Graduation date: 1996

Graduation date: 1996

Not available / text

published_or_final_version / Biochemistry / Master / Master of Philosophy

This thesis describes the synthesis of novel analogs of the unique opioid receptor modulator mitragynine along with pharmacological and behavioral studies of a subset of its novel analogs. In Chapter 1, a general overview of opioid receptors and the importance of the mu opioid receptor (MOR) for the treatment of pain is provided. The rise of the opioid epidemic is discussed which brings into attention the need to develop safer opioid therapeutics for the treatment of pain. In this regard the Mitragyna indole alkaloids, isolated from kratom leaves are of great interest as they are considered to be “atypical” opioid ligands and represent novel molecular scaffolds for the development of safer opioid receptor modulators. The introductory chapter includes a brief description of the pharmacological profile of mitragynine as a prelude to the work that follows – examining unexplored positions on the alkaloid by devising new methodology and synthetic routes to synthesize novel analogs to study its structure-activity relationship (SAR) at the opioid receptors. Chapter 2 describes the development of a new synthetic method for selective functionalization of the unexplored C11 position of the MG scaffold (C6 position in indole numbering). The method takes advantage of an indole-ethylene glycol adduct as a key intermediate which can undergo subsequent iridium-catalyzed borylation at the desired position. This late-stage C(sp2)-H functionalization approach provides a practical route to novel C11-analogs of mitragynine and related scaffolds starting from the natural product, thus allowing a systematic SAR exploration of the C11 position. Chapter 3 directly builds on Chapter 2, summarizing the neuropharmacological and behavioral studies on the C11 analogs of 7-hydroxymitragynine (7OH) and mitragynine ethylene glycol (MG-EG). Through these studies we discover that the C11 position represents a key locant for fine-tuning opioid receptor signaling efficacy. We also discuss that the parent 7-hydroxymitragynine (7OH), a low efficacy agonist, is transformed to an even lower efficacy agonist by introducing a fluorine substituent at the C11 position (11-F-7OH). This is demonstrated in vitro at both mouse and human mu opioid receptors (mMOR/hMOR) and in vivo in mouse analgesia tests. Low efficacy opioid agonists are of high interest as candidates for generating safer opioid therapeutics with mitigated side effects. Thus, this section concludes with the identification 11-F-7OH as lead compound for future investigation. Chapter 4 describes our attempts towards the functionalization of another unexplored and vital position for the activity of mitragynine at the mu opioid receptor (MOR) – the ethyl group at the C20 position. This chapter illustrates our extensive efforts towards the late-stage functionalization of the ethyl group in the C20 position of mitragynine via directed C(sp3)-H activation. Various strategies including using the mitragynine ethylene glycol (MG-EG) as a bidentate ligand or manipulating the acrylate ester group on mitragynine as a directing group are discussed in the chapter. Also described are all the screened reaction conditions using palladium catalysts and various ligands starting from pyridine-based to mono-protected amino acid-based ligands. The outcomes and hypotheses for the failures of each strategy employed are also presented in the chapter. Chapter 5 describes our efforts towards the de novo synthesis of the C20 analogs, as an alternative strategy to the failed late-stage functionalization from Chapter 4. We present a strategy to synthesize the C20 analogs through a diversification strategy from a common intermediate. We further discuss the results of our efforts towards the formal synthesis of this common intermediate. The chapter concludes with a discussion of an alternate strategy for the synthesis of the C20 analogs.

Major Depressive Disorder (MDD) is a common and debilitating mental illness primarily characterized by depressed mood and anhedonia, as well as a heterogenous mixture of other somatic symptoms. Existing therapies for MDD act primarily on monoamine transporters and receptors, with only partial success. A more recent advancement in depression treatment is the identification of the N-methyl-d-aspartate receptor (NMDAR) antagonist (R,S)-ketamine, which has shown efficacy in individuals with treatment resistant depression. Studies have raised the possibility of a mu opioid receptor (MOR)-dependent component to the actions of (R,S)- ketamine, through direct activation of MOR or indirect effects of NMDAR antagonism on endogenous opioids. Considering the ongoing global opioid epidemic, in which over prescribing of opioid painkillers and greater availability of illicit opioids has caused significant morbidity and mortality, studies suggesting an opioid component to ketamine’s actions have caused concern. We undertook the current experiments to better understand 1) the contribution of MORs to the behavioral effects of ketamine, and 2) how very low efficacy agonism of MOR may lead to a better separation between the desirable and undesirable effects of opioid drugs. Previous work from collaborators had established that a single dose of (R,S)-ketamine, specific hydroxynorketamine (HNK) metabolites, and the memantine derivative fluoroethylnormemantine (FENM), given one week before a contextual fear conditioning stressor could prevent the development of learned fear and behavioral despair. Moreover, some of these drugs also prevented the development of learned fear and behavioral despair when given shortly after the contextual fear conditioning stressor. We were interested in how (R,S)- ketamine’s reported MOR activity may contribute to these behavioral effects. We began by testing these compounds in cell-based signaling assays to determine their ability to directly activate MORs. We found that the parent (R)- and (S)-ketamine enantiomers could activate MORs with low potency in a high amplification G protein activation assay. In contrast, in low amplification miniG-based assays, the compounds tested showed little to no activation of MOR, suggesting that (R,S)-ketamine activates MOR with low potency and low efficacy. We moved to experiments in mice with the pseudo-irreversible MOR antagonist methocinnamox (MCAM) and attempted to block various behavioral effects of (R,S)-ketamine and the more selective NMDAR antagonist (FENM). We found that MCAM pretreatment blocked effects of (R,S)-ketamine on antinociception, behavioral despair, and learned fear, while other effects such as anesthesia and changes in hippocampal electrophysiology were not prevented by MCAM. With FENM the antinociceptive effects were less potent and less impacted by MCAM; however, the effects on behavioral despair and learned fear were still MORdependent. These results suggest that the protective effects of (R,S)-ketamine and FENM against stress may be an indirect effect of NMDAR antagonism on endogenous opioids. In a separate line of experiments, we used a range of doses of MCAM to antagonize the behavioral effects of morphine, the MOR agonist antidepressant tianeptine, and the MOR partial agonist 7-OH mitragynine. 7-OH mitragynine is a metabolite of mitragynine, the major alkaloid in kratom, which some reports suggest may have antidepressant and anxiolytic properties. Based on fundamentals of pharmacology, we hypothesized that inherent differences in the presence of “spare receptors” or receptor reserve between brain circuits could be revealed by differential inhibition by MCAM across behaviors and agonists. We assessed the inhibitory potency of MCAM against these drugs in tests of antinociception, hyperlocomotion, behavioral despair, respiration, and gastrointestinal motility. We found that MCAM pretreatment more potently inhibited the low efficacy agonist 7-OH mitragynine in the tests of antinociception, behavioral despair, and constipation. These data suggest that in circuits modulating antinociception, behavioral despair, and constipation, differences in receptor reserve likely facilitate the response to low efficacy agonists. However, our data also argue that the wider therapeutic window of G protein biased, low intrinsic efficacy MOR agonists is not solely a result of differences in the number of “spare” MORs regulating the effects of opioids in different circuits.

