1 = OCF 3 , 90% AcOH 100°C, 15h 5.1 5.2 149 A similar scale up was performed for the trifluoromethyl- and trifluoromethoxy- benzimidazole conjugates with a thiophene linker (Scheme 5.3). To begin, thiophene-2,5- dicarboxylic acid was diesterified using methanol with catalytic sulfuric acid to yield 5.7. After a selective reduction and halogenation, an alkylation reaction was used to attach uracil to yield intermediate 5.10. After saponification, an amide coupling reaction was performed with either trifluoromethyl- or trifluoromethoxy-substituted dianiline compounds. Finally, heating overnight in acetic acid provided efficient condensation conditions to provide the substituted benzimidazole final products with thiophene linkers (5.12, 5.13). A dUTPase inhibitor anticancer compound was discovered through extensive structure- activity relationship studies by Taiho Pharmaceuticals and is currently in Phase I clinical trials in Japan. 29-33 This lead compound (5.25) was synthesized on a large scale in order to use it as a positive control compound in biological studies. 4-Fluoro-3-hydroxybenzoic acid was treated with bromomethyl cyclopropane in the presence of potassium carbonate and potassium iodide S OH O HO O S O O O O S O O HO S O O Br HN N O O S O O HN N O O S OH O HN N O O S N H N R 1 H 2 SO 4 , MeOH reflux, 15h 88% 2.0M LiBH 4 , THF 0°C to rt, 4h 45% Imidazole PPh 3 , Br 2 CH 2 Cl 2 , rt, 1h 92% Uracil, K 2 CO 3 DMSO, rt, 1h 49% 2M NaOH MeOH, rt, 4h 82% 1) Diamine, HATU, DIPEA DMF, rt, 17h 2) AcOH, 100°C, 13h 5.12, R 1 = CF 3 , 88% 5.13, R 1 = OCF 3 , 84% 5.7 5.8 5.9 5.10 5.11 Scheme 5.3. Synthesis of lead compounds with thiophene linker. 150 in DMF to yield intermediate 5.14 (Scheme 5.4). A selective ester reduction with DIBAL followed by oxidation with manganese dioxide yields aldehyde 5.16. A Grignard reaction with ethylmagnesium bromide followed by a functional group conversion and hydrogenation yields benzylamine 5.19. An alkylation with 3-chloropropane sulfonyl chloride, acetylation reaction, and acetyl deprotection reaction give alcohol intermediate 5.22. Upon reaction with MOM-Cl, intermediate MOM-ether 5.23 is achieved. This MOM-ether can then be reacted with a TMS- protected uracil (Scheme 5.5) to yield the final dUTPase inhibitor compound (5.25) as a white foam (Scheme 5.6). OH F HO O Br K 2 CO 3 , KI DMF, 90°C, 16h 87% O F O O DIBAL-hexanes Toluene, 0°C, 2h 80% O F HO MnO 2 , CH 2 Cl 2 rt, 24h 87% O F O EtMgBr/THF 0°C to rt, 2h 99% O F HO DPPA, DBU Toluene 0°C to rt, 16h 71% O F N 3 H 2 , Pd/C rt, 16h MeOH, rt O F H 2 N O F N H S Cl O O Cl S Cl O O Et 3 N, CH 2 Cl 2 0°C to rt, 16h 96% O F N H S O O O O NaOAc, NaI DMF, 80°C, 18h 50% O F N H S HO O O HCl, MeOH reflux, 20h 80% MOM-Cl, DIPEA CH 2 Cl 2 , 0°C to rt, 18h 63% O F N H S O O O O 5.14 5.15 5.16 5.17 5.18 5.19 5.20 5.21 5.22 5.23 Scheme 5.4. Synthesis of positive control dUTPase inhibitor that is in Phase I in Japan. 151 5.4. Thymine and 5-Fluorouracil Derivatives of Lead Compounds As mentioned before, the active site of the dUTPase enzyme is very specific to uracil. A dUTPase inhibitor containing a thymine instead of uracil would not be able to bind effectively in the active site. This specificity permits for quick validation that the cytotoxic effects exhibited by the computationally designed inhibitors is the direct result of dUTPase inhibition and not the results of off-target effects. A literature inhibitor of dUTPase containing a trityl group that targets the upper allosteric area of the binding pocket was synthesized with both a uracil and a thymine to test if it only functions as a dUTPase inhibitor. As can be seen in Figure 5.4 (data collected by Dr. Marcos Sainz), this compound clearly has other targets apart from dUTPase because it still is cytotoxic to the HCT116 colorectal carcinoma cells when the uracil is replaced with thymine, but N N OTMS TMSO HN N H O O BSA, CH 2 Cl 2 rt, 1.5h 5.24 Scheme 5.5. Synthesis of TMS- protected uracil. 1) BCl 3 , CH 2 Cl 2 , 0°C to rt, 1h 2) 5.24, TBAI, rt, 23h, 31% O F H N S O O O N HN O O O F N H S O O O O 5.23 5.25 Scheme 5.6. Final step in synthesis of positive control dUTPase inhibitor. 152 Scheme 5.7. Synthesis of thymine and 5-fluorouracil conjugates of lead inhibitors. Br OMe O HN N O O O OMe HN N O O HN N R 1 HN N O O O OH HN N O O H N O NH 2 R 1 5-FU or thymine K 2 CO 3 DMSO rt, 19h 2M NaOH MeOH/H 2 O rt, 14h HATU DIPEA DMF rt, 20h 5.30, R 1 = CF 3 , R 2 = F, 67% 5.31, R 1 = OCF 3 , R 2 = F, 61% 5.32, R 1 = CF 3 , R 2 = CH 3 , 67% 5.33, R 1 = OCF 3 , R 2 = CH 3 , 100% AcOH 100°C, 15h R 2 R 2 R 2 R 2 5.26, R 2 = F, 47% 5.27, R 2 = CH 3 , 55% 5.28, R 2 = F, 88% 5.29, R 2 = CH 3 , 96% 5.34, R 1 = CF 3 , R 2 = F, 91% 5.35, R 1 = OCF 3 , R 2 = F, 90% 5.36, R 1 = CF 3 , R 2 = CH 3 , 95% 5.37, R 1 = OCF 3 , R 2 = CH 3 , 73% Figure 5.4. Comparison of cytotoxicity of thymine- and uracil-based dUTPase inhibitors. 153 designed dUTPase inhibitor 5.5 was not, suggesting the cytotoxic effects are coming only from dUTPase inhibition. It was also of interest to synthesize 5-fluorouracil conjugates of the lead inhibitors due to the propensity of fluorine groups to participate in electrostatic interactions, enhance metabolic stability and increase half-life of several known drugs. 5-fluorouracil alone is an FDA approved TS inhibitor drug, and the body has the potential to metabolize 5-FU containing dUTPase inhibitors into the TS inhibitor, effectively making one drug have dual purposes. The lead compounds with the phenyl linker were chosen for the synthesis of thymine and 5-FU conjugates due to ease of synthesis and scalability when compared to the thiophene linker. The four-step synthesis is analogous to that of the uracil-based compounds, and the thymine and 5-FU derivatives were made in great yields (Scheme 5.7). The derivatives of dUTPase inhibitor compound developed by Taiho Pharmaceuticals that is in Phase I clinical trials in Japan were also synthesized. Interestingly, the conditions that worked well for uracil coupling were only applicable to the thymine derivative (Scheme 5.10). In order to couple 5-FU to MOM-ether 5.23, tin (iv) chloride was needed (Scheme 5.9). N N OTMS TMSO HN N H O O BSA, CH 2 Cl 2 rt, 1.5h R R 5.38, R = F 5.39, R = CH 3 Scheme 5.8. Synthesis of TMS- protected thymine and 5- fluorouracil. 154 5.5. Conclusions In conclusion, a library of dUTPase compounds was first computationally designed and then was synthesized and tested for cytotoxicity against a colorectal carcinoma cell line. Lead dUTPase inhibitors were identified and their synthesis was found to be scalable and inexpensive. Thymine and 5-fluorouracil derivatives of lead inhibitors were synthesized in a step towards elucidating whether the mechanism of cytotoxicity is through the desired inhibition of the dUTPase enzyme. In the future, an in vitro enzyme assay and further biological analysis will confirm dUTPase inhibition. O F N H S O O O O SnCl 4 , CH 2 Cl 2 5.38, rt, 1.5h 11% O F H N S O O O N HN O O F 5.40 Scheme 5.9. Synthesis of 5-fluorouracil conjugate of positive control dUTPase inhibitor. Scheme 5.10. Synthesis of thymine conjugate of positive control dUTPase inhibitor. 1) BCl 3 , CH 2 Cl 2 , 0°C to rt, 1h 2) 5.39, TBAI, rt, 17h, 26% O F H N S O O O N HN O O O F N H S O O O O CH 3 5.41 155 5.6. Experimental methyl 4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzoate (5.1) Potassium carbonate (10.860g, 78.58 mmol) was added to a stirred solution of uracil (8.809g, 78.58 mmol) in DMSO (200 mL), and the suspension was stirred at room temperature for 20 minutes. A solution of methyl 4-(bromomethyl)benzoate (6.00g, 26.19 mmol) in DMSO (30 mL) was added dropwise, and the mixture was allowed to stir overnight at room temperature. After 19 hours, the reaction mixture was cooled to 0°C and quenched with H 2O (450 mL) and extracted with EtOAc (4 x 150 mL). The combined organic layers were washed with brine (150 mL), dried over Na 2SO 4 and concentrated in vacuo to yield a white powdery solid. The solid was dissolved in a 1:1 mixture of hexane and ethyl acetate (300mL) and stirred at room temperature for 40 minutes. The suspension was filtered to yield a powdery white solid (3.9665g, 59% yield). 1 H NMR (600 MHz, DMSO-d6) δ 11.36 (s, 1H), 7.95 (d, J = 8.0 Hz, 2H), 7.78 (d, J = 7.9 Hz, 1H), 7.41 (d, J = 8.1 Hz, 2H), 5.62 (d, J = 7.8 Hz, 1H), 4.96 (s, 2H), 3.84 (s, 3H). 13 C NMR (400 MHz, DMSO-d6) δ 165.91, 163.64, 150.98, 145.63, 142.32, 129.50, 128.84, 127.44, 101.52, 52.14, 50.11. 156 4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzoic acid (5.2) To a stirred solution of 5.1 (3.9665g, 15.24 mmol) in 1:1 H 2O/MeOH (100 mL) was added 61mL of a 2M NaOH(aq) solution, and the resulting mixture was stirred at room temperature overnight. After 14 hours, the methanol was removed under reduced pressure and the remaining aqueous solution was acidified to pH 1 with 12M HCl (~8mL). The resulting precipitate was filtered, washed with H 2O, and dried in a vacuum oven for 4 hours to afford a white powder (3.5762g, 95%). 1 H NMR (400 MHz, DMSO-d6) δ 12.95 (s, 1H), 11.35 (s, 1H), 7.93 (d, J = 6.8 Hz, 2H), 7.78 (d, J = 7.8 Hz, 1H), 7.38 (d, J = 6.8 Hz, 2H), 5.62 (d, J = 7.9 Hz, 1H), 4.95 (s, 2H). 13 C NMR (400 MHz, DMSO- d6) δ 166.98, 163.66, 151.00, 145.65, 141.82, 130.04, 129.66, 127.30, 101.50, 50.12. N-(2-amino-4-(trifluoromethyl)phenyl)-4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)benzamide (5.3) 157 A 50mL round bottom flask containing a solution of 5.2 (0.2357g, 0.96mmol) and DIPEA (0.334mL, 1.92mmol) in DMF (5mL) was charged with HATU (0.365g, 0.96mmol) in one portion. The resulting yellow solution was stirred at room temperature for ten minutes, then was charged with 3,4-diaminobenzotrifluoride (0.6746g, 3.83mmol) and stirred at room temperature overnight. After 20 hours, the reaction was quenched with water (10mL) and extracted with ethyl acetate (4 x 5mL). The combined organics layers were concentrated under reduced pressure and dry-loaded onto celite. The crude mixture was then purified using flash column chromatography with the product eluting in a broad peak in 90% ethyl acetate/hexane. The relevant fractions were combined and concentrated under reduced pressure to yield a light brown solid (0.3394g, 87%) that was taken directly on to the next step without further characterization. 1-(4-(5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)benzyl)pyrimidine-2,4(1H,3H)-dione (5.5) An 8-dram vial was charged with 5.3 (0.3394g, 0.839mmol) and glacial acetic acid (8mL) and was stirred at 100°C overnight. After 15 hours, the acetic acid was removed under reduced pressure, and the resulting tan solid was charged with saturated Na 2CO 3(aq) (15mL) and stirred at room temperature for 1 hour. The pH was adjusted to pH 8, and the tan solid was collected by filtration and washed with cold water (200mL) and then dried for several hours in a vacuum oven to yield 158 a flaky tan solid (0.2847g, 88%). 1 H NMR (400 MHz, DMSO-d6) δ 13.35 (s, 1H), 11.36 (s, 1H), 8.19 (d, J = 8.3 Hz, 2H), 7.95 (s,1H), 7.82 (d, J = 7.8 Hz, 1H), 7.77 (s, 1H), 7.64 – 7.41 (m, 3H), 5.64 (d, J = 7.9 Hz, 1H), 4.97 (s,2H). 13 C NMR (400 MHz, DMSO-d6) δ 163.80, 153.87, 151.13, 145.64, 143.47, 143.45, 140.82, 138.72, 138.36, 129.74, 127.92, 126.89, 120.39 (q, J = 254.8 Hz), 115.37, 108.51, 101.50, 50.15. 19 F NMR (400 MHz, DMSO-d6) δ -57.18. N-(2-amino-4-(trifluoromethoxy)phenyl)-4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)benzamide (5.4) A 50mL round bottom flask containing a solution of 5.2 (0.236g, 0.96 mmol) DIPEA (0.334mL, 1.92mmol) in DMF (5mL) was charged with HATU (0.365g, 0.96mmol) in one portion. The resulting yellow solution was stirred at room temperature for ten minutes, then was charged with 4-(trifluoromethoxy)benzene-1,2-diamine (0.735g, 3.83mmol) and was stirred at room temperature for an additional 20 hours. the reaction was quenched with water (10mL) and extracted with ethyl acetate (4 x 5mL). The combined organics layers were concentrated under reduced pressure and dry-loaded onto celite. The crude mixture was then purified using flash column chromatography with the product eluting in a broad peak in 90% ethyl acetate/hexane. 159 The relevant fractions were combined and concentrated under reduced pressure to yield a light- brown powder that was used directly in the following step (0.326 g, 84%). 1-(4-(5-(trifluoromethoxy)-1H-benzo[d]imidazol-2-yl)benzyl)pyrimidine-2,4(1H,3H)-dione (5.6) An 8-dram vial was charged with 5.5 (0.326 g, 0.776 mmol) and glacial acetic acid (9 mL) and was stirred at 100°C overnight. After 15 hours, the acetic acid was removed under reduced pressure, and the resulting tan solid was charged with saturated Na 2CO 3(aq) (20mL) and stirred at room temperature for 1 hour. The pH was adjusted to pH 8, and the tan solid was collected by filtration and washed with cold water (200mL) and then dried for several hours in a vacuum oven to yield an off-white solid (0.281 g, 90%). 1 H NMR (400 MHz, DMSO-d6) δ 13.19 (s, 1H), 11.36 (s, 1H), 8.16 (d, J = 8.3 Hz, 2H), 7.81 (d,J = 7.8 Hz, 1H), 7.65 (s, 2H), 7.48 (d, J = 8.4 Hz, 2H), 7.20 (d, J = 9.0 Hz, 1H), 5.63 (d, J = 7.9Hz, 1H), 4.96 (s, 2H). 13 C NMR (400 MHz, DMSO-d6) δ 163.80, 153.87, 151.13, 145.64, 143.47, 143.45, 140.82, 138.72, 138.36, 129.74, 127.92, 126.89, 120.39 (q, J = 254.8 Hz), 115.37, 108.51, 101.50, 50.15. 19 F NMR (400 MHz, DMSO-d6) δ -56.98. 160 dimethyl thiophene-2,5-dicarboxylate (5.7) An oven-dried 100mL round-bottom flask was charged with thiophene-2,5-dicarboxylic acid (8.49g, 49mmol) and placed under Argon. Anhydrous methanol (50 mL) and concentrated sulfuric acid (1 mL) were added, and the resulting white slurry was refluxed for 15 hours. After cooling to room temperature, the mixture was cooled in an ice bath and filtered. The recovered solid was washed with cold methanol to yield a white powder (8.690g, 88%). 1 H NMR (400 MHz, DMSO-d6) δ 7.82 (s, 2H), 3.86 (s, 6H). 13 C NMR (400 MHz, DMSO-d6) δ 161.22, 138.09, 133.75, 52.81. methyl 5-(hydroxymethyl)thiophene-2-carboxylate (5.8) An oven-dried 250mL round-bottom flask was charged with 5.7 (6.00 g, 30 mmol) and anhydrous THF (100 mL) and was cooled to 0°C. The solution was then charged dropwise with LiBH 4 (2.0 M in THF, 8.5mL), and allowed to warm to room temperature and stir for 4 hours. The solution was concentrated under reduced pressure, treated with water (100mL) and extracted with EtOAc (3 x 100mL). The combined organic layers were washed with brine (10mL), dried over Na 2SO 4 and dry-loaded onto celite. The crude material was purified by automated column chromatography (30% EtOAc/hexanes) to yield a colorless oil (2.31g, 45%). 1 H NMR (400 MHz, CDCl 3 ) δ 7.66 (d, J = 3.7 Hz, 1H), 6.97 (d, J = 3.8 Hz, 1H), 4.84 (d, J = 0.9 Hz, 2H), 3.87 (s, 3H). 13 C NMR (400 MHz, CDCl 3) δ 162.85, 151.95, 133.68, 132.85, 125.44, 60.34, 52.32. 161 methyl 5-(bromomethyl)thiophene-2-carboxylate (5.9) An oven-dried 100mL round-bottom flask was charged with imidazole (1.2g, 17.5mmol), PPh 3 (4.6g, 17.5mmol), anhydrous CH 2 Cl 2 (45mL), and bromine (0.9mL, 17.5mmol) and placed under Argon. After stirring at room temperature for 5 minutes, the solution was charged dropwise with a solution of 5.8 (2.00g, 11.7mmol) in anhydrous CH 2Cl 2 (12mL). The resulting orange mixture was stirred at room temperature for 1 hour, and then concentrated under reduced pressure and dry-loaded onto celite. The residue was purified by automated column chromatography (5-11% EtOAc/hexanes). The relevant fractions were combined and concentrated under reduced pressure to yield a colorless oil (2.53g, 92%). 1 H NMR (400 MHz, CDCl 3) δ 7.63 (d, J = 3.8 Hz, 1H), 7.09 (d, J = 3.8 Hz, 1H), 4.67 (s, 2H), 3.88 (s, 3H). 13 C NMR (400 MHz, CDCl 3) δ 162.42, 147.53, 134.36, 133.55, 128.55, 52.45, 25.59. methyl 5-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)thiophene-2-carboxylate (5.10) A 100mL round-bottom flask was charged with potassium carbonate (2.35g, 17mmol), uracil (1.91g, 17mmol) and DMSO (40mL) and stirred at room temperature for 15 minutes. The suspension was then charged with a solution of 5.9 (2.00g, 8.5mmol) in DMSO (10mL) and was stirred at room temperature for 1 hour. The reaction mixture was quenched with H 2O (30 mL) 162 and extracted with EtOAc (3 x 50mL). The combined organic layers were washed with brine (30mL), dried over Na 2SO 4 and dry-loaded onto celite. The residue was purified by autocolumn chromatography (70-80% EtOAc/hexanes) to yield a white solid (1.11g, 49%). 1 H NMR (400 MHz, DMSO-d6) δ 11.40 (s, 1H), 7.81 (d, J = 7.9 Hz, 1H), 7.69 (d, J = 3.8 Hz, 1H), 7.20 (d, J = 3.7 Hz, 1H), 5.62 (d, J = 7.9 Hz, 1H), 5.07 (s, 2H), 3.80 (s, 3H). 13 C NMR (400MHz, DMSO-d6) δ 163.51, 161.62, 150.75, 146.58, 144.88, 133.53, 132.55, 128.18, 101.76, 52.25, 45.50. 5-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)thiophene-2-carboxylic acid (5.11) A 250mL round-bottom flask was charged with 5.10 (0.85g, 3.192mmol), EtOH (100mL), 2M NaOH(aq) (6.5mL, 12.8mmol), and H 2O (10mL), and the resulting solution was stirred at room temperature for 4 hours. The reaction mixture was concentrated under reduced pressure to remove the remaining ethanol, and the resulting aqueous solution was acidified to pH 1 using 2M HCl(aq). The white precipitate was collected by vacuum filtration, washed with copious amounts of cold H 2O and cold ether, and dried in the vacuum oven overnight to yield a white solid (0.659g, 82%). 1 H NMR (400 MHz, DMSO-d6) δ 13.09 (brs, 1H), 11.39 (s, 1H), 7.80 (d, J = 7.9 Hz, 1H), 7.60 (d, J = 3.7 Hz, 1H), 7.16 (d, J = 3.8 Hz, 1H), 5.62 (d, J = 7.8 Hz, 1H), 5.06 (s, 2H). 13 C NMR (400 MHz, DMSO-d6) δ 163.54, 162.64, 150.75, 145.92, 144.92, 134.54, 133.01, 128.05, 101.73, 45.52. 163 N-(2-amino-4-(trifluoromethyl)phenyl)-5-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)thiophene-2-carboxamide A 50mL round bottom flask containing a solution of 5.11 (0.250g, 0.991mmol) and DIPEA (0.31mL, 1.802mmol) in DMF (5mL) was charged with HATU (0.377g, 0.991mmol) and stirred at room temperature for 10 minutes. The yellow solution was then charged with 3,4- diaminobenzotrifluoride (0.525g, 2.97mmol) and allowed to stir at room temperature overnight. After 17 hours, the reaction was quenched with saturated NaHCO 3 (10 mL) and extracted with EtOAc (3 x 8mL). The combined organic layers were washed with brine (5mL), dried over Na 2SO 4 and dry-loaded onto celite. The crude mixture was purified by automated column chromatography (100% EtOAc) to yield a light brown solid that was used directly in the next step (0.360g, 97% yield). 1-((5-(5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)thiophen-2-yl)methyl)pyrimidine- 164 2,4(1H,3H)-dione (5.12) An 8-dram vial with N-(2-amino-4-(trifluoromethyl)phenyl)-5-((2,4-dioxo-3,4-dihydropyrimidin- 1(2H)-yl)methyl)thiophene-2-carboxamide from the previous step (0.360 g, 0.877 mmol) was charged with glacial acetic acid (10 mL), and the resulting orange solution was stirred at 100°C overnight. After 13h, the reaction mixture was allowed to cool to room temperature and then was concentrated under reduced pressure. The solid was treated with water (10mL) and the pH was adjusted to pH 12 with 2M NaOH(aq). The resulting solid was extracted with EtOAc (3 x 20mL), and the combined organic layers were dry-loaded onto celite. The residue was purified by automated column chromatography (100% EtOAc) to yield an off-white solid (0.310g, 90%). 1 H NMR (400 MHz, DMSO-d6) δ 11.45 (s, 1H), 7.88 (s, 1H), 7.84 (d, J = 7.9 Hz, 1H), 7.76 (d, J = 3.7 Hz, 1H), 7.72 (d, J = 8.4 Hz, 1H), 7.50 (d, J = 8.1 Hz, 0H), 7.24 (d, J = 3.7 Hz, 1H), 5.64 (d, J = 7.9 Hz, 1H), 5.10 (s, 1H). 13 C NMR (400 MHz, DMSO-d6) δ 163.60, 150.82, 149.31, 144.97, 142.42, 133.03, 128.41, 127.46, 126.37, 123.25, 122.93, 122.61, 118.97, 109.57, 101.74, 45.47. N-(2-amino-4-(trifluoromethoxy)phenyl)-5-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)thiophene-2-carboxamide A 50mL round bottom flask containing a solution of 5.11 (0.279g, 1.1mmol) and DIPEA (0.35mL, 2.0mmol) in DMF (6mL) was charged HATU (0.420g, 1.105mmol) in one portion and stirred at 165 room temperature for 10 minutes. The yellow solution was then charged with 4- (trifluoromethoxy)benzene-1,2-diamine (0.633g, 3.3mmol) and allowed to stir at room temperature overnight. After 17 hours, the reaction mixture was diluted with EtOAc (10mL) and washed with saturated NaHCO 3 (10mL). The aqueous layer was then extracted with EtOAc (3 x 10mL), and the combined organic layers were washed with brine (10mL), and dry-laoded onto celite. The crude mixture was purified by automated column chromatography (80% EtOAc/hexanes) to yield a brown solid (0.45g, 96%) that was used directly in the next step. 1-((5-(5-(trifluoromethoxy)-1H-benzo[d]imidazol-2-yl)thiophen-2-yl)methyl)pyrimidine- 2,4(1H,3H)-dione (5.13) An 8-dram vial with N-(2-amino-4-(trifluoromethoxy)phenyl)-5-((2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)methyl)thiophene-2-carboxamide from the previous step (0.45g, 1.06mmol), was charged with glacial acetic acid (10mL) and stirred at 100°C overnight. After 13h, the reaction mixture was allowed to cool to room temperature and then was concentrated under reduced pressure. The solid was treated with water (10mL) and the pH was adjusted to pH 12 with 2M NaOH(aq). The resulting solid was extracted with EtOAc (3 x 20mL), and the combined organic layers were dry-loaded onto celite. The residue was purified by automated column chromatography (100% EtOAc) to yield a light-brown solid (0.375g, 87%). 1 H NMR (400 MHz, 166 DMSO-d6) δ 13.23 (s, 1H), 11.41 (s, 1H), 7.84 (d, J = 7.9 Hz, 1H), 7.72 (d, J = 3.7 Hz, 1H), 7.61 (d, J = 8.7 Hz, 1H), 7.53 (s, 1H), 7.23 (d, J = 3.7 Hz, 1H), 7.18 (d, J = 8.1 Hz, 1H), 5.64 (d, J = 7.9 Hz, 1H), 5.09 (s, 2H). 13 C NMR (400 MHz, DMSO-d6) δ 163.56, 150.79, 144.94, 142.01, 133.21, 128.31, 127.00, 101.70, 45.43. cyclopropylmethyl 3-(cyclopropylmethoxy)-4-fluorobenzoate (5.14) A 100mL round bottom flask containing a solution of 4-fluoro-3-hydroxybenzoic acid (2.984g, 19.12mmol) in DMF (27mL) was charged sequentially with bromomethyl cyclopropane (3.75mL, 38.67mmol), potassium carbonate (5.341g, 38.64mmol), and potassium iodide (0.3231g, 1.946mmol), and then heated to 90°C overnight. After 22 hours, the reaction was incomplete by TLC, so more bromomethyl cyclopropane (1.85mL, 19.12mmol) was added and the reaction was allowed to continue stirring at 90°C overnight again. After a total of 41 hours, the reaction was cooled to 0°C and quenched with water (30mL). The aqueous layer was extracted with ethyl acetate (3 x 30mL), and the combined organic layers were washed with brine (30mL), dried over sodium sulfate, and concentrated under reduced pressure to yield a orange semi-solid (4.4074g, 87%, rf=0.8 in 80:20 EtOAc:Hexane) that was used directly in the next step. (3-(cyclopropylmethoxy)-4-fluorophenyl)methanol (5.15) 167 A 100mL round bottom flask containing ester 5.14 (4.4074g, 16.68mmol) and a stir bar was placed under Argon and charged with dry Toluene (20mL). The resulting orange solution was cooled to 0°C and charged dropwise with a 1.0M solution of DIBAL in hexanes (42mL, 41.69mmol). The then colorless solution was stirred at 0°C for 2 hours, and then quenched with water (5mL) and 1.0 NaOH(aq) (5mL) resulting in a white precipitate. The solid byproduct was collected by filtration and washed with ethyl acetate (250mL), and the filtrate was concentrated under reduced pressure. The residue was charge with water (50mL) and extracted with ethyl acetate (3 x 30mL), and the combined organic layers were washed with brine (30mL), dried over sodium sulfate, concentrated under reduced pressure, and dry-loaded onto celite. The crude product was then purified using flash column chromatography with the product eluting in a broad peak between 40-45% ethyl acetate/hexane. The relevant fractions were combined and concentrated under reduced pressure to yield a white, shiny crystalline solid (2.6215g, 80%, rf=0.15 in 80:20 EtOAc:Hexane). 3-(cyclopropylmethoxy)-4-fluorobenzaldehyde (5.16) A 250mL round bottom flask containing a solution of alcohol 5.15 (2.6215g, 13.36mmol) and CH 2Cl 2 (100mL) was charged with manganese dioxide and the resulting black mixture was stirred at room temperature overnight. After 24 hours, the mixture was vacuum filtered through a cake of celite and washed with CH 2Cl 2 (200mL). The filtrate was washed with brine (50mL), dried over 168 sodium sulfate, and concentrated under reduced pressure to yield a white solid (2.2488g, 87%, rf=0.7 in 80:20 EtOAc:Hexane) that was used directly in the following step. 1-(3-(cyclopropylmethoxy)-4-fluorophenyl)propan-1-ol (5.17) A 100mL round bottom flask containing aldehyde 5.16 (2.2488g, 11.58mmol) and a stir bar was placed under Argon and charged with dry THF (30mL). The resulting clear, colorless solution was cooled to 0°C and charged dropwise with a 1.0M solution of EtMgBr in THF (12.2mL, 12.16mmol). The resulting pale yellow solution was warmed to room temperature and stirred for 2 hours, and then recooled to 0°C and quenched with saturated NH 4Cl(aq) (30mL). The cloudy mixture was extracted with ethyl acetate (3 x 50mL), and the combined organic layers were washed with brine (30mL), dried over sodium sulfate, concentrated under reduced pressure to yield a pale yellow oil (2.597g, 100%, rf=0.25 in 80:20 EtOAc:Hexane) that was used directly in the following step. 4-(1-azidopropyl)-2-(cyclopropylmethoxy)-1-fluorobenzene (5.18) A 100mL round bottom flask containing alcohol 5.17 (2.7724g, 12.36mmol) and diphenylphosphoryl azide (DPPA, 3.223mL, 15mmol) and dry toluene (12mL) was placed under Argon and cooled to 0°C. The cooled solution was charged dropwise with 1,8- 169 Diazabicyclo[5.4.0]undec-7-ene (DBU, 2.24mL, 15mmol), stirred at 0°C for 2 hours, and then at room temperature overnight. After 16 hours, the amber reaction mixture was quenched with water (20mL), and the aqueous layer was extracted with toluene (3 x 15mL). The combined organic layers were washed with brine (30mL), dried over sodium sulfate, concentrated under reduced pressure, and dry-loaded onto celite. The crude product was then purified using flash column chromatography with the product eluting in a broad peak between 40-45% ethyl acetate/hexane. The relevant fractions were combined and concentrated under reduced pressure to yield a white, shiny crystalline solid (2.6215g, 80%, rf=0.85 in 80:20 EtOAc:Hexane). 1-(3-(cyclopropylmethoxy)-4-fluorophenyl)propan-1-amine (5.19) A 100mL round bottom flask containing azide 5.18 (2.0608g, 8.27mmol) and MeOH (25mL) was charged with Pd/C (5wt%, 0.4122g, 3.87mmol) in one portion. The flask was placed under hydrogen atmosphere and stirred vigorously at room temperature overnight. After 16 hours, the reaction still contained unreacted starting material, so an additional amount of Pd/C (0.103g, 0.97mmol) was added and the reaction was allowed to continue stirring at room temperature for one hour. The mixture was then filtered through a cake of celite and washed with copious amounts of methanol (200mL). The filtrate was then concentrated under reduced pressure to yield a clear oil (1.820g, 99%, rf=0.05 in 80:20 EtOAc:Hexane) that was used directly in the next step. 170 3-chloro-N-(1-(3-(cyclopropylmethoxy)-4-fluorophenyl)propyl)propane-1-sulfonamide (5.20) A 100mL round bottom flask containing amine 5.19 (1.82g, 8.15mmol) was placed under Argon atmosphere and dissolved in dry CH 2Cl 2 (27mL). The solution was cooled to 0°C and charged with triethylamine (2.4mL, 17mmol) and 3-chloropropane sulfonyl chloride (1.0mL, 8.3mmol) in dropwise fashion. The pale yellow reaction mixture was stirred at 0°C for 15 minutes and then allowed to warm to room temperature and stir overnight. After 17 hours, the reaction quenched with water (30mL) and extracted with CH 2Cl 2 (3 x 30mL). The combined organic layers were washed with 1.0M HCl(aq) (40mL) and brine (40mL), dried over sodium sulfate, and concentrated under reduced pressure to yield a viscous yellow oil (2.836g, 96%, rf=0.70 in 70:30 EtOAc:Hexane) that was used directly in the following step. 3-(N-(1-(3-(cyclopropylmethoxy)-4-fluorophenyl)propyl)sulfamoyl)propyl acetate (5.21) A 100mL round bottom flask containing alkyl chloride 5.20 (2.836g, 7.79mmol), sodium acetate (1.407g, 17.15mmol), and sodium iodide (2.570g, 17.15mmol) was placed under Argon atmosphere and charged with dry DMF (30mL). The yellow solution was heated to 80°C overnight. After 18 hours, the reaction was quenched with water (30mL) and extracted with ethyl acetate (3 x 30mL). The combined organic layers were washed with brine (30mL), dried 171 over sodium sulfate, concentrated under reduced pressure, and dry-loaded onto celite. The crude product was then purified using flash column chromatography with the product eluting in 40% ethyl acetate/hexane. The relevant fractions were combined and concentrated under reduced pressure to yield a clear, colorless oil (1.5109g, 50%, rf=0.50 in 70:30 EtOAc:Hexane) that was used directly in the next step. N-(1-(3-(cyclopropylmethoxy)-4-fluorophenyl)propyl)-3-hydroxypropane-1-sulfonamide (5.22) A 100mL round bottom flask containing ester 5.21 (1.5109g, 3.9mmol) and 1.25M HCl in MeOH (20mL) was refluxed (75-80°C) overnight. After 20 hours, the reaction was allowed to cool to room temperature and was then concentrated under reduced pressure and dry-loaded onto celite. The crude product was then purified using flash column chromatography with the product eluting between 70-80% ethyl acetate/hexane. The relevant fractions were combined and concentrated under reduced pressure to yield a clear, colorless oil (1.0816g, 80%, rf=0.30 in 50:50 EtOAc:Hexane) that was used directly in the next step. N-(1-(3-(cyclopropylmethoxy)-4-fluorophenyl)propyl)-3-(methoxymethoxy)propane-1- sulfonamide (5.23) 172 A 100mL round bottom flask containing alcohol 5.22 (1.0816g, 3.13mmoL) was placed under Argon atmosphere and dissolved in dry CH 2Cl 2 (16mL). The clear solution was charged with N,N’- diisopropylethylamine (DIPEA, 1.12mL, 6.26mmol) and cooled to 0°C. Chloromethyl methyl ether (MOM-Cl, 0.29mL, 3.76mmol) was added dropwise at 0°C. The reaction was stirred at 0°C until the fumes dissipated, and then was allowed to warm to room temperature and stir overnight. After 18 hours, the pale pink/orange reaction mixture was quenched slowly with saturated NH 4Cl(aq) (35mL) and extracted with CH 2Cl 2 (3 x 20mL). The combined organic layers were washed with brine (30mL), dried over sodium sulfate, concentrated under reduced pressure, and dry-loaded onto celite. The crude product was then purified using flash column chromatography with the product eluting in 40% ethyl acetate/hexane. The relevant fractions were combined and concentrated under reduced pressure to yield a clear, colorless oil (0.7636g, 63%, rf=0.80 in 50:50 EtOAc:Hexane) that was used directly in the next step. 2,4-bis((trimethylsilyl)oxy)pyrimidine (5.24) An oven-dried 10mL round bottom flask containing uracil (0.069g, 0.613mmol) dissolved in dry CH 2Cl 2 (2mL) was placed under Argon atmosphere. The white suspension was charged dropwise with N,O-Bis(trimethylsilyl)acetamide (BSA, 0.37mL, 1.53mmol) at room temperature and allowed to stir at room temperature for 1.5 hours until the solution was clear. Solution was used directly in the following step. 173 N-(1-(3-(cyclopropylmethoxy)-4-fluorophenyl)propyl)-3-((2,4-dioxo-3,4-dihydropyrimidin- 1(2H)-yl)methoxy)propane-1-sulfonamide (5.25) An oven-dried 5mL pear bottom flask containing MOM ether 5.23 (0.2386g, 0.613mmol) dissolved in CH 2Cl 2 (1.5mL) was placed under Argon atmosphere and cooled to 0°C. The solution was charged dropwise with boron trichloride (1.0M in CH 2Cl 2, 0.23mL, 0.23mmol) and stirred at 0°C for 15 minutes, then allowed to warm to room temperature and stir for 1 hour. The solution was canula transferred to the 10mL round bottom flask containing TMS-protected uracil from the previous step. The reaction mixture was charged with a catalytic amount of N-tetrabutyl ammonium iodide (0.0058g, 0.023mmol), purged with Argon, and allowed to stir at room temperature overnight. After 23 hours, the reaction was quenched with saturated NaHCO 3(aq) (5mL) and extracted with ethyl acetate (4 x 10mL). The combined organic layers were washed with brine (10mL), dried over sodium sulfate, concentrated under reduced pressure, and dry- loaded onto celite. The crude product was then purified using two sequential flash columns: 1) product eluted in 20% MeOH in CH 2 Cl 2 , but with byproducts seen in 19 F NMR; 2) product eluted in 98-100% ethyl acetate/hexane. The relevant fractions were combined and concentrated under reduced pressure to yield a white, sticky foam (0.0881g, 31%, rf=0.55 in 100% EtOAc). 1 H NMR (400 MHz, Chloroform-d) δ 8.34 (s, 1H), 7.23 (d, J = 7.9 Hz, 1H), 7.06 (dd, J = 11.0, 8.2 Hz, 1H), 6.88 (dd, J = 8.0, 2.1 Hz, 1H), 6.82 (ddd, J = 8.3, 4.2, 2.1 Hz, 1H), 5.77 (dd, J = 7.9, 2.3 Hz, 1H), 5.03 174 (s, 2H), 4.71 (d, J = 6.7 Hz, 1H), 4.27 (q, J = 7.2 Hz, 1H), 3.88 (d, J = 7.0 Hz, 2H), 3.50 (dd, J = 6.6, 5.4 Hz, 2H), 2.75 (ddd, J = 14.8, 9.2, 6.0 Hz, 1H), 2.61 (ddd, J = 14.3, 9.1, 5.8 Hz, 1H), 1.91 (ddd, J = 15.0, 9.0, 6.1 Hz, 1H), 1.87 – 1.79 (m, 1H), 1.75 (td, J = 14.0, 7.1 Hz, 1H), 1.35 – 1.22 (m, 1H), 0.93 – 0.81 (m, 4H), 0.71 – 0.60 (m, 2H), 0.36 (dt, J = 5.9, 4.7 Hz, 2H). 13 C NMR (400 MHz, cdcl 3) δ 163.37, 153.44, 151.04, 150.99, 147.26, 147.15, 143.26, 137.92, 137.88, 119.27, 119.20, 116.44, 116.25, 114.12, 103.30, 77.33, 77.01, 76.69, 76.56, 74.57, 66.92, 59.41, 50.39, 31.56, 30.68, 23.77, 22.63, 14.10, 10.73, 10.24, 3.31, 3.29. 19 F NMR (400 MHz, DMSO-d6) δ -134.4. methyl 4-((5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzoate (5.26) A 500mL round-bottom flask was charged with 5-fluorouracil (8.517g, 65.5mmol), DMSO (170mL) and K 2CO 3 (9.05g, 65.5mmol). The resulting suspension was stirred at room temperature for 30 minutes and then charged dropwise over 1 hour with a solution of methyl 4- (bromomethyl)benzoate (5.0g, 21.8mmol) in DMSO (30mL) and then stirred at room temperature overnight. After 19 hours, the reaction mixture had solidified. The flask was cooled to 0°C, charged with H 2O (400mL), and extracted with EtOAc (4 x 150mL). The combined organic layers were washed with brine (150mL) and concentrated under reduced pressure. The resulting white solid was charged with 1:1 EtOAc/hexanes (250 mL) and was stirred at room temperature for 1 hour. The precipitate was collected by filtration, washed with cold water and cold 1:1 175 EtOAc/hexanes, and dried in the vacuum oven overnight to yield a white solid (8.55g, 47%). 1 H NMR (400 MHz, DMSO-d6) δ11.88 (s, 1H), 8.24 (d, J = 6.7 Hz, 1H), 7.95 (d, J = 8.6 Hz, 2H), 7.45 (d, J = 8.7 Hz, 2H), 4.91 (s, 2H), 3.85 (s, 3H). 13 C NMR (400 MHz, DMSO-d6) δ 165.92, 157.58, 157.33, 149.65, 141.94, 141.00, 138.72, 130.24, 129.91, 129.46, 128.89, 127.50, 52.16, 50.46. 19 F NMR (376 MHz, DMSO-d6) δ -168.95. methyl 4-((5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzoate (5.27) A 500mL round bottom flask containing thymine (7.2307g, 57.34mmol) and DMSO (200mL) was charged with K 2CO 3 and stirred for 20 minutes at room temperature. The suspension was charged dropwise with a solution of methyl 4-(bromomethyl)benzoate (4.378g, 19.11mmol) in DMSO (25mL) over a period of 30 minutes and then stirred at room temperature overnight. After 22 hours, the reaction mixture had developed a white precipitate. The reaction was cooled to 0°C, quenched with H 2O (400mL), and extracted with ethyl acetate (4 x 150mL). The combined organic layers were washed with brine (150mL) and concentrated under reduced pressure to yield a white solid. The solid was dissolved in a 1:1 mixture of hexane and ethyl acetate (250mL) and stirred at room temperature for one hour. The suspension was filtered to yield a powdery white solid (3.4472g, 55% yield). 1 H NMR (400 MHz, DMSO-d6) δ 11.35 (s, 1H), 7.95 (d, J = 8.6 Hz, 2H), 7.65 (d, J = 1.3 Hz, 1H), 7.41 (d, J = 8.3 Hz, 2H), 4.92 (s, 2H), 3.84 (s, 3H), 1.76 (s, 3H). 13 C 176 NMR (400 MHz, DMSO-d6) δ 165.92, 164.24, 150.98, 142.48, 141.29, 129.49, 128.81, 127.47, 109.17, 52.14, 49.90, 11.95. 4-((5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzoic acid (5.28) A 500mL round bottom flask containing 5.26 (7.0g, 25.2mmol) in 1:1 MeOH/H2O (200 mL) was charged dropwise with 2M NaOH(aq) (100mL) and stirred at room temperature overnight. The methanol was removed under reduced pressure, and the resulting aqueous solution was acidified to pH 1 with concentrated HCl. The precipitate was collected by vacuum filtration, washed with cold water and dried in the vacuum oven to yield a white solid (5.85g, 88%). 1 H NMR (400 MHz, DMSO-d6) δ 12.96 (s, 1H), 11.88 (d, J = 5.1 Hz, 1H), 8.23 (d, J = 6.7 Hz, 1H), 7.93 (d, J = 8.5 Hz, 2H), 7.42 (d, J = 8.6 Hz, 2H), 4.90 (s, 2H). 13 C NMR (400 MHz, DMSO-d6) δ 167.01, 157.60, 157.34, 149.67, 141.44, 141.01, 138.73, 130.25, 130.08, 129.92, 129.63, 127.37, 50.48. 19 F NMR (400 MHz, DMSO-d6) δ -168.95. 177 4-((5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)benzoic acid (5.29) A 250mL round bottom flask with methyl ester 5.27 (3.4472g, 12.57mmol) dissolved in 1:1 MeOH/H 2O (90mL) was charged dropwise with 50mL of 2M NaOH(aq) solution, and the resulting mixture was stirred at room temperature overnight. After 16 hours, the methanol was removed under reduced pressure and the remaining aqueous solution was acidified to pH 1 with 12M HCl (~10mL). The resulting precipitate was filtered, washed with H 2 O, and dried in a vacuum oven for 4 hours to afford a fine white powder (2.5202g, 77% yield). 1 H NMR (400 MHz, DMSO-d6) δ 12.94 (s, 1H), 11.35 (s, 1H), 7.92 (d, J = 8.2 Hz, 2H), 7.64 (s, 1H), 7.38 (d, J = 8.1 Hz, 2H), 4.91 (s, 2H), 1.76 (s, 3H). 13 C NMR (400 MHz, DMSO-d6) δ 166.99, 164.25, 151.00, 141.99, 141.30, 130.00, 129.65, 127.32, 109.16, 49.90, 11.96. N-(2-amino-4-(trifluoromethyl)phenyl)-4-((5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)benzamide (5.30) 178 An 100mL round bottom flask containing 5.28 (1.50g, 5.68mmol) and DIPEA (2.0mL, 11.35mmol) in DMF (20mL) was charged with HATU (2.2g, 5.68mmol), and the resulting yellow solution was stirred at room temperature for 10 minutes. The mixture was then charged with 3,4- diaminobenzotrifluoride (4.0g, 22.7mmol) and allowed to stir at room temperature overnight. After 14 hours, the reaction mixture was quenched with saturated NaHCO 3 (30mL) and extracted with EtOAc (3 x 30mL). The combined organic layers were washed with brine (30mL), and dry- loaded onto celite. The residue was purified by autocolumn chromatography (70% EtOAc/hexanes) to yield a yellow solid (1.602g, 67%) that was used directly in the next step. N-(2-amino-4-(trifluoromethoxy)phenyl)-4-((5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)benzamide (5.31) An 100mL round bottom flask containing 5.28 (1.50g, 5.68mmol) and DIPEA (2.0mL, 11.35mmol) in DMF (20mL) was charged with HATU (2.2g, 5.68mmol), and the resulting yellow solution was stirred at room temperature for 10 minutes. The mixture was then charged with 4- (trifluoromethoxy)benzene-1,2-diamine (4.36g, 22.7mmol) and allowed to stir at room temperature overnight. After 14 hours, the reaction mixture was quenched with saturated NaHCO 3 (30mL) and extracted with EtOAc (3 x 30mL). The combined organic layers were washed 179 with brine (30mL), and dry-loaded onto celite. The residue was purified by autocolumn chromatography (70% EtOAc/hexanes) to yield a brownish yellow solid (1.515g, 61%) that was used directly in the next step. N-(2-amino-4-(trifluoromethyl)phenyl)-4-((5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)benzamide (5.32) An 100mL round bottom flask containing 5.29 (0.900g, 3.45mmol) and DIPEA (1.2mL, 6.9mmol) in DMF (15mL) was charged with HATU (1.34g, 3.45mmol), and the resulting yellow solution was stirred at room temperature for 10 minutes. The mixture was then charged with 3,4- diaminobenzotrifluoride (2.43g, 13.8mmol) and allowed to stir at room temperature overnight. After 14 hours, the reaction mixture was quenched with saturated NaHCO 3 (30mL) and extracted with EtOAc (3 x 30mL). The combined organic layers were washed with brine (30mL), and dry- loaded onto celite. The residue was purified by autocolumn chromatography (70% EtOAc/hexanes) to yield a tan solid (0.968g, 67%) that was used directly in the next step. 180 N-(2-amino-4-(trifluoromethoxy)phenyl)-4-((5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)benzamide (5.33) A 50mL round bottom flask containing a solution of 5.29 (0.700g, 2.69mmol) and DIPEA (0.94mL, 10.76mmol) in DMF (5mL) was charged with HATU (1.0227g, 2.69mmol) in one portion. The resulting yellow solution was stirred at room temperature for ten minutes, then was charged with 4-(trifluoromethoxy)benzene-1,2-diamine (2.067g, 10.76mmol) and stirred at room temperature overnight. After 15 hours, the reaction was quenched with water (30mL) and extracted with ethyl acetate (4 x 10mL). The combined organics layers were concentrated under reduced pressure and dry-loaded onto celite. The crude mixture was then purified using flash column chromatography with the product eluting in a broad peak between 70-90% ethyl acetate/hexane. The relevant fractions were combined and concentrated under reduced pressure to yield a brown solid (1.168g, 100%) that was taken directly on to the next step without further characterization. 181 5-fluoro-1-(4-(5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)benzyl)pyrimidine- 2,4(1H,3H)-dione (5.34) An 8-dram vial with 5.30 (1.602g, 3.8mmol) was charged with glacial acetic acid (10mL) and stirred at 100°C overnight. After 15 hours, the reaction mixture was allowed to cool to room temperature and then was concentrated under reduced pressure. The solid was treated with water (10mL) and the pH was adjusted to pH 12 with 2M NaOH(aq). The resulting solid was extracted with EtOAc (3 x 20mL), and the combined organic layers were dry-loaded onto celite. The residue was purified by automated column chromatography (100% EtOAc) to yield a yellow- orange solid (1.40g, 91%). 1 H NMR (400 MHz, DMSO-d6) δ 13.36 (s, 1H), 11.89 (s, 1H), 8.27 (d, J = 6.7 Hz, 1H), 8.19 (d, J = 8.3 Hz, 2H), 8.03 (s, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.52 (d, J = 8.3 Hz, 2H), 4.92 (s, 2H). 19 F NMR (376 MHz, DMSO-d6) δ -58.76, -168.85. 5-Fluoro-1-(4-(5-(trifluoromethoxy)-1H-benzo[d]imidazol-2-yl)benzyl)pyrimidine- 182 2,4(1H,3H)-dione (5.35) An 8-dram vial with 5.31 (1.515g, 3.41mmol) was charged with glacial acetic acid (10mL) and stirred at 100°C overnight. After 15 hours, the reaction mixture was allowed to cool to room temperature and then was concentrated under reduced pressure. The solid was treated with water (10mL) and the pH was adjusted to pH 12 with 2M NaOH(aq). The resulting solid was extracted with EtOAc (3 x 20mL), and the combined organic layers were dry-loaded onto celite. The residue was purified by automated column chromatography (100% EtOAc) to yield a light- brownish-yellow solid (1.45g, 90% yield). 1 H NMR (400 MHz, DMSO-d6) δ 13.21 (s, 1H), 11.87 (s, 1H), 8.26 (d, J = 6.6 Hz, 1H), 8.16 (d, J = 8.3 Hz, 2H), 7.77 – 7.57 (m, 2H), 7.50 (d, J = 8.1 Hz, 2H), 7.22 – 7.17 (m, 1H), 4.91 (s, 2H). 13C NMR (126 MHz, DMSO-d6) δ 157.61, 157.40, 153.01, 149.72, 143.71, 140.80, 138.97, 138.70, 130.22, 129.95, 129.02, 128.03, 126.85, 121.37, 119.34, 50.50. 19 F NMR (400 MHz, DMSO-d6) δ -57.00, -168.94. 5-methyl-1-(4-(5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)benzyl)pyrimidine- 2,4(1H,3H)-dione (5.36) An 8-dram vial with 5.32 (0.968g, 0.231mmol) was charged with glacial acetic acid (10mL) and stirred at 100°C overnight. After 15 hours, the reaction mixture was allowed to cool to room 183 temperature and then was concentrated under reduced pressure. The solid was treated with water (10mL) and the pH was adjusted to pH 12 with 2M NaOH(aq). The resulting solid was extracted with EtOAc (3 x 20mL), and the combined organic layers were dry-loaded onto celite. The residue was purified by automated column chromatography (100% EtOAc) to yield an off- white solid (0.881g, 95%). 1 H NMR (400 MHz, DMSO-d6) δ 13.36 (s, 1H), 11.36 (s, 1H), 8.19 (d, J = 8.0 Hz, 2H), 8.02 (s, 1H), 7.83 (s, 1H), 7.68 (s, 1H), 7.51 (m, 3H), 4.93 (s, 2H), 1.78 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 164.28, 151.05, 141.31, 128.74, 128.04, 127.08, 109.16, 49.93, 11.98. 5-methyl-1-(4-(5-(trifluoromethoxy)-1H-benzo[d]imidazol-2-yl)benzyl)pyrimidine- 2,4(1H,3H)-dione (5.37) An 8-dram vial was charged with 5.33 (1.168g, 2.69mmol) and glacial acetic acid (10mL) and was stirred at 100°C overnight. After 15 hours, the acetic acid was removed under reduced pressure, and the resulting tan solid was charged with saturated Na 2CO 3(aq) (75mL) and stirred at room temperature for 1 hour. The pH was confirmed to be pH 8, and the solid was collected by filtration and washed with cold water (200mL) and then dried for several hours in a vacuum oven to yield a light brown solid (0.821g, 73%). 1 H NMR (400 MHz, DMSO-d6) δ 13.20 (s, 1H), 11.36 (s, 184 1H), 8.16 (d, J = 8.2 Hz, 2H), 7.68 –7.59 (m, 3H), 7.47 (d, J = 8.1 Hz, 2H), 7.20 (d, J = 7.7 Hz, 1H), 4.92 (s, 2H), 1.77 (s, 3H). 13 C NMR (125 MHz, DMSO-d6) δ 164.29, 153.04, 151.06, 143.74, 141.32, 139.26, 128.94, 128.01, 126.89, 121.38, 119.35, 109.17, 49.93, 11.99. 19 F NMR (470 MHz, DMSO- d6) δ -57.00. 5-fluoro-2,4-bis((trimethylsilyl)oxy)pyrimidine (5.38) An oven-dried 25mL round bottom flask containing 5-fluorouracil (0.0379g, 0.291mmol) dissolved in dry CH 2Cl 2 (5mL) was placed under Argon atmosphere. The white suspension was charged dropwise with N,O-Bis(trimethylsilyl)acetamide (BSA, 0.19mL, 0.771mmol) at room temperature and allowed to stir at room temperature for 1 hour until the solution was clear. Solution was used directly in the following step. 5-methyl-2,4-bis((trimethylsilyl)oxy)pyrimidine (5.39) An oven-dried 10mL round bottom flask containing thymine (0.033g, 0.26mmol) dissolved in dry CH 2Cl 2 (2mL) was placed under Argon atmosphere. The white suspension was charged dropwise with N,O-Bis(trimethylsilyl)acetamide (BSA, 0.16mL, 0.65mmol) at room temperature and 185 allowed to stir at room temperature for 1.5 hours. Solution was used directly in the following step. (R)-N-(1-(3-(cyclopropylmethoxy)-4-fluorophenyl)propyl)-3-((5-fluoro-2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)methoxy)propane-1-sulfonamide (5.40) A solution of MOM ether 5.23 (0.136g, 0.349mmol) dissolved in CH 2Cl 2 (3mL) was added to the 25mL round bottom flask containing TMS-protected 5-Fluorouracil 5.38 from the previous step. The reaction mixture was charged dropwise over a period of 1.5 hours with a solution of Tin (IV) chloride (0.0228mL, 0.195mmol) in CH 2Cl 2 (3mL). The clear solution was stirred for an additional 10 minutes at room temperature after addition of SnCl 4 was complete, and then was concentrated under reduced pressure. The white residue was dissolved in chloroform (10mL), washed with brine (10mL), dried over sodium sulfate, concentrated under reduced pressure, and dry-loaded onto celite. The crude product was then purified by flash column chromatography with the product eluting between 50-55% ethyl acetate in hexane. The relevant fractions were combined and concentrated under reduced pressure to yield a white, sticky foam (0.0158g, 11%). 1 H NMR (400 MHz, Chloroform-d) δ 9.54 (d, J = 4.7 Hz, 1H), 7.35 (dd, J = 5.3, 1.2 Hz, 1H), 7.04 (ddd, J = 10.9, 8.2, 1.2 Hz, 1H), 6.91 (dt, J = 8.0, 1.6 Hz, 1H), 6.83 (ddd, J = 8.5, 3.8, 1.8 Hz, 1H), 5.37 – 5.27 (m, 1H), 5.01 (s, 2H), 4.23 (q, J = 7.3 Hz, 1H), 3.88 (dd, J = 7.0, 1.1 Hz, 2H), 3.49 (h, J = 186 4.6 Hz, 2H), 2.75 (ddd, J = 14.7, 8.8, 6.1 Hz, 1H), 2.60 (ddd, J = 14.3, 8.8, 5.9 Hz, 1H), 2.21 – 2.06 (m, 0H), 1.86 (tt, J = 14.5, 8.2 Hz, 2H), 1.78 (s, 2H), 1.83 – 1.66 (m, 1H), 1.42 – 1.18 (m, 4H), 1.01 – 0.80 (m, 4H), 0.64 (dtd, J = 6.2, 4.9, 2.3 Hz, 2H), 0.35 (qd, J = 6.1, 5.6, 3.2 Hz, 2H). 13 C NMR (400 MHz, CDCl 3) δ 157.38, 157.12, 153.46, 151.02, 149.79, 147.27, 147.16, 142.02, 139.64, 137.94, 137.90, 127.32, 127.00, 119.34, 119.27, 116.41, 116.22, 114.14, 77.33, 77.02, 76.88, 76.70, 74.62, 66.98, 59.43, 50.23, 30.66, 29.68, 23.72, 10.73, 10.22, 3.29, 3.26. 19 F NMR (400 MHz, DMSO-d6) δ -135.6, -164.2. (R)-N-(1-(3-(cyclopropylmethoxy)-4-fluorophenyl)propyl)-3-((5-methyl-2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)methoxy)propane-1-sulfonamide (5.41) An oven-dried 5mL pear bottom flask containing MOM ether 5.23 (0.085g, 0.22mmol) (0.2386g, 0.613mmol) dissolved in CH 2Cl 2 (1mL) was placed under Argon atmosphere and cooled to 0°C. The solution was charged dropwise with boron trichloride (1.0M in CH 2Cl 2, 0.1mL, 0.1mmol) and stirred at 0°C for 15 minutes, then allowed to warm to room temperature and stir for 1 hour. The solution was canula transferred to the 10mL round bottom flask containing TMS-protected thiamine 5.39 from the previous step. The reaction mixture was charged with a catalytic amount of N-tetrabutyl ammonium iodide (0.002g, 0.01mmol), purged with Argon, and allowed to stir at room temperature overnight. After 17 hours, the reaction was quenched with saturated 187 NaHCO 3(aq) (5mL) and extracted with ethyl acetate (4 x 10mL). The combined organic layers were washed with brine (10mL), dried over sodium sulfate, concentrated under reduced pressure, and dry-loaded onto celite. The crude product was then purified using autocolumn chromatography (98-100% EtOAc/hexane). The relevant fractions were combined and concentrated under reduced pressure to yield a white foam (0.0271g, 26%). 1 H NMR (400 MHz, Chloroform-d) δ 8.34 (s, 1H), 7.23 (d, J = 7.9 Hz, 1H), 7.06 (dd, J = 11.0, 8.2 Hz, 1H), 6.88 (dd, J = 8.0, 2.1 Hz, 1H), 6.82 (ddd, J = 8.3, 4.2, 2.1 Hz, 1H), 5.77 (dd, J = 7.9, 2.3 Hz, 1H), 5.03 (s, 2H), 4.71 (d, J = 6.7 Hz, 1H), 4.27 (q, J = 7.2 Hz, 1H), 3.88 (d, J = 7.0 Hz, 2H), 3.50 (dd, J = 6.6, 5.4 Hz, 2H), 2.75 (ddd, J = 14.8, 9.2, 6.0 Hz, 1H), 2.61 (ddd, J = 14.3, 9.1, 5.8 Hz, 1H), 1.91 (ddd, J = 15.0, 9.0, 6.1 Hz, 1H), 1.87 – 1.79 (m, 1H), 1.75 (td, J = 14.0, 7.1 Hz, 1H), 1.35 – 1.22 (m, 1H), 0.93 – 0.81 (m, 4H), 0.71 – 0.60 (m, 2H), 0.36 (dt, J = 5.9, 4.7 Hz, 2H). 