The Design, Synthesis, and BiologicalEvaluation of Novel Peptidic Ligands for

the Treatment of Chronic Neuropathic Pain

Item Type text; Electronic Dissertation

Authors Remesic, Michael Vincent

Publisher The University of Arizona.

Rights Copyright © is held by the author. Digital access to this materialis made possible by the University Libraries, University of Arizona.Further transmission, reproduction or presentation (such aspublic display or performance) of protected items is prohibitedexcept with permission of the author.

Download date 19/05/2018 23:04:13

Link to Item http://hdl.handle.net/10150/625593

1

THE DESIGN, SYNTHESIS, AND BIOLOGICAL EVALUATION OF NOVEL PEPTIDIC

LIGANDS FOR THE TREATMENT OF CHRONIC NEUROPATHIC PAIN

by

Michael Vincent Remesic

__________________________

Copyright © Michael Vincent Remesic 2017

A Dissertation Submitted to the Faculty of the

DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY

In Partial Fulfillment of the Requirements

For the Degree of

DOCTOR OF PHILOSOPHY

WITH A MAJOR IN CHEMISTRY

In the Graduate College

THE UNIVERSITY OF ARIZONA

2017

2

THE UNIVERSITY OF ARIZONA

GRADUATE COLLEGE

As members of the Dissertation Committee, we certify that we have read the dissertation

prepared by Michael Vincent Remesic, titled The Design, Synthesis, and Biological Evaluation

of Novel Peptidic Ligands for the Treatment of Chronic Neuropathic Pain and recommend that it

be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy.

_______________________________________________________________________ Date: July 10th, 2017

Victor J. Hruby, Ph.D.

_______________________________________________________________________ Date: July 10th, 2017

Yeon Sun Lee, Ph.D.

_______________________________________________________________________ Date: July 10th, 2017

Richard Glass, Ph.D.

_______________________________________________________________________ Date: July 10th, 2017

Edita Navratilova, Ph.D.

Final approval and acceptance of this dissertation is contingent upon the candidate’s submission

of the final copies of the dissertation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my direction and recommend

that it be accepted as fulfilling the dissertation requirement.

________________________________________________ Date: July 10th, 2017

Dissertation Director: Victor J. Hruby, Ph.D.

3

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of the requirements for an advanced

degree at the University of Arizona and is deposited in the University Library to be made

available to borrowers under rules of the Library.

Brief quotations from this dissertation are allowable without special permission, provided that an

accurate acknowledgement of the source is made. Requests for permission for extended

quotation from or reproduction of this manuscript in whole or in part may be granted by the head

of the major department or the Dean of the Graduate College when in his or her judgment the

proposed use of the material is in the interests of scholarship. In all other instances, however,

permission must be obtained from the author.

SIGNED: Michael Vincent Remesic

4

ACKNOWLEDGEMENTS

I would like to take the time to give my sincere gratitude to Dr. Victor J. Hruby for his

support over the years. He always asked me insightful questions and gave great feedback that

pushed my research forward. Most importantly, he was always confident in my abilities and

always drove me to make my mark in the scientific community. Dr. Hruby’s kindness also

extended outside of the lab as I share many great memories with him. I am immensely grateful to

be a part of his research group.

Dr. Yeon Sun Lee contributed greatly to my successes here as a graduate student. Had I

not had the opportunity to collaborate with her and have her take on a mentor-like role, I am

confident in saying that I would not have been exposed to as many techniques nor acquired the

skillset in the lab that I currently have. Arguably the most significant thing she has done for me

is to try and bring out my best and be sure that I tried to achieve my full potential. When I was

satisfied with a given result, she always said that I could do better and that making mistakes are

ok, as long as you are not making the same ones. She was always most welcoming to my

questions and was great company to have as we would converse about the many intricacies of

science and life.

I would like to also thank Dr. Richard Glass on having such a positive impact on me. I

found his lectures inspirational and his approach to teaching to be most beneficial. Instead of

giving me answers to questions that I would have, he forced me to think critically about the

given problem and work through the answer.

5

Thank you to Dr. Edita Navratilova for providing great feedback and advice throughout

the duration of my graduate pursuits. She has been great company and I have had many great

discussions throughout our many encounters.

I also would like to express my thanks to Dr. Frank Porreca for allowing me to attend his

group meetings and be a part of the great discussions. Also, Dr. Porecca was kind enough to let

me use his lab space and gave me great advice on how to go about tackling my ambitions.

Last, but not least, I would like to thank Hruby group members past and present,

especially Christine Hiner, as they have contributed greatly to my happiness, and to the friends

who have shared this journey with me.

6

DEDICATION

Dedicated to my parents for their generous support, love, and encouragement in the pursuit of

my education. If it were not for them, I do not know what would have been;

the loving memory of my grandfather, who despite no longer being with us, has never left my

side.

7

Table of Contents

LIST OF FIGURES ................................................................................................... 11-12

LIST OF TABLES ..................................................................................................... 13-14

ABSTRACT ..................................................................................................................... 15

ABBREVIATIONS .................................................................................................... 16-20

Chapter 1: Pain and the Opioid Receptors (ORs) ....................................................... 21

1.1 Pain ................................................................................................................. 21-25

1.2 ORs and Their Role in Pain States .............................................................. 25-28

1.2.1 Biased Signaling ...................................................................................... 28-29

1.2.2 Orthosteric and Allosteric Interactions ................................................ 29-31

1.2.3 Endogenous Ligands for the ORs.......................................................... 31-32

1.3 Mu-Opioid Receptor (MOR) ........................................................................ 32-35

1.4 Delta-Opioid Receptor (DOR) ...................................................................... 35-37

1.5 Kappa-Opioid Receptor (KOR) ................................................................... 37-39

1.6 Peptide Therapeutics ..................................................................................... 39-41

1.7 Workflow ........................................................................................................ 41-42

Chapter 2: Neuroexcitatory Effects of Dynorphin A (DYN A) .................................. 43

2.1 DYN A's Paradoxical Behavior .................................................................... 43-44

2.2 DYN A at the Bradykinin Receptors (BRs) ................................................ 44-46

2.3 The BRs .......................................................................................................... 46-48

8

Chapter 3: Novel Peptidic Ligands at the Bradykinin-2 Receptor (B2R) ................. 49

3.1 Previous SARs of DYN A .............................................................................. 49-56

3.2 Increasing the Stability of lead ligand [des-Arg7]-DYN A-(4-11) ............. 56-58

3.3 Experimental .................................................................................................. 58-64

3.4 Results and Discussion .................................................................................. 64-68

3.5 Summary and Future Directions ................................................................. 68-69

Chapter 4: Multifunctional Opioid Ligands for the Treatment of Chronic Pain ..... 70

4.1 Benefits of a Multifunctional Ligand ........................................................... 70-71

4.2 The Synergistic Effect Between the MOR and DOR ................................. 71-72

4.3 Recent SARs for MOR/DOR Multifunctional Ligands ............................. 72-80

4.4 Enkephalin (ENK) Analogues ........................................................................... 80

4.4.1 Rationale and Design ................................................................................... 80

4.4.2 Experimental ........................................................................................... 81-84

4.4.3 Results and Discussion ........................................................................... 84-88

4.4.4 Summary and Future Directions........................................................... 88-89

4.5 [DAla2, DLys4]-dermorphin (DALDA) Analogues .......................................... 89

4.5.1 Introduction to [Dmt1]DALDA ............................................................. 89-90

4.5.2 Rationale and Design .............................................................................. 90-91

4.5.3 Results and Discussion ........................................................................... 92-93

4.5.4 Summary and Future Directions........................................................... 93-94

4.6 Biphalin Analogues ............................................................................................ 94

4.6.1 Introduction to Biphalin ........................................................................ 94-99

9

4.6.2 Rationale and Design .......................................................................... 100-101

4.6.3 Experimental ....................................................................................... 101-103

4.6.4 Results and Discussion ....................................................................... 103-105

4.6.5 Summary and Future Directions.............................................................. 105

4.7 Endomorphin (EM) Analogues ....................................................................... 106

4.7.1 Introduction to EMs ........................................................................... 106-108

4.7.2 Rationale and Design .......................................................................... 108-109

4.7.3 Experimental .............................................................................................. 110

4.7.4 Results and Discussion .............................................................................. 111

4.7.5 Summary and Future Directions.............................................................. 112

Appendix A: Publications ............................................................................................ 113

A.1 Amphipathic Non-opioid Dynorphin A Analogs to Inhibit Neuroexcitatory Effects

at Central Bradykinin Receptors ..................................................................... 114-115

A.2 Blockade of non-opioid excitatory effects of spinal dynorphin A at bradykinin

receptors ............................................................................................................. 116-119

A.3 Cyclic non-opioid dynorphin A analogues for the bradykinin receptors...120-123

A.4 Cyclic Opioid Peptides .............................................................................. 124-139

A.5 Modification of amphipathic non-opioid dynorphin A analogues for rat brain

bradykinin receptors ......................................................................................... 140-143

A.6 Recent Advances in the Realm of Allosteric Modulators for Opioid Receptors for

Future Therapeutics .......................................................................................... 144-155

10

A.7 Structure-Activity Relationships of [des-Arg7]Dynorphin A Analogues at the κ

Opioid Receptor ................................................................................................. 156-163

A.8 Various modifications of the amphipathic dynorphin A pharmacophore for rat

brain bradykinin receptors ............................................................................... 164-168

REFERENCES ....................................................................................................... 169-193

11

List of Figures

Figure 1. The difference of chronic pain prevalence in gender of developed and undeveloped

countries ............................................................................................................................ 22

Figure 2. (A) The economic cost of various diseases in 2010. (B) The amount of money the NIH

categorically funded in 2015 ............................................................................................. 23

Figure 3. Schematic of a GPCR signaling through the inhibitory Gi/Go protein ............. 27

Figure 4. Structures of biased ligands at the MOR .......................................................... 29

Figure 5. MOR-selective small molecule drugs based on the morphine scaffold ........... 34

Figure 6. DOR small molecule agonists and antagonists................................................. 36

Figure 7. KOR small molecule agonists and antagonists................................................. 37

Figure 8. Examples of cyclizations used for peptide ligands ........................................... 40

Figure 9. Drug discovery workflow ................................................................................. 42

Figure 10. Cellular responses of DYN A at the B2R in the spinal cord under normal (A) and

chronic pain (B) conditions ............................................................................................... 46

Figure 11. Pharmacophore of DYN A at the BRs............................................................ 51

Figure 12. Dose-dependent reversal of thermal hyperalgesia (left, radiant heat test) and tactile

hypersensitivity (right, von Frey test) using [des-Arg7]-DYN A-(4-11) (14 in the figure) in SNL-

injured rats ........................................................................................................................ 52

Figure 13. Structures of selected SPPS reagents: A) Fmoc-Lys(Boc)-Wang resin B) 2-

Chlorotrityl chloride resin C) HBTU D) HOBt ................................................................ 59

Figure 14. General scheme of SPPS to generate DYN A analogues ............................... 59

Figure 15. Synthesis of olefin or alkyl bridged cyclic DYN A analogues ....................... 62

12

Figure 16. N-terminus to side chain cyclization scheme of DYN A analogues .............. 63

Figure 17. Chemical structure of fentanyl and employed derivatives ............................. 73

Figure 18. ENK-like tetrapeptide analogues with various C-terminal modifications ...... 74

Figure 19. Antihyperalgesic and antiallodynic effects tested in the thermal hypersensitivity (left)

and von Frey filaments (right) assays of 57 (i.th. injection, 6 in the figure) using SNL-injured rats

........................................................................................................................................... 79

Figure 20. Structures of Fmoc-rink amide resin, BOP, and NMM .................................. 81

Figure 21. General scheme of LPPS to synthesize multifunctional analogues ................ 84

Figure 22. Structures of dimeric ENK analogues ............................................................ 88

Figure 23. Original design of bivalent ENK analogues ................................................... 95

Figure 24. Structures of other diamine bridges used with biphalin ................................. 99

Figure 25. Synthetic scheme of cyclic biphalin analogues ............................................ 103

Figure 26. Competition binding assays of MR239 at the MOR (left, [3H]DAMGO) and KOR

(right, [3H]U69593)......................................................................................................... 104

13

List of Tables

Table 1. Differences between acute and chronic pain syndromes ................................... 21

Table 2. Examples of chronic pain assessments to investigate pain symptoms ............... 24

Table 3. Drugs used to combat chronic neuropathic pain ................................................ 25

Table 4. Selectivity profiles of endogenous opioid peptides at the ORs.......................... 32

Table 5. Binding affinities of selected DYN A fragments at the human ORs ................. 44

Table 6. Selected examples of peptidic agonists and antagonists at the BRs .................. 48

Table 7. Binding affinities of select DYN A analogues at rat brain BRs ........................ 50

Table 8. Binding affinities of DYN A analogues at BRs in rat brain .............................. 55

Table 9. Analytical data of cyclic DYN A analogues ...................................................... 61

Table 10: Binding affinities of DYN A analogues with Aib substitutions at the BRs in rat brain

........................................................................................................................................... 66

Table 11. Binding affinities of cyclic DYN A analogues at the BRs in rat brain ............ 67

Table 12. Bioactivities of the ENK-like tetrapeptide analogues ...................................... 76

Table 13. Structure of lipophilic Enk-like tetrapeptide analogues ................................... 77

Table 14. Bioactivities of lipophilic opioid ligands ......................................................... 78

Table 15. Analytical data of synthesized multifunctional ligands for MOR/DOR/KOR 83

Table 16. In vitro biological activities of multifunctional peptide ligands at MOR/DOR/KOR

........................................................................................................................................... 85

Table 17. Analytical data of DALDA analogues ............................................................. 91

Table 18. Binding affinities of DALDA analogues at the MOR/DOR/KOR .................. 93

Table 19. Binding affinities of bivalent ENK analogues with varying linker length....... 96

14

Table 20. Analytical data of cyclic biphalin analogues ................................................. 101

Table 21. Binding affinities of cyclic biphalin analogues at the MOR/DOR/KOR ....... 105

Table 22. Analytical data of EM analogues ................................................................... 109

Table 23. Binding affinities of EM analogues at the MOR/DOR/KOR ........................ 111

15

Abstract

Chronic neuropathic pain is a disease that impacts the livelihood of millions of people in the

United States with no effective pain treatments and limited information pertaining to the underlying

mechanisms. Opioid therapy is considered the gold standard for pain therapeutics, but chronic use of

these medications brings about serious side effects such as tolerance, addiction, and respiratory

depression which limit their overall therapeutic potential. Herein, two approaches are discussed to

circumvent these issues: i) a multifunctional approach using N-phenyl-N-piperidin-4-yl-propionamide

(Ppp) coupled to various endogenous opioid ligand scaffolds, and ii) non-opioid dynorphin A (DYN A)

ligands at the Bradykinin-2 receptor (B2R).

The μ-opioid receptor (MOR) upon agonist stimulation provides analgesia and concomitant

activation of the δ-opioid receptor (DOR) leads to an increased antinociceptive effect. Chronic activation

of the MOR has been correlated with an upregulation of the κ-opioid receptor (KOR) and KOR-

associated side effects such as anxiety and depression. The discovery of a new class of opioid receptor

(OR) ligands that have the biological profile of MOR/DOR agonists and KOR antagonists would be

beneficial considering they would have an increased analgesic effect, leading to a lower dosage being

administered and thus lower overall side effects, and block symptoms elicited from KOR stimulation.

Discussed are various structure activity relationships (SARs) of numerous scaffolds that present novel

biological profiles. Ultimately, we discovered a compound that, to our knowledge, is the 1st MOR/DOR

agonist and KOR antagonist.

DYN A is the endogenous ligand for the KOR and its [des-Tyr1]-DYN A fragment interacts with

the B2R, but not the KOR, promoting hyperalgesia. Peptidomimetic non-opioid DYN A analogues were

synthesized and evaluated at the B2R. A minimum pharmacophore was identified and antagonists with

both improved biological stability and affinity were discovered.

16

Abbreviations

1-Nal – 1-naphthylalanine

Ac – acetyl

Acm – acetamidomethyl

AcOH – acetic acid

Aib – α-aminoisobutyric acid

B1R – bradykinin-1 receptor

B2R – bradykinin-2 receptor

BBB – blood brain barrier

BK – bradykinin

BMEC – bovine brain microvessel endothelial cells

Boc – t-butyloxycarbonyl

BOP – (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate

BR – bradykinin receptor

BSA – bovine serum albumin

Bzl – benzyl

cAMP – cyclic adenosine monophosphate

CHO – Chinese hamster ovaries

CNS – central nervous system

CPP – conditioned place preference

CSF – cerebral spinal fluid

DALA – [DAla2]-Met-enkephalinamide

17

DALDA – [DArg2, Lys4]-dermorphin

Dap – 2,3-diaminopropionic acid

DCM – dichloromethane

DER – dermorphin

DIC – N,N’-diisopropylcarbodiimide

DIPEA – diisopropylethylamine

DLT – deltorphin

DMF – N,N-dimethylformamide

Dmt –2′,6′-dimethyltyrosine

DOR – delta-opioid receptor

DRG – dorsal root ganglion

DYN – dynorphin

EM – endomorphin

END – endorphin

ENK – enkephalin

ESI – electronspray ionization

FAB-MS – fast atom bombardment mass spectrometry

Fmoc – 9-fluorenylmethyloxycarboxy

GDP – guanosine diphosphate

GPI – guinea pig ileum

GRK – G-protein-coupled receptor kinase

GTP – guanosine triphosphate

18

h – hour

HBTU – 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate

HEK – human embryonic kidney

HOBt – N-hydroxybenzotriazole

HR-MS – high resolution-mass spectrometry

Hyp – (2S,4R)-4-hydroxyproline

i.c.v. – intracerebroventricular

i.p. – intraperitoneal

i.th. – intrathecal

i.v. – intravenous

KO – knockout

KOR – kappa-opioid receptor

LCQ – liquid chromatography quadrupole

LPPS – liquid phase peptide synthesis

M – molarity

MALDI-TOF – matrix-assisted laser desorption/ionization-time of flight

MeOH – methanol

min – minute

MOR – mu-opioid receptor

MVD – mouse vas deferens

n.r. – no response

n.s. – not saturated

19

NEO – neoendorphin

NMDAR – N-Methyl-D-Aspartate receptor

NMM – N-methylmorpholine

Oic – octahydroindolecarboxylic acid

OR – opioid receptor

Pbf – 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl

PhiPr – 2-phenylisopropyl

PKA – protein kinase A

PMSF – phenylmethylsulfonylfluoride

ppm – parts per million

Ppp – N-phenyl-N-piperidin-4-yl-propionamide

RP-HPLC – reversed phase-high performance liquid chromatography

rt – room temperature

SEM – standard error of the mean

SNL – spinal nerve ligation

SPPS – solid phase peptide synthesis

TFA – trifluoroacetic acid

Thi – β-(2-thienyl)alanine

Tic – 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

TIS – triisopropylsilane

TLC – thin layer chromatography

tR – retention time

20

WT – wild type

21

Chapter 1: Pain and the Opioid Receptors (ORs)

1.1 Pain

Pain may be classified into three distinct categories. The first of which describes pain to

be physiologically protective with a purpose to minimize exposure to noxious stimuli and is

dubbed nociceptive pain.1 This may be thought of as a high-threshold pain that one experiences

due to intense stimuli such as placing a hand upon a hot stove. In response to this, a withdrawal

reflex is activated and an unpleasant feeling is experienced. The second type of pain, often

referred to as inflammatory pain, is protective and provides the individual with allodynic-like

effects in injured tissues brought about by the immune system via tissue injury or infection.

Heightened sensitivity and pain response in the localized area of injury discourages avoidable

use and contact to aid in the healing process. These first two categories are commonly associated

with acute pain, a type of pain that goes away after a short period of time and the person can

proceed with their life unaffected afterwards (Table 1).