published_or_final_version / abstract / Anaesthesiology / Master / Master of Philosophy

There is a dogma in molecular biology that biological functions of peptides are determined by their structure ("function" code), coded in their primary structure ("structure" code). This work describes a new approach that attempts to elucidate these relationships by peptide topology design based on intriguing conformational properties of pipecolic acid based amino acids--like 1,2,3,4 tetrahydroisoquinoline (Tic). Opioid peptides, owing to the heterogeneity of opioid receptors, display a wide variety of physiological actions. The mu opioid receptor selective octapeptide I (D-Tic-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂) is a model compound for topographical modifications induced by sequential substitutions by Tic residue. Thus, the closely related peptides I and II (Gly-D-Tic-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂, obtained by coupling Gly residue to I) have contrasting affinities for the mu opioid receptor (IC₅₀ = 1.2 and 278 nM, respectively). Conformational analysis of I and II by means of 1D and 2D ¹H NMR spectroscopy allowed to determine dramatic differences in the side chain orientation of D-Tic in both peptides and to propose features of the bioactive conformation. The extended conformation of I (due to g(-) side chain conformation of D-Tic) is well recognized by the mu receptor in contrast to the folded conformation of II (due to a g(+) side chain conformation of D-Tic¹, that places the aromatic ring on the opposite side of the molecule), which is not. Peptide III (D-Phe-Cys-Tic-D-Trp-Orn-Thr-Pen-Thr-NH₂), featuring replacement of Tyr³ by Tic³, binds very weakly to the mu opioid receptor, due to rotation of the Tic aromatic side chain to the opposite side of the molecule (Tic side chain is in a g(+) conformation again). As these substitutions conserve the conformation of the backbone, constrained cyclic amino acids (picolic acid derivatives) can modify the topography of the peptide in a predictable manner, and (in conjunction with biological data) disclose structural elements of bioactive conformations. The mechanisms of pipecolic acid side chain rotamer selection, will be discussed in the context of design principles.

Opioid/adrenergic interactions were studied to evaluate two hypotheses: (1) naloxone potentiates the effect of epinephrine on cardiac contractility by increasing circulating epinephrine concentrations; and (2) endogenous and exogenous opioids alter left cardiac nerve stimulationinduced norepinephrine release and cardiac function. A canine isolated heart-lung preparation was used for the first study. Plasma epinephrine was determined and myocardial epinephrine uptake was calculated during intravenous epinephrine infusion. Naloxone (4 mg) was given and the epinephrine infusion was repeated. Naloxone increased cardiac contractility, coronary blood flow, and the coronary sinus epinephrine concentration. When coronary blood flow was subsequently held constant (100% above resting), naloxone increased only contractility. This result indicated that the previously observed increase in coronary sinus epinephrine was flow dependent. Corticosterone (an uptake II blocker) was employed as a positive control. Corticosterone increased the contractile response to epinephrine, but unlike naloxone, corticosterone was accompanied by a clear decrease in myocardial epinephrine uptake. The stereospecificity of the response to naloxone was investigated and (+) naloxone equaled or exceeded (-) naloxone in potentiating the inotropic effect of epinephrine. In the second study, the left cardiac nerve was isolated and electrically stimulated in intact dogs. Norepinephrine overflow gradually declined during successive control stimulations. Pretreatment with naloxone (100 Mg/kg) prevented or delayed the decline. An intracoronary dynorphin 1-9 infusion (2 nmol/min/kg for 20 minutes) reduced both norepinephrine overflow and cardiac performance, and both effects were prevented by pretreatment with naloxone (100 /xg/kg) . To summarize, naloxone potentiated the inotropic effect of infused epinephrine without altering circulating epinephrine concentrations or myocardial epinephrine uptake. This effect of naloxone was not stereospecific and probably not mediated through a traditional opiate receptor. Endogenous and exogenous opioids inhibited the left cardiac nerve stimulation-induced norepinephrine overflow, suggesting that opiate receptors may regulate cardiac excitability by modulating norepinephrine release.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences