Novel Proteasome Inhibitors

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TECHNISCHE UNIVERSITT MNCHEN

DEPARTMENT CHEMIE UNIVERSITY OF SOUTHAMPTON

SCHOOL OF CHEMISTRY

REVERSIBLE INHIBITORS OF THE PROTEASOME

MChem Dissertation

Thomas A. Fleming

Supervisors:

Philipp Beck M.Sc.

Professor Dr Michael Groll

Dr Ali Tavassoli


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Abstract

In the development of next generation inhibitors of the proteasome, a target for the effective

treatment of certain cancers, a series of sulphonamide structures have been synthesised and their

anti-proteasomal activity assessed. The structural design of these compounds was guided bycrystallographic information from a fragment-based structure discovery experiment wherein a

sulphonamide fragment was found to bind selectively in the S3 pocket of the 2 (trypsin-like) active

site. This 2 subunit selectivity is currently, to the best of our knowledge, a unique finding. Structural

variation of these sulphonamides could exploit differences between the topography of the immuno-

(2i) and constitutive (2c) proteasome 2-S3 pocket, creating selective inhibitionfor the immuno-

or constitutive proteasome, thus enhancing target specificity. Their syntheses are explored and future

directions have been proposed.

A fragment merging approach to the development of a hydroxyurea compound was based on

the superposition of two structures found binding in a novel manner to a 6 S3 subpocket. An

adamantly hydroxyurea inhibitor and a derivative of the substructure Palauamine Phakellin of the

complex natural product proteasome inhibitor Palauamine were merged to create a structural hybrid

incorporating the key binding elements of both compounds. The findings and future directions are

discussed.


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Table of Contents

ABSTRACT I

ACKNOWLEDGEMENTS III

ABBREVIATIONS, ACRONYMS AND CONTRACTIONS VII

INTRODUCTION 1

THE UBIQUITIN-PROTEASOME SYSTEM 1PROTEASOME STRUCTURE,ASSEMBLY &NOMENCLATURE 4CONSEQUENCES OF PROTEASOME INHIBITION 8INHIBITORS OF THE PROTEASOME 10OVERVIEW 10PEPTIDE ALDEHYDES 13PEPTIDE BORONIC ACIDS 14-KETOALDEHYDES 15,-EPOXYKETONES 15-LACTONES 15BACTERIA SPECIFIC 16VINYLSULPHONES 16SYRBACTINS 16CYCLIC PEPTIDES 17CAPPED PEPTIDES 17NON-PEPTIDE 17SULPHONAMIDE INHIBITOR DEVELOPMENT [FRAGMENT GROWING] 19SULPHONAMIDE SYNTHESIS PLAN 21HYDROXYUREA INHIBITOR DEVELOPMENT [FRAGMENT MERGING] 25HYDROXYUREA SYNTHESIS PLAN 28

RESULTS & DISCUSSION 29

SULPHONAMIDES 29SYNTHESIS OF 2-(4-ETHOXYPHENYL)QUINOLINE-4-CARBOXYLIC ACID 29DIVERSITY IN EXTENSIONS TOWARDS THREONINE 1 29SYNTHESIS OF C3ALKYL SPACER SULPHONAMIDES 3 33SYNTHESIS OF AMINO ACID SULPHONAMIDES 7,10&13 35SYNTHESIS OF BORONIC ACID SULPHONAMIDES 4,18&20 37HYDROXYUREA 40HYDROXYUREA SYNTHESIS 40HEADGROUP SYNTHESIS 43

CONCLUSIONS & FUTURE WORK 45

SULPHONAMIDES PROJECT 45HYDROXYUREA PROJECT 46

EXPERIMENTAL DETAILS 49

GENERAL METHODS &MATERIALS 49MATERIALS 49SYNTHESIS 49QUALITATIVE THIN-LAYER CHROMATOGRAPHY 49CHROMATOGRAPHIC PURIFICATION 49NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY 50HIGH PERFORMANCE LIQUID CHROMATOGRAPHY -ELECTROSPRAY IONISATION MASS SPECTROMETRY 50ORGANIC SYNTHESIS PROCEDURES 51SULPHONAMIDE SYNTHESES 51HYDROXYUREA SYNTHESIS 76

REFERENCES 81

APPENDICES 85


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Abbreviations, Acronyms and Contractions

19S Regulatory particle, 19 Svedbergs

20S Core particle, 20 Svedbergs

26S Proteasome, 26 Svedbergs

Angstrom, 10-10metres

AAA ATPases Associated with diverse

cellular Activities

Ac Acyl

AMP Adenosine monophosphate

bar Unit of pressure, 105 Pascals

BAX Bcl-2-associated X protein

BCL2 B-cell lymphoma 2 gene

Bcl-2 B-cell lymphoma 2 protein

Bcl-xL Bcl-2-associated protein

Boc N-tert-butoxycarbonyl (protecting

group)

BrAAP Branched chain amino acid

preferring

BSA N,O-Bis(trimethylsilyl)acetamide

c Constitutive (e.g. cCP)

ChTL Chymotrypsin like

CL Caspase like

COSY Correlation spectroscopy

CP Core particle (20S)

CYP450 Cytochrome P450

d Doublet (spectroscopic)

Chemical shift/relative resonance

frequency

Da Dalton, unified atomic mass unit

DCM Dichloromethane

DEPT Distortionless enhancement by

polarisation transfer

DMF Dimethylformamide

E1,2,3 Ubiquitin activating, conjugating

and ligase enzymes of the UPS

eq. Stoichiometric equivalent

ER Endoplasmic reticulum

ESI Electrospray ionisation

Et Ethyl

FCC Flash column chromatography

FDA Food and Drug Administration

g Gram

HIF Hypoxia inducible factor

h Hour

hex Hexane

HPLC High pressure liquid

chromatography

Hz Hertz, unit of frequency, s-1

i Immuno (e.g. iCP)

IAP Inhibitor of apoptosis proteins

IC50 Concentration achieving 50%

inhibition

IB() Nuclear factor of kappa light

polypeptide gene enhancer in

B-cells inhibitor (alpha)

k Kilo, 103

Ki Dissociation constant

L Litre

LMP Low molecular mass protein

LPE Light petroleum ether (40-60C)

LQC Low quality control

Continued overleaf


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m Milli, 10-3; metre; multiplet;

murine

M Molarity,

Mcl1 Myeloid leukemia cell

differentiation protein

Mdm2 Mouse double minute 2 homolog;

E3 ubiquitin ligase

MECL Multicatalytic endopeptidase

complex-like

MHC Major histocompatibility complex

mol mol

MS Mass spectrometry

N Normality

n Nano, 10-9

NBC N-bromosuccinimide

NCS N-chlorosuccinimide

NF-B Nuclear factor kappa-light-chain-

enhancer of activated B cells

NMM N-methylmorpholine

NMR Nuclear magnetic resonance

Noxa From Latin, damage;pro-

apoptotic BCL-2 protein

Ntn N-terminal nucleophilic

P1,2,3 Peptide residue 1, 2, 3 (non-

primed)

P1,2,3 Peptide residue 1, 2, 3 (primed)

p27 Cyclin-dependent kinase inhibitor

1B

p53 Tumour suppressor protein 53

p Pentet (spectroscopic)

Pin Pinacolato (boronic acid

protecting group)

POP Phosphorous-oxygen-

phosphorous reagent

ppm Parts per million

q Quartet (spectroscopic)

Rf Retention factor

(chromatographic)

RMS Root mean squared

ROS Reactive oxygen species

RP Regulatory particle

Rpn Regulatory particle non-ATPase

protein

Rpt Regulatory particle triple A (AAA)

protein

Rt Retention time (chromatographic)

rt Room temperature (20C)

rtp Room temperature & pressure

(20C, 1 atmosphere)

S1,2,3 Specificity pocket 1, 2, 3 (non-

primed)

S1,2,3 Specificity pocket 1, 2, 3

(primed)

S Svedberg sedimentation

coefficient

s Singlet (spectroscopic)

SICLOPPS Split-intein cyclic ligation of

peptides and proteins

Smac Second Mitochondria-Derived

Activator of Caspases protein

SN2 Bimolecular nucleophilic

substitution

SnAAP Small, neutral amino acid

preferring

t Triplet (spectroscopic), thymo

(e.g. tCP)

TEA Triethylamine

TFA Trifluoroacetic acid

TL Trypsin like

TLC Thin-layer chromatography


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TMS Trimethylsilane (protecting

group), Tetramethylsilane (NMR

calibration shift)

UMP Ubiquitin maturation protein

UPR Unfolded protein response

UPS Ubiquitin-Proteasome system

UV Ultra-violet

y Yeast (e.g. yCP)


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Introduction

The proteasome is a large multi-subunit protease found in archaea and in some bacteria as

well as in the cytoplasm and nucleus of eukaryotes.1 The proteasome plays a central role in non-

lysosomal protein regulation and, thereby, cellular homeostasis in a system known as the Ubiquitin-

Proteasome System (UPS).2Proteins that are earmarked for degradation viacovalent attachment of

polyubiquitin chains are recognised by the proteasome, unfolded and translocated through the pore of

the barrel-shaped core particle (CP) where three proteolytic active sites reside.3The system is tightly

regulated and displays remarkable specificity towards its broad spectrum of protein substrates.3

Protein degradation via the UPS does not serve simply to destroy proteins at the end of their lifecycle.

It matures, activates and mediates proteins that are implicated in many basic cellular processes from

organelle biogenesis; differentiation, development and cell cycle progression to circadian rhythms;

inflammatory response and antigen processing; gene transcription and tumour suppression.4-7 The

Ubiquitin-Proteasome System is involved in the fundamental pathways of cell life and death health

or disease. With such broad and important function, it is no surprise that modulation of proteasomal

activity is an appealing target for medicinal chemists, but what may initially appear counter-intuitive

is that inhibition of such a central system could have a therapeutic window at all.8-10 Indeed, two

inhibitors of the proteasome are currently on market for the frontline treatment of haematological

cancers whilst others hold promise for the treatment of autoimmune disorders.11-13The majority of

proteasome inhibitors have an irreversible or slowly reversible mode of action, a consequence of the

reactive electrophilic head groups that are a common design principle.14-19This reactive mechanism

of inhibition produces dose-limiting side effects due to off-target activity and poor systemic

distribution as a result of their high bonding propensity.20, 21 Advanced generation proteasome

inhibitors of a readily-reversible nature will require fine structural optimisation to achieve sufficient

ligand stabilisation to compensate for the lack of an anchoring covalent bond. The development of

such inhibitors will be rewarded with improved pharmacological profiles, reduced side effects and

higher target selectivity.