Table 1. Differences between acute and chronic pain syndromes.

Acute Chronic

• Protective

• Adaptive

• Tissue injury/inflammation

• Pain does not persist

• May have no physiological purpose

• Maladaptive

• Abnormal nerve function

• Pain duration > 6 months

The third category, pathological pain, is the consequence of anomalous functioning of the

nervous system and is viewed as a disease state, typically associated with chronic pain.

22

Pathological pain can be elicited when there is an absence of noxious stimuli and inflammatory

response (dysfunctional pain) or by suffering damage to the nervous system (neuropathic pain)

and is the clinical pain syndrome with the largest unmet need.2

Figure 1. The difference of chronic pain prevalence in gender of developed and undeveloped

countries.

Chronic pain is highly prevalent no matter if you are a male or female, or in a developed

country or a developing country (Figure 1).3 In 2008, it was estimated that 100 million adults in

the United States were afflicted by chronic pain which in turn translated to costing a conservative

estimate of $560-635 billion in 2010 dollars.4,5 The National Institute of Health (NIH) reported

that the costs of chronic pain is greater than the economic costs of the 6 most costly major

diagnoses, and in lieu of these results, the NIH underfunded pain research relative to other

afflictions such as cancer and diabetes (Figure 2).6

0

10

20

30

40

50

60

70

80

1 2 3 4

Pre

vale

nce

(%

)

Age

Prevalence of Chronic Pain

Females in DevelopingCountries

Males in DevelopingCountries

Females in DevelopedCountries

Males in DevelopedCountries

18-35 36-50 51-65 66+

23

Figure 2. (A) The economic cost of various diseases in 2010. (B) The amount of money the NIH

categorically funded in 2015.

Providing symptomatic relief to chronic neuropathic pain is typically difficult, but a

plethora of assessments based on positive and negative sensory symptoms have been established

(Table 2).7-9 Classifying the type of pain based on its location comes with limitations considering

nervous system dysfunction can bring about pain distributions that do not coincide with their

respective nerve, root, segmental, or cortical regions.9 This observation has forced alternative

approaches to treatments with the paradigm shifting towards treating the underlying mechanism.

Although most of these mechanisms largely remain unknown, the advent of discoveries

pertaining to novel molecular targets, biotechnological advancements regarding gene therapies

and stem cells, and neural mechanisms of neuropathic pain pathways have fortified this thought

process.

0 200 400 600 800

Cardiovascular Diseases

Neoplasms

Injury and Poisoning

Endocrine/Nutritional/Metabolic

Digestive System Diseases

Pain

Billions of Dollars

A

0 1 2 3 4 5 6

Cardiovascular Disease

Pain

Cancer

Rare Disorders

Digestive Diseases

Diabetes

Billions of Dollars

B

24

Table 2. Examples of chronic pain assessments to investigate pain symptoms.

Symptom Definition Test Expected Response

Hypoalgesia Weakened feeling to

noxious stimuli Prick skin with a pin Reduced perception

Cold allodynia Non-painful cold

stimuli elicit pain

Touch skin with

objects of 20 °C

Painful temperature

sensation

Paroxysmal pain Electrical attacks for

seconds

Number of attacks

per episode -

Superficial pain Ongoing sensation of

pain Grade scale (0-10) -

Mechanical

allodynia

Painful sensation

caused by innocuous

stimuli

Touch objects Painful sensation

A vast number of drugs are used to alleviate the symptoms of chronic neuropathic pain,

but opioids are the most commonly prescribed class (Table 3).10,11 Such classes employed for

therapies include antidepressants, anticonvulsants, and non-steroidal anti-inflammatories

(NSAIDs), but apart from NSAIDs, their use is evaluated on a case-by-case basis as what is

deemed appropriate for one patient may not be for another. Although opioid therapies are

thought to be safe when dealing with acute pain scenarios, 9.6 to 11.5 million people within the

adult population were prescribed opioid therapy for long-term use.12 Morphine remains to be the

gold-standard for providing analgesia, but opioid analgesics have been responsible for a national

epidemic of addiction and overdose deaths. In 2013, out of 44,000 drug-overdose deaths, 56%

were attributed to pharmaceutical opioids and heroin.13 It is paramount that a new approach to

pain therapy is rendered as a means to provide safer treatment options.

25

Table 3. Drugs used to combat chronic neuropathic pain.

Drug

Structure Mechanism of Action Side Effects

Dextromethorphan

NMDA-antagonist Stomach pain, nausea,

constipation

Carbamazepine

Na+-channel blockade

Motor impairment,

migraines, alters

production of red and

white blood cells

Lamotrigine

Na+-channel blockade Rash, fever, fatigue,

anxiety, dizziness

Gabapentin

Na+- and Ca2+-channel

blockade

Ataxia, tremor,

peripheral edema,

fatigue

Baclofen

GABA-agonist

Withdrawal,

hallucinations,

insomnia, seizures

Oxycodone

MOR agonist Addiction, tolerance,

respiratory depression

1.2 ORs and Their Role in Pain States

The perception of pain commonly starts through the stimulation of peripheral receptors

where their function is to detect tissue-damaging stimuli.14 These primary afferent nociceptors,

26

once triggered, signal through the dorsal horn where it is then relayed to the thalamus and

cortex.15 The ORs are distributed throughout the pain-modulating circuit in which their ligands

regulate numerous behaviors such as feeding and pain.14 Postsynaptic inhibition of neurons in the

rostral ventromedial medulla and periaqueductal gray is brought about by MOR agonists which

activate inwardly rectifying potassium channels.16 The DOR has been observed on axon

terminals where antagonists for this receptor have been shown to block analgesia that is evoked

from activation of the periaqueductal gray.17,18 KOR agonists have demonstrated two presynaptic

roles in the rostral ventromedial medulla: i) hyperpolarization of neurons that are unaffected by

MOR agonists; and ii) block excitatory glutamatergic signaling to neurons including those

affected by MOR agonists.14

The ORs belong to the class A (Rhodopsin) G-protein-coupled receptor (GPCR) family.

GPCRs are made up of seven hydrophobic transmembrane domains, an extracellular N-terminal

domain, and an intracellular C-terminal tail, that mediate signaling through heterotrimeric G-

proteins which are on the intracellular surface to elicit a slew of signaling cascades and

responses.19 The ORs share about 60% sequence homology, most of which reside within their

transmembrane domains.20 Various kinds of stimuli such as small molecules, lipids, amino acids,

and peptides, can interact with their complementing GPCR and bring about a ligand-dependent

conformational alteration to recruit various proteins or stimulate others. ORs along with other

GPCRs can trigger responses via mechanisms that are independent of the G-protein, an example

being the β-arrestin pathway. The ORs exhibit signal transduction through the heterotrimeric

Gi/Go proteins as both G-protein subunits, α and βγ, interact with numerous cellular effector

networks, causing the inhibition of cyclic adenosine monophosphate (cAMP) formation from

27

adenosine triphosphate, a reaction catalyzed by the enzyme adenylyl cyclase. This leads to a

number of cellular events: the inhibition of cyclic adenosine monophosphate formation,

inhibition of Ca2+ internalization which reduces neuronal activity providing an antinociceptive

effect, the activation of K+ channels, and also modulation of gene transcription (Figure 3).20-22

Figure 3. Schematic of a GPCR signaling through the inhibitory Gi/Go protein.

The G-protein activation cycle is dependent upon guanosine triphosphate (GTP) and

guanosine diphosphate (GDP) exchange within the G-protein heterotrimeric complex. When

GDP is bound to the α-subunit, it forms a complex with the βγ-subunits and is inactive. When the

GPCR binds an agonist, the receptor gains affinity for the G-protein allowing for GDP release

from the α-subunit. GTP is then picked up by the α-subunit which reduces its affinity to the βγ-

subunits. These now dissociated subunits go on to activate or inhibit cellular effector systems.23

28

After the GPCR is activated, it is subjected to several events to regulate its signaling. The

receptor becomes phosphorylated by G protein-coupled receptor kinases (GRKs) which aid in

the uncoupling of the G-protein from the receptor (receptor desensitization) and eventually β-

arrestin is recruited to initiate receptor internalization via endocytosis into an intracellular

compartment.20 However, the fate of the receptor post GRK and β-arrestin activity does not

always lead to receptor recycling. Although the mu-opioid receptor (MOR) is often recycled

after undergoing endocytosis, the delta-opioid receptor (DOR) has been shown to be processed

further in the endocytic pathway where it is eventually degraded by lysosomes.24,25

1.2.1 Biased Signaling

Formerly, the GPCR paradigm included only an inactive and active form of the receptor,

but studies have concluded that there exist innumerable conformations that may provide a

distinct signaling outcome.26,27 Typically, agonists bind to the orthosteric site of their respective

receptor to trigger downstream events in a similar manner where the differences reside in

coupling efficacy.28 The MOR is a GPCR that provides analgesia upon agonist binding.

However, if β-arrestin 2 is recruited to the receptor, a different signaling cascade results and this

signaling pathway has been shown to negatively impact antinociception and is implicated in

augmenting MOR-related side effects.29 This pathway is independent of the G-protein and these

results were initially observed by seeing an increased analgesic response of morphine in β-

arrestin 2 knockout (KO) mice along with attenuated side-effects such as addiction and tolerance

which are associated with the MOR.30-34 On the basis of these results, efforts are shifting towards

the identification of functionally selective ligands, so called-biased ligands, that promote G-

protein-mediated signaling instead of β-arrestin 2 recruitment. Thus far, a few compounds have

29

been identified to show increased analgesia with reduced side-effect profiles which are parallel

with β-arrestin 2 KO mice (Figure 4).35-38 Herkinorin, an agonist at the MOR, was shown not to

promote β-arrestin 2 translocation or MOR internalization, but was instead observed to only

evoke signaling through the MAP kinase pathway and not influence others.36 Contralateral

administration of herkinorin was unable to reduce flinching brought about by formalin, showing

that its effects are limited to the periphery.39 After 5-day administration of herkinorin,

antinociceptive efficacy was maintained, unlike that of morphine, which shows evidence that this

compound may have reduced tolerance liability. PZM21 was shown not to elicit addictive

behavior in mice as was shown in the conditioned place preference (CPP) assay and compounds

PZM21 and TRV130 exhibited attenuated side effects in the form of respiratory depression and

constipation.35,37

Figure 4. Structures of biased ligands at the MOR.

1.2.2 Orthosteric and Allosteric Interactions

Along with biased signaling, another approach to increasing the therapeutic potential of

opioids is through targeting the allosteric site(s) on the receptor. A myriad of drugs bind the

orthosteric site, the binding location of the endogenous ligand, and either agonize or antagonize

30

the receptor to some degree. The design of therapeutics that target the orthosteric site seem

logical considering that therapeutics are often designed after the endogenous receptor ligand,

almost ensuring some degree of specificity. Within the past decade, functional screening is

beginning to outdo radioligand competition assays as the method of choice for high-throughput

screening. This has ultimately led to the increased detection of biologically active compounds. In

the previous paradigm, a potential candidate would displace the binding of a radioligand, but

some compounds, despite having a vast effect on orthosteric binding, would have little effect on

the modulative activity of the receptor. With the latter approach, compounds can be identified

that mediate receptor function even if they have little impact on radioligand binding against a

specific ligand.

Research focused on targeting the allosteric site(s) of a receptor has recently been gaining

momentum where allosteric ligands that do not potentiate activity without the presence of an

orthosteric ligand is the overarching goal.40 The allosteric site is typically found in the

nonconserved sequences of a given GPCR which gives rise to the potential of having augmented

selectivity among GPCR subtypes, and thus, lowering off-target activity. This is especially

advantageous for the ORs considering their orthosteric ligands commonly interact with multiple

ORs seeing as they have high sequence homology among them.41 Due to the allosteric site being

in a unique environment, this allows for new scaffolds and chemical space to be explored.

Allosteric compounds for GPCRs have the capability of differentially controlling

signaling pathways, especially away from that of β-arrestin 2 mediated effects.42 These ligands

also exhibit a maximal effect in the presence of an orthosteric ligand. This thereby allows for a

decreased liability of overdosing.43,44 A major complexity to this approach resides within the

31

probe dependence that is observed. Allosteric ligands elicit unique effects dependent upon the

orthosteric agonist.45,46 This complication is further enhanced when dealing with the ORs

considering one must acknowledge multiple endogenous ligands interact with multiple ORs,

including their biologically active metabolites, and the vast differentiation between active

scaffolds (i.e. small molecules to peptides of varying lengths).

The exploration of the ORs and viable therapeutic ligands have predominantly been

performed using an SAR approach and through the employment of rhodopsin’s crystal structure

to help render docking studies for high-throughput screening efforts. Recently, antagonist-bound

crystal structures of the ORs have been made available, but due to the nature of the small

molecule antagonists, there may be consequences going after a design for an antagonist-based

GPCR conformation, especially at the MOR.47-50

1.2.3 Endogenous Ligands for the ORs

Opioids have been used clinically for over 5,000 years and deliver their pharmacological

effects through the ORs. In the 1970s and 1980s, the endogenous peptidic ligands for the ORs

were discovered: enkephalin (ENK), endorphin (END), and dynorphin (DYN).50-52 Three

precursor proteins that are expressed in the nucleus and then translocated to the terminals of

nerve cells are hydrolyzed by various proteases to afford these ligands: 1) Proenkephalin gives

four Met-ENKs and one Leu-ENK, Met-ENK-Arg6-Phe7, and Met-ENK-Arg6-Gly7-Leu8; 2)

Prodynorphin yields three ENK peptides, DYN A and B, and α- and β-neoendorphin (NEO); 3)

Proopiomelanocortin gives rise to β-END.53-56 These compounds have varying selectivity

profiles at the ORs (Table 4).57 Later in the 1990s, the discovery of the highly selective MOR

ligands endomorphin (EM)-1 and -2 brought forth the idea that these were the true endogenous

32

MOR orthosteric ligands.58 The peptides deltorphin (DLT) and dermorphin (DER) are DOR- and

MOR-selective, respectively, and were isolated from the skin of frogs from the genus

Phyllomedusa.59 They contain the D-isomer of their respective amino acids at the 2-position.

Table 4. Selectivity profiles of endogenous opioid peptides at the ORs.

Peptide Structure Selectivity

ENKs

YGGFL DOR > MOR

YGGFM DOR > MOR

YGGFMRF MOR > DOR > KOR

YGGFMRGL MOR > DOR > KOR

β-END YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE MOR > DOR

DYN A YGGFLRRIRPKLKWDNQ KOR > MOR > DOR

DYN B YGGFLRRQFKVVT KOR > MOR > DOR

α-NEO YGGFLRKYPK KOR

β-NEO YGGFLRKYP KOR

EM-1 YPWF-NH2 MOR

EM-2 YPFF-NH2 MOR

DERs

YaFGYPS-NH2 MOR

YaFGYPK MOR

YaFWYPN MOR

DLT-1 YaFDVVG-NH2 DOR

DLT-2 YaFDVVG-NH2 DOR

- Lower case amino acid abbreviations denote D-chirality at the α-Carbon.

1.3 MOR

Opioids mostly exert their antinociceptive effects by binding to the MOR. These

receptors are densely localized in brain regions that mediate the perception of pain such as the

33

periaqueductal gray, thalamus, cingulate cortex, and insula, but are also heavily represented in

the amygdala and nucleus accumbens which are associated with pleasure states and well-

being.13,60 Although the MOR in the central nervous system (CNS) has garnered the most

attention, the MOR has been observed in the periphery and is responsible for common opioid

effects. It has been shown that the MORs residing in the brain stem are accountable for

respiratory depression.53,61 Since the 1970s the MOR has been implicated in other serious side

effects such as sedation, reduced blood pressure, constipation, tolerance, and addiction.62-65

These side effects are not observed when MOR antagonists are given nor in MOR-KO animals

which suggest that these effects are originating from the MOR.66

The compound commonly known as morphine was first isolated from opium in 1805, but

it took over 150 years for chemists to synthesize the molecule owing to its structural

intricacy.67,68 Codeine also is prevalent at high levels in opium, and ever since the structural

elucidation of both codeine and morphine, synthetic derivatives were being established long

before their structures were known (i.e. heroin). As morphine’s apparent respiratory depressive

effects were uncovered, efforts were geared towards coming up with a derivative devoid of this

effect, and thus, N-allylnorcodeine was synthesized and shown to increase respiration and

antagonized the effect elicited by morphine (Figure 5).69 Nalorphine is shown with an N-allyl

substituent and reversed morphine’s effect, yet exhibited withdrawal symptoms in test

subjects.70,71 However, this compound played a key role in the history of ORs. At enhanced

dosages, nalorphine was observed to be analgesic and was later theorized that this compound

was an antagonist at the MOR and an agonist at the KOR.72-74 This spearheaded the idea of OR

subtypes and the synthesis of multifunctional ligands. It has been documented that a hydroxyl

34

Figure 5. MOR-selective small molecule drugs based on the morphine scaffold.

group at the 3 position is critical for biological activity at the MOR.75 A number of potent drugs

for the MOR are shown to have a different moiety at this position and thus it is postulated that

their potencies are accredited to enzymatic processes that yield a more active metabolite (i.e.

demethylation of codeine’s methoxy to morphine). Other modifications at the 3 and/or 6th

position have also been shown to be fruitful. Heroin is di-acetylated and more effective than

morphine despite the moiety at the 3rd position. This may be partly due to these modifications

making

heroin more biologically available, stemming from an augmented ability to cross the blood-brain

barrier (BBB). In addition, the enzymatic process of deacetylation of the 3rd position yields a

powerful metabolite that may contribute to its biological activity.76,77 Scaffold B of figure 5 is

Drug A R1 R2 R3 Drug B R1 R2 R3 R4

Morphine H CH3 H Oxymorphone H CH3 OH H

Codeine CH3 CH3 H Hydromorphone H CH3 H H

Heroin Ac CH3 Ac Oxycodone CH3 CH3 OH H

Nalorphine H Allyl H Hydrocodone CH3 CH3 H H

N-Allylnorcodeine CH3 Allyl H Naloxone H Allyl OH H

35

different from that of morphine due to the oxidation at position 6 to the corresponding ketone

and a reduction of the C7-C8 bond. This scaffold has afforded numerous drugs that are presently

on the market such as oxycodone and hydrocodone. Other opioid frameworks have been

explored extensively such as morphinans, benzomorphans, and oripavines.78

The first selective peptidic MOR agonists to be derivatized from ENK were Tyr-DAla-

Gly-MePhe-Met(O)-ol (FK33-824), Tyr-DAla-Gly-MePhe-Gly-ol (DAMGO), and Tyr-

c2,4[DCys-Phe-DPen]-NH2 (JOM-5) with the latter peptides showing augmented selectivity and

affinity.79,80 The DERs have been employed as a scaffold to uncover potent and selective MOR

agonists such as Tyr-DArg-Phe-βAla-OH (TAPA) and Tyr-DArg-Phe-Lys-NH2 (DALDA).81,82

Some classical selective antagonists at the MOR include the cyclic opioids DPhe-c2,7[Cys-Tyr-

DTrp-Arg-Thr-Pen]-Thr-NH2 (CTAP) and DPhe-c2,7[Cys-Tyr-DTrp-Orn-Thr-Pen]-Thr-NH2

(CTOP) which are based on a somatostatin scaffold.83,84

1.4 DOR

The DOR has exhibited promise in becoming a therapeutic target for its antidepressive

and antinociceptive properties, but much is still unknown regarding this receptor’s

pharmacological behavior. These receptors are most abundantly found in the CNS, more

specifically the olfactory tubercle, nucleus accumbens, and the caudate putamen.60 There are

opposing results implicating the DOR’s role in respiratory depression. Large doses of

[DPen2,DPen5]-enkephalin (DPDPE) and SNC-80, both DOR agonists, brought about respiratory

depression in sheep.85,86 On the other hand, DLT-2 and (+)-BW373U86 augmented respiratory

function and inhibited the MOR agonist alfentanil’s induction of respiratory depression.87 Other

36

studies have agreed upon the DOR’s role in other biological activities such as controlling mood

states, providing analgesia, and catalyzing convulsive and seizurogenic behavior.88-91

The first nonpeptidic DOR selective antagonist was naltrindole (Figure 6).92 The rationale

of this synthetic achievement was in part due to the message-address notion where the

naltrexone-like moiety acted as the message component, and the benzene component simulated

the Phe motif which was thought to be the address structure of the enkephalins.93 Other notable

small molecules selective for the DOR include the agonists (+)-BW373U86 and SNC80. These

two ligands presented novel scaffolds that, at the time, were unlike typical opioid ligands.94,95

Further exploration of this area lead to numerous compounds such as SIOM which is based on

morphinan, TAN-67 which has an isoquinoline core, and DPI2505 which includes a

diarylmethylpiperazine skeleton.87,96,97

Figure 6. DOR small molecule agonists and antagonists.

37

In the realm of peptides, the endogenous ligands Leu-ENK and Met-ENK both display

selectivity for the DOR along with the DLT-1 and -2.50,59 Classic peptidic opioid ligands that are

selective for the DOR include the agonists [DAla2,DLeu5]-ENK (DADLE), [DSer2,Leu5]-ENK-

Thr6 (DSLET), and the cyclic peptides c[DPen2,5]-ENK (DPDPE), c[DPen2,Pen5]-DPLPE, and

Tyr-c2,4[DCys-Phe-DPen]-OH (JOM-13).98-101 Historical peptidic antagonists for the DOR are H-

Tyr-Tic-Phe-Phe-OH (TIPP) and [DAla2,Leu5,Cys6]-ENK (DALCE).102,103

1.5 KOR

Figure 7. KOR small molecule agonists and antagonists.

The KOR has gained recent notoriety as a therapeutic target due to its implications in

analgesia without the side effects associated with MOR activation, anti-inflammatory response,

treatment of addiction, and treatment of HIV-1 encephalopathy.104-107 Despite these promising

38

traits, KOR agonists have been shown to be less effective in bringing forth analgesia relative to

that of MOR agonists and elicit both dysphoric and sedative effects.108-110 Studies have shown

that the KOR is expressed widely in both the peripheral and CNS as autoradiographical studies

in rats have shown that the most populous area in the CNS of the KORs are in the endopiriform

nucleus and medial preoptic area.111

Salvinorin A, a naturally occurring diterpene, represents the only known non-nitrogenous

KOR agonist and was found to be highly selective for the KOR, being slightly more efficacious

than DYN A-(1-13) or U-50488 (Figure 7).112-114 It produces an experience described as

‘spatiotemporal dislocation’ when consumed.112 Due to KOR function appearing to have a great

impact on behavioral states, this led to interest in targeting this receptor for potential mood

disorders. Nor-BNI, a classical KOR antagonist, produced antidepressant-like effects in rats

along with JDTic, a peptidic antagonist, which made it into Phase I clinical trials.115-118 It was

also thought that dysphoric effects required arrestin recruitment to the receptor, as opposed to the

analgesic effects which do not. Therefore, a push to identify functionally selective KOR ligands

was initiated. The small molecule 6′-GNTI was found to be a potent partial agonist at the KOR

that does not recruit arrestin and thus provided a promising lead.119

Proposed endogenous ligands for the KOR include DYN and both α- and β-NEO. DYN

A, DYN B, their endogenous variants, withholding DYN-(1-6), show minor selectivity for the

KOR.120-121 Numerous SAR studies have been performed on the DYN scaffold and have yielded

highly selective and potent compounds such as [DPro10]-DYN A-(1-11) and

[NMeTyr1,NMeArg7,DLeu8]-DYN A-(1-8)-ethylamide (E-2078).122,123 Various peptide

antagonists have been discovered at the KOR: DYN A-(4-11)-NH2 (Dynantin),

39

[AcPhe1,Phe2,Phe3,Arg4,DAla8]-DYN A-(1-11)-NH2 (Arodyn), and the cyclic peptide

c5,8[BzlTyr1,DAsp5,Dap8]-DYN A-(1-11)-NH2 (Zyklophin).124-126

1.6 Peptide Therapeutics

Employing peptides over small molecules for therapeutics has several advantages such as

a higher specificity and selectivity for its target, relative ease of synthesis for modifying

scaffolds, and excellent biocompatibility. Disadvantages include low bioavailability from

susceptibility to enzymatic degradation and weak BBB penetration. Approaches to circumvent

these issues have been used for decades, including, but not limited to, backbone modification of

the peptide, the incorporation of unnatural amino acids and D-chiral centers, cyclization of linear

peptides, and rendering desired ligands into a prodrug. Increasing the rigidity of an amino acid’s

side chain at the β-position lowers the peptide’s dynamic nature (i.e. Val, Ile, and Thr).

Unnatural amino acids have been designed with this notion in mind such as β-methyl-2’,6’-

dimethyl tyrosine which has much more reduced ability to rotate about its Cβ-Cγ bond.127 Nature

uses modifications at the α-position such as α-aminoisobutyric acid (Aib) which takes part in

turn-structures. In addition, N-methylations can be used to increase the bioavailability by

disrupting intermolecular and intramolecular hydrogen bonding networks along with augmenting

lipophilicity of the peptide.128

Cyclization is a common strategy to increase the positive attributes of a peptidic ligand.

When a linear compound is rendered into a cyclic ligand, the peptide’s dynamics are restricted,

lipophilicity is increased, and both its hydrogen bonding capability and hydrodynamic radius are

lowered.129,130 These constraints are typically formed through a disulfide or amide bond, but

many variations are seen and they can be from side chain to side chain, N-terminus to C-

40

terminus, or a combination of the two (Figure 8). Typically, synthetic approaches must

incorporate some sort of orthogonal protection strategy to achieve regioselective cyclization(s).

Figure 8. Examples of cyclizations used for peptide ligands.

Numerous ways to circumvent the issue of BBB permeation for peptides have been

achieved. A ligand’s ability to diffuse across the BBB is mostly attributed to its lipid solubility,

molecular size, and charge, albeit there are also explicit transporters at the BBB that enable

nutrients and waste products to enter and exit.131,132 There are a dense population of efflux

transporters at the BBB that discard compounds from the cytoplasm of endothelial cells prior to

entering the brain parenchyma. These BBB capillary endothelia are sealed via tight junctions

which disallow any significant paracellular transport.133 In addition, not only do the endothelia

have lower vesicular transport capabilities, the BBB has an increased concentration of peptide-

seeking enzymes such as aminopeptidase A, aminopeptidase M, and angiotensin converting

enzyme.134 In lieu of this, chemists have developed methods to counteract or take advantage of

these conditions. For example, glycosylation of a ligand may be used to allow compounds to use

the GLUT transporters. Other thought processes are centered around increasing the overall

41

lipophilicity of a compound to increase passive diffusion across the BBB, the inclusion of

charged moieties, modifications at the termini, and using unnatural stereocenters.

One example of a peptide drug that takes advantage of some of these solutions is

Ziconotide, a conotoxin from snail venom that has been approved for the treatment of intractable

chronic pain.135,136 However, this drug must be intrathecally (i.th.) administered due to its

cardiovascular effects caused through the intravenous (i.v.) route.137 Overall, there is great

interest in bringing forth peptide drugs as 5 of the top 15 global pharmaceutical products are

peptide or protein drugs (Enbrel, Remicade, Humira, Avastin, and MabThera).138

1.7 Workflow

Our approach to the drug discovery process starts with the design of compounds that can

be based off novel scaffolds and approaches, present ideas that are found in the literature, and/or

with the aid of computer modeling and docking studies (Figure 9). Once a series has been

generated, then these compounds are synthesized efficiently in various ways using standard

liquid phase (LPPS) and/or solid phase peptide synthesis (SPPS). Once these compounds are

synthesized and validated for their structures, they are first tested for their affinities at the ORs.

Compounds showing high affinity in the nanomolar range will be then moved into in vitro

functional assays to evaluate their functional activities at the ORs. Even if a compound turns out

to not bind to the receptors strongly, the SAR results will have been utilized for drug design.

Functional assays along with the binding assays will select lead ligands for in vivo assays and

pharmacokinetic studies to assess their antinociceptive effects and bioavailability such as BBB

permeability. Through the cycle, new compounds that fulfill all of requirements including high

42

affinity, efficacy, potency, and bioavailability can be identified and processed for drug

development prior to clinical trials.

Figure 9. Drug discovery workflow.

43

Chapter 2: Neuroexcitatory Effects of DYN A

2.1 DYN A’s Paradoxical Behavior

There is literature precedent regarding the non-opioid effects of opioid peptides.139 The

N-terminal Tyr residue is known to be critical for OR recognition, but when DYN A is released

in the synapse, aminopeptidases can degrade the ligand to its des-tyrosyl fragments which no

longer interact with the ORs (Table 5).140,141 The concept of DYN A’s pharmacological

inconsistency was first elucidated when the fragment DYN A-(1-13) was injected into the brain

and brought about motor and behavioral side effects that were unlike those elicited by the EMs

nor could the effect be blocked by the non-selective opioid antagonist naloxone.142 Further

scrutiny revealed that these results were similar to the des-Tyr1 fragment DYN A-(2-13) which

does not bind to the ORs. Other studies have shown that intracerebroventricular (i.c.v.) injection

of DYN A or DYN A-(1-13) brings about repeated lateral rolling (barrel rotation) in rats and i.th.

injection of high dosages brings about paralysis and long lasting allodynia (greater than 60

days).143-149 Naloxone was not able to inhibit these motor impairment effects, whereas a DYN A

antiserum is able to reverse DYN A induced effects suggesting that these results are being

mediated through a non-opioid mechanism.150

An upregulation of DYN A has been observed in subjects that are subjected to both

neuropathic and inflammatory pain.151,152 In the latter, DYN A catalyzed the release of histamine

in rat mast cells and plasma extravasation, effects that were not attenuated when naloxone was

administered.153,154 Upregulation of DYN A was also discovered in the CNS in an arthritic

chronic pain model and other pathological pain conditions such as spinal cord trauma, bone

44

cancer pain, and chronic pancreatitis.155-158 Interestingly, it was shown that DYN A is not

necessary to bring about the initial experience of pain, but for the preservation of pain. These

results were demonstrated in prodynorphin-KO mice in a chronic pain model via L5/L6 spinal

nerve ligation (SNL).159 These mice had comparable pain thresholds and paw withdrawal

latencies compared to wild type (WT) mice < 1 week after injury affliction, but after 10 days, the

prodynorphin-KO mice responses were brought to baseline whereas the WT mice stayed below

baseline. Based on these results, it was hypothesized that DYN A can provide pronociceptive

effects through an independent signaling cascade of the ORs in chronic pain states.

Table 5. Binding affinities of selected DYN A fragments at the human ORs.

Ki (nM)

KORa MORb DORc

DYN A-(1-17) 5.3 6.9 182

DYN A-(1-13) 6.4 16.4 141

DYN A-(2-17) >10,000 >10,000 >10,000

DYN A-(2-13) >10,000 >10,000 >10,000

-Values shown are the mean from two independent experiments. aKOR: [3H]U69,593; Kd =

1.4 nM. bMOR: [3H]DAMGO; Kd = 0.37 nM. cDOR: [3H]pCl-DPDPE; Kd = 1.3 nM.

2.2 DYN A at the BRs

The non-opioid fragment DYN A-(2-17) was found to increase Ca2+ in cultured neurons

from the neonatal rat cortex.160 This was not blocked by naloxone or by dizocilpine, a

noncompetitive antagonist at the N-Methyl-D-Aspartate receptor (NMDAR). Dizocilpine was

used because previous studies suggested that the non-opioid mechanism of action from DYN A

could be through the NDMARs, but there is contradictory data in the literature depending on

45

which cell lines are employed.149,161-163 It was later deciphered that DYN A is able to interact at

the BRs and produce neuroexcitatory effects.164 DYN A-(2-13) promoted an increase in Ca2+

concentration mediated by P/Q- and L-type Ca2+ channels in rat dorsal root ganglion (DRG) and

in the F11 cell line which is a hybridoma of rat DRG and mouse neuroblastoma cells. HOE-140,

a bradykinin-2 receptor (B2R) antagonist, was found to block the effects of DYN A-(2-13) and

not dizocilpine. The protein kinase A (PKA) inhibitor, H89, could inhibit DYN A’s

neuroexcitatory effects and thus supported that the BRs mediate signaling through the PKA

pathway to activate P/Q- and L-type Ca2+ channels through the G-protein Gs (Figure 10).165

The concentration of mRNA correlating to the B2R was quantified in the DRG and was

shown to be more expressed after affliction of nerve-injury in rats.165 In addition, similar results

were observed for the transcripts of prodynorphin, whereas the levels of the parent protein of

bradykinin (BK), the endogenous ligand for the B2R, were low. A crucial piece of evidence for

the BRs being responsible for DYN A’s non-opioid action is that BK or its parent protein are not

found in the spinal cord whereas the B2R and Dyn A are. Therefore, it was postulated that DYN

A is the endogenous ligand for the B2R in the spinal cord during chronic pain states. DYN A’s

ability to interact with the B2R was further investigated and it was able to displace [3H]BK in a

competitive radioligand binding assay with an IC50 of 4 µM. It was later found that DYN A-(2-

13) exhibits an IC50 of 170 nM in rat brain membranes.166 Taken altogether, it is alleged that

B2R antagonists can be a clinically viable treatment for those experiencing chronic pain.

46

Figure 10. Cellular responses of DYN A at the B2R in the spinal cord under normal (A) and

chronic pain (B) conditions.

2.3 The BRs

The BRs belong to the rhodopsin-like family of GPCRs and have two subtypes: the

bradykinin-1 receptor (B1R) and the B2R, sharing approximately 36% sequence identity.167 Both

receptor subtypes populate both the CNS and the periphery on sensory neurons that are a part of

the nociceptive signaling cascade.168 The B1R is primarily expressed during pain states as it has

been found to be upregulated following inflammation or injury, but it is present at low levels

47

when the subject is healthy.167 On the other hand, tissue expression of the B2R is ubiquitous and

constitutively expressed and mediates signaling through the Gq protein which then signals the

effector phospholipase C.169

The endogenous ligands for the BRs are the agonists BK and kallidin (KD) and they have

been implicated in inflammatory responses (Table 6).170 BK and KD are endogenously

synthesized from either high or low molecular mass kinogen primarily in the liver.171 A subgroup

of serine proteases called kallikreins act on kinogen to yield these endogenous ligands, and along

with other peptidases such as carboxypeptidase, other metabolites can be rendered such as [des-

Arg9]BK. BK exhibits a Ki = 0.54 nM and a Ki >10,000 to the human B2R and B1R,

respectively.172 Due to this large selectivity profile, the design of antagonists for the B2R

typically employ a BK scaffold.

Antagonists have been synthesized in an attempt to inhibit the hyperalgesic effects

brought about by BR stimulation, but BK is also cardioprotective and involved in blood pressure

regulation, thus providing a major complication in targeting only the hyperalgesic response.172-174

Further investigative efforts showed that this cardioprotective function is restricted to the

peripheral B2Rs. Considering that DYN A’s neuroexcitatory effects are regulated through the

BRs in the CNS, the design of an antagonist that is centrally selective is a viable approach to

blocking DYN A’s non-opioid effect due to avoiding peripheral BR side effects. SAR studies

have revealed several influential modifications to the agonist BK scaffold to yield an antagonist

at the B2R. The substitutions of DPhe for Pro7 and β-(2-thienyl)alanine (Thi) for both Phe5 and

Phe8 changed BK to a formidable antagonist.175 Other such modifications include exchanging

both Pro2 and Pro3 with trans-4-hydroxyproline (Hyp) or by lengthening the peptide at the N-

48

terminus with the fragment Lys-Lys or DArg have afforded quality antagonists.176,177 The cyclic

peptidomimetic drug, HOE140, incorporated a combination of D-chirality and unnatural amino

acids such as Hyp, Thi, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), and (2S,3aS,7aS)-

Octahydro-1H-indole-2-carboxylic acid (Oic) to ultimately become an antagonist with 100-1000-

fold greater potency than previous antagonists.178

Table 6. Selected examples of peptidic agonists and antagonists at the BRs.

Ligand Receptor and Function

Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (KD)a B1R/B2R Agonist

Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (BK)a B2R Agonist

[des-Arg9]BK (DABK)a B1R Agonist

[des-Arg10]KD (DAKD)a B1R Agonist

[des-Arg10,Leu9]KD (DALKD a B1R Antagonist

[des-Arg9,Leu8]BK (DALBK)a B1R Antagonist

DArg-c[Hyp3,Thi5,DTic7,Oic8] (HOE 140)b B2R Antagonist

Lys-Lys-[Hyp2,3,Thi5,8,DPhe7]BK (NPC414)b B2R Antagonist

[DNMePhe7]BK (DNMFBK)b B2R Antagonist

aendogenous; bsynthethic.

49

Chapter 3: Novel Peptidic Ligands at the B2R

3.1 Previous SARs of DYN A

The strong affinity of DYN A at the ORs is mainly attributed to the N-terminal Tyr

residue considering its des-tyrosyl fragments exhibit very weak affinity for the ORs.141 These

des-tyrosyl derivatives have been shown to bring about neuroexcitatory effects such as motor

impairment, paralysis, and hyperalgesia.143,146,163 It was previously observed that after nerve

injury there is an upregulation of DYN A which interacts at the spinal BRs promoting

neuropathic pain.164,165 This was a serendipitous discovery as there is a lack of structural

similarity among the endogenous BR ligands, BK and kallidin, with DYN A. Due to these

results, it is believed that an antagonist at the BRs can attenuate or ablate the side effects elicited

by BR stimulation.

A previous study showed the results of an SAR using DYN A-(2-13) as a scaffold at the

BRs to render an effective antagonist. The SAR was performed systematically by truncating an

amino acid at the C- or N-terminus due to earlier studies showing the implications of these amino

acids in neuroexcitatory non-opioid effects mediated by the BRs.141,160,164 As a result, a key

pharmacophore was identified, DYN A-(4-11), which exhibited a similar IC50 of DYN A-(2-13)

(Table 7).166 In addition, the SAR uncovered that a basic residue at the C-terminus is important

for B2R recognition suggesting an electrostatic interaction at the receptor. Also, other

modifications at the C-terminus such as amidation were not tolerated, but acetylation of various

N-termini were. Subsequent truncations from the N-terminus up to the 4th position had little

effect on B2R interactions.

50

Table 7. Binding affinities of select DYN A analogues at rat brain BRs.

Structure DYN A Fragment

BRa,

[3H]DALKD

IC50 (nM)

H-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-

Leu-Lys-OH DYN A-(2-13) 170

H-Gly-Phe-Leu-Arg-Arg-Ile-Arg-OH DYNA-(3-9) 780

H-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-OH DYN A-(3-11) 130

H-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-OH DYN A-(5-13) 470

H-Gly-Phe-Leu-Arg-Arg-Ile-OH DYN A-(3-8) 2300

H-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-OH DYN A-(3-10) 810

H-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-OH DYN A-(5-12) 7100

H-Leu-Arg-Arg-Ile-Arg-Pro-Lys-OH DYN A-(5-11) 280

H-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-OH DYN A-(4-11) 140

H-Phe-Leu-Arg-Ile-Arg-Pro-Lys-OH [des-Arg7]-DYN A-(4-11) 190

H-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-NH2 DYN A-(4-11)-NH2 6500

Ac-Phe-Leu-Arg-Ile-Arg-Pro-Lys-OH Ac-[des-Arg7]-DYN A-(4-11) 120

H-Phe-Ala-Arg-Ala-Arg-Pro-Arg-OH [Ala5,8, des-Arg7, Arg11]-DYN

A-(4-11) 450

H-Phe-Nle-Arg-Nle-Arg-Pro-Arg-OH [Nle5,7, des-Arg7, Arg11]-DYN

A-(4-11) 140

aCompetition assays were carried out at pH 7.4 using rat brain membranes. DALKD: IC50 = 76

nM.

Now that a minimum pharmacophore was identified, modifications to the scaffold were

performed to elucidate what is critical for receptor interaction and to augment in vivo stability

and potency. Ablation of Arg7 resulted in what would become the lead ligand: H-Phe-Leu-Arg-

51

Ile-Arg-Pro-Lys-OH ([des-Arg7]-DYN A-(4-11)). In an effort to further investigate the role of

the hydrophobic amino acids proximal to the Arg residues, Leu and Ile were substituted with an

Ala which resulted in weaker affinities, but when replaced with Nle, the affinities were

recovered. This suggested that both hydrophobicity and size of the hydrophobic moiety are

important in ligand design. To better understand the topology of the lead ligand, NMR studies

were performed and evidence suggested that a type 1 β-turn is at the C-terminus. In summary of

the SAR and NMR studies, it appeared that the pharmacophore for the B2R is amphipathic,

needs a basic amino acid at the C-terminus, and a turn structure towards the C-terminus (Figure

11).

Figure 11. Pharmacophore of DYN A at the BRs.

The lead ligand was then subjected to an assay that evaluates a ligand’s ability to

stimulate the hydrolysis of [3H]inositol phosphates in human embryonic kidney (HEK) 293 cells

expressing human B2R by quantifying the production of inositol triphosphates, a secondary

messenger, that is part of the signaling cascade induced by BK at the B2R.179 The lead ligand did

not show receptor stimulation up to a dose of 10 µM and it was thus confirmed that the ligand

does not possess agonist activity unlike DYN A-(2-13).

52

Figure 12. Dose-dependent reversal of thermal hyperalgesia (left, radiant heat test) and tactile

hypersensitivity (right, von Frey test) using [des-Arg7]-DYN A-(4-11) (14 in the figure) in SNL-

rats.

[des-Arg7]-DYN A-(4-11) blocked thermal and mechanical hyperalgesia of L5/L6 SNL

animals in a dose-dependent manner (Figure 12). Prior to nerve injury, paw withdrawal latencies

and thresholds were in the range of 19.3-20.9 s and 15 g, respectively, and following SNL injury,

these values decreased to 8.6-11.3 s and 2.2-2.9 g. SNL-injured animals that were administered

[des-Arg7]-DYN A-(4-11) exhibited antihyperalgesic effects in a dose dependent manner in both

assays with the greatest effect being observed at 3 nmol/10 µL. [des-Arg7]-DYN A-(4-11) also

did not exhibit toxicity or motor impairment, and furthermore, inhibited DYN A-(2-13) elicited

paralysis. Importantly, these antagonist activities were subjected only to the BRs in the CNS

considering no peripheral activity was observed in both the paw edema and plasma extravasation

tests.

Our research group took the lead ligand, [des-Arg7]-DYN A-(4-11), and performed

additional modifications on [des-Arg7]-DYN A-(4-11) to further investigate the key structural

feature of amphipathic DYN A analogues at the B2R using the template (Figure 11).180 In the

preceding study that generated the lead ligand [des-Arg7]-DYN A-(4-11), it was shown that

53

receptor recognition was greatly dependent on a basic amino acid being at the C-terminus and

that any modification to the carboxylic acid moiety would drastically reduce affinities.166 In this

SAR, evidence further suggests this notion as compounds 2, 3, and 23-25 showed a severe

reduction in binding to the BRs despite the length of the peptide providing more evidence that

electrostatic interactions between the ligand and receptor are important (Table 8).180 All ligands

in this SAR had their affinities evaluated at pH 6.8. Competitive binding studies of DYN A-(2-

13) in rat brain membranes showed that its affinity varies in a small pH window with the optimal

pH being that of 6.8 (IC50 = 22 nM).181 At pH 7.4, the IC50 increases to 48 nM, and at a pH of

8.5, it increases to 1,700 nM. This pH effect has also been congruently demonstrated on BK

which also has an optimal affinity at pH 6.8. Due to this pH being close to biological pH, it is

believed to be physiologically relevant, especially considering the role of the BRs and that tissue

injury also evokes a drop in pH around that of 6.8 which can be sustained for multiple days.182

Ligand 1 exhibited near micromolar affinity (950 nM) while having a basic amino acid at

the C-terminus, but did not contain an amphipathic fragment (m = 0). This shows the importance

of having both an amphipathic moiety of at least 1 unit along with having a basic amino acid at

the C-terminus. Ligands 2 and 3 showed an IC50 of 2,300 nM and 4,800 nM, respectively,

suggesting that fulfilling the basic amino acid requirement at the C-terminus is more critical than

having an amphipathic moiety. In general, as the number of amphipathic units increase, the

ligands exhibit a similar range of affinities. Except for 14, the ligands for where B = 2 had the

same range of affinities as analogues 19-22 which corresponded to B = 3. In addition, when

comparing analogue 10 (B = 2, IC50 = 58 nM) with analogues 20 (B = 3, IC50 = 83 nM) and 21

(B = 3, IC50 = 65 nM), the same range of affinity for the BRs is observed despite different

54

amphipathic units. This suggests that the number of amphipathic units is not critical as long as it

is >1 with a basic amino acid at the C-terminus. Substitution of the N-terminal hydrophobic unit

did not influence binding affinities when B = 2, 3, or 4. Analogues 15-17 gave similar binding

affinities despite having two hydrophobic residues at the N-terminus with various residue

combinations. In analogues 19-21 where B = 3, the length of this hydrophobic unit fluctuates

from 4, 1, and 0, and yet, a similar range of affinities is observed. In view of these data, it is not

understood if the hydrophobic moieties at the N-terminus are significant in having BR

recognition.

Truncation of DYN A to DYN A-(8-13) (9) exhibited an IC50 = 49 nM, similar to that of

the previous lead ligand, [des-Arg7]-DYN A-(4-11) (IC50 = 69 nM). Exchanging the C-terminal

Lys residue with an Arg in analogues 15-17 gave the same range of affinities as 13. Yet, when

two Arg residues were substituted by two Lys residues (14), affinity was lost approximately 9-

fold relative to other analogues within the B = 2 category even though these replacements were

tolerated in 11 and 22 as they exhibited IC50 = 78 nM and IC50 = 85 nM, respectively.

The reason for this effect may be explained due to the Pro residue spatially orienting

these basic amino acids differently. Previous studies have shown that using retro-inverso

synthesis can maintain high correlation of spatial orientation with the parent ligand and increase

the stability of the peptide mainly due to enhanced resistance to enzymatic degradation while

exuding similar side chain orientations.184-186 Retro-inverso modifications incorporate both an

inversion of chirality with a reversal of the peptide’s primary sequence. This approach was

applied to analogue 17 to afford 27, but a complete loss of affinity was observed which may be

attributed to no longer having a basic amino acid at the C-terminus. Analogue 18, which contains

55

a Lys residue at this position, was tested, but this too lost affinity for the BRs. Therefore, it was

postulated that D-amino acids are not tolerated in the amphipathic structure.

Table 8. Binding affinities of DYN A analogues at BRs in rat brain.

Ligand AA1 B (AA2-AA1)B AA2 BR,a [3H]DALKD

IC50 (nM)b

1 Gly-Phe-Leu 0 Arg 950c

2 Gly-Phe-Leu Arg-Arg-Ile 2300c

3 Pro-Leu 1 Arg-Arg-Ile 4800c

4 Pro Lys-Leu Lys 210d

5 Phe Lys-Leu Lys 350c

6 Phe(NH2) Lys-Leu Lys 450c

7 Pro Lys-Nle Lys 71c

8 Nle Lys-Nle Lys 110c

9 Ile 2 Arg-Pro-Lys-Leu Lys 49

10 Arg-Pro-Lys-Leu Lys 58

11 Nle Lys-Pro-Lys-Nle Lys 78d

12 Nle Arg-Pro-Arg-Nle Lys 83

13 Phe-Leu Arg-Ile-Arg-Pro Lys 69d

14 Nle Lys-Ile-Lys-Pro Lys 460

15 Phe-Ile Arg-Ile-Arg-Pro Arg 110

16 Phe-Leu Arg-Leu-Arg-Pro Arg 100

17 Phe-Nle Arg-Nle-Arg-Pro Arg 87d

18 DPhe-DNle DArg-DNle-DArg-DPro DLys n.c

19 Gly-Gly-Phe-Leu 3 Arg-Ile-Arg-Pro-Lys-Leu Lys 41d

20 Phe Arg-Ile-Arg-Pro-Lys-Leu Lys 83

21 Arg-Ile-Arg-Pro-Lys-Leu Lys 65

22 Lys-Nle-Lys-Pro-Lys-Leu Lys 85d

23 Lys-Leu-Lys-Pro-Arg-Ile 6300

24 Phe-Leu Arg-Arg-Ile-Arg-Pro-Lys-Leu 5200

25 Lys-Pro-Arg-Ile-Arg-Leu 930

26 Lys-Pro-Arg-Ile-Arg-Leu Phee 50

27 DArg-DPro-DArg-DNle-DArg-DNle DPhee n.c.

28 Leu-Leu Lys-Pro-Arg-Ile-Arg-Arg-Leu Phe-Gly-Glye 93

29 4 Lys-Leu-Lys-Pro-Arg-Ile-Arg-Leu Phe-Gly-Glye 150

30 Lys-Leu-Lys-Pro-Arg-Ile-Arg-Arg-Leu Phe-Gly-Glye 78

-n.c.: no competition. aCompetition assays done at pH 6.8. bValues determined from nonlinear

regression analysis of data from at least two independent experiments in duplicate. cpH 7.4. dIC50

values from references 166 and 183. eAA2 definition does not pertain to these analogues.

56

Irrespective of the retro-inverso modifications, retro modification of analogue 13 to yield

26 gave similar binding affinities (IC50 = 69 nM and IC50 = 50, respectively). This result was

unexpected considering 26 has a Phe residue instead of a basic amino acid at the C-terminus

which possibly suggested that BRs need just one terminal basic amino acid. To further assess this

result, 26’s C-terminal Phe residue was truncated to afford 25. In opposition to the proposed

idea, 25 had greatly lost affinity at the BRs (IC50 = 930 nM). A possible explanation for this

could be that an electron dense moiety is sufficient to participate in electrostatic interactions at

the BRs. Retro synthesis was also performed on the longer DYN A fragments [des-Arg7]-DYN

A-(2-13) and DYN A-(2-13) and they maintained the same range of affinities as their parent

ligands showing that this type of modification is tolerated in longer analogues. Fascinatingly,

analogues 28-30 did not have a basic amino acid or a Phe residue at the C-terminus, yet exhibited

good affinity for the BRs.

In summary of these results, this SAR may likely only be applied to longer chained

ligands considering they are more likely to conform to an ordered structure. The dynamics of

shorter chained peptides may provide reasoning for why a basic amino acid at the C-terminus is

essential, whereas with longer sequenced ligands it is not. However, in most cases, the basicity

of the C-terminus is critical with an amphipathic core sequence where B > 1 to afford strong

binding at the BRs.

3.2 Increasing the Stability of lead ligand [des-Arg7]-DYN A-(4-11)

[des-Arg7]-DYN A-(4-11) despite showing high potency and affinity at the BRs,

exhibited low metabolic stability in plasma and was degraded within 4 hours of incubation (t1/2 =

0.7 hours). This low stability was observed despite enduring a truncation of an Arg residue so a

57

dual Arg-Arg motif would not be present in the structure to evade enzymatic degradation.

Therefore, there was a need to develop novel ligands based off [des-Arg7]-DYN A-(4-11) to

augment its biological stability.

C-terminal amidation is a straightforward method to increase stability, but it was found

not to be tolerated.180 Substitution of basic and/or hydrophobic amino acids with unnatural basic

and/or hydrophobic amino acids were tolerated in most ligands on the condition that

amphipathicity is maintained. Finally, N-terminal modifications were explored and did not alter

the range of affinities for ligands whereas those in the C-terminal region did. Retroinverso-

ligands show potential to increase stability and to retain affinity by reversing their stereocenters,

and thus, are not easily recognized by enzymes despite having similar structure. In some cases,

retro-peptides, due to the reversal of peptide backbone –NHCO- to –CONH-, can stabilize

secondary structures such as a helix that are dependent on the peptide backbone’s orientation.187

Thusly, a new comparative SAR was generated to investigate the effects of retro-peptides

relative to their parent ligands to gain further information on the structural component necessary

for good binding at the BRs (Table 9).188 Also, further scrutiny of the correlation between a turn-

like structure and ligand interaction with the BRs was performed by incorporating multiple Aib

residues in the hydrophobic positions of alternating Arg-Aib sequences (35-37) whilst

maintaining an Arg residue at the C-terminus. Incorporation of multiple Aib residues have been

observed to stabilize 310 helices in short peptidic sequences by restricting conformational

space.189-191

In addition, another SAR was generated that included cyclic DYN A analogues to

augment ligand stability. Cyclizations to both [des-Arg7]-DYN A-(4-11) (IC50 = 69 nM)166 and

58

another potential lead ligand, Nle-Lys-Pro-Lys-Nle-Lys (38, IC50 = 78 nM)183, were employed

and their binding affinities were evaluated at the BRs (Tables 10 and 11).192 Furthermore, cyclic

ligands were subjected to a stability assay to assess their degradation profiles in rat plasma.

3.3 Experimental

DYN A analogues were synthesized by using standard SPPS with an Nα-Fmoc (9-

fluorenylcarboxy) protecting group approach on preloaded Wang resin (100-200 mesh,

Novabiochem) with >30% overall yields (Figure 13).193 Coupling of amino acids were

performed using 3 equivalents of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium

hexafluorophosphate (HBTU), 3 equivalents of N-hydroxybenzotriazole (HOBt), and 6

equivalents of diisopropylethylamine (DIPEA) in N,N-dimethylformamide (DMF) for 1 hour at

room temperature (rt) and the Nα-Fmoc-moiety was deprotected using 20% piperidine in DMF

for 20 mins at rt (Figure 14). After the coupling of amino acids was complete, the resin was dried

under vacuum for 3 hours and then cleaved from the resin using a 95% trifluoroacetic acid (TFA)

solution containing 2.5% triisopropylsilane (TIS) and 2.5% water for 3 hours at rt to afford crude

peptides with high purity (70-90%). Crude peptides were then subjected to RP-HPLC using a

preparative column with a gradient of 10-40% acetonitrile in 0.1% TFA/water in 15 mins to

afford >97% purity. The purified analogues were then validated by analytical RP-HPLC and HR-

MS in positive ion mode.

59

Figure 13. Structures of selected SPPS reagents: A) Fmoc-Lys(Boc)-Wang resin B) 2-

Chlorotrityl chloride resin C) HBTU D) HOBt.

Figure 14. General scheme of SPPS to generate DYN A analogues.

Cyclic DYN A analogues were initially synthesized by using standard SPPS with an Nα-

Fmoc protecting group approach on preloaded Fmoc-Lys(Boc)-Wang resin (100-200 mesh,

Novabiochem) with 2-phenylisopropyl (PhiPr), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-

60

sulfonyl (Pbf), and t-butyloxycarbonyl (Boc) used as side chain protecting groups for Glu, Arg,

and Lys, respectively with overall yields >40%.193 Cyclization was induced via ring closing

metathesis using a microwave (100 °C) with second generation Grubbs catalyst in

dichloromethane (DCM) containing 10% of 0.4 M LiCl in DMF for 1 hour (Figure 15).194 Half

of the resin was then cleaved by 90% TFA, 5% thioanisole, 3% 1,2-ethanedithiol, and 2%

anisole and then purified by preparative RP-HPLC using a gradient of 10-50% acetonitrile in 20

mins to give the pure (>95%) dicarba cyclic analogue 39. The other half of the resin was then

reduced using the Wilkinson’s hydrogenation method with the catalyst Rh(PPh3)3Cl in 90%

DCM and 10% methanol (MeOH) at rt for 1 day (Figure 15).192 The peptide was then cleaved

from the resin using the previously described cocktail and purified via preparative RP-HPLC to

give the pure (>95%) dicarba cyclic analogue 40. Analogues 41-43 were subjected to 5% TFA to

deprotect the PhiPr from Glu after completion of linear amino acid coupling. The peptides were

then cyclized using 5 equivalents of N,N’-diisopropylcarbodiimide (DIC) and 5 equivalents of

HOBt (Figure 16). The peptides were then cleaved from the resin using the previously described

TFA cocktail and purified using preparative RP-HPLC to afford >97% pure cyclic DYN A

analogues. All analogues were validated using analytical RP-HPLC and HR-MS in positive ion

mode (Table 10).

61

Table 9. Analytical data of cyclic DYN A analogues.

Ligand Molecular Formula MSa HPLCb

(tR, min)

Purity

(%) aLogPsc

Calculated Observed

39 C31H55N9O7 666.4 666.4 15.0 95 -2.25

40 C31H57N9O7 668.4 668.5 11.6 95 -2.17

41 C44H74N14O9 943.6 943.7 14.1 98 -2.24

42 C41H77N15O9 924.6 924.5 11.9 98 -2.35

43 C35H65N13O8 796.5 796.5 11.7 98 -2.74 a(M+H)+, ESI method (Finnigan, Thermoelectron, LCQ classic). bPerformed on a Hewlett

Packard 1100 (C-18, Microsorb-MVTM, 4.6 mm x 250 mm, 5 µM) using gradient system (10-

100% acetonitrile containing 0.1% TFA within 45 min, 1 mL/min).

chttp://www.vcclab.org/lab/alogps.

62

Figure 15. Synthesis of olefin or alkyl bridged cyclic DYN A analogues.

63

Figure 16. N-terminus to side chain cyclization scheme of DYN A analogues.

64

To assess stability of a given ligand, ligands were tested in rat plasma at 37 °C, and after

various incubation times (1 h, 2 h, 4 h, 8 h), the quenched sample was analyzed via RP-HPLC.

The half-life was calculated using t1/2 = 0.693/b, where b is the slope of the linear fit of the

natural logarithm of the fraction remaining of the parent compound vs. incubation time.195

Radioligand competition assays to assess binding affinities of the DYN A analogues at

the BRs were performed using [3H]DALKD in rat brain membranes (non-specific binding was

defined by 10 µM kallidin).166 Crude rat brain membranes were pelleted and then resuspended in

50 mM tris buffer with 50 µg/mL bacitracin, 10 µM captopril, 100 µM PMSF, and 5 mg/mL

BSA. 10 concentrations of a DYN A analogue were incubated along with 50 µg of rat brain

membranes and [3H]DALKD (1 nM, 76.0 Ci/mmol) at 25 °C for 2 hours. Nonspecific binding

was determined using 10 µM KD in all tests. Reactions were stopped by rapid filtration through

Whatman GF/B filters that were presoaked in 1% polyethylenimine and then washed four times

with 2 mL of cold saline. The presence of radioactivity was quantified by liquid scintillation

counting using a Beckman LS5000 TD. The collected data was then analyzed using GraphPad

Prism 4 (GraphPad Software, Inc., La Jolla, CA, USA) via a nonlinear least-squares analysis.

Logarithmic values were obtained from nonlinear regression analysis of data taken from at least

two independent experiments.