13 C NMR (400 MHz, CdCl 3) δ 163.37, 153.44, 151.04, 150.99, 147.26, 147.15, 143.26, 137.92, 137.88, 119.27, 119.20, 116.44, 116.25, 114.12, 103.30, 77.33, 77.01, 76.69, 76.56, 74.57, 66.92, 59.41, 50.39, 31.56, 30.68, 23.77, 22.63, 14.10, 10.73, 10.24, 3.31, 3.29. 19 F NMR (400 MHz, DMSO-d6) δ -134.6. 188 5.7. 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Cancer Chemother Pharmacol 2014, 73 (3), 577–583. 218 Appendix: Selected Spectra 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 f1 (ppm) -100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 PROTON_01 2.66 1.83 1.01 1.95 2.04 1.00 1.48 1.02 1.04 1.06 1.13 1.15 1.17 1.22 1.24 1.25 2.48 dmso 2.48 dmso 2.49 dmso 2.49 dmso 2.50 dmso 3.32 HDO 4.08 4.10 4.14 4.15 4.17 4.19 6.06 6.09 6.73 6.73 6.73 6.74 6.82 6.82 6.83 6.84 6.84 6.87 7.48 7.48 7.52 7.52 7.54 7.56 8.31 8.42 10.90 1 2 3 4 5 6 O 7 8 9 1 0 O H 1 1 O 1 2 1 3 1 4 1 5 O 1 6 O 1 7 1 8 C H 3 1 9 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 f1 (ppm) -4 -2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 CARBON_01 14.61 14.64 39.33 dmso 39.54 dmso 39.75 dmso 39.87 39.96 dmso 40.17 dmso 40.38 dmso 40.58 dmso 60.52 102.39 102.41 111.98 114.48 116.73 120.19 131.35 145.95 145.98 155.81 159.66 163.31 166.70 1 2 3 4 5 6 O 7 8 9 1 0 O H 1 1 O 1 2 1 3 1 4 1 5 O 1 6 O 1 7 1 8 C H 3 1 9 244 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 f1 (ppm) -200 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 PROTON_01 2.77 1.94 1.06 1.01 0.95 1.06 1.06 1.00 1.23 1.25 1.27 1.89 2.20 2.29 2.30 2.31 2.48 dmso 2.48 dmso 2.49 dmso 2.49 dmso 2.50 dmso 3.28 3.28 3.28 3.29 3.29 3.29 3.29 3.29 3.29 3.29 3.29 3.30 3.30 3.30 3.30 3.30 3.30 HDO 3.31 3.31 4.16 4.18 4.20 4.21 6.91 6.92 6.96 7.19 7.20 7.21 7.22 7.32 7.32 7.32 7.32 7.33 7.52 7.52 7.56 7.56 7.75 7.78 8.55 8.56 1 2 3 4 5 6 O 7 8 9 1 0 O 1 1 1 2 1 3 1 4 O 1 5 O 1 6 1 7 C H 3 1 8 O 1 9 O 2 0 2 1 C H 3 2 2 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 f1 (ppm) -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 CARBON_01 14.58 39.35 dmso 39.56 dmso 39.77 dmso 39.98 dmso 40.19 dmso 40.40 dmso 40.60 dmso 60.73 109.99 117.27 120.89 122.23 154.15 154.28 159.13 166.41 169.15 1 2 3 4 5 6 O 7 8 9 1 0 O 1 1 1 2 1 3 1 4 O 1 5 O 1 6 1 7 C H 3 1 8 O 1 9 O 2 0 2 1 C H 3 2 2 245 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 f1 (ppm) -10 0 10 20 30 40 50 60 70 80 90 100 110 120 CARBON_01 14.57 14.61 21.19 21.21 21.29 21.34 25.32 39.33 dmso 39.54 dmso 39.75 dmso 39.96 dmso 40.17 dmso 40.38 dmso 40.58 dmso 60.20 60.74 70.03 70.13 71.80 110.17 110.25 110.75 114.02 117.37 119.21 120.83 128.44 129.58 139.80 152.82 153.49 156.80 159.96 162.19 169.28 170.78 172.22 190.76 1 2 3 4 5 6 O 7 8 9 1 0 O 1 1 1 2 1 3 1 4 O 1 5 O 1 6 1 7 C H 3 1 8 O H 1 9 O H 2 0 O 2 1 O 2 2 2 3 C H 3 2 4 246 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 f1 (ppm) -200 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 PROTON_01 3.36 1.30 1.15 1.29 1.19 1.00 2.30 2.31 2.31 2.31 2.31 2.32 2.48 dmso 2.48 dmso 2.49 dmso 2.49 dmso 2.50 dmso 2.50 dmso 3.25 3.27 3.28 3.28 3.28 3.28 3.28 3.28 3.28 3.28 3.28 3.28 3.29 3.29 3.29 3.29 3.29 3.29 3.29 3.29 3.29 3.29 3.30 3.30 3.30 3.30 3.30 HDO 3.31 3.31 3.31 3.31 3.31 3.31 3.31 3.32 3.32 3.32 3.32 3.35 7.24 7.24 7.26 7.26 7.37 7.37 7.38 7.38 7.38 7.38 8.02 8.02 8.02 8.04 8.04 8.68 8.68 10.01 10.01 1 2 3 4 5 6 O 7 8 9 1 0 O 1 1 1 2 O 1 3 O 1 4 O 1 5 1 6 C H 3 1 7 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 f1 (ppm) 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 PROTON_01 1.22 1.36 1.34 1.26 1.00 1.14 1.16 1.18 1.22 1.74 1.74 1.89 1.97 1.99 2.48 dmso 2.48 dmso 2.49 dmso 2.49 dmso 2.50 dmso 2.51 dmso 3.15 3.16 3.31 HDO 4.00 4.02 4.08 6.65 6.76 6.77 6.77 6.77 6.85 6.86 6.87 6.88 7.25 7.80 7.82 8.57 8.57 9.95 11.28 11.88 1 2 3 4 5 6 O 7 8 9 1 0 O H 1 1 O 1 2 1 3 O 1 4 247 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 f1 (ppm) -5 0 5 10 15 20 25 30 35 40 45 50 55 60 CARBON_01 27.85 27.88 27.90 27.99 28.43 30.20 76.68 cdcl3 77.00 cdcl3 77.32 cdcl3 77.49 79.70 80.99 152.12 O 1 2 N 3 O 4 5 C H 3 6 C H 3 7 C H 3 8 C H 3 9 1 0 1 1 N H 1 2 C H 3 1 3 248 249 250 251 252

Abstract (if available)

Abstract N-formylation is an important biological process and an interesting and vital synthetic strategy in organic chemistry and drug discovery. The N-formyl functional group connects to each ensuing chapter of this dissertation, and therefore its prevalence in disease and drug discovery is discussed in Chapter 1. Then, a novel mechanism for N-formylation by N,N’-dimethylformamide (DMF) facilitated by water and oxygen is proposed in Chapter 2. N-formylated peptides from bacteria are responsible for activating formyl peptide receptors (FPR) and consequently other pro-inflammatory processes that have been linked to Alzheimer’s disease, cancer, chronic inflammatory diseases, and many others. Cyclosporine, a cyclic peptide that is traditionally used as an immunosuppressant drug, is a potent FPR1 inhibitor that has also demonstrated a protective effect on mitochondrial ultrastructure and function in cases of traumatic brain injury (TBI). A light-based drug delivery system for targeted and controlled delivery of cyclosporine and another lead candidate, gabapentin, in cases of TBI is discussed in Chapter 4 along with many synthetic methods for diversification of the delivery system discussed in Chapter 3. Finally, one of the ten FDA approved drugs that contain N-formyl functionality, all discussed in Chapter 1, is Leucovorin, also called folinic acid, and it is used to stabilize the binding of 5-fluorouracil (5-FU), an anticancer drug, to its target enzyme thymidylate synthase. Increased activity of another enzyme, deoxyuridine triphosphatase (dUTPase), has been implicated in several 5-FU-resistant cancers, including colorectal cancer, breast cancer, and non–small cell lung cancer. The computational design, synthesis, and evaluation of novel dUTPase inhibitors for 5-fluorouracil-resistant cancers are discussed in Chapter 5.

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Accessible cytotoxic and antiviral drug analogues: improved synthetic approaches to isoindolinones and bioisosteric difluoromethylated nucleotides, and the search for therapeutic organotelluranes

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Creator DeAngelo, Caitlin M. (author)

Core Title Chemical investigations in drug discovery and drug delivery

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 10/19/2020

Defense Date 08/28/2018

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag 5-fluorouracil,computational drug design,cyanine dye,deoxyuridine triphosphatase,dimethylformamide,DMF,dUTPase,heptamethine,inhibitor,light-based drug delivery,near-IR light,N-formyl,N-formyl functional group,N-formyl peptide receptor,N-formylation,NIR light,OAI-PMH Harvest,photocage,secondary injury,structure-based drug design,TBI,traumatic brain injury,uncaging

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Advisor Petasis, Nicos A. (committee chair), Prakash, G.K. Surya (committee member), Zhang, Yong (committee member)

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