The Ubiquitin-Proteasome System

The ubiquitin-proteasome system (UPS) is the sophisticated and highly-regulated process by

which cellular protein degradation is achieved. Proteins in the cell are diverse and perform a vast

range of functions. The simultaneous synthesis and destruction of such proteins is poised in a

dynamic equilibrium that responds to physiological conditions, thus maintaining cellular homeostasis.


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The half-lives of these various proteins vary from minutes, such as tumour suppressor p53 or

ornithine decarboxylase, to the lifetime of the organism, such as crystallin, a protein in the lens of the

eye.22Errors in translation, oxidative stress and other changes that may occur during the lifetime of

the protein all contribute to defective proteins that must be degraded. The process for protein

degradation is strictly controlled in order for proteins to coexist in the cytosol alongside the

destructive proteasome. Regulation ensures that hydrolysis occurs at the correct rate and at the

appropriate position. It is via the highly-specific ligation of ubiquitin tags to targeted proteins that

control of UPS-mediated degradation is achieved.23This pathway is outlined in figure 1, A.

Figure 1 Targeted protein degradation via the Ubiquitin-Proteasome System (UPS) A) 1) Ubiquitin activating enzyme E1 activatesubiquitin towards conjugation via adenylation, forming a high-energy thioester intermediate; 2) A cysteine thiol on E1 forms a high-energy thioester bond with the activated ubiquitin adenylate, releasing adenosine monophosphate (AMP); 3) The activated ubiquitinmoiety is transferred to ubiquitin conjugating enzyme (UBC) E2; 4) Protein substrates bind specifically to E3, ubiquitin-protein ligaseenzymes that, 5) directly or indirectly, catalyse the transfer of the ubiquitin moiety to the substrate forming covalent isopeptide linkagebetween a substrate -amino group and ubiquitin C-terminal carboxylate.24Several cycles produce polyubiquitination, for example atetraubiquitin unit (B ii)), which is the primary signal for protein degradation; 6) Recognition of and binding of the polyubiquitin unit tothe RP, unfolding of the substrate and substrate entry into the catalytic chamber; 7) Recycling of ubiquitin, release of oligopeptidedegradation products. B) i) Crystal structure of the ubiquitin protein; ii) Crystal structure of one form of tetraubiquitin;25 iii)Highlighted isopeptide bond in magenta formed between ubiquitin C-terminal carboxylate and -amino group of protein substratelysine residue compared with standard peptide bonds in green involving -amino groups

The three enzyme groups, E1, 2 &3, that participate in the targeted ligation of ubiquitin (figure

B, i) to protein substrates operate in a hierarchal manner.2At the tip of the hierarchy, there exists only

a few E1 ubiquitin activating isozymes that are responsible for the adenylation of ubiquitin. These E1


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enzymes interact with all E2 ubiquitin conjugating enzymes, in which there is greater isozymatic

diversity.26 In turn, most E2 ubiquitin conjugating enzymes interact with several of the even more

numerous and diverse E3 ubiquitin-protein ligases.27The E3 enzymes themselves are capable of the

specific recognition of several substrates. The overlapping and complex combinatronics of these

enzymatic interactions directs protein degradation with great sophistication and specificity. Once a

protein is tagged with ubiquitin, it is recognised by the proteasome as a substrate for degradation.


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Proteasome Structure, Assembly & Nomenclature

The protein units of the proteasome are regularly referred to by their Svedberg sedimentation

coefficients (S). The entire proteasome complex is the 26S proteasome (figure 1, A). The 20S core

particle (figure 2), a 700 kDa multi-subunit cylindrical structure, with a 13 diameter pore (alphaannulus) through the centre that accommodates the proteolytic active sites is capped at either end by

700 kDa, 19S regulatory particles (RPs) (figure 1, A).

The 20S core particle comprises four rings, each with 7 homologous subunits creating an

overall subunit stoichiometry of 1-71-71-71-7 (figure 2).

Figure 2 Subunit organisation of the eukaryotic 20S core particle; proteolytic subunits labelled in red: a) Exploded view of the four rings- , , & ; b) End-on view of the CP; c) Side-on view of the CP; d) Ribbon diagram representation of eukaryotic Saccharomyces

cerevisiaeCP crystal structure.

Archaebacterial proteasome core particles are composed of 14 identical - and 14 identical -

subunits. This simple subunit composition permits autonomous assembly. However, eukaryotic core

particles are constructed from related, but subtly different (homologous) and subunits, 1-7 and

1-7. This additional complexity is reflected in the requirement for a more sophisticated assembly

mechanism involving proteasome-assembling chaperones28and maturation factors such as ubiquitin

maturation protein 1 (UMP1).29All catalytically active subunits (1, 2, & 5) include an N-terminal

threonine (Thr1), the nucleophilic oxygen atom (Thr1O) of which is responsible for the initiation of


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proteolysis, thus classifying the proteasome as an N-terminal nucleophilic hydrolase (Ntn).30, 31

Whereas all archaebacterial subunits are catalytically active, eukaryotic core particles contain only

three active subunits: 1, 2, & 5.32 In vertebrates, three distinct forms of CP have evolved: the

constitutive; thymo- and immunoproteasome, each distinguished by the active subunits they

incorporate.

The somatic distribution of the constitutive proteasome (cCP) is near ubiquitous, whilst

immunoproteasome (iCP) and thymoproteasome (tCP) are predominantly found in mono- and

lymphocytes and in cortical thymic epithelial cells, respectively.33, 34 In the immune response,

cytotoxic T cells are activated to destroy cells whose major histocompatibility complex class I (MHC-I)

proteins extracellularly present peptides of foreign bacterial or viral origin (antigens).33The activated

cytotoxic T cell releases cytokines into the local tissue, such as necrosis factor- and interferon-.These shift the predominant proteasome subtype to the immunoproteasome by inducing the

expression of the three catalytically active immuno subunits: 1i (LMP2), 2i (MECL-1) and 5i

(LMP7) that are incorporated into the de novo production of proteasomes (table 1).35 Variation in

subunit composition produces altered cleavage specificities. Immunoproteasome subunits

preferentially cleave the peptide amide post non-polar amino acids and the hydrophobic C-termini of

the subsequent degradation products are ideal for docking in complementary hydrophobic grooves of

the MHC-I proteins.36, 37

The role of the immunoproteasome in the processing of peptides for antigenpresentation and in the production of cytokines would suggest selective immunoproteasome

inhibition may offer clinical benefit in autoimmune and inflammatory disorders and this has been

verified in mouse models of such maladies.38-40

Table 1 Four eukaryotic core particles (CP) and their constituent active subunits. The CP of yeast: (yCP), the three vertebral CPclasses: constitutive proteasome CP (cCP); immunoproteasome CP (iCP) and the thymoproteasome CP (tCP).

Core particle

(CP) class

Incorporated subunit | Activity descriptor

1 2 5

yCP y1 CL y2 TL y5 ChTL

cCP 1c CL 2c TL 5c SnAAP

iCP 1i BrAAP 2i TL 5i ChTL

tCP 1i CL 2i TL 5t ChTL

1 subunits preferentially cleave peptide bonds succeeding acidic side chains, earning the

activity descriptor of caspase-like (CL).411i has a smaller active site then other 1 subunits, altering


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the cleavage preference towards a branched chain amino acid preferring (BrAAP) activity.41 2

subunits preferentially cleave peptides succeeding basic side chains, earning the descriptor of trypsin-

like (TL),41 although a larger substrate binding pocket broadens its substrate profile. The apolar

environment of 5 subunit active site produces preferential cleavage of peptide bonds succeeding

hydrophobic residues and is therefore termed chymotrypsin-like (ChTL).41

5c forms a smaller activesite that modifies its substrate preference towards an elastase-like or small neutral amino acid

preferring (SnAAP) activity.42Unfolded proteins are passed through the alpha annulus towards the

catalytic centres and are stabilised by forming an ant-parallel -pleated sheet with the substrate

binding channel and through interactions with the peptide amino acid residues protruding into

recesses known as specificity pockets (figure 3).

Figure 3 Schematic representation of substrate binding channels within subunits; positioning of the active site and the nucleophilicthreonine 1 relative to the scissile peptide bond of the substrate. S denotes Specificity pockets, numbered ascendingly with increasingdistance from the active site. Peptide residues are numbered according to the same pattern

The primed notation indicates position in relation to the three proteolytic active sites of

subunits 1, 2, & 5. Substrate residues (P1, P2, P3, ...Pn and P1, P2, P3, Pn) interact with

corresponding specificity pockets (S1, S2, S3, ...Sn and S1, S2, S3, Sn) in the substrate binding

channel, located within the alpha annulus (figure 3).43The N-terminal threonine (Thr1) of the active

subunits bears the nucleophilic oxygen atom (Thr1O) that initiated cleavage of peptide substrates.

Peptide cleavage is processive and produces oligopeptides with a length distribution between three to

twenty five amino acid monomers, placing the proteasome amongst the endopeptidases. A general

mechanism of peptide cleavage is shown in figure 4.


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Figure 4 Mechanism of substrate hydrolysis by the N-terminal nucleophilic hydrolase subunits of the proteasome CP: A) Thr1Nassisted deprotonation of Thr1Oinitiates nucleophilic attack at peptide carbonyl carbon atom resulting in a tetrahedral intermediate,44stabilised by the oxyanion hole; B) Collapse of the intermediate affords the first peptide degradation product and produces an acyl-enzyme intermediate; C)the ester is attacked by a water molecule, cleaving the second degradation product from the enzyme and D)regenerating the active site with the assistance of a defined cluster of water molecules that act as proton shuttle between Thr1OandThr1N.45, 46

The internal catalytic sites are protected from the external cellular environment by the outer

structure of the core particle, thus preventing uncontrolled peptide hydrolysis. Entrance to the alpha

annulus is guarded by the 19S RPs. In the absence of the 19S RPs, the pore is shielded by the tailing N-

termini of the alpha subunits. Upon association of the 19S RP, a conformational change is induced,

exposing the alpha annulus.

The eukaryotic 19S RP is itself a multi-subunit complex that can be grouped into two structural

features: a base and a lid.47The base forms a contact with the 20S CP and comprises a hexameric ring

of AAA (ATPases associated with various cellular activities) type ATPase subunits referred to by

their common notation as regulatory particle triple A proteins (Rpt) 1-6 and by three regulatory

particle non-ATPase proteins (Rpn)1, 3 & 13. The lid of the 19S RP is connected to the base viaRpn 10

and consists of nine Rpn subunits: 3, 5-9, 11, 12 & 15.

The 19S RP serves to recognise (poly)ubiquitinated protein substrates, to unfold,

deubiquitinate and translocate the protein substrate through the alpha annulus to the hydrolytically

active core of the proteasome.48

O NH2

OHO NH3

O

HN

P1

HN

O P1'

NH

O

HN

P1

HN

O P1'

NH

O

O NH2

O

HN

P1

NHO

P1'

H2N

OO

H H

O NH2

OHP1

HN

O

OH

A) B)

C) D)

Peptide

substrate

Product 1 Product 2

Thr1


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Consequences of Proteasome Inhibition

Considering the numerous important cellular processes in which proteasome substrates and

products are involved, such as cell cycle regulation, it is perhaps surprising that inhibition of this keyenzyme has a therapeutic window at all.49Indeed, the therapeutic dose of bortezomib is 1.3 mg m -2,

but administration of 1.5 mg m-2 produced dose-limiting toxicity in phase I clinical studies.50 This

demonstrates the subtlety required in the development of proteasome inhibitors and their

administration regimes. The selective targeting of malignant cells exploits small differences in cell

behaviour, such as rate of division and chromosomal stability.8

During early exploration of proteasome inhibitor potential as anti-inflammatory agents, rapidand selective proteasome inhibitor-induced apoptosis of transformed cells from cultures of different

cancers redirected the clinical focus towards their antineoplastic potential. Malignant cells that divide

more rapidly than healthy cells are affected more by proteasome inhibition-based interference in cell

cycle progression. Normally, a regulatory protein, cyclin, must be removed by the UPS before the cell

may enter anaphase.51Prevention of cyclin degradation thereby prevents cell proliferation, inducing

cell cycle arrest. The chromosomal instability of malignant transformed cells manifests as

uncontrolled protein synthesis. The proteasome is also responsible for the clearing and recycling ofmisfolded proteins that form the endoplasmic reticulum (ER).52The accumulation of proteins leads to

ER stress and impair ER function.53, 54 Disruption of normal ER function causes apoptosis since it

prevents the production of correctly-folded proteins, causing ER stress and inducing the unfolded

protein response (UPR).55-57 Proteasome inhibitors also appear to interfere with mitochondria,

resulting in the generation of reactive oxygen species (ROS), changes in mitochondrial membrane

potential, the release of cytochrome C into the cytosol and activation of apoptosis-related cysteine

peptidase, caspase 8 and pro-caspase 9.58

Bortezomib is known to prevent IB degradation by theproteasome leading to accumulation of IB. IB is an inhibitory protein of NF-B, a factor controlling

transcription of anti-apoptotic target genes. When the proteasome is inhibited, IB accumulates and

NF-B is thereby inactivated and its target genes are not transcribed. 59

Tumour suppressor p53 is earmarked for proteasomal degradation by Mdm2, an E3 ligase.

Proteasome inhibition therefore leads to raised levels of p53. Tumour suppressor p53 has anticancer

effects that are mediated through many mechanisms, particularly in the initiation of cell cycle arrest

or apoptosis. For example, p53 binds DNA and promotes the expression of many genes. One such

protein whose expression is activated by p53 is a pro-apoptotic protein of the BCL-2 family called


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Noxa. BCL-2 proteins regulate apoptosis; heterodimerisation of pro-apoptotic Noxa with anti-

apoptotic BCL-2 proteins, such as Bcl-xL and Bcl-2 silences the anti-apoptotic signal and leads to

apoptotic cell death in malignant cells. In addition to raised levels of the tumour suppressor protein

p53, other pro-apoptotic proteins such as p27, Bcl-2-associated X protein (BAX) and Second

Mitochondria-derived Activator of Caspases (Smac) are also more abundant under proteasome

inhibition, whilst levels of anti-apoptotic proteins such as hypoxia-inducible factor (HIF1), inhibitor

of apoptosis (IAP) and myeloid leukemia cell differentiation protein (Mcl1) are decreased.

The successful positioning of proteasome inhibitors against multiple myeloma and other

haematological disorders has basis in their ability to access the malignant cells.


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Inhibitors of the Proteasome

Overview

Proteasome inhibitors, although structurally diverse, can be easily categorised by mode ofaction at the proteasome active sites i.e. covalent or non-covalent interaction with Thr1O. All non-

covalent inhibitors are reversible (cyclic peptides, capped peptides and some non-peptides) as are

selected covalent inhibitors (aldehydes, -keto-oxadiazoles, -keto-aldehydes, -lactones and boronic

acids). Covalent, irreversible inhibitors are dominated by the bivalently reacting ,-epoxyketone

structural class. The design principle common to many proteasome inhibitors consists of a peptide

backbone attached to an electrophilic head group. The reasoning is that the proteasome recognises

the peptidic structure as a potential substrate, whilst the electrophilic moiety intercepts for thenucleophilic attack from Thr1.


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Figure 5 Inhibitors of the proteasome organised by structural family and colour coordinated according to binding mode. In blue -REVERSIBLE COVALENT: aldehydes, -ketoaldehydes, boronic acids, -lactones and -keto-oxadiazoles; in green NON-COVALENT:

Non-peptides, capped peptides and cyclic peptides; in red IRREVERSIBLE: ,-epoxyketones, bacteria specific and syrbactins.

Proteasome inhibitors encompass a wide range of molecular structures (figure 5) with varied

mechanisms of inhibition (figure 6). It is noteworthy that many natural product and inhibitors of

bacterial origin exist. That natural potent inhibitors of exquisite mechanism have evolved vindicates

the proteasome as a target of real biological consequence.


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Figure 6 Mechanisms of covalent proteasome inhibition: Blue REVERSIBLE COVALENT: Aldehydes, boronic acids, -ketoaldehydes

and -lactones; Red IRREVERSIBLE COVALENT: Marizomib (unique -lactone), ,-epoxyketones, oxathiazol-2-ones, syrbactins andvinylsulphones; Colour coding represents: inhibitor structures in dark green, proteasome structures in black, new bonds formed inmagenta. Non-covalent inhibitors are not shown as they interact purely via electrostatics, Van der Waals forces, hydrophobic andentropic stabilisations.


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Finding the next therapeutically effective proteasome inhibitor is a tantalising prospect for

industry and academia. Clinical trials of proteasome inhibitors in mono and combination therapy,

various dosing regimes and against numerous malignancies are performed continuously. A summary

of current clinical developments is shown in table 2.

Table 2 Proteasome inhibitors in clinical development as anticancer drugs

ProteasomeInhibitorDeveloper

StructureStructural

ClassType

InhibitionProfile

Route ofadministratio

n

Types of cancerstreated

Clinicalstatus

BortezomibMillenium

Pharmaceuticals

Boronic acidReversibleCovalent

ChTL, CL,iCP

IntravenousSubcutaneous

Multiple myeloma,recurrent multiple

myeloma and mantlecell lymphoma.

Approved

CarfilzomibOnyx

Pharmaceuticals

,-epoxyketone

Irreversible ChTL, iCP Intravenous

Advanced multiplemyeloma,

monotherapy

Approved

Recurrent multiplemyeloma, non-

Hodgkins lymphoma

and solid tumours

Phase III

OprozomibOnyx

Phamaceuticals

,-epoxyketone

Irreversible ChTL Intravenous

Haematologicalmalignancies - mono

and combinationtherapy, solid

tumours -monotherapy

Phase II

Marizomib-lactone--

lactamIrreversible

ChTL, CL, TL,iCP

IntravenousRecurrent multiple

myeloma, solid

tumours, lymphomas,leukaemias

Phase II

CEP-18770Cephalon


ChTLIntravenous

oral

Recurrent multiplemultiple myeloma,

advanced stage solidtumours,

lymphoblasticleukaemia, non-

Hodgkins lymphoma

Phase II

MLN-9708Millenium

Pharmaceuticals


ChTLIntravenous

oral

Lymphoma and solidtumours Phase III

Peptide Aldehydes

The earliest investigated proteasome inhibitors were of the aldehyde family. Although none

have had clinical success, they are simple to synthesise and represent useful tools for the research

scientist.60 The peptidic component of peptide aldehyde structures, as with most other peptide-

mimicking inhibitors, forms an anti-parallel -pleated sheet with the substrate binding channel and

must be at least a dipeptide to achieve sufficient stabilisation.32, 44 The aldehyde group is then

orientated identically to a normal peptide substrate carbonyl group and nucleophilic attack by Thr1O


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proceeds as normal at the aldehyde carbonyl carbon atom. Subsequent formation of the hemiacetal

adduct is rapidly reversible and the inhibitors dissociate rapidly (figure 6). Additionally, in vivo they

are subject to deactivation viaCYP450 oxidation to their corresponding carboxylic acids.61

For the reasons just mentioned, the aldehyde family are poorly suited to the clinical setting, but

as research tools they remain useful for investigating the in vitro activities of subunits and as

uncomplicated standards against which new inhibitors can be compared.

Peptide Boronic Acids

Peptide boronic acids (or peptide boronates) are a class of proteasome inhibitors exemplified

by the first FDA-approved proteasome inhibitor, by bortezomib (generic) or Velcade (proprietary).

Generally, these covalent reversible inhibitors form boronate-proteasome adducts with slower

dissociation rates than directly comparable aldehyde-proteasome adducts.62 Taking a peptide

backbone (Z-Leu-Leu-Leu-[headgroup]) and replacing the aldehyde headgroup (MG132, figure 5) with

a boronic acid headgroup (MG262) produces 100-fold greater inhibition of the ChTL activity of rabbit

proteasome with a Kiof 30 nM.16Boronic acids offer enhanced selectivity for and potency against the

proteasome relative to their aldehyde counterparts due to the binding mechanism which creates a

hydrogen bond with the N-terminal threonine nucleophile, Thr1O.63Additional stabilisation unique

to the boronic acids is the second hydrogen bonding interaction between the second boronic acid

hydroxy group and Gly47NH (figure 6). The hard electrophilic boron atom also reacts preferentially

with the hard nucleophilic character of the proteasome Thr1 oxygen atom over the softer

nucleophilic character of cysteine protease sulphur atoms in accordance with Pearsons Acid Base

Concept.64

The first approved proteasome inhibitor, bortezomib, is still in clinical use today. In many

aspects, bortezomib is a success. Bortezomib achieves selective toxicity towards tumour cells, the

primary criterion for a cancer drug. It has been shown to have efficacy in patients who have not

responded to several established therapies.65Additionally, bortezomib operates synergistically with

other chemotherapy agents and radiation treatments and its inclusion in treatment regimes improves

patient outcomes such as overall response rate and time to progression.66Unfortunately bortezomib

produces unpleasant side effects; required intravenous or subcutaneous administration; is hindered

by the development of resistance and is completely ineffective against solid tumours. 67 These


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shortcomings must be addressed if the full potential of proteasome inhibitors as anticancer drugs is to

be realised.

-Ketoaldehydes

When this family of compounds was discovered, initially little interest was raised as it was not

immediately apparent that there was anything advantageous about them.68 However, X-ray

crystallographic elucidation of their exact binding mode revealed something very interesting.69The -

ketoaldehyde group reacts with Thr1Obivalently forming an oxazine ring, an adduct accessible only

upon reaction with Ntn proteases (figure 6). This selective interaction with the proteasome is

reflected in a 1000-fold greater potency of proteasome inhibition over trypsin or chymotrypsin.68

,-Epoxyketones

This structural class of inhibitors are the most specific proteasome inhibitors currently known.

Highly active natural product proteasome inhibitors epoxy- and eponomycin with ,-epoxyketone

functionality were shown to react with the proteasome active site to form a morpholine ring between

Thr1 and the epoxyketone group (figure 6).70-72 Hundreds of synthetic peptide epoxyketones have

since been synthesised and one, carfilzomib (generic) or Kyprolis (proprietary, Onyx

Pharmaceuticals, Inc), a tetrapeptide, has been approved for advanced multiple myeloma

monotherapy. Two other epoxyketone inhibitors ONX-0912 (generic) or Oprozomib (proprietary,

Onyx) and ONX-0914 (previously PR-957, Onyx) are in phase I clinical trials for haematological

malignancies. Both are tripeptides and the former, ONX-0912 is remarkably bioavailable; the latter a

highly selective inhibitor for the immunoproteasome.42, 73

-lactones

The -lactone family of proteasome inhibitors originated as natural products from the

microbial world and many synthetic compounds have since been derived, such as the natural product,

omuralide (clastolacocystin--lactone) and its synthetic analogue, PS-519.74, 75 This group of

compounds inhibit the proteasome by covalently inactivating the active site, forming a hydrolytically

reversible ester bond with Thr1O(figure 6).76


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Of the many -lactone proteasome inhibitors known, by far the most potent is marizomib (NPI-

0052, Salinosporamide A). Marizomib is a natural product of the marine microorganism Salinospora

tropicathat has developed an additional level of complexity to its secreted chemical weapon. The P2

residue that projects from the -lactam ring is a seemingly unimportant chloroethyl group. Upon

marizomib:CP adduct formation, the structure is perfectly aligned to undergo an intramolecular S N2,

assisted initially by a deprotonation by the N-terminal amino group (figure 6). The reaction forms a

tetrahydrofuran ring that stabilises marizomib binding but, more importantly, that displaces the

water molecule from the active site normally involved in ester cleavage thereby explaining the

irreversible nature of marizomibs inhibition.77-79Indeed, this impressive product of nature could also

produce cytotoxicity in Salinospora tropica itself, except that within the same gene that encodes the

marizomib biosynthesis operon, lies the code for a marizomib-resistant proteasome subunit fifty

times less sensitive than the normal subunits encoded elsewhere in the microorganisms genome.80

Bacteria Specific

The oxathiazol-2-one compound series inhibit mycobacterial but not human proteasomes and

therefore offer clinical benefit from a different angle.20 The foundation of this bacteria-specific

inhibition is structural differences between the bacterial and human proteasome. In humans, the

residues that are critical for inducing and stabilising large conformational changes caused upon

oxathiazol-2-one binding in the bacterial proteasome are different. Oxathiazol-2-one proteasome

inhibitors are the second class of drug capable of effectively killing non-replicating bacteria.

Proteasome inhibitors also display antimalarial and trypanocidal activity, but gaining selectivity for

the proteasomes of these lower eukaryotes is yet to be achieved.81, 82

Vinylsulphones

Vinylsulphones have mainly been used to explore the effect of varying amino acid residues in

the backbone of peptide epoxyketone inhibitors since they are easier to synthesise, allowing for

simpler fine-tuning of the peptide backbone without requiring synthesis of the epoxyketone

headgroup.83The vinylsulphones inhibit irreversibly viaMichael addition of Thr1O(figure 6).84

Syrbactins


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17

Of the syrbactin class, glidobactin A was first observed to display anti-tumour activity in 1988,

but the significance of its structure and its cellular target were not realised until structural similarities

with syringolin A were recognised two decades later. Syrbactins is the unifying term for 12-

membered lactam-peptide structures of natural product origin, encompassing glidobactins,

syringolins and cepafungins.85 The mechanism of proteasome inhibition is common amongst all

syrbactins as it is their shared lactam core that participates in irreversible Michael addition with Thr1,

analogous to the vinyl sulphone inhibition mechanism (figure 6).18

Cyclic Peptides

Cyclic peptides bind through non-covalent interactions to the proteasome active sites, blocking

substrate access.86 Cyclic peptides are stabilised in the active site by multiple hydrogen bondinginteractions. Natural products have populated the majority of this structural class, but synthetic

structural derivatives have also been prepared. Combination of chemical biology techniques such as

SICLOPPS peptide library formation and reverse two-hybrid active compound identification enables

rapid discovery of new active cyclic peptides.87 Cyclic peptides are extremely resistant to digestive

proteases due to the steric shielding of their cyclic form, offering promise for orally bioavailable

inhibitor development.88 Another benefit of their constrained form is the reduction in entropic

penalty that occurs upon binding.86, 89 Achieving low nanomolar Ki values, this category of inhibitordemonstrates that a reactive headgroup is not the sole source of potency.90

Capped Peptides

An alternative to cyclic peptides are linear synthetic peptides and peptide bioisosteres that

mimic natural proteasome peptide substrates. Tuning the peptide sequence and the possibility ofincorporation of unnatural amino acids allows for the fine modifications that result in subunit

selectivity and high affinities.91Tripeptides allow for P1, 2 and 3 residues to be selected that project

into and complement S1, 2 and 3 pockets of the desired subunit. The S4 is less pocket like and

shallower; bulky P4 residues project mostly towards the open binding channel rather than into a

proteasome pocket contributing little towards selectivity or binding affinity, producing unspecific

interactions.92

Non-peptide


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Non-peptidic inhibitors offer advantages over the more common peptide-like proteasome

inhibitor format, such as increased bioavailability due to increased biological and chemical stability.

Foreign peptidic structures are also more likely to have undesirable immunogenic properties.

Members of this inhibitor class may be discovered through in-vitroor in-silicoscreening of compound

and/or fragment libraries. A worryingly large number of inhibitors are reported in literature that

have no crystallographic data to confirm their mode of action and frequently, these are later shown to

have been false positive readings when the crystal structure is elucidated. One must be cautious when

reviewing such literature and use common sense when considering if a proposed inhibitor, such as a

large porphyrin ring, could even enter the alpha annulus of the proteasome.93


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Sulphonamide Inhibitor Development [Fragment Growing]

The sulphonamide inhibitor development project was a process of fragment growing. The lead

structure was selected from a 2009 in silico and in vitro compound screening (Basse et al.).94 Until

now, all known inhibitors bind to the non-primed substrate binding channel of the proteasome.Therefore the computational modelling used in this screening only included the non-primed substrate

binding channel and made predictions that this sulphonamide fragment would dock there. However,

the crystal structure of the fragment in complex with yeast proteasome reveals a novel binding mode.

As depicted in figure 7, the fragment sits entirely and only in the primed region of the 2 subunit. Of

particular interest is the observation that quinoline ring system protrudes deep into the S3 pocket.

Figure 7 Representations of the spatial arrangement of the original, acyl-capped sulphonamide fragment, binding in 2-S3 site: A)

simplified binding schematic; B) sulphonamide fragment surrounded by stick representations of surrounding residues within 5 proximity, including selected polar contact measurements; C)electrostatic surface mesh diagram of residues within 5 proximity; 2 isrepresented with blue, 1in magenta


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The positioning of the fragment suggests that extension of the sulphonamide structure beyond

the acyl cap could achieve interactions with the nucleophilic Thr1O. Additionally, modifications made

to the quinoline ring could exploit topographical differences between the immuno and constitutive

proteasomes since they differ significantly at the S3 specificity pocket in which the quinoline ring

resides (figure 8).

Figure 8 Schematic representations of three perspective highlighting the topographical differences between the cCP (tan, left-handside) and iCP (blue, right-hand side) 2-S3 pocket. The residues of the iCP that come together to form a much smaller 2-S3 pocketthan in the cCP. Exploiting this difference by synthesising a sulphonamide with a smaller heterocycle in place of the quinoline ring couldgenerate selectivity between the inhibition of the immuno or constitutive proteasomes. The schematic representation was based on thesuperposition of murine iCP and cCP crystal structures.

The significance of binding in the primed site is that there are greater differences in the

topographies of the primed regions of the proteasome amongst subtypes than in the non-primed. The

S3 specificity pocket is composed of different amino acid residues in the immunoproteasome core

particle, forming a smaller pocket with subtly altered electronic character. Using structurally guided

drug design, a synthesis plan was formed in an attempt to convert the inactive sulphonamide


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Scheme 1 Synthesis plan for C3 alkyl spacer sulphonamide: i) sulphonamide coupling; ii) palladium catalysed nitro reduction; iii) amidecoupling viaactivated carboxylic acid; iv) borylation; installation of pinocolato-protected boronic acid

In the next series of syntheses (scheme 2), amino acid methyl esters were incorporated to

experiment with various linker structures. The chosen amino acids were simple and sterically

unhindered: Ala-(L), Ala-(D) and Gly in order to suit the narrow channel in which they were expected

to bind. Their synthesis followed paths analogous to the C3 alkyl spacer sulphonamide synthesis,

excluding the borylation, since the boronic acid head group was not a feature of these structures.

Scheme 2 Synthesis plan for amino acid sulphonamides: i) sulphonamide coupling; ii) Palladium catalysed nitro reduction; iii) amidecoupling viaactivated carboxylic acid

Early inclusion of a bromine atom (in place of a chlorine atom) into the C3 alkyl spacer

sulphonamide structure was performed in order to assist in the final borylation step that is more

amenable to the larger halogen, bromine which is is more polarisable and a better leaving group(scheme 3). In parallel, compound 15 was used to create variation of the heterocyclic body of the

sulphonamide structures. Smaller isonicotinic acid derivatives were incorporated in place of the

S

O

OCl

N

O

O

S

O

ONH

N

O

O

Cl

S

O

ONH

NH2

Cl

S

O

ONH

HN

Cl

N

OO S

O

ONH

HN

B

N

OO

O

O

1 2

3 4

i) ii)

iii) iv)

S

O

OCl

N

O

O

S

O

ONH

N

O

O

S

O

ONH

NH2

S

O

O

HN

N

OO

6 , 9 , 1 2

7, 10 , 13

i) ii)

iii)

O

O

O

O

O

O

NH

R

R R

5, 8, 11

= (R)-Me = 8, 9, 10 (D-Ala)

= H = 11, 12, 13 (Gly)

R =(S)-Me = 5, 6, 7 (L-Ala)


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original bulky quinoline ring. 2-methoxyisonicotinic acid and 2-chloro-6-methoxyisonicotinic acids

were incorporated into the synthesis as these heterocycles retain the nitrogen atom in the 1-position,

a stabilising aspect of the original sulphonamide.

Scheme 3 Gaining selectivity for iCP/cCP. Synthetic approach to C3 alkyl spacer sulphonamides with alternative heterocyclic bodies (18& 20) and incorporation of bromine atom (16) in place of chlorine atom (3) to assist borylation i) sulphonamide bond formation viacoupling of bromoalkylamine and sulphonyl chloride, ii) palladium catalysed reduction of nitro group to amino group, iii) amidecoupling of acid chloride activated carboxylic acid derivatives of heterocyclic body structures with previously produced amine, iv)borylation to install pinocolato protected boronic acid

Experimentation with a synthetic approach to achieve rapid diversification of threonine

extension groups was performed. This divergent synthetic route allowed the majority of the target

structure to be prepared in bulk and late diversification to occur with minimal synthetic steps

(scheme 4).

S

O

ON

H

HN

Br

N

OO

16

S

O

OCl

N

O

O

S

O

ONH

N

O

O

Br

S

O

ONH

NH2

Br

S

O

ONH

HN

Br

i) ii)

iii)

N

O

O

Cl

S

O

ONH

HN

O

N

O

14 15

17Br

S

O

ONH

HN

N

O

O

Cl

B

O

O

19

S

O

ONH

HN

O

N

O

B

O

O

20

18

4iv)

iv)

iv)


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Scheme 4 Extending towards Thr1O. Divergent synthetic approach for rapid and efficient incorporation of R groups: i) amide coupling

viaactivated carboxylic acid to sulphanillic acid, followed by ii) Sulphonamide coupling viasulphonyl chloride activated sulphonic acidor direct coupling method with desired R-NH2

An attempt to install a C2 alkyl spacer was made (scheme 5). This was prevented by a

significant intramolecular SN2 degradation reaction an unavoidable consequence of the structural

nature of the compound. The planned synthetic route is shown below; the encountered problems are

discussed in more depth in Results & Discussion.

Scheme 5 Synthetic approach for the synthesis of C2 alkyl spacer sulphonamide: i) Sulphonamide coupling between sulphonyl chlorideand 2-bromoethylamine; ii) Reduction of nitro group to amino group viaPalladium catalysis; iii) Amide coupling between acid chloride

activated carboxylic acid and amine 23; iv) Borylation to install pinocolato protected boronic acid

S

O

HO

HN

N

OO

N

O

OHO

O

i)

21

S

O

ONH

HN

R

N

OO

ii)

S

O

ONH

NH2

BrS

O

ONH

N

O

O

Br

S

O

ONH

HN

Br

N

OO

S

O

ONH

HN

B

N

OO

O

O

22 23

24

25

S

O

OCl

N

O

O

i) ii)

iii) iv)


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Hydroxyurea Inhibitor Development [Fragment Merging]

In a separate project, a novel hydroxyurea inhibitor was designed based on a hybrid of two

molecules found binding in the same pocket of the proteasome CP. One molecule is from a series of

non-peptidic, non-covalent N-hydroxyurea inhibitors of the proteasome that were recently developedby Gallestegui et al. One of the compounds is a known lipoxygenase inhibitor and studies have shown

it to have advantageous pharmacological properties, such as cell accessibility and good clearance rate,

a promising outlook for a new structural class.95The hydroxyurea inhibitors were found to bind in a

novel fashion, sitting in subpocketsS1suband S3subadjacent to specificity pockets 5 S1 and 6 S3,

respectively, figure 10.

Figure 10 Hydroxyurea inhibitor HU8 displaying novel binding mode in S3 and S1 subpockets. A) Schematic of amino acid spatialarrangement forming subpockets; B) sulphonamide fragment surrounded by stick representations of surrounding residues within 5 proximity, including selected polar contact measurements; C) electrostatic surface mesh diagram of residues within 5 proximity;selected polar contact measurements in Angstroms. 6 is represented with blue, 5in magenta


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Numerous hydrogen bonding interactions between the hydroxyurea functional group and the

substrate binding channel indicate that the hydroxyurea headgroup is likely a key binding element.

The second molecule is an analogue of the phakellin substructure of a highly complex oroidin

alkaloid marine sponge secondary metabolite, Palauamine.96 Palauamine is itself a proteasome

inhibitor, but its structural complexity precludes any clinical application.97The Phakellin substructure

is more synthetically accessible and has been shown to retain inhibitory activity.98, 99An analogue of

the Phakellin substructure was also found to bind in the 5 S1 and 6 S3 subpockets, figure 11.

Figure 11 Three representations of the phakellin derivative binding in the 20S CP 5-S1, 6-S3 subpockets: A)Spatial organisation ofcertain amino acid residues within a 5 radius of the ligand, selected polar interaction distances included (); B)Stick representation ofthe crystal structure of the bound ligand; C)Electrostatic surface mesh representation of the binding pocket. 6 is represented withblue, 5in magenta


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The crystal structure of the phakellin analogue reveals proximity of the 2-aminoimidazole ring

(or guanidine group) nitrogen atoms to several hydrogen bonding sites in the subpocket, suggesting

that this is a key structural element for high affinity binding.

Figure 12 Crystallography-guided structural design: A) i) 3D conformation of adamantyl hydroxyurea (HU10) upon binding to CP; B)3D conformation of phakellin derivative upon binding to CP; C) Superposition of aligned crystal structures (PyMol, RMS = 0.376 (23352to 23352 atoms)) revealing almost perfect overlap (0.4 separation) of an HU10 adamantyl group carbon atom and a bromophakellin

derivative dihydroimidazole ring carbon atom; D) i) Proposed structural hybrid overlaid onto original crystal structure conformations,the adamantly group of HU10 is replaced by the bromophakellin derivative aminoimidazole ring; ii) separated structure for clarity

When superimposed and computationally aligned, it is apparent that the two structures share

almost perfectly the position of the carbon atom highlighted with the red arrow, figure 12, C. Either

side of the shared carbon atoms lie key binding elements of the two structures. Thus a proposed

hybrid was designed, combining the key binding structures of both compounds, merging at the shared

carbon atom (figure 12, D). For synthetic simplicity, the specific chirality of the hydroxyurea methyl

group was discarded and the racemate synthesised, despite the original (R)-enantiomer displaying

approximately twice the potency of its racemic form.100The synthesis follows a convergent route and,


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in theory, is very simple. The majority of the structure (compound 27) can be prepared separately to

the headgroup and the two joined in the penultimate synthetic step, a Sonogashira cross-coupling

reaction.

Hydroxyurea Synthesis Plan

The synthetic route was designed with the incorporation of the hydroxyurea group left until

last due to the additional complexity to purification that it was predicted to cause.

Scheme 6 Hydroxyurea synthesis approach: i) Allylation with allyl source; ii) Generation of bromonium ion, followed by opening of thebromonium by N-Boc-guanidine and then intramolecular substitution of bromine; iii) Sonogashira coupling to attach hydroxyureaheadgroup; iv) Acidic Boc deprotection revealing guanidine functionality

Starting with commercially available 3-iodophenol, the desired structure was built on this

frame in three synthetic transformations followed by a simple Boc deprotection, scheme 6.

HO I O I O IN

HNHN

Boc

ON

HN

HNBoc

N

OH

NH2

O

ON

HN

H2N

N

OH

NH2

O

26 27

28

29

iv)

i) ii)

iii)

O

O

Boc =


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Results & Discussion

Sulphonamides

Synthesis of 2-(4-ethoxyphenyl)quinoline-4-carboxylic acid

The core quinoline structure was synthesised via the Pfitzinger reaction, figure 13.101 This

quinoline heterocycle centred molecule forms the body of the majority of the sulphonamide

structures.

Figure 13 The mechanism of the Pfitzinger reaction: hydrolysis of isatin, imine (Schiff base) formation and dehydrative cyclisation

Diversity in Extensions Towards Threonine 1

In an attempt to gain structural variety in threonine extensions (R groups, scheme 7), with

fewest synthetic steps (scheme 7, path A)), the quinoline - sulphonic acid intermediate (scheme 7,

dashed outline), which can be prepared in bulk for subsequent conjugation to amine components (R-

NH2) of choice, was constructed (scheme 8).

NH

O

O

N

HO O

O

O

O

KOH

NH2

O

O

HO

KOH

O

N

O

O

HO

O

NH

O

O

HO

-H2O

-H2O


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Scheme 7 General synthetic approaches to sulphonamide inhibitors:A)Attempted direct coupling method that enables rapid syntheticdiversification with various R groups coupled to sulphonic acid (dashed box) that could be prepared in bulk i) amide coupling viaacidchloride-activated carboxylic acid and sulphanillic acid; ii) Direct Caddick coupling utilising Hendrickson POP reagents; B)Longer,more tolerant route chosen due to practical considerations, requires early incorporation of R group and further synthetic steps appliedto each R-variant separately; iii) Sulphonamide bond formation viachlorine-nitrogen substitution at sulphonyl chloride sulphur atom;iv) palladium mediated catalytic reduction under hydrogen; v) amide coupling via acid chloride-activated carboxylic acid andsulphanillic acid

The sulphonic acid intermediate (scheme 7, A, dashed outline) could then be coupled to R-

groups of choice in a divergent manner, scheme 7, A. Initially, the coupling was attempted by

activation of the sulphonic acid to the sulphonic chloride (scheme 8, steps ii), iii) & iv)), but without

success. Subsequently, a method of direct sulphonic acid to amine coupling was performed

successfully (scheme 8, step v)).102

S

O

O

HN

N

OO

NH

R

S

O

O

NH2

NH

RS

O

O

NO2

NH

RS

O

O

NO2

Cl iii) iv) v)

R NH2

S

O

O

NH2

HO i)

S

O

O

HN

N

OO

HO

ii)

R NH2

S

O

O

HN

N

OO

N

H

R

A)

B)


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Scheme 8 Attempted approach to rapid efficient structural diversity in extensions towards Thr1O Y: i) thionyl chloride (SOCl2, excess),sulphanilic acid; ii) SOCl2 (excess); iii) N-chlorosuccinimide (NCS, 1.2 eq.); iv) NCS (2.0 eq.) HCl aq. (2 M); v) Caddick - R-NH2/R-NH3+Cl-(excess), triflic anhydride (1.0 eq.), triphenylphosphine (2.5 eq.), TEA (2.0 eq.), DCM (anhydrous, degassed)102

This application of the reagent combination known as the Hendrickson POP reagent to

sulphonamide bond formation was pioneered by Caddick et al. and the mechanism is illustrated in

figure 14.

S

O

OHO

HN

N

OO

N

O

OHO

S

O

ONH

HN

N

OO

R

i)

v)R-NH2

ii)

S

O

OCl

iii)

R-NH2

iv)


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Figure 14 Direct synthesis of sulphonamides from sulphonic acids. A) P=O lone pair attack at highly electrophilic sulphur atomresulting in release of trifluoromethanesulphonate and B)electron deficient phosphorous atom with a strong leaving group, attacked bya second triphenylphosphine oxide P=O lone pair, releasing the second triflate and forming the active coupling agent, which C)activatesthe sulphonic acid to D)nucleophilic substitution by the desired amine. Due to the highly reactive reagent trifluoromethanesulphonicanhydride, anhydrous conditions and degassed solvents are essential. Triethylamine is frequently included in the reaction mixture toneutralise acidity due to the formation of the two equivalents of trifluoromethanesulphonic acid. The metal or organic salt of the initialsulphonic acid is also compatible with these reaction conditions and, with the use of a polystyrene-supported triphenylphosphine oxidereagent, the reaction mixture can be purified simply by filtration to remove solid-supported OPPh3 and an aqueous wash to removesalts.

The direct Caddick coupling approach was accomplished, but several factors reduced itsappeal as a convenient route for coupling various amines to the main body of the target molecule.

These included difficulty in purification of the sulphonic acid starting material; the meticulous

preparation required for the sensitive sulphonamide bond forming reaction and the poor shelf life of

the triflic anhydride reagent. Ultimately, it was deemed easier to synthesise each compound with

variant threonine extension groups viaa longer, but more tolerant synthetic pathway, scheme 7, B.

P O

S

O

O

F F

FS

O

O

O

FF

FP

O

S

O

O

F F

F

S

HO O

O

F

F

F

Ph3P-O=PPh3

2

S

O

OOH

Ph3P-O-PPh3S

O

OO

PPh3 OPPh3

S

O

OO

PPh3

H2N R

S

O

O HN

R

F3CSO3H

OPPh3

F3CSO3H

A)

B)

C)

D)

PO

S

HO O

F

F

F

PO

S

O

O

F F

FO

S

O

O

F F

FO

Ph3P-O-PPh3


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Synthesis of C3 Alkyl Spacer Sulphonamides 3

Scheme 9 Synthetic approach to sulphonamide fragment 3, 4: i)3-chloropropylamine hydrochloride (250 mg, 1.9 mmol, 1.0 eq.), para-

chlorosulphonylnitrobenzene (533 mg, 2.4 mmol, 1.2 eq.), NMM (0.5 mL, 4.3 mmol. 2.3 eq.), DCM, 0-20C, 3 h, 93%; ii) N-(3-chloropropyl)benzenesuphonamide (450 mg, 1.6 mmol, 1.0 eq.), palladium on carbon (25 mg, 0.024 mmol, 0.015 eq.), hydrogen (3 L,excess), DCM, 20C, 18 h, 92%); iii)2-(4-ethoxyphenyl)quinolin-4-oic acid (232 mg, 0.8 mmol, 1.5 eq.); thionyl chloride (1 mL, 13.7mmol, 30 eq.), 4-amino-N-(3-chloropropyl)benzenesulphonamide (131 mg, 0.5 mmol, 1.0 eq.), NMM (0.1 mL, 1 mmol, 2 eq.), DCM, 67C,20C, 85%

The sulphonamide bond formation (scheme 9, i) between amino and sulphonyl chloride

functional groups was consistently rapid, clean and high-yielding, both in this example and in

analogous reactions. Incorporation of the non-nucleophilic nitro group serves as a convenient

installation of an amino group, with protected nucleophilicity exposable via palladium-mediatedcatalytic reduction, scheme 9, ii). Subsequent coupling to the substituted quinoline moiety (scheme 9,

iii) was achieved via an acid chloride, prepared in-situfrom the corresponding carboxylic acid.

S

O

OCl

N

O

O

S

O

ONH

N

O

O

Cl

S

O

ONH

NH2

Cl

S

O

ONH

HN

Cl

N

OO

1 2

3

i) ii)

iii)


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Scheme 10 Attempted synthetic approach to the boronic ester sulphonamide 4: i)3-chloropropylamine hydrochloride (250 mg, 1.9mmol, 1.0 eq.),para-chlorosulphonylnitrobenzene (533 mg, 2.4 mmol, 1.2 eq.), NMM (0.5 mL, 4.3 mmol. 2.3 eq.), DCM, 0-20C, 3 h, 93%;ii)N-(3-chloropropyl)benzenesuphonamide (450 mg, 1.6 mmol, 1.0 eq.), palladium on carbon (25 mg, 0.024 mmol, 0.015 eq.), hydrogen(3 L, excess), DCM, 20C, 18 h, 92%); iii)2-(4-ethoxyphenyl)quinolin-4-oic acid (232 mg, 0.8 mmol, 1.5 eq.); thionyl chloride (1 mL, 13.7mmol, 30 eq.), 4-amino-N-(3-chloropropyl)benzenesulphonamide (131 mg, 0.5 mmol, 1.0 eq.), NMM (0.1 mL, 1 mmol, 2 eq.), DCM, 67C,20C, 85%; iv) 3 (150 mg, 0.3 mmol, 1.0 eq.), bis(pinacolato)diboron (109 mg, 0.4 mmol, 1.5 eq.), triphenylphosphine (10 mg, 0.04mmol, 0.13 eq.), copperIiodide (5 mg, 0.03 mmol, 0.1 eq.), lithium methanolate (22 mg, 0.6 mmol, 2 eq.), DMF (10 mL), 20C no reaction;v)3(44 mg, 0.08 mmol, 1.0 eq.), sodium bromide (8.6 mg, 0.08 mmol, 1.0 eq.), acetone (5 mL) 20C, partial conversion (HPLC-MS); vi)3*

(42 mg, 0.08 mmol, 1.0 eq.), bis(pinacolato)diboron (30 mg, 0.12 mmol, 1.5 eq.), triphenylphosphine (3 mg, 11 mol, 0.13 eq.), copperIiodide (1.5 mg, 8 mol, 0.1 eq.), lithium methanolate (6 mg, 0.16 mmol, 2.0 eq.), DMF (5 mL), 20C, no reaction.

Following an unsuccessful borylation (scheme 10, iv), a halogen exchange (Finkelstein)

reaction was employed with partial success in order to convert R-Cl into the more labile R-Br. The

Finkelstein operates via SN2 and exploits the preferential solubility of the bromide salt over the

chloride salt in acetone, thus tipping the equilibrium in favour of the R-Br formation as sodium

chloride precipitates from the reaction mixture. Some salt formation was observed and HPLC-MS

confirmed that some reaction progress has been made. The subsequent borylation also proved

unsuccessful and it was decided that the bromine would be better installed by beginning the synthesis

with a sulphonamide coupling of 3-bromopropylamine and para-chlorosulphonylnitrobenzene,

scheme 10, i). This was achieved in later syntheses. Sulphonamide fragment 3 was soaked onto

proteasome crystals and X-ray crystallography performed, however no electron density

corresponding to the fragment was observed.

= No electron density observedin crystal structure

S

O

OCl

N

O

OS

O

ONH

N

O

O

Cl S

O

ONH

NH2

Cl

S

O

ONH

HN

Cl

N

OO

S

O

O

N

H

HN

B

N

OO

O

O

1 2

3

4

i) ii)

iii)

iv)

v) Br

vi)


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Synthesis of Amino Acid Sulphonamides 7, 10 & 13

Replacing the acyl cap of the original sulphonamide with structures of simple peptidic nature

was a logical structural modification. The methyl esters of the chosen amino acids (alanine R& Sand

glycine) were employed to confer favourable physical properties, attenuating the hydrophilicity of the

standard carboxylic acid terminus.

Scheme 11 Synthetic approach to sulphonamide 7: i) (L)-alanine methyl ester hydrochloride (252 mg, 1.8 mmol, 1.0 eq.), para-chlorosulphonylnitrobenze (500 mg, 2.3 mmol, 1.25 eq.), NMM (411 L, 4.1 mmol, 2.3 eq.), DCM (10 mL), 0-20C, 3 h, 97%; ii)5(480

mg, 1.7 mmol, 1.0 eq.), palladium on carbon (35 mg, 0.03 mmol, 0.2 eq.), hydrogen (3 dm3

), DCM (20 mL), 20C, 24 h, 97%; iii)6(147mg, 0.6 mmol, 1.0 eq.), 2-(4-ethoxyphenyl)quinolin-4-oic acid (250 mg, 0.9 mmol, 1.5 eq.), thionyl chloride (1.2 mL, 17 mmol, 30 eq.),DCM (20 mL), reflux, 0-20C, 10%

Steps i) & ii) were both achieved in high yield and purity, but step iii) suffered a poor yield. The

acid chloride, formed in-situ, is a highly reactive and water-sensitive intermediate; it was soon

realised that this must be handled quickly and with utmost care with respect to anhydrous conditions.

Later analogous reactions attained yields of 85%, using carefully dried glassware, molecular sieves,

rapid transfers and inert atmospheres. Sulphonamide 7was soaked onto proteasome crystals and X-

ray crystallography performed, however no electron density corresponding to the fragment was

observed.

S

O

OCl

N

O

O

S

O

ONH

N

O

O

S

O

ONH

NH2

S

O

O

HN

N

OO

5 6

7

i) ii)

iii)

(S)(S)O

O

(S)(S)O

O

(S)(S)O

O

NH

No electron density observedin crystal structure


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Scheme 12 Synthetic approach to compound 10: i) (D)-alanine methyl ester hydrochloride (252 mg, 1.8 mmol, 1.0 eq.), para-chlorosulphonylnitrobenze (500 mg, 2.3 mmol, 1.25 eq.), NMM (411 L, 4.1 mmol, 2.3 eq.), DCM (10 mL), 0-20C, 3 h, 94%; ii)8(480mg, 1.7 mmol, 1.0 eq.), palladium on carbon (35 mg, 0.03 mmol, 0.2 eq.), hydrogen (3 dm 3), DCM (20 mL), 20C, 24 h, 97%; iii)9(147

mg, 0.6 mmol, 1.0 eq.), 2-(4-ethoxyphenyl)quinolin-4-oic acid (250 mg, 0.9 mmol, 1.5 eq.), thionyl chloride (1.2 mL, 17 mmol, 30 eq.),DCM (20 mL), reflux, 0-20C, 28%

Sulphonamide 10was soaked onto proteasome crystals and X-ray crystallography performed,

however no electron density corresponding to the fragment was observed.

Scheme 13 Synthetic approach to compound 13: i) glycine methyl ester hydrochloride (227 mg, 1.8 mmol, 1.0 eq.), para-chlorosulphonylnitrobenzene (500 mg, 2.3 mmol, 1.25 eq.), NMM (411 L, 4.1 mmol, 2.3 eq.), DCM (10 mL), 0-20C, 3 h, 99%; ii)11(480 mg, 1.7 mmol, 1.0 eq.), palladium on carbon (35 mg, 0.03 mmol, 0.2 eq.), hydrogen (3 dm 3), DCM (20 mL), 20C, 24 h, 91%; iii)12(147 mg, 0.6 mmol, 1.0 eq.), 2-(4-ethoxyphenyl)quinolin-4-oic acid (250 mg, 0.9 mmol, 1.5 eq.), thionyl chloride (1.2 mL, 17 mmol, 30eq.), DCM (20 mL), reflux, 0-20C, 16%.

The incorporation of the glycine methyl ester into the sulphonamide structure was as straight-

forward as was the previous alanine methyl ester sulphonamide syntheses, with the slightly increasedpolarity of the glycine compounds relative to the corresponding alanine compounds being the only

discernable distinction. Sulphonamide 13 was soaked onto proteasome crystals and X-ray

S

O

OCl

N

O

O

S

O

ONH

N

O

O

S

O

ONH

NH2

S

O

O

HN

N

OO

8 9

10

i) ii)

iii)

(R)(R)O

O

(R)(R)O

O

(R)(R)O

O

NH


S

O

OCl

N

O

O

S

O

ONH

N

O

O

S

O

ONH

NH2

S

O

O

HN

N

OO

11 12

13

i) ii)

iii)

O

O

O

O

O

O

NH



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crystallography performed, however no electron density corresponding to the molecule was

observed.

Synthesis of Boronic Acid Sulphonamides 4, 18 & 20

Scheme 14 i) Synthetic approach to compounds 4, 18 &20: i)3-bromopropylamine hydrobromide (3.74 g, 17 mmol, 1.0 eq.), para-chlorosulphonylnitrobenzene103(3.79 g, 17 mmol, 1.0 eq.), NMM (4.2 mL, 38 mmol, 2.0 eq.), DCM (25 mL), 0-20C, 3 h, 97%; ii)14(4.2g, 13 mmol, 1.0 eq.), palladium on carbon (200 mg, 0.2 mmol, 0.015 eq.), hydrogen (6 dm 3), DCM (20 mL), 20C, 24 h, 99%; iii)15(456mg, 1.55 mmol, 1.0 eq.), 2-(4-ethoxyphenyl)quinoline-4-carboxylic acid (500 mg, 1.7 mmol, 1.1 eq.), thionyl chloride (3.4 mL, 46 mmol,30 eq.), DCM (10 mL), 0-20C, 12 h, 28%; iv) 16 (512 mg, 0.9 mmol, 1.0 eq.), copper I iodide (17 mg, 0.09 mmol, 10 mol%),triphenylphosphine (31 mg, 0.12 mmol, 13 mol%), lithium methanolate (68 mg, 1.8 mmol, 2.0 eq.), bispinacolato diboron (343 mg, 1.35mmol, 1.5 eq.), DMF (4 mL), 20C, 18 h, 24%; v)15(440 mg, 1.5 mmol, 1.0 eq.), 2-methoxyisonicotinic acid (287 mg, 1.9 mmol, 1.25 eq.),thionyl chloride (5 mL, 70 mmol, 45 eq.), DCM (20 mL), reflux, 0-20C, 42%; vi)17(50 mg, 0.1 mmol, 1.0 eq.), bis(pinacolato)diboron

(41 mg, 0.16 mmol, 1.5 eq.), triphenylphosphine (4 mg, 0.014 mmol, 0.13 eq.), copper I iodide (2 mg, 11 mol, 0.1 eq.), lithiummethanolate (8 mg, 0.2 mmol, 2.0 eq.), acetone (5 mL), 20C, 48 h, 14%; vii) 15 (440 mg, 1.5 mmol, 1.0 eq.), 2-chloro-6-methoxyisonicotinic acid (352 mg, 1.9 mmol, 1.25 eq.), thionyl chloride (5 mL, 70 mmol, 45 eq.), DCM (20 mL), reflux, 0-20C, 33%; viii)19(50 mg, 0.1 mmol, 1.0 eq.), bis(pinacolato)diboron (41 mg, 0.16 mmol, 1.5 eq.), triphenylphosphine (4 mg, 0.014 mmol, 0.13 eq.),copperIiodide (2 mg, 11 mol, 0.1 eq.), lithium methanolate (8 mg, 0.2 mmol, 2.0 eq.), acetone (5 mL), 20C, 48 h, 8%.

Following crystallographic studies that revealed no electron density in the proteasome crystal

structure relating to compounds 4, 7, 10or 13, the more elaborate alterations, such as variations in

heterocycle (compounds 18& 20), were abandoned. Returning to more minor structural alterations,

the two carbon linker version of compound 4was attempted.

SO

ONH

HN

Br

N

OO

16

S

O

OCl

N

O

O

S

O

ONH

N

O

O

Br

S

O

ONH

NH2

Br

S

O

ONH

HN

Br

i) ii)

iii)

N

O

O

Cl

S

O

ONH

HN

O

N

O

14 15

17Br

S

O

ONH

HN

N

O

O

Cl

B

O

O

19

S

O

ONH

HN

O

N

O

B

O

O

20

18

4

iv)

vii)

v) vi)

viii)


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Scheme 15 Synthetic route to C2 linker derivative of originally planned compound, compound 4: i)2-bromoethylamine hydrobromide(1 g, 4.9 mmol, 1.0 eq.), NMM (1.2 mL, 11 mmol, 2.25 eq.), para-nitrobenzenesulphonyl chloride (1.19 g, 5.4 mmol, 1.1 eq.), DCM (10mL), 0-20C, 6 h, 96%; ii) 22(1.45 g, 4.7 mmol, 1.0 eq.), palladium on carbon (75 mg, 0.07 mmol, 15 mol%), MeOH:EtOAc (1:1, 20 mL),hydrogen (excess, multiple cycles), 20C, 24 h, 82%; iii) 2-(4-ethoxyphenyl)quinoline-4-carboxylic acid (568 mg, 1.9 mmol, 1.1 eq.),thionyl chloride (5.7 mL, 80 mmol, 45 eq.), 23 (491 mg, 1.9 mmol, 1.0 eq.), NMM (193 L, 3.7 mmol, 2.0 eq.), DCM (10 mL), 0-20C, 12 h,22%; iv) 24 (500 mg, 0.9 mmol, 1.0 eq.), copperI iodide (17 mg, 0.09 mmol, 10 mol%), triphenylphosphine (30 mg, 0.12 mmol, 13mol%), lithium methanolate (70 mg, 1.8 mmol, 2.0 eq.), bispinacolato diboron (340 mg, 1.3 mmol, 1.5 eq.), DMF (4 mL), 20C, 12 h,extensive degradation

During the synthesis of C3 alkyl spacer boronic acid sulphonamide 4, it was observed that the

compound was unstable to an intramolecular SN2 reaction involving nucleophilic attack of thesulphonamide nitrogen lone pair on the carbon neighbouring the boron atom, resulting in expulsion

of the entire pinocolato boron moiety and formation of an azetidine ring. This degradative reaction

was not anticipated, but its occurrence did not cause great surprise due to the entropic favourability

of an intramolecular reaction regarding orbital orientation and atomic proximity. These factors were

magnified in the case of the C2 linker derivative (compound 25) due to the reduced degrees of

freedom and thereby increased statistical likelihood of the compound adopting an S N2-enabling

orientation.104The result of this side-reaction caused many difficulties for purification and isolation.

In addition, the behaviour of the sulphonamide compounds on silica gel meant troublesome

purifications. Due to time restrictions, the sulphonamides project was put on indefinite hold. The

results of the final compounds are summarised below in figure 15.

S

O

ONH

NH2

BrS

O

ONH

N

O

O

Br

S

O

ONH

HN

Br

N

OO

S

O

ONH

HN

B

N

OO

O

O

22 23

24

25

S

O

OCl

N

O

O

i) ii)

iii) iv)


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Figure 15 An overview of the results obtained in the sulphonamides project: Structural modifications extending towards Thr1O(threonine extension structures) compounds 3, 7, 10 & 13 gave negative results in crystallographic experiments; boronic acidcompounds 4& 25and modified heterocycle (iCP/cCP selectivity) boronic acid compounds 18& 20were subject to an intramoleculardegradation process

The outcome of these logically planned structures was unexpected. The original sulphonamide

fragment from which development began was a weak-binding structure and even minor modifications

were not tolerated by the proteasome. Future design alternatives for enhanced ligand stabilisation or

avoiding degradation are proposed in Conclusions & Future Work.


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Hydroxyurea

Hydroxyurea Synthesis

The hydroxyurea structure, excluding the hydroxyurea headgroup was synthesised in a four-

step synthesis as shown in scheme 16.

Scheme 16 Synthetic pathway to hydroxyurea compound 29: i) 3-iodophenol (475 mg, 2 mmol, 1.0 eq.), 2,4,6-trivinyl-1,3,5,2,4,6-trioxatriborinane:pyridine complex (1:1, 500 mg, 2 mmol, 3 vinyl eq.), copper II acetate monohydrate (415 mg, 2 mmol, 1 eq.), DCM (8

mL), rt, 48 h, 36%; ii)1-iodo-3-(vinyloxy)benzene (500 mg, 2 mmol, 1.0 eq.), N-bromosuccinimide (NBS) (713 mg, 4 mmol, 2.0 eq.), N-Boc-guanidine (954 mg, 6 mmol, 3 eq.), DCM:DMF (8:1, 10 mL), 0-20C, 3 h, 41%; iii) 27 (340 mg, 0.84 mmol, 1.0 eq.),triphenylphosphine (11 mg, 0.04 mmol, 5 mol%), copperI iodide (8 mg, 0.04 mmol, 5 mol%), diacetonitriledichloropalladium(5 mg,0.02 mmol, 2 mol%), triethylamine (120 L, 1 mmol, 1.2 eq.), EtOAc (6 mL),rt, 3 h; iv)28(10 mg, 24 mol, 1.0 eq.), trifluoroacetic acid(50 L, excess), DCM:H2O (1:1, 1 mL)

Building on the frame of 3-iodophenol, the target molecule was constructed in three synthetic

steps and a final Boc deprotection. Installation of the hydroxyurea moiety was performed last due to

the predicted difficulties it would cause in terms of purification a consequence of its extensive

hydrogen bonding capacity and high polarity. The first synthetic transformation achieved vinylation

at the phenol oxygen atom. This involved an unusual reagent, a trivinylcyclotriboroxane-pyridine

complex, that served as the vinyl source and although its mechanism of vinyl transfer remains to be

elucidated.103 A proposed mechanism of the vinyl transfer envisages oxygen-copper complexation,

assisted by the increased boron-oxygen bond lengths and reduced carbon-boron-oxygen bond angles

produced by the tetrahedral geometry of the organic amine-coordinated boron atom, figure 16.

HO I O I O IN

HN

HNBoc

ON

HN

HNBoc

N

OH

NH2

O

ON

HN

H2N

N

OH

NH2

O

26 27

28

29

iv)

i) ii)

iii)

O

O

Boc =


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The Boc-protected 2-aminoimidazole product proved difficult to purify and therefore

purification by reaction was attempted since the next step, the Sonogashira coupling, was known to be

a reliable and robust reaction, the product of which should be of much greater polarity and

hydrophilicity and possible to purify by reverse phase HPLC. Coupling of the alkyne hydroxyurea

headgroup with the aryl iodide employed standard Sonogashira coupling conditions and proceeds via

a mechanism of two metal atom mediated catalytic cycles, the palladium and the copper cycle, figure

18.

Figure 18 General mechanism of the Sonogashira cross-coupling reaction

Purification of the Sonogashira product was attempted several times by HPLC. Initially the

results were very low yielding and subsequently yielded no product. Normal phase silica gel column

chromatography was attempted with various high polarity eluent systems and with neutralised silica

but to no avail. Testing of crude aliquots indicated that the target compound was no longer present

and therefore a degradation reaction was suspected, although no obvious mass peaks that related to

Pd0L2

Oxidativeaddition

Transmetallation

tr an s-c isIsomerisation

Reductiveelimination

ThePalladium Cycle

Pd0L4

TheCopper Cycle

-Coordination

-Coordination

PdII X

L

R'

L

PdIIL

R'

L

R

PdII

L

L

R'

R

CuIX

R HDeprotonation

Cu X

R H

R3N

R3NH X

Ligandexchange R' X

R R'

R Cu


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logical degradation products were found. The highly substituted carbon centre linking the ether

bridge to the 2-aminodihydroimidazole ring was a suspected point of structural instability. Modified

syntheses and future improvements are discussed in Conclusions & Future Work.

The final synthetic step uses trifluoroacetic acid (TFA) as a proton source to initiate the

removal of the tert-butyloxycarbonyl (Boc) protecting group, figure 19. Excess acid is used and both

acid and the deprotection side products can be separated from the reaction mixture by concentration

in vacuo without the need for chromatographic purification.

Figure 19 Quantitative acidic deprotection of the Boc-protected aminoimidazole. Formation of a tertiary isobutane carbocation rapidlyrearranges to isobutene and can be removed with excess TFA and CO2, the remaining reaction mixture components under reducedpressure leaving pure product, or its TFA salt

The final deprotection step was attempted once with excess TFA, but mass spectrometry didnot indicate successful deprotection. The troubleshooting of this final deprotection was limited by the

small quantity of material available to work with. Future success can only be achieved if degradation

processes are circumvented or minimised.

Headgroup Synthesis

O

HN

NHN

N

OH

NH2

O

O

O

O

HN

NHN

N

OH

NH2

O

O

O H

H

O

HN

NHN

NOH

NH2

O

O

OH

H

O

HN

NH2N

NOH

NH2

O

O

O

O

HN

NH2N

N

OH

NH2

O

-CO2


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The hydroxyurea-alkyne headgroup of the hydroxyurea compounds was synthesised in-house

as described in Hydroxyureas as Non-covalent Proteasome Inhibitors,Groll et. al.2012.

Figure 20 Two-step synthesis of the hydroxyurea headgroup viathe mesylate-activated SN2 substitution of but-1-yn-3-ol hydroxy groupwith hydroxylamine to form the N-hydroxy functional group, followed by reaction with isocyanate, forming the urea functional group

The mechanism involves the activation of the hydroxy group as a leaving group by conversion

to the mesylate, followed by displacement by hydroxylamine. Finally, addition to isocyanate yields thehydroxyurea functional group and the headgroup is ready for Sonogashira coupling.

OHNH

OH

S

O

O

Cl

NEt3

S

O

O

Cl

O

S OO

OS

O

OH

H2N OH

NH2

HO-HCl

SO

OHO

-

N

OH

KOCN

i)

ii)

via)

NH

C

O

NH2

O

HCl

H

via)

SN2

i)


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Conclusions & Future Work

Sulphonamides Project

The sulphonamides project began with a known proteasome-binding fragment and only small

and logical structural changes were made. No electron density corresponding to ligand binding was

observed in soaking experiments with three amino acid derivative sulphonamides and a C3

chloroalkyl spacer sulphonamide. In addition, an entire series of structures were abandoned due to a

shared degradation reaction that was promoted by the addition of the boronic acid headgroup in their

final stage of synthesis. Further logically designed structures are shown in figure 21.

Figure 21 Summary of proposed future sulphonamide structures: Irreversible headgroups the well-documented epoxyketone

headgroup with phenylalanine P1 residue; Multiple combinations of linker groups and electrophilic headgroups with different P1residues; Alternative reversible Thr1 extension structures offering extra hydrophobic stabilisation; Alkyne spacer to preventintamolecular SN2 by conformational restriction

The original sulphonamide structure bound only weakly to the proteasome. This was reflected

in the rejection of structures bearing only minor modifications. Inspection of the crystal structure of

the original fragment revealed an additional point of ligand stabilisation that is not exploited in the

original fragment, or in any of the structures synthesised so far, figure 22. Although a subtle

modification, such changes can drastically alter the potency of an inhibitor and should be investigated.


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Figure 22 Crystallo

Novel Proteasome Inhibitors

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