3.4 Results and Discussion

The lessened affinity of ligand 3 was not improved by the replacement of the Leu residue

at the C-terminus to the N-terminus as it resulted in about a five-fold reduction in affinity despite

fulfilling the pharmacophoric requirements (33, IC50 = 4,300 nM) (Table 10).188 Additional

modifications through truncating the C-terminal Arg residue also did not recover binding affinity

65

at the BRs (34, IC50 = 6,000 nM). Placing a Leu residue between the Lys and Pro residues

marginally resulted in stronger affinity (32, IC50 = 320 nM). These data suggested that Pro’s turn

making ability must be indispensable for BR recognition. Not only is Pro known to cause

ordered disruption of secondary structures, but it is also commonly found at the end regions of

helices and helps peptides adopt β-turn conformations.196,197 Sequence analysis has shown that

89% of Pro residues that reside within α-helices are in the 1st turn with the most frequented

position being the second. Also, hydrogen bonding between the 1st and 5th positions are critical to

stabilize helical turn structure, and consequently, Pro residues are not observed at this position.185

In longer sequenced peptides, Pro has been observed all the way to the 4th position without

disturbing secondary structure, but it is rarely found beyond that. This may be the reason for

attenuated affinities for ligands 6 and 7 as the Pro residue is now in the middle of the sequence

and is able to interrupt the β-turn or 310 helix structure that is required for recognition at the BRs.

Evidence for this reasoning can be seen when comparing ligands 33 with 31 and 32 as the Pro

residue resides more in the middle of ligand 33, as opposed to ligands 31 and 32 where it is more

towards the N-terminus.

Ligands 35-37 contained the Aib design and maintained the Arg residue at the C-

terminus. Unfortunately, these ligands were not shown to interact at the BRs. This is likely

attributed to the ligands having adopted a drastically different conformation with the positively

charged Arg residues being optimally positioned. Moreover, the need for the hydrophobic

residues to have flexible hydrophobic side chains may be key for BR interaction.

66

Table 10: Binding affinities of DYN A analogues with Aib substitutions at the BRs in rat brain.

Ligand Structure

BRa,

[3H]DALKD

IC50b

31 Lys-Pro-Arg-Ile-Arg-Phe, retro of [des-Leu5,Arg7]-DYN A-(4-11) 590

32 Lys-Leu-Pro-Arg-Ile-Arg-Leu 320

33 Leu-Lys-Pro-Arg-Ile-Arg, retropeptide of DYN A-(7-12) 4300

34 Leu-Lys-Pro-Arg-Ile, retropeptide of DYN A-(8-12) 8100

35 Arg-Aib-Arg-Aib-Arg n.c.

36 Aib-Arg-Aib-Arg-Aib-Arg n.c.

37 Arg-Aib-Arg-Aib-Arg-Aib-Arg n.c.

-n.c.: no competition. aCompetition assays were performed using rat brain membranes.

bValues determined from non-linear regression analysis of data collected from at least two

independent experiments in duplicate using GraphPad Prism.

Analogues 39 and 40 were designed based on the scaffold of 38 (Table 11). The two Nle

residues were picked as cyclization points due to being branched hydrophobic amino acids as it

was determined that cyclizations at these points would likely be tolerated. These two residues

were substituted with allyl glycine to form the cyclic moiety. Analogues 41-43 retained their

hydrophobic alkyl side chains as their design incorporated the scaffold of [des-Arg7]-DYN A-(4-

11). The Pro residue was substituted with a Glu to form a point of cyclization with the N-

terminal amino group as it was previously shown to not be essential for BR

interaction.166,180,183,188 Both series of ligands displayed amphipathic character with a Lys residue

as the C-terminal basic amino acid.

Analogues 39 and 40 do not have exposed hydrophobic moieties and resulted in no

observed binding at the BRs despite having a C-terminal basic amino acid and an overall net

67

charge of +3, fulfilling the electrostatic component of the BR pharmacophore (Table 11).

Previous studies suggested that appropriate orientation of basic amino acids via hydrophobic

amino acid insertions and amphipathicity were optimal for receptor recognition, but the

hydrophobic positions were utilized as cyclization points leading to no hydrophobic side chains

being present on the ring.

Table 11. Binding affinities of cyclic DYN A analogues at the BRs in rat brain.

Ligand Sequence Ring Size BR, [3H]BKa

IC50 (nM)b

38c Nle-Lys-Pro-Lys-Nle-Lys - 78

39 c1,5(-cisCH=CH-)[Ala-Lys-Pro-Lys-Ala]-Lys 17 n.c.

40 c1,5(-CH2CH2-)[Ala-Lys-Pro-Lys-Ala]-Lys 17 n.c

[des-Arg7]-DYN

A-(4-11)c Phe-Leu-Arg-Ile-Arg-Pro-Lys - 69

41 cN,6[Phe-Leu-Arg-Ile-Arg-Glu]-Lys 20 191

42 cN,6[Lys-Leu-Arg-Ile-Arg-Glu]-Lys 20 302

43 cN,6[Leu-Arg-Ile-Arg-Glu]-Lys 17 1510

-n.c.: no competition. aRadioligand competition assays were carried out using [3H]BK in rat

brain membranes at pH 6.8. bValues determined from nonlinear regression analysis of data from

at least two independent experiments in duplicate. cReference #166 and #183, [3H]DALKD.

Cyclic analogues 41-43 were based on the [des-Arg7]-DYN A-(4-11) scaffold and

contained two hydrophobic amino acids, Leu and Ile, within the ring structure. The amino group

at the N-terminus was used to initiate cyclization with a Glu residue to yield 20- and 17-

membered rings. Analogues 41 and 42 retained similar affinity for the BRs as their linear parent

(IC50 = 191 and 302 nM, respectively) showing that cyclization between the Pro residue and the

N-terminus is well tolerated. Analogue 43 on the other hand exhibited attenuated affinity by

68

approximately 22-fold, signifying that a 20-membered ring is an ideal size for the BRs. The loss

of affinity may be attributed to the ring’s constraint imposing improper orientation of the

amphipathic motifs. Considering that peptides are typically degraded by proteolytic enzymes in

plasma, cyclic analogue 41 was subjected to a stability assay in rat plasma at 37°C and was

shown to be more stable than its linear parent (t1/2 = 5.5 h vs 0.7 h).

3.5 Summary and Future Directions

We have previously shown that DYN A-(2-13) exhibited an IC50 of 22 nM in rat brains

where predominantly the B2R is expressed with low levels of B1R and an IC50 of 62 nM in cells

expressing the B2R at a pH of 6.8. The binding affinity of DYN A-(2-13) at the B1R remained in

the µM range, showing selectivity for the B2R, and thus, presented itself as a viable scaffold for

antagonist design to inhibit DYN A’s action at the B2R. Through SAR analysis, a clear

pharmacophore has been uncovered: a basic amino acid at the C-terminus, amphipathic moieties,

and a Pro residue to bring about a turn structure. A minimum pharmacophore was also

developed, [des-Arg7]-DYN A-(4-11), which was found to inhibit DYN A’s neurotoxic effects in

vivo and showed strong antihyperalgesic effects in a neuropathic pain model.166 Despite these

positive attributes, this ligand is susceptible to enzymatic degradation (t1/2 = 0.7 hours). To

augment the stability of this peptide, various modifications were performed such as incorporation

of unnatural amino acids, terminal modifications, retroinverso-variants, and cyclization. It was

observed that modification of the C-terminus is not well tolerated, but changes at the N-terminus

are. Also, spatial orientation of the basic amino acid appears to be crucial for strong interaction at

the BRs. In the cyclic variants, the effect of cyclization on stability was shown as 41 had a half-

life of 5.5 hours, approximately 8-fold greater than its linear parent. Evidence suggests that a 20-

69

membered ring size is ideal. However, future SARs could increase the ring size of the ligands.

Only 17- and 20- membered rings were adjudicated at the BRs so it may be worthwhile to

increase the size, especially considering longer peptides were well tolerated in linear ligands. In

addition, it may be worthwhile to explore stability assays that use whole blood as opposed to rat

and human plasma considering that there are peptidases present and may degrade the analogues

differently. In an effort to further elucidate the binding modality of these ligands, lead ligands

can be radiolabeled using [125I] to quantify binding kinetics. Furthermore, future work could

incorporate docking studies to understand the optimal orientation of these basic amino acids to

better aid in the design of future analogues.

70

Chapter 4: Multifunctional Opioid Ligands for the Treatment of Chronic

Pain

4.1 Benefits of a Multifunctional Ligand

Current design strategies often encompass a blueprint that has the ability to interact with

multiple receptors.198,199. Multifunctional compounds are ligands that interact in a monovalent

fashion with multiple targets. They often display better potency due to synergistic effects and/or

produce fewer side effects relative to ligands that only interact at one target. Another advantage

of a multifunctional approach is that their pharmacokinetic and pharmacodynamic profiles are

more in sync with the convenient administration of one drug. Variance in metabolic rates based

on an individual can give rise to intricate pharmacokinetic and pharmacodynamic relationships in

scenarios where multicomponent drugs are given which can lead to unforeseeable variability

among individuals. Other benefits of targeting multiple receptors with one compound is that

there is a greater patient compliance and an attenuated risk involving drug-drug interactions

compared to the multicomponent drug approach. Numerous bifunctional drugs that treat

depression, inflammation, and metabolic diseases are on the market or in clinical trials.198

There are three traditional approaches for designing multifunctional ligands that have

functionally dissimilar pharmacophores: i) pharmacophores can be linked using a linker; ii) there

can be an overlapping of the pharmacophores; iii) highly integrated pharmacophores.200 The

latter, ligands with integrated pharmacophores, are typically discovered serendipitously or by

screenings from compound libraries, whereas methods i and ii can be rationally designed. This

approach usually requires preexisting knowledge and special attention to the SARs that have

been previously performed on the individual pharmacophores to design novel multifunctional

71

ligands. Upon combining pharmacophores together, biological activities such as binding affinity

and functionality at the receptor may be altered upon rendering of the multifunctional construct.

Because of this possibility, it is important that ligands are tested for their functional activities

before they are subjected to in vivo studies because a compound that was once an antagonist may

become a partial antagonist or agonist when combined with another or other pharmacophores.

4.2 The Synergistic Effect between the MOR and DOR

Evidence of a synergistic effect in vivo between the MOR and DOR was revealed in the

1970s just as the endogenous ligands for the ORs were being discovered.201 Administration of

i.c.v. Leu-ENK 15 mins prior or intraperitoneal injection (i.p.) 15 mins after morphine

administration showed a dose-dependent augmentation of the analgesic property of morphine

which was quantified by the mouse tail-flick assay. Correspondingly, co-administration of these

compounds resulted in a faster rate of acquiring acute tolerance and dependence. Methadone and

levorphanol, small molecules that are agonists at the MOR, likewise had their effects enhanced

by Leu-ENK which was also observed in the mouse tail-flick assay. It is worthwhile to mention

that the dosage of Leu-ENK was not enough to bring about observable analgesia without the

presence of morphine. Other studies followed and exposed the modulatory effect of the DOR on

the MOR.202,203 Co-administration of morphine with a non-antinociceptive dose of DPDPE or

[DAla2,Glu4]DLT exhibited a positive modulatory leftward shift of the morphine dose-response

curve and a negative modulatory rightward shift when morphine was administered with Met-

ENK.204 This provided further evidence for the DOR being capable of modulating MOR activity.

On the basis of the aforementioned results, it was then hypothesized that highly selective

MOR agonists may provide analgesia without acquiring tolerance because it was shown that the

72

DOR was involved.204 H-2′,6′-dimethyltyrosine1 (Dmt)[DALDA] is a highly selective MOR

agonist with a Ki = 143 pM (Kiδ/Ki

µ > 14,000).205 Daily dosage of [Dmt1]DALDA for a week (5

times ED50; subcutaneous (s.c.)) enhanced the ED50 3.6-fold. A dosage of 10 times the ED50

every 12 hours for only 2.5 days gave an ED50 that increased by approximately 12-fold.

Tolerance was observed for both scenarios by a 3.4- and 15.1-fold shift in the morphine ED50,

respectively. The effects of supraspinal and spinal tolerance were also scrutinized following

systematic s.c. [Dmt1]DALDA administration. Five doses of the ligand, 10 times the ED50 every

12 hours gave a 3.4-fold shift in the i.c.v. ED50, however a 44-fold shift was seen in i.th. ED50.

Upon co-administration of [Dmt1]DALDA with naltriben, a potent antagonist at the DOR, spinal

tolerance was abridged by 50%. When spinal tolerance had already been recognized, both

administration of naltriben or another antagonist at the DOR, H-Tyr-Ticψ[CH2NH]Phe-Phe-OH,

increased the potency of i.th. administered [Dmt1]DALDA by 2- to 4-fold. Not only do these

data support the notion of a synergistic effect, it also displays that the DOR’s role is not

necessary to develop tolerance, but the mediation of the tolerant state.

4.3 Recent SARs for MOR/DOR Multifunctional Ligands

The ENKs are endogenous pentapeptide ligands for the DOR and MOR. Previous SARs

of the ENKs have unveiled that the incorporation of an additional aromatic moiety strongly

enhanced binding affinities and that the carboxylic acid moiety of the C-terminus is responsible

for DOR selectivity.206-209 Lee et al. sought to increase the potency of ligands at the MOR and

DOR by modifying the C-terminus of an ENK-based scaffold with various fentanyl derivatives

(Figure 17).210 Previous studies suggested that the propionyl and phenethyl moieties of fentanyl,

a MOR agonist, play important roles in OR interaction.211,212 In addition, the sequence Tyr-

73

DAla-Gly-Phe exhibited strong opioid affinity and bioactivity, and thus, was used to design

subsequent ligands along with the corresponding amide and hydrazine derivatives (Tyr-DAla-

Gly-Phe-NH2 and Tyr-DAla-Gly-Phe-NH-NH2, respectively). Various ligands were rendered

with different fentanyl derivatives coupled to the C-terminus either with or without a linker and

an SAR was generated (Figure 18). Also, the Tyr residue was substituted with a Dmt. These

ligands attain greater lipophilic character relative to their parent ligand which was theorized to

strengthen their biological profiles.213

Figure 17. Chemical structure of fentanyl and employed derivatives.

Most ligands in the SAR increased their affinities to the DOR with the coupling of the

fentanyl moiety to the C-terminus, but displayed varied selectivity. Analogue 44 which

incorporated 1-phenethyl-piperidin-4-ylamine at the C-terminus showed MOR selectivity as

opposed to the Ppp moiety which displayed DOR selectivity and agonist activity in the mouse

vas deferens assay (MVD) (48, Table 12). This tendency was observed throughout the analogue

series with the exception of 51 which has a hydrazine linker between the fentanyl derivative and

the peptidic sequence. This ligand showed MOR selectivity, but it is likely attributed to the

hydrazine linker as opposed to the C-terminal modification. Analogues 45 and 46 had an

74

increased selectivity to the MOR, relative to analogue 44. The additional aromatic moiety in

ligand 46 did not strengthen biological activities at the ORs, whereas analogue 45 which has a

more dynamic and less bulky structure, showed greater affinity for the ORs.

Figure 18. ENK-like tetrapeptide analogues with various C-terminal modifications.

Analogue 48, which has the Ppp moiety at the C-terminus, gave highly selective

biological activities in both binding and functional experiments. For analogues 47 and 49, the

Phe residue was ablated to elucidate the role of the aromatic moiety of the Ppp motif. Both

analogues lost their agonist activities in the MVD and GPI assays, but 49 still maintained

respectable affinities at the MOR and DOR (Ki = 40 nM and 180 nM, respectively). These

affinities are likely attributed to the Dmt substitution, and thus, the Phe residue is irreplaceable

by the Ppp group.214 Overall, analogue 48 was determined to be a lead compound owing to its

strong binding affinities and biological agonist efficacies despite its significant selectivity for the

DOR. Dmt has previously been shown to enhance ligand affinity for the ORs with the MOR

being more greatly impacted than the DOR.214-216 Therefore, Dmt was used as a substitution for

75

Tyr to provide a better balanced ligand at the MOR and DOR along with increasing OR affinity.

It was indeed shown that this substitution had a marked effect on affinity. 50 had a 2-fold and

60-fold increase to the DOR and MOR, respectively, relative to 48. Also, 50 was 13-fold and 24-

fold more potent than 48 in the MVD and GPI assays, respectively. Overall, analogue 50

exhibited picomolar affinity and EC50 values to both the MOR and DOR and was determined to

be a new template for further SAR studies.

76

Table 12. Bioactivities of the ENK-like tetrapeptide analogues.

No

hDORa rMORa [35S]GTP-γ-S binding IC50 (nM)e

[3H]DPDPEb [3H]DAMGOc hDORd rMORd

Kif (nM) Ki

f (nM) EC50 (nM) Emaxg (%)

EC50

(nM)

Emaxg

(%)

MVD

(DOR)

GPI

(MOR)

44 14 14 510 92 125 53 380 ± 80 160 ± 50

45 3.7 1.2 29 78 16 52 250 ± 50 47 ± 12

46 6.1 1.1 110 32 11 52 290 ± 70 95 ± 6

47 7000 5700 ns ns ns ns 3% 3%

48 0.69 23 37 72 41 63 24 ± 2 200 ± 60

49 180 40 170 15 82 32 10% 15%

50 0.36 0.38 0.77 24 0.88 50 1.8 ± 0.2 8.5 ± 3.3

51 3.2 5.7 150 71 24 43 250 ± 70 120 ± 50

n.s.: not saturated. aCompetition analyses were performed using membrane preparations from

transfected HN9.10 cells that constitutively expressed the respective receptor types. bKd = 0.50 ±

0.1 nM. cKd = 0.85 ± 0.2 nM. dExpressed from CHO cells. eConcentration at 50% inhibition of

muscle contraction at electrically stimulated isolated tissues. fValues determined from the

nonlinear regression analysis of data collected from at least two independent experiments. gNet

total bound / basal binding x 100 ± SEM.

Analogue 50, owing to its C-terminal modification, should have an improved

bioavailability considering its aLogP = 2.96. Improvement of a compound’s lipophilicity can

improve its analgesic properties since it greatly influences the rate of a compound being able to

cross the BBB. Another way to increase cellular penetration, is to increase a ligand’s methylation

and halogenation which reduces overall hydrogen bonding. An example of this was shown when

a chlorine was added to the para position of a Phe4 residue in DPDPE and displayed a substantial

77

increase in cell permeability in in vivo and in vitro assays.217,218 This SAR conserved the C-

terminal fentanyl derivative and the Dmt1 residue to generate a series of ligands with enhanced

lipophilic character that are nonselective at the MOR and DOR (Table 13).219

Table 13. Structure of lipophilic Enk-like tetrapeptide analogues.

Ligand 1 2 3 4 aLogP

50 Dmt DAla Gly Phe 2.96

52 Dmt DNle Gly Phe 3.66

53 Dmt DNle - Phe 4.20

54 Dmt DAla - Phe 3.52

55 Dmt DNle Gly Phe(Cl) 4.18

56 Dmt DNle Gly Phe(F) 3.74

57 Dmt DAla Gly Phe(Cl) 3.47

58 Dmt DNle - Phe(Cl) 4.74

59 Dmt DAla - Phe(Cl) 3.97

60 Dmt DAla Gly Phe(F) 3.01

61 Dmt DTic Gly Phe(F) 4.02

62 Dmt DTic Gly Phe(Cl) 4.46

DAla replacement with DNle at the second position along with halogenation of the Phe at

the para position augmented ligand affinities at both the MOR and DOR along with increasing

their lipophilic character. The DNle substitution had more of an impact on receptor activities and

efficacies rather than the ligand’s binding affinities which supports the notion that the change of

functional output was due to increased lipophilicity (Table 14). Ligands 53, 54, 58, and 59,

78

endured a Gly truncation to reduce the size of the pharmacophore from 52, 50, 55, and 57,

respectively, but brought forth a reduction in bioactivities at the MOR and DOR with slight

favoring of selectivity for the MOR in binding and functional assays.

Table 14. Bioactivities of lipophilic opioid ligands.

No

hDORa rMORa [35S]GTP-γ-S binding IC50 (nM)e

[3H]DPDPEb [3H]DAMGOc hDORd rMORd

Kif (nM) Ki

f (nM) EC50

(nM)

Emaxg

(%)

EC50

(nM)

Emaxg

(%)

MVD

(DOR)

GPI

(MOR)

50 0.36 0.38 0.77 24 0.88 50 1.8 ± 0.2 8.5 ± 3.3

52 0.18 0.39 0.10 47 0.11 77 0.69 ± 0.09 1.6 ± 0.1

53 1.1 0.36 2.8 61 1.9 29 8.5 ± 1.7 4.7 ± 1.2

54 1.7 0.15 n.r. - 0.94 33 15 ± 1 3.6 ± 0.8

55 0.08 0.10 0.07 37 0.14 58 1.9 ± 0.5 1.3 ± 0.3

56 0.40 0.02 0.07 48 0.29 98 0.37 ± 0.12 0.26 ± 0.14

57 0.14 0.14 0.16 58 0.31 55 0.70 ± 0.38 2.6 ± 0.8

58 19 5.0 30 36 19 15 250 ± 90 220 ± 40

59 1.6 0.33 26 27 4.7 29 180 ± 50 93 ± 30

60 0.03 0.01 0.12 23 0.69 50 1.8 ± 0.8 1.0 ± 0.1

61 0.48 0.35 0.09 24 0.72 51 0.029 ± 0.0007 0.96 ± 0.13

62 0.11 0.15 0.20 31 0.06 37 0.21 ± 0.06 4.8 ± 0.7

- n.r.: no response. aCompetition analyses performed using membrane preparations from transfected

HN9.10 cells that constitutively expressed receptor types. bKd = 0.50 ± 0.1 nM. cKd = 0.85 ± 0.2 nM.

dExpressed from CHO cells. eConcentration at 50% inhibition of muscle contraction at electrically

stimulated isolated tissues. fValues determined from the nonlinear regression analysis of data

collected from at least two independent experiments. gNet total bound / basal binding x 100 ± SEM.

79

Halogenation of the Phe residue proved to be beneficial in both binding and functional

assays. Ligand 56, which contains a Phe(p-F) at the 4th position and a DNle at the 2nd position,

exhibited the most potent biological profile and had great efficacy at both the MOR and DOR.

However, in Gly truncated variants with halogenation by a chlorine, binding affinities and

functional activities were greatly attenuated (58 and 59). The dipeptide Dmt-Tic is a selective

and potent DOR antagonist and thus its pharmacophore was incorporated to investigate

functional effects in the present scaffold with DTic at the 2nd position (61 and 62).214 These

analogues exhibited parallel profiles to other ligands in the series which demonstrated that the

dynamically constrained and hydrophobic DTic residue could be a viable amino acid at the 2nd

position. Both compounds showed to be DOR selective with no antagonistic property.

Figure 19. Antihyperalgesic and antiallodynic effects tested in the thermal hypersensitivity (left)

and von Frey filaments (right) assays of 57 (i.th. injection, 6 in the figure) using SNL-injured

rats.

80

Due to the biological profile that analogue 57 displayed, it was selected to undergo in

vivo studies (6 in figure 19). This compound demonstrated potent antihyperalgesic and

antiallodynic effects in the SNL chronic pain model. 57 demonstrated significant efficacy at

doses 3, 10, and 30 µg in the von Frey assay with peak effects observed 30 mins after i.th.

administration with no effect shown in vehicle treated animals. Parallel results were exhibited in

the thermal hypersensitivity assay as peak effects were witnessed at 30 mins after i.th.

administration at doses of 3, 10, and 30 µg with no effect observed in vehicle treated animals.

4.4 ENK Analogues

4.4.1 Rationale and Design

As previously mentioned, chronic activation of the MOR has been linked with an

upregulation of the KOR and KOR-associated side effects such as anxiety and depression.

Analogue 55 underwent a general receptor binding assay that tested the ligand’s affinity at other

receptors. It was shown to also associate with the KOR (Ki = 1.4 nM) in which it was then re-

evaluated for its functionality which showed as a full antagonist. To our knowledge, this is the

first known compound to possess MOR/DOR agonist, KOR antagonist properties. Based on this

result, many analogues from the previous SAR were synthesized and examined at the KOR along

with their functional activities. In order to establish a complete SAR, selected ligands from Table

14, along with the addition of new analogues to further investigate the roles of various positions

and moieties, were synthesized and their binding affinities at the KOR were tested. Additionally,

their functional activities were also examined to determine agonist or antagonist properties.

81

4.4.2 Experimental

Analogues LYS729, MR106, MR107, and MR109, due to having the corresponding

amide at the C-terminus, were synthesized using standard SPPS with an Nα-Fmoc protecting

group approach on rink amide MBHA resin (100-200 mesh, Novabiochem) with >40% overall

yields (Figure 20, Table 15).171 The resin was first deprotected using 20% piperidine in DMF for

20 mins at rt. Coupling of amino acids were performed using 3 equivalents of HBTU, 3

equivalents of HOBt, and 6 equivalents of DIPEA in DMF for 1 hour at rt. After the coupling of

amino acids was complete, the resin was dried under vacuum for 3 hours and then cleaved from

the resin using a 95% TFA solution containing 2.5% TIS and 2.5% water for 3 hours at rt to

afford crude peptides with high purity (70-90%). Crude peptides were then subjected to RP-

HPLC using a preparative column (C-18, Microsorb-MV, 10 x 250 mm, 10 μm) with a gradient

of 10-100% acetonitrile in 0.1% TFA/water in 45 mins to afford >95% purity. The purified

analogues were then validated by analytical RP-HPLC and HR-MS in positive ion mode.

Figure 20. Structures of Fmoc-rink amide resin, BOP, and NMM.

82

All other analogues of the series were synthesized by liquid phase peptide synthesis

(LPPS) using Boc-chemistry. Coupling reactions were monitored by thin layer chromatography

(TLC) using the following solvent system: chloroform/methanol/acetic acid = 90:10:1 with

ninhydrin spray being used for detection. Analogues were synthesized by stepwise synthesis

using an Nα-Boc protecting group approach starting from Ppp (Figure 21). 1.0, 1.1, 1.2, 1.2, and

2.0 equivalents of Ppp, the respective amino acid, HOBt, (Benzotriazol-1-

yloxy)tris(dimethylamino) phosphonium hexafluorophosphate (BOP), and 4-methylmorpholine

(NMM), respectively, were dissolved in DMF and cooled in an ice bath for 30 mins and stirred at

rt for 2 hours. After the disappearance of the starting amine was observed via TLC, the reaction

was diluted 10x with ethyl acetate and extracted against equal volume of 5% NaHCO3 x3, 5%

citric acid x2, brine x1, and water x1, consecutively. The organic layer was then dried using

anhydrous Na2SO4 or MgSO4 and filtered. The resulting solution was then concentrated under

reduced pressure and triturated with cold diethyl ether or hexanes to yield a Boc-protected

peptide as a white powder. The Boc protecting group was deprotected using 95% TFA/5% TIS

for 30 mins in an ice bath for 30 mins and monitored by TLC. The solution was then diluted with

toluene and concentrated under reduced pressure and triturated with cold diethyl ether or hexanes

to yield a target peptide as a white powder. After the completion of the chain elongation, the

analogues were subjected to preparative RP-HPLC on a Hewlett-Packard 1100 (C-18,

Microsorb-MV, 10 x 250 mm, 10 μm) to afford >95% purity using a gradient of 10-100%

acetonitrile containing 0.1% TFA/water within 45 minutes, 3 mL/min. Analytical HPLC was

performed on a Hewlett-Packard 1090 (C-18, Vydac, 4.6 x 250 mm, 5 μm) at 1 mL/min and

validated using HR-MS.

83

Table 15. Analytical data of synthesized multifunctional ligands for MOR/DOR/KOR.

Analogue Structure HR-MSa (M+H)+ HPLCb

(tR, min) aLogPc

Observed Calculated

LYS729 Tyr-DAla-Gly-Phe-NH2 456.2239 456.2246 14.0 0.32

MR106 Tyr-DNle-Gly-Phe(4-F)-NH2 n.d. 515.2544 14.8 1.36

MR107 Dmt-DNle-Gly-Phe(4-F)-NH2 n.d. 543.2857 15.7 1.75

48 Tyr-DAla-Gly-Phe-Ppp 671.3579 671.3557 19.1 2.80

50 Dmt-DAla-Gly-Phe-Ppp 699.3852 699.3870 20.1 2.96

52 Dmt-DNle-Gly-Phe-Ppp 741.4325 741.4340 19.3 3.66

56 Dmt-DNle-Gly-Phe(4-F)-Ppp 759.4247 759.4245 20.0 3.74

55 Dmt-DNle-Gly-Phe(4-Cl)-Ppp 775.3995 775.3951 21.8 4.18

MR119 Dmt-DNle-Gly-Phe(4-Br)-Ppp 821.34156 819.3445 25.4 4.25

MR109 Dmt-DNle-Gly-Phe(4-Br)-NH2 606.2104 604.2135 19.7 2.49

LYS702 Dmt-DTic-Phe(4-Cl)-Ppp 764.3632 764.3579 23.0 4.91

57 Dmt-DAla-Gly-Phe(4-Cl)-Ppp 733.3494 733.3480 23.1 3.47

62 Dmt-DTic-Gly-Phe(4-Cl)-Ppp 821.3799 821.3793 25.7 4.46

61 Dmt-DTic-Gly-Phe(4-F)-Ppp 805.4055 805.4090 24.3 4.02

MR126 Dmt-DTic-Gly-Phe-Ppp 787.41677 787.4184 23.7 3.91

MR127 Dmt-Tic-Gly-Phe-Ppp 787.41646 787.4184 24.0 3.91

MR128 Dmt-Tic-Gly-Phe(4-F)-Ppp 805.40757 805.4090 24.4 4.02

MR129 Dmt-Tic-Gly-Phe(4-Cl)-Ppp 821.37849 821.3795 26.1 4.46

MR111 (Dmt-DNle-Homocys-Phe(4-F)-Ppp)2 1635.5d 1635.8324 26.1 5.58

MR112 (Dmt-Homocys-Gly-Phe(4-F)-Ppp)2 1524.4d 1524.7151 23.4 4.89

aFAB-MS (JEOL HX110 sector instrument) or MALDI-TOF. bHewlett Packard 1100 [C-18,

Vydac, 4.6 mm x 250 mm, 5 m, 10-100% of acetonitrile containing 0.1% TFA within 45 min,

1 mL/min]. chttp://www.vcclab.org/lab/alogps/. dLow resolution-Mass. n.d. not determined.

84

Figure 21. General scheme of LPPS to synthesize multifunctional analogues.

4.4.3 Results and Discussion

LYS729, an enkephalin-like tetrapeptide amide, showed mid-nanomolar affinity to the

KOR with no functional activity observed (Table 16). MR106 and MR107, fluorinated

analogues without Ppp at the C-terminus and a Tyr residue and Dmt1 substitution, respectively,

increased affinities at the ORs with slight MOR selectivity. MR106 showed weak agonist

function, whereas MR107 was shown to be a partial agonist/antagonist in the [35S]GTPγS

recruitment assay. 48, despite having mid-nanomolar affinity at the KOR, did not show any functional

activity at this receptor. 50 showed a Ki = 21 nM and partial agonist/antagonist activity, but was

not as efficacious as MR107, possibly owing to the nature of the elongated hydrophobic chain at

the 2nd position and halogenation of the Phe residue. In fact, analogues 52, 56, and 55 only vary

in their halogenations of the Phe residue and change from a partial agonist/antagonist, to a more

efficacious partial agonist/antagonist, and finally to a potent antagonist, respectively, with mostly

85

picomolar affinities at the ORs. This interesting result showed how only halogenation could

markedly enhance and switch receptor functionality.

Table 16. Biological activities of multifunctional peptide ligands at MOR/DOR/KOR.

Ligand Ki, nMa

KORe

[35S]GTPγS-binding KOR function

MORb DORc KORd EC50, nM Emax, %f IC50, nM Imax, %

g

LYS729 2.8 300 190 - < 30h - < 10h No function

MR106 2.3 9.3 30.0 - 70h - - Weak agonist

MR107 0.2 0.6 1.0 10 62 250 37 Partial

agonist/antagonist

48 23 0.69 220 - < 10h - < 10h No function

50 0.38 0.36 21 540 40 630 49 Partial

agonist/antagonist

52 0.39 0.18 174.4 260 53 290 70 Partial

agonist/antagonist

56 0.02 0.40 0.70 21 39 60 65 Potent partial

agonist/antagonist

55 0.10 0.08 1.4 - < 10%h 52 122 Potent antagonist

LYS702 0.45 0.76 890 - < 10%h - - No agonist activity

MR126 - - - - - - - -

MR127 32.0 >10000 2.1 - - - - -

MR128 57.0 9.2 4.8 - - - - -

MR129 59.0 18.0 16.0 - - - - -

MR111 1.3 1.8 8.1 - - - - -

MR112 23.0 11.0 67.0 - - - - -

-n.c.: no competition aCompetition analyses were carried out using membrane preparations from

transfected HNB9.10 cells that constitutively expressed the respective receptor types.

b[3H]DAMGO, Kd = 0.85 nM. c[3H]DPDPE, Kd = 0.50 nM. d[3H]U69,593, Kd = 5.3 nM.

eExpressed in CHO cells. fMean ± SEM of the % relative to 10 μM U50,488 stimulation. gMean

± SEM of the % relative to 10 μM naloxone inhibition of 100 nM U50,488. hat 10 μM.

To further assess the role of the halogen, analogues MR119 and MR109, were

synthesized with a bromination of the Phe residue with and without the Ppp moiety at the C-

86

terminus, respectively. Our current hypothesis is that the halogen interacts with the receptor via a

σ-hole. Halogen bonding involves non-covalent interactions, and in the case of the σ-hole,

halogens have a positive electrostatic potential on the outermost portion of their surface, centered

on the R-X axis, where X = I, Br, and Cl.220 The unshared pairs of electrons form a belt of

negative electrostatic potential around the central part of the halogen, leaving the outermost

region positive (the σ-hole). Due to the small size, fluorine does not experience this type of

bonding because it contains a high degree of electronegativity, which coupled with significant

sp-hybridization, enables the σ-hole to become neutralized. If the ligand’s affinity at the KOR

turns out to be through the electrostatic interaction between the ligand’s σ-hole related positive

ion and the receptor’s negative ion, bromination may show the same pattern of functional

activity as the chlorinated analogue (55). However, bromine is a bigger atom than that of

chlorine and fluorine, and thus a steric factor may result in a reduction of affinity.

Analogue 62, which incorporates a DTic residue at the 2nd position, exhibited picomolar

affinity with slight selectivity for the DOR over the MOR with no antagonistic properties at these

receptors. Analogues were generated from this scaffold to further assess a highly constrained

amino acid at this position coupled with halogenation of the Phe residue. LYS702, an analogue

with an ablated Gly residue, displayed slight selectivity for the MOR over the DOR (Ki = 0.45

nM and 0.76 nM, respectively), with near micromolar affinity at the KOR (Ki = 890 nM). No

agonist activity at the KOR was observed. Analogues 61 and MR126-129 were synthesized and

vary only in their Tic residue chirality and Phe halogenation. The non-halogenated derivative,

MR127, displayed the strongest affinity at the MOR (Ki = 32.0 nM) with the L-Tic residue at the

2nd position. Interestingly, this ligand completely lost interactive capabilities at the DOR and was

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rendered a KOR-selective ligand (Ki = 2.1 nM). It would be interesting to see how MR126 binds

to the ORs considering that its only variance is the D-Tic chirality, but this ligand is currently

undergoing affinity assays. Overall, the halogenated derivatives along with MR127 displayed the

same range of affinity at the MOR. MR128 and MR129 also displayed nanomolar affinities at

the DOR and KOR as halogenation of the Phe residue appeared to be critical for DOR binding

with L-chirality at the 2nd position. 61, which displayed Ki = 0.48 nM and 0.35 nM at the DOR

and MOR, respectively, exhibited the strongest affinity at the MOR and DOR within the Tic-

analogues owing to the augmentative effect of OR binding when employing D-chirality at the 2nd

position.

Enkephalin-based dimeric peptides were also rendered and evaluated at the ORs. MR111

and MR112 are linked by a disulfide bond from homocysteine at the 3rd and 2nd position,

respectively (Figure 22). MR111 showed low nanomolar affinity for all the ORs with no

apparent selectivity. MR112 showed slight selectivity at the DOR and overall had approximately

10-fold decreased affinities at the ORs in respect to MR111. This loss of interaction at the ORs

may be due to the homocysteine being of L-chirality as opposed to the D-isomer at the 2nd

position.

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Figure 22. Structures of dimeric ENK analogues.

4.4.4 Summary and Future Directions

A series of analogues based on ligand 50 from a previous SAR were synthesized and

evaluated at the KOR. The most prominent discovery was that halogenation of the Phe residue

with a chlorine converted a partial agonist/antagonist to a potent antagonist at the KOR. To our

knowledge, this is the first compound to have demonstrated potent agonist characteristics at the

MOR and DOR while being an antagonist at the KOR. In an effort to further elucidate positional

effects and various moieties, a subsequent series of analogues was designed and synthesized.

Most of these analogues still need to be evaluated for their functional activities at the ORs.

Another interesting takeaway was the effect of the homocysteine placement within the

enkephalin-based dimers. It was postulated that the order of magnitude loss of affinity was due to

the L-chirality at the 2nd position. One way to scrutinize this would be to incorporate the D-

isomer of homocysteine at the 2nd position. Our current hypothesis for the functional activity

switch based on halogenation at the KOR is that the substituent takes part in a σ-hole interaction

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with the receptor. One way to further investigate this notion in addition to testing ligands MR109

and MR119 is to test an analogue where the Phe residue is para-substituted with a methyl group.

Relative to the brominated analogue, these substituents are similar in size, but the methyl group

lacks the ability to participate in a σ-hole interaction or any form of halogen bonding. This would

be a straightforward way to assess whether steric factors are more important than electrostatic

factors at this position.

4.5 DALDA Analogues

4.5.1 Introduction to [Dmt1]DALDA

DALDA is a tetrapeptidic multifunctional ligand that is based on DER. The analogue

[Dmt1]DALDA is a MOR selective ligand with picomolar affinity (Ki = 0.143 nM at the MOR

with a Ki ratio = 1/14,700/156 to the MOR/DOR/KOR) with a half-life of 1.8 hours.205,221 It

showed an antinociceptive response 3000-fold greater than that of morphine in the rat tail-flick

assay and 100- to 200-fold more potent in the mouse tail-flick assay via i.th. and i.c.v.

administration, respectively, despite only exhibiting a 7-fold MOR affinity increase relative to

morphine’s.204,222,223 Additionally, when co-administered with morphine, the peptide brought

about an analgesic response that was 4-fold greater than that of morphine alone at equipotent

doses. This implicated a synergistic effect taking place upon administration of [Dmt1]DALDA.

This compound was shown to inhibit norepinephrine uptake in rat spinal cord synaptosomes

(IC50 = 4.1 µM) ultimately leading to further activation of the MOR and α2-adrenergic receptors,

a circumstance that has been shown to augment analgesic effects.224 Other evidence supports this

synergistic idea considering that the antinociceptive response by [Dmt1]DALDA was weakened

by pretreatment of nor-BNI, a KOR antagonist, or with antiserum against dynorphin A(1-17) or

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Met-ENK.225 This revealed the possibility that [Dmt1]DALDA may induce endogenous release

of ligands that interact at the KOR and DOR to contribute to the overall analgesic profile.

A drawback to this compound is that it elicits rapid tolerance following chronic i.th.

administration.204,226 However, when [Dmt1]DALDA was given s.c. in morphine-tolerant mice,

only minimal cross-tolerance was observed and thus making this compound possibly viable in

pain treatment where morphine has been previously exhausted.223,227 When [Dmt1]DALDA was

administered s.c., its effects were shown to be greater (36-218 fold) relative to that of morphine’s

with a 4-fold greater duration of action.204,223 The observed s.c. potency suggested that this

ligand is able to appreciably cross the BBB. Indeed, [3H][Dmt1]DALDA was able to cross Caco-

2 cell monolayers in a concentration-dependent manner until equilibrium is reached which

provides evidence that it is effluxed by a transporter.228 Caco-2 cells are a human epithelial

colorectal adenocarcinoma cell line that mimics the lining of the small intestine and is the

standard used to adjudicate membrane permeability. It has still yet to be determined how the

cells uptake [Dmt1]DALDA, but results have shown that it is not by a transporter, receptor-

mediated endocytosis, or absorptive endocytosis. Due to the +3 charge of the ligand, it is

theorized that the ligand interacts with the negatively charged functionalities on the cell surface

and somehow migrates across the lipid bilayer.

4.5.2 Rationale and Design

Due to the KOR being implicated in producing side effects during chronic MOR agonist

administration, there is therapeutic promise in developing ligands that are MOR/DOR agonists

and KOR antagonists. The peptides DALDA and [Dmt1]DALDA are incredibly MOR selective.

DALDA exhibits Ki = 1.69 nM, 19,200 nM, and 4,230 nM at the MOR, DOR, and KOR, and

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[Dmt1]DALDA displays a Ki selectivity ratio of 1/14,700/156 to the MOR/DOR/KOR,

respectively.205,221 The half-life of DALDA was measured to be 1.5 hours as [Dmt1]DALDA

showed 1.8 hours.221 By strengthening KOR affinity and producing ligands with increased

biological stability, the therapeutic potential would be augmented. By incorporating halogenation

of the Phe residue and attaching the Ppp moiety at the C-terminus, we hypothesized that KOR

affinity and antagonist activity could be increased along with biological stability. Additionally,

the increased lipophilicity should aid in cellular permeability. Therefore, a series of ligands

employing DALDA as a scaffold were designed and synthesized by the same LPPS method as

4.4 (Figure 21, Table 17), and evaluated for their biological activities at the ORs (Table 18).

Table 17. Analytical data of DALDA analogues.

Analogue Structure HR MSa (M-TFA + H)+ HPLCb

(tR, min) aLogPc

Observed Calculated

MR110 Dmt-DArg-Phe-Lys-Ppp 855.52386 855.5246 13.9 1.89

MR232 Dmt-DArg-Phe(4-Cl)-Lys-Ppp 889.48402 889.4856 17.0 2.40

MR233 Dmt-DArg-Phe(4-F)-Lys-Ppp 873.51465 873.5152 16.2 1.92

MR124 Dmt-DArg-Phe-Gly-Ppp 784.45068 784.4511 16.6 1.92

MR125 Dmt-DArg-1Nal-Gly-Ppp 834.4661 834.4667 19.9 2.83

MR120 Dmt-DArg-1Nal-Lys-Ppp 905.53896 905.5402 17.4 2.67

MR122 Dmt-DArg-1Nal-Ppp 777.44518 777.4453 21.4 3.25

MR121 Dmt-DArg-Phe(4-Cl)-Ppp 761.39009 761.3906 20.3 3.00

aFAB-MS (JEOL HX110 sector instrument) or MALDI-TOF. bPerformed on a Hewlett

Packard 1100 [C-18, Vydac, 4.6 mm x 250 mm, 5 m, 10-90% of acetonitrile containing 0.1%

TFA within 40 min, 1 mL/min]. chttp://www.vcclab.org/lab/alogps/.

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4.5.3 Results and Discussion

Compared to [Dmt1]DALDA, MR110 has the Ppp moiety coupled at the C-terminus

which decreased affinity for the MOR approximately by an order of magnitude (Table 18). Even

though it was shown not to interact with the KOR, it is unclear whether this compound is purely

MOR selective considering it has yet to be tested at the DOR. MR232 and MR233 are

halogenated analogues of MR110 with a chlorine and fluorine, respectively, but have not been

tested at the ORs. MR124 substitutes the Lys residue at the 4th position with a Gly residue,

making it more resemble an enkephalin-like peptide. The ablation of this positive charge may

enhance the permeation characteristics of this peptide thereby making it more biologically active.

To further elucidate the role of the 3rd position, the Phe residue was substituted with a 1-

naphthylalanine (1-Nal), a bulkier aromatic residue, in analogues MR125, MR120, and MR122.

MR125 still maintains the Gly substitution at the 4th position and shows mid to high nanomolar

binding at the ORs with slight selectivity for the DOR. MR122 is an analogue without the 4th

position amino acid, and shows slight recovery of affinity at the DOR and KOR with slight

selectivity for the latter. Overall, it is unclear whether the 1-Nal substitution is responsible for the

loss of affinity or if it is more attributed to the Lys to Gly substitution relative to that of ligand

MR110 at the MOR, but determining the affinities at the ORs for ligand MR120, which

maintains the Lys residue with the 1-Nal substitution, would help shed some light on these

effects. MR121 shows a para-chloro substituted Phe residue without a 4th amino acid and yields

a low nanomolar KOR selective ligand with a reduction in MOR and DOR affinities. Compared

to DALDA and [Dmt1]DALDA, analogues MR125, MR122, and MR121, not only have

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recovered affinities for the DOR and KOR, but have also switched the selectivity away from the

MOR to the KOR.

Table 18. Binding affinities of DALDA analogues at the MOR/DOR/KOR.

Ligand Structure Ki, nMa

MORb DORc KORd

[Dmt1]DALDAe Dmt-DArg-Phe-Lys-NH2 0.14 2100 22

MR110 Dmt-DArg-Phe-Lys-Ppp 0.06 - n.c.

MR232 Dmt-DArg-Phe(4-Cl)-Lys-Ppp - - -

MR233 Dmt-DArg-Phe(4-F)-Lys-Ppp - - -

MR124 Dmt-DArg-Phe-Gly-Ppp - - -

MR125 Dmt-DArg-1Nal-Gly-Ppp 610 450 770

MR120 Dmt-DArg-1Nal-Lys-Ppp - - -

MR122 Dmt-DArg-1Nal-Ppp 760 350 130

MR121 Dmt-DArg-Phe(4-Cl)-Ppp 1100 960 62

aCompetition analyses were carried out using membrane preparations from transfected

HNB9.10 cells that constitutively expressed the respective receptor types. b[3H]DAMGO,

Kd = 0.85 nM. c[3H]DPDPE, Kd = 0.50 nM. d[3H]U69,593, Kd = 5.3 nM. eReference #205.

n.c. non-competitive.

4.5.4 Summary and Future Directions

MR110 decreased its potency at the MOR relative to [Dmt1]DALDA by approximately

10-fold, but lost its affinity at the KOR. To determine the selectivity of this ligand, its affinity at

the DOR must be assessed. MR122 and MR121 are small tripeptide analogues in which a Lys

residue at position 4 is deleted and thus 1Nal and Phe(4-Cl) at position 3 are connected to the

Ppp group, respectively, showed moderate binding affinity with a slight selectivity for KOR over

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MOR and DOR. The SAR result indicates the role of aromatic ring at the C-terminus along with

the Ppp group in KOR recognition. MR125 is a non-selective opioid ligand with moderate

affinity. The remaining DALDA analogues are undergoing binding assays so that the substitution

effects, especially halogen effect, can be identified which can then be further applied to the

development of potent MOR/DOR agonists and KOR antagonists. To evaluate their functional

activities, [35S]GTPγS assays will be followed, and the results will inform if a MOR/DOR agonist

profile is conserved for these analogues along with KOR antagonism at ligands that interact at

the KOR. Lastly, the biological stabilities of these ligands will be determined. Due to the

increased hydrophobicity and decreased overall charge for ligands where a Lys is substituted

with a non-basic amino acid, these analogues are expected to exhibit enhanced stabilities and

BBB/cellular penetration.

4.6 Biphalin Analogues

4.6.1 Introduction to Biphalin

At the time of the synthesis of biphalin, the following was known about ENK SARs: i)

the amino terminal Tyr residue was critical for the compound’s activity; ii) the substitution of D-

amino acids at the 2nd position in place of Gly typically enhances the activity of ENK analogues;

iii) the C-terminal Met or Leu residues of ENK could be replaced by a large array of substituents

without attenuating the ligand’s ability to interact at the ORs.229 Based on this information, the

C-terminal Leu or Met residues of ENK were substituted by a second ENK analogue fragment

which were connected by a diamine bridge of varying length (Figure 23). This proposed design

was theorized to increase the biological stability due to the C-terminus being shielded against

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enzymatic hydrolysis and any single hydrolysis of the proposed analogues would yield a

different form of an active ENK sequence.

Figure 23. Original design of bivalent ENK analogues.

The bivalent enkephalin analogues where n = 0 (biphalin) and n = 3 (ENK-3) yielded the

most intriguing results. Biphalin was 90-fold and 12-fold more potent than Met-ENK and

[DAla2]-Met-enkephalinamide (DALA) on GPI, respectively, whereas ENK-3 was 4-fold and

0.5-fold more potent than Met-ENK and DALA, respectively. I.p. route of administration of

biphalin provided significant analgesia at 10 and 20 mg/kg of body weight doses at both 30 and

60 min post injection. The 20 mg dosage increased the response latency 60 mins post injection of

paw withdrawal in the hot plate assay by 186% relative to the control and 82% to that of

equimolar morphine hydrochloride. ENK-3 was shown to be ineffective. The effects of biphalin

were antagonized by pretreatment with naloxone showing that these effects are mediated through

the ORs.

Shortly thereafter, another SAR was developed that further looked at the size of the linker

between the two ENK fragments and the substitution of DPhe with DAla at the 2- and 2′-

positions (Table 19).230 The SAR revealed that ligands with DPhe at the 2nd positions have

weaker affinities than D-alkyl substituents. The tetrapeptidic analogues showed a reduced

affinity at the ORs relative to biphalin and the hydrazide linker appeared to be optimal for OR

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interaction whether it was a tetrapeptide or an octapeptide. This reasoning for this trend may be

due to the hydrazine linker being more rigid than the other proposed bridging agents.

Table 19. Binding affinities of bivalent ENK analogues with varying linker length.

Sequence Ki (nM)

MORa DORb KORc

(Tyr-DPhe-NH-)2=CH2 690 ± 48 1,480 ± 49 14,000 ± 390

(Tyr-DPhe-NH-)2 31 ± 2 187 ± 15 360 ± 34

(Tyr-DPhe-NH-CH2-)2 720 ± 170 820 ± 97 14,000 ± 1,200

(Tyr-DPhe-Gly-Phe-NH-)2 190 ± 28 150 ± 13 2,320 ± 480

(Tyr-DAla-Gly-Phe-NH-)2 12 ± 2 4.6 ± 0.2 270 ± 15

Morphine 38 ± 4 510 ± 55 1,900 ± 93

-Brain membranes from male Hartley guinea pigs. aMOR: [3H]naloxone; Kd = 0.98 nM.

bDOR: [3H]DADLE; Kd = 0.64 nM. cKOR: [3H]ethylketocyclozocine; Kd = 0.62 nM.

The tetrapeptide with the hydrazine bridge was tested for its antinociceptive effect via i.p.

and i.th. administration in mice. I.p. administration afforded high antinociceptive effect in both

visceral and thermal nociceptive tests, similar to that of biphalin. However, i.th. administration

afforded much less of an antinociceptive effect relative to biphalin. Considering that biphalin has

approximately 40-fold greater affinity for the DOR than the tetrapeptide, there may be a greater

synergistic effect between simultaneous activation of the MOR and DOR to provide analgesia.

The analgesic activity of biphalin was more closely scrutinized by studying its effects in

the tail flick and tail pinch assays post s.c., i.v., and i.th. administration in rats.231 S.c.

administration showed limited analgesic activity relative to morphine in both assays, but was

more potent when given via i.v. despite still being lesser than morphine’s and of shorter duration.

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I.th. administration afforded more potent analgesia stemming from biphalin in the tail flick assay,

but not in the tail pinch. I.c.v. administered biphalin was incredibly potent, about 7-fold greater

than that of etorphine, a highly potent opioid agonist, with only 20 mins necessary to reach its

peak effect.232 Morphine was found to be 245-fold less potent than biphalin. Mice were

pretreated with a variety of MOR, DOR, and KOR antagonists prior to biphalin administration.

A rightward shift of antinociceptive dose-response curves were observed for i.c.v. biphalin

except for DALCE and nor-BNI which failed to antagonize the antinociceptive effects.

Following i.c.v. biphalin, a dose-related inhibition of gastrointestinal transit was observed.

Equimolar morphine showed an effect that was 20-fold less than biphalin’s. Acute physical

dependence was also tested by dosing the mice with biphalin and then antagonizing its effects

with naloxone. Withdrawal-related jumping was then counted for a 10 min period. Little jumping

was observed with i.p. injected or 3-day continuous i.p. infused biphalin. No other signs of

physical dependence were observed. Continuous i.c.v. infusions, on the other hand, exhibited

signs of physical dependence manifested as agitation, diarrhea, and urination.

Molecular entry into the CNS is dependent upon the size, charge, hydrophobicity, and/or

access to carriers that are at the BBB and blood-cerebral spinal fluid (CSF) barriers. ENKs have

limited ability to cross this barrier due to peptidases being present in the blood, at the blood-CNS

interface, and within the endothelial and/or epithelial cytosolic compartments, but once present,

there is also an efflux system for N-tyrosinated peptides.233-235 The metabolic stability of biphalin

was determined to be 87 ± 2 and 112 ± 6 mins in serum and brain membrane homogenates,

respectively.232 It has been shown that halogenation of ENK analogues increases brain uptake

and specificity for the DOR.217,218,236 On the basis of these results, various biphalin analogues

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with halogenations at the Phe-4,4′ positions with either chlorine or fluorine have been

characterized for their stability and CNS uptake.

An in vitro BBB model using bovine brain microvessel endothelial cells (BMEC) was

used to assess the halogenated biphalin analogues’ BBB permeability.237 Both chlorine and

fluorine derivatives passed the BMEC monolayers more appreciably than that of [14C]sucrose.

[125I]biphalin and [125I] [Phe(4-Cl)4,4′]biphalin uptake plots in the brain and CSF revealed that

both compounds entered the brain and CSF much quicker than that of [14C]sucrose with the

chlorinated analogue exhibiting the greatest extent of uptake in both regions. [Phe(4-

Cl)4,4′]biphalin’s enzymatic stability was tested in brain membrane homogenates and was

observed to have a half-life greater than that of biphalin (173 vs. 310 mins).

Other more constrained diamine linkers have been investigated in place of the hydrazine

bridge (Figure 24).238 Use of benzene-1,4-diamine afforded low nanomolar affinities at MOR

and DOR with 36 nM and 40 nM IC50 values in the MVD and GPI functional assays. The

benzene-1,2-diamine bridge afforded a compound with 0.19 nM and 1.9 nM of binding affinities

at the DOR and MOR with 0.72 nM and 40 nM IC50 values in the MVD and GPI functional

assays, respectively. Employing the piperazine bridge gave rise to a ligand that was

approximately 5-fold stronger in binding to the MOR and DOR to that of biphalin and in the

MVD and GPI assays (IC50 = 9.3 nM and 2.5 nM, respectively). Fluorination of the para position

on the Phe residues of biphalin analogues and with bridging benzene-1,2-diamine and piperazine

were also synthesized.239 Halogenation of the ligand encompassing the benzene-1,2,-diamine

bridge showed Ki = 13 nM and 0.51 nM at the DOR and MOR, respectively, whereas the

piperazine bridged analogue afforded low picomolar affinities (0.09 nM and 0.11 nM at DOR

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and MOR, respectively) with an Emax = 94.1% at 0.8 nM at the DOR and an Emax = 77% at 1.0

nM at the MOR in the [35S]GTPγS binding assay.

Figure 24. Structures of other diamine bridges used with biphalin.

Few attempts have been made to cyclize biphalin and investigate the subsequent ligands’

biological profiles.240,241 In one study, two cyclic compounds were synthesized via a disulfide

bridge from substituting the DAla residues with two Cys or DCys residues at the 2 and 2′

positions giving 22-membered cycles with a hydrazine bridge.240 The L-Cys analogue lost both

affinity and efficacy at the DOR and MOR, but the D-Cys variant maintained picomolar affinity

at the DOR and MOR (0.87 nM and 0.60 nM, respectively). This ligand was also very

efficacious at the DOR (IC50 = 0.87 nM with an Emax = 100%) and partially at the MOR (Emax =

47% with an IC50 = 0.2 nM). A subsequent study synthesized these same biphalin analogues, but

instead, Pen or DPen residues were used to form the cyclization. Again, the analogue that

contained the Pen residues with L-chirality was not found to be active at the ORs, but the DPen

analogue showed strong affinities (Ki = 5.2 nM, 1.9 nM, and 260 nM at the DOR, MOR, and

KOR, respectively) and Emax values >100% for the ORs relative to their corresponding

radioligand (DOR = [Ile5,6]DLT II; MOR = DAMGO; KOR = U69593).

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4.6.2 Rationale and Design

Biphalin was designed to have increased stability at the ORs by shielding the C-terminus

and making it more resistant to enzymatic degradation by incorporation of D-amino acids.

Additionally, the palindromic double ENK sequence enables active fragments of the peptide

even when enduring hydrolysis or cleavage via enkephalinase. Despite biphalin’s unique and

potent antinociceptive profile, its half-life was measured to be 87 and 112 mins in serum and

brain membrane homogenates in one study, and 173 mins in brain membrane homogenates in

another.232,237 Halogenation of biphalin increased its half-life to 310 mins.237 Another common

method to increase stability is to cyclize peptides. The only cyclic series of biphalin that have

been generated are those with a hydrazine linker. On top of that, only diamine bridges of various

carbon lengths and moieties have been investigated as a linker system for the two ENK-like

tetrapeptides. By exploring other types of diamine linkers and formulating a design based on the

literature precedent of both the ENKs and biphalin, it was postulated that more potent analogues

may be rendered.

It has been previously established that substitution of Tyr with Dmt augments ligand-

receptor interaction for the ORs, and justly, this substitution was incorporated into the design.

Based on the literature precedent, it was shown that using piperazine as a bridge for the two

ENK-like tetrapeptides was ideal, but this moiety was either not employed, or not successfully

synthesized, in cyclic ligands. Also, other non-alkyl diamino bridges have not been adjudicated

in a biphalin scaffold at the ORs. Here, we employed both piperazine and the more dynamic

cystamine in our ligands, both of which have yet to be tested in a cyclic scaffold (Table 20).

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Table 20. Analytical data of cyclic biphalin analogues.

Analogue Structure HR MSa (M-TFA + H)+

HPLCb

Retention Time aLogPc

Observed Calculated

MR234 (Tyr-c[DCys-Gly-Phe-Piperazine])2 1025.40081 1025.4014 17.7 1.62

MR235 (Dmt-c[DCys-Gly-Phe-Piperazine])2 1081.46647 1081.4640 18.3 1.92

MR236 (Tyr-c[DPen-Gly-Phe-Piperazine])2 1081.46206 1081.4640 18.7 2.56

MR238 (Tyr-c[DCys-Gly-Phe-Cystamine])2 1091.35949 1091.3612 20.4 1.68

MR239 (Dmt-c[DCys-Gly-Phe-Cystamine])2 1147.42226 1147.4238 21.2 2.11

MR241 (Tyr-c[DPen-Gly-Phe-Cystamine])2 1147.42410 1147.4238 21.4 2.93

aFAB-MS (JEOL HX110 sector instrument) or MALDI-TOF. bPerformed on a Hewlett

Packard 1100 [C-18, Vydac, 4.6 mm x 250 mm, 5 m, 10-90% of acetonitrile containing

0.1% TFA within 40 min, 1 mL/min]. chttp://www.vcclab.org/lab/alogps/.

4.6.3 Experimental

Analogues from the cyclic biphalin series were synthesized using LPPS. Coupling

reactions were monitored by TLC using chloroform/methanol/acetic acid = 90:10:1 with

ninhydrin spray being used for detection. Analogues were synthesized by stepwise synthesis

using an Nα-Boc protecting group approach starting from either piperazine or cystamine (Figure

25). 1.0, 2.2, 2.4, 2.4, and 4.0 equivalents of piperazine or cystamine, the respective amino acid,

HOBt, BOP, and NMM, respectively, were dissolved in DMF and cooled in an ice bath for 30

mins and stirred at rt for 4 hours. For the DCys and DPen amino acids, the acetamidomethyl

(Acm) protecting group was employed to allow for in situ deprotection and cyclization. After the

disappearance of the starting amine was observed via TLC, the reaction was diluted 10x with

ethyl acetate and extracted against equal volume of 5% NaHCO3 x3, 5% citric acid x2, brine x1,

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and water x1, consecutively. The organic layer was then dried using anhydrous Na2SO4 or

MgSO4 and filtered. The resulting solution was then concentrated under reduced pressure and

triturated with cold diethyl ether or hexanes to yield a powdered precipitate. The Boc protecting

group was removed using 95% TFA/5% TIS in an ice bath for 30 mins and monitored by TLC.

The solution was then diluted with toluene and concentrated under reduced pressure and

triturated with cold diethyl ether or hexanes to yield a powdered precipitate. For ligands MR234,

MR235, MR238, and MR239, coupling was stopped at Boc-DCys(Acm)-R to induce

cyclization. Simultaneous deprotection of Acm and cyclization to afford a disulfide bond was

performed in situ using 0.06 M I2 in MeOH added dropwise to a 0.1 mg/mL peptide in an 80/20

MeOH:AcOH solution at rt for 5 days. The remaining I2 was quenched using ascorbic acid and

the resultant solution was concentrated under reduced pressure and triturated with cold diethyl

ether to afford a white powder. The final amino acid coupling and Boc deprotection then took

place to afford crude product with 30-40% overall yield. For the DPen analogues MR236 and

MR241, attempts to cyclize these two analogues in a similar manner were not successful.

Therefore, the full length linear variant was synthesized before cyclization was initiated. The

Boc-Tyr(Boc)-R peptide was then subjected to the same conditions as previously described for

simultaneous deprotection of Acm and cyclization. The last Boc deprotection then took place to

afford crude peptide with 20% overall yield. Analogues were subjected to preparative RP-HPLC

on a Hewlett-Packard 1100 (C-18, Microsorb-MV, 10 mm, 250 mm, 10 μm) to afford >95%

purity using a gradient of 10-90% acetonitrile containing 0.1% TFA/water within 40 mins, 3

mL/min. Analytical HPLC was performed on a Hewlett-Packard 1090 (C-18, Vydac, 4.6 mm,

250mm, 5μm) at 1 mL/min and validated using HR-MS.

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Figure 25. Synthetic scheme of cyclic biphalin analogues.

4.6.4 Results and Discussion

All analogues but MR241 have been tested for their binding affinities at the ORs. These

ligands displayed similar nanomolar affinities for the MOR, apart from MR239, which exhibited

picomolar affinities at the ORs (Table 21, Figure 26). Overall, a balanced affinity profile was

observed for all analogues. Comparisons of analogues MR234 with MR235 and MR238 with

MR239 show the effect of Dmt substitution as it enhanced affinities at the ORs by about an

order of magnitude. There were slight effects going from a DCys bridge (MR234) to a DPen

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bridge (MR236) and from a piperazine bridge (MR235) to a cystamine bridge (MR239) as those

variants were well tolerated at the ORs. However, if MR241 displays picomolar affinity at the

ORs, then one may draw the conclusion that the cystamine linker employed is responsible for the

tighter binding. It would be worthwhile to explore the cyclic biphalin derivates that have Dmt

residues with DPen-bridging for both cystamine and piperazine linkers considering that a slight

augmentation in OR affinities have been observed in this series.

Figure 26. Competition binding assays of MR239 at the MOR (left, [3H]DAMGO) and KOR

(right, [3H]U69593).

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Table 21. Binding affinities of cyclic biphalin analogues at the MOR/DOR/KOR.

Analogue

Ki (nM)*

MOR

[3H]DAMGOa

DOR

[3H]DADLEb

KOR

[3H]U69593c

MR234 8.9 27 19

MR235 2.5 2.0 4.9

MR236 2.9 1.9 5.6

MR238 2.4 4.1 18

MR239 0.3 0.5 0.9

MR241 n.d. n.d. n.d.

*Assays were performed in Bryan Roth Laboratory at the University of North

Carolina at Chapel Hill by the NIMH PDSP program.

4.6.5 Summary and Future Directions

The ligands from this series have been sent to have their functional activities assessed at

the ORs. Ideally, we would like to see compounds that are MOR/DOR agonists and KOR

antagonists. Biological stabilities should also be determined. Due to the cyclization, an enhanced

stability is anticipated. It would be interesting to see the impact of halogenation on these cyclic

compounds considering that they have yet to be investigated. Following up from the ENK SAR

where halogenation could switch functional activities at the KOR, a similar result may be

observed. Lastly, a linker-type similar to that of cystamine has yet to be evaluated in a series

where functional activities were determined. Despite previous data suggesting that more

dynamically accessible bridges hinder affinities for the ORs, in our series, we see strong

affinities at all the ORs, and thus, the sulfur atoms within the cystamine bridge may have an

unforeseen impact on affinities and perhaps functional activities.

106

4.7 Endomorphin Analogues

4.7.1 Introduction to the Endomorphins

Many believe that EM-1 and EM-2 are the true endogenous ligands for the MOR due to

their high selectivity (EM-1 Ki = 0.36, 1,506, 4,183 at the MOR/DOR/KOR; EM-2 Ki = 0.69,

9,233, >10,000 at the MOR/DOR/KOR) despite not having found the gene responsible for

producing these peptides.58 EM-1 was observed to mostly inhabit in the brain, whereas EM-2

was mostly discovered in the terminal regions of primary afferent neurons of the dorsal horn in

the spinal cord.242,243 These peptides brought about antinociception in various pain tests and have

also been implicated to play a role in mood, behavior, and maintenance of a variety of

physiological functions.58,244-247 The message-address notion is believed to also hold true for

these ligands and that the N-terminal tripeptide unit is thought to be the message sequence that is

required for receptor interaction and the address region belongs to the C-terminal Phe. Despite

the simple structure of these molecules, they meet the defined requirements for OR activity: i) a

phenolic hydroxyl group; ii) an amino group at the N-terminus; iii) and an additional aromatic

moiety. A C-terminal amide has been shown to be required in EM analogues with the Pro residue

at the 2nd position thought to be used as a tool to properly orient the pharmacophore.248,249

Due to the C-terminal amidation of the EMs, these endogenous ligands are the most

stable of the known endogenous opioids (t1/2: 1 hour for EM-1 and 2-3 hours for EM-2) with

their metabolic products being pharmacologically inactive.250 Extensive studies have shown their

pharmacological profiles to displace the OR antagonist naloxone, DAMGO, and other MOR

ligands.245 In reference to their functional activities, it is generally agreed upon that the EMs are

partial agonists at the MOR, coupled to the inhibitory Gi/Go protein, but some studies have noted

107

full agonist capabilities.58,251-253 Their efficacies are significantly lower than that of DAMGO’s

and are not notably influenced by DOR or KOR antagonists.

Electrophilic substitutions of the aromatic ring have generally afforded high-affinity

MOR ligands. Alkylation of the Tyr residue to afford Dmt has also been used to increase

affinities at the ORs.254 Lengthening of the N-terminus has been met with some success as N-

allylation of Dmt1 converted the partial agonist activity to antagonist with a lessened selectivity

profile, but weakened MOR interaction.255 Ablation of the N-terminal amino group, which

usually converts agonist activity to antagonist, or substitution with an alkyl substituent, gave rise

to such an effect, but at the cost of affinity at the MOR and efficacy at both the MOR and

DOR.256

A number of SARs only looked at the Trp3 and Phe3 residues of EM-1 and EM-2,

respectively, by incorporating either natural or unnatural amino acids.257 Ala substitution resulted

in severe loss of affinity at the MOR with similar data observed using cyclohexylalanine. This

demonstrated the importance of having aromatic functionality whether it is due to π electrons or

planarity of the ring. To further elucidate this effect, substitution with other aromatic residues

such as His, Tyr, and pentafluoro-Phe, and then with branched alkyl amino acids (Val, Leu, and

Nle) were tested and displayed parallel results (loss of MOR affinity). This led to the idea that

both the size and aromaticity matters in MOR-ligand recognition. Alkylation of the Phe3 residue

at the 3′ and 5′ positions led to functionally inactive compounds, although alkylating the 2′ and 6′

positions were well tolerated.258 Other substitutions with more bulky residues at the 3rd position

such as 1-D-Nal, 2-D-Nal, and DPhe(p-Cl) generally yielded moderate affinities at the MOR

with antagonistic properties in in vivo assays.259

108

Phe4 and C-terminal modifications have also been investigated for the EMs. Amide

conversion to an alcohol at the C-terminus decreased the potency, but maintained agonist

behavior.260,261 In another study, EM-2 analogues following the scaffold Tyr-Pro-Phe-NH-X

where X is an array of bulky and/or aromatic substituents (i.e. phenylethyl, benzyl, phenyl,

naphthyl, cyclohexyl, and adamantyl), which were largely accepted, and concomitant

modification with Dmt1 resulted in a picomolar range of affinities at the MOR with MOR/DOR

agonist or DOR antagonist functional profiles.262 Incorporation of various amino and alkyloxy

substituents at the same position were also scrutinized. Many of the derivatives that included

small, polar moieties (i.e. methoxy, methanolic, or hydrazine) yielded potent and selective

analogues at the MOR, but like the rest of the ligands, ultimately resulted in poor efficacy in the

in vitro assays relative to EM-2.263 Hydrophobic, bulky substituents were not favorable for MOR

interaction.

4.7.2 Rationale and Design

The EMs are very potent endogenous opioid peptides that are effective in their regulation

of pain, but like other selective MOR agonists, they come with serious side effects such as

physical dependence and respiratory depression. The use of peptides in a clinical setting has

promise, yet some of its major drawbacks are poor bioavailability, possessing low metabolic

stability towards proteolysis and low permeability to the BBB. Comparing with other

endogenous opioid ligands, the EMs have relatively longer half-lives under metabolic conditions,

but their profiles can still be improved upon. Moreover, it is important to avoid the unwanted

side effects caused by MOR agonist activity. Considering that the KOR is more likely related

with side effects caused by agonists at the MOR, it will be valuable to develop MOR/DOR

109

agonist and KOR antagonist ligands. For this purpose, the EM scaffold can be utilized with the

addition of KOR antagonist function to its MOR agonist function.

A series of EM-1 and -2 analogues were designed (Table 22). These analogues include a

Phe(4-Cl) residue and a Ppp moiety at the 4th position and the C-terminus, respectively, which

were shown to be important for KOR antagonist activity in our previous work. In an effort to

further improve potency at the ORs and evoke KOR antagonist functionality, Dmt was

substituted for the Tyr1 residue. Therefore, the SAR study can also investigate role of the 3rd

positioned residue for opioid activity along with the discovery of highly integrated

multifunctional opioid ligands. These modifications were expected to improve the metabolic

stability and the BBB permeability due to increased lipophilicity and resistance to enzymatic

degradation.

Table 22. Analytical data of EM analogues.

Analogue Structure HR MSa (M-TFA + H)+ HPLCb

Retention Time aLogPc

Observed Calculated

MR114 Dmt-Pro-Trp-Phe(4-Cl)-Ppp 888.42087 888.4216 26.0 5.20

MR115 Dmt-Pro-Phe-Phe(4-Cl)-Ppp 849.40945 849.4107 22.5 4.78

MR116 Dmt-Pro-Phe(4-Cl)-Phe(4-Cl)-Ppp 883.36932 883.3717 23.9 5.23

MR123 Dmt-Pro-Gly-Phe(4-Cl)-Ppp 759.36358 759.3636 23.0 3.68

aFAB-MS (JEOL HX110 sector instrument) or MALDI-TOF. bPerformed on a Hewlett

Packard 1100 [C-18, Vydac, 4.6 mm x 250 mm, 5 m, 10-100% of acetonitrile containing

0.1% TFA within 45 min, 1 mL/min]. chttp://www.vcclab.org/lab/alogps/.

110

4.7.3 Experimental

EM-1 and EM-2 analogues were synthesized using LPPS. Coupling reactions were

monitored by TLC using chloroform/methanol/acetic acid = 90:10:1 with ninhydrin spray being

used for detection. Analogues were synthesized by stepwise synthesis using an Nα-Boc

protecting group approach starting from Ppp (Figure 21). 1.0, 1.1, 1.2, 1.2, and 2.0 equivalents of

Ppp, the respective amino acid, HOBt, BOP, and NMM, respectively, were dissolved in DMF

and cooled in an ice bath for 30 mins and stirred at rt for 2 hours. After the disappearance of the

starting amine was observed via TLC, the reaction was diluted 10x with ethyl acetate and

extracted against equal volume of 5% NaHCO3 x3, 5% citric acid x2, brine x1, and water x1,

consecutively. The organic layer was then dried using anhydrous Na2SO4 or MgSO4 and filtered.

The resulting solution was then concentrated under reduced pressure and triturated with cold

diethyl ether or hexanes to yield a powdered precipitate. The Boc protecting group was

deprotected using 95% TFA/5% TIS in an ice bath for 30 mins and monitored by TLC. The

solution was then diluted with toluene and concentrated under reduced pressure and triturated

with cold diethyl ether or hexanes to yield a powdered precipitate. After the completion of the

sequence, the analogues were subjected to preparative RP-HPLC on a Hewlett-Packard 1100 (C-

18, Microsorb-MV, 10 mm, 250 mm, 10 μm) to afford >95% purity using a gradient of 10-90%

acetonitrile containing 0.1% TFA/water within 40 mins, 3 mL/min. Analytical HPLC was

performed on a Hewlett-Packard 1090 (C-18, Vydac, 4.6 mm, 250 mm, 5μm) at 1 mL/min and

validated using HR-MS.

111

4.7.4 Results and Discussion

A brief SAR based on the EM scaffold was generated (Table 23). All of the analogues

showed balanced binding affinities at the ORs, indicating that the modifications at the 4th

position and the C-terminus with chlorination and Ppp substitution, respectively, enhanced the

DOR and KOR affinities as expected. This result demonstrated that highly integrated

multifunctional opioid ligands could be developed using the EM scaffold. Compared to MR116

and MR123, MR114 and MR115 showed better affinities, and so it is considered that an

aromatic residue without a halogen substitution at position 3 is preferred as Gly substitution or

the addition of a chlorine resulted in slight losses of binding affinities. This may be due to an

electronic repelling effect with the chlorine and an acidic amino acid in the binding environment.

Table 23. Binding affinities of EM analogues at the MOR/DOR/KOR.

Ligand

Ki (nM)a

MOR

[3H]DAMGO

DOR

[3H]DADLE

KOR

[3H]U69593

EM-1 0.36b 1,500b 4,200b

EM-2 0.69b 9,200b >10,000b

MR114 9.6 15 16

MR115 3.9 11 5.5

MR116 37 49 21

MR123 21 59 23

aAssays were performed in Bryan Roth Laboratory at the University of North

Carolina at Chapel Hill by the NIMH PDSP program. bReference #58.

112

4.7.5 Summary and Future Directions

Overall, the employed modifications on the EM scaffold were successful as incorporation

of chlorine and attachment of the Ppp moiety appeared to have a dramatic change on binding

affinities as the parent compound went from a MOR selective ligand to a multifunctional ligand.

All ligands recovered KOR and DOR interactions which were greatly improved. Although

ligands at the MOR showed at least an order of magnitude decrease in binding affinity relative to

the EMs, the binding profiles shown are still good enough to justify running functional assays at

the ORs. Stability assays of lead ligands should also be performed to help quantify their

biological availability. Ligands that exhibit strong efficacies in the functional assays will then be

sent for in vivo studies. It would be interesting to see if, despite having similar affinities at the

ORs, halogenation evokes a different functional profile at the KOR as was observed in the ENK

series. It may be worthwhile to synthesize various aromatic residue containing analogues by

substituting the 3rd position with 1-Nal, cyclohexyl, and para-methylated Phe derivatives to

further assess whether it is a steric or electronic effect that is being observed with MR116 to

cause a slight loss of affinity at the ORs. Substitution of Phe3 with a Gly residue in MR123 was

tolerated at the ORs with slight loss of affinity. Nevertheless, a more comprehensive SAR studies

should be generated to further evaluate the effects of the employed modifications. Despite

conflicting studies owing to the EMs being full or partial agonists at the MOR, it would be

attractive to see the impact of these types of substitutions and alterations.

113

Appendix A. Publications

1. Yeon Sun Lee; Sara M. Hall; Cyf Ramos-Colon; Michael Remesic; Alexander Kuzmin;

David Rankin; Todd W. Vanderah; Frank Porreca; Josephine Lai; Victor J. Hruby “Amphipathic

Non-opioid Dynorphin A Analogs to Inhibit Neuroexcitatory Effects at Central Bradykinin

Receptors” Proceeding of the 24th American Peptide Symposium, 2015, doi:

10.17952/24APS.2015.

2. Yeon Sun Lee; Sara M. Hall; Cyf Ramos-Colon; Michael Remesic; David Rankin; Todd W.

Vanderah; Frank Porreca; Josephine Lai; Victor J. Hruby “Blockade of non-opioid excitatory

effects of spinal dynorphin A at bradykinin receptors” Receptors Clin Investig 2015, doi:

10.14800/rci.517.

3. Yeon Sun Lee; Michael Remesic; Cyf Ramos-Colon; Sara M. Hall; Alexander Kuzmin;

David Rankin; Frank Porreca; Josephine Lai; Victor J. Hruby “Cyclic non-opioid dynorphin A

analogues for the bradykinin receptors” Bioorganic. Med. Chem. 2016, 26(22), 5513-5516.

4. Michael Remesic; Yeon Sun Lee; Victor J. Hruby “Cyclic opioid peptides” Curr. Med.

Chem., 2016, 23(13), 1288-1303.

5. Yeon Sun Lee; Sara M. Hall, Cyf Ramos-Colon, Michael Remesic, Lindsay LeBaron, Ann

Nguyen, David Rankin, Frank Porreca, Josephine Lai, and Victor J. Hruby “Modification of

Amphipathic Non-opioid Dynorphin A Analogues for Rat Brain Bradykinin Receptors” Bioorg.

Med. Chem. Letts 2015, 25(1), 30-33.

6. Michael Remesic; Victor J. Hruby; Frank Porreca; Yeon Sun Lee “Recent Advances in the

Realm of Allosteric Modulators for Opioid Receptors for Future Therapeutics” ACS Chem.

Neurosci. 2017, 8(6), 1147-1158.

7. Cyf N. Ramos-Colon; Yeon Sun Lee; Michael Remesic; Sara M. Hall; Justin LaVigne; Peg

Davis; Alexander J. Sandweiss; Mary I. McIntosh; Jessica Hanson; Tally M. Largent-Milnes;

Todd W. Vanderah; John Streicher; Frank Porreca; and Victor J. Hruby. “Structure Activity

Relationships of [des-Arg7]- Dynorphin A Analogues at the Kappa Opioid Receptor” J. Med.

Chem. 2016, 59(22), 10291-10298.

8. Yeon Sun Lee, Robert Kupp, Michael V. Remesic, Cyf Ramos-Colon, Sara M. Hall,

Christopher Chan, David Rankin, Frank Porreca, Josephine Lai and Victor J. Hruby “Various

modifications of the amphipathic dynorphin A pharmacophore for rat brain bradykinin

receptors” Chemical Biology & Drug Design, 2016, DOI: 10.1111/cbdd.12789.

114

Appendix A.1

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Appendix A.2

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Appendix A.3

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Appendix A.4

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Appendix A.5

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Appendix A.6

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Appendix A.7

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Appendix A.8

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169

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