Allin1 - Project CUT and REVISED

Structure-based Design and Synthesis of Novel

Inhibitors of Hepatitis C Virus NS3 Helicase

An MPharm Degree Project in Medicinal Chemistry

Matthew Courtney-Smith

The Welsh School of Pharmacy

Cardiff University

2011

Declaration

This thesis is the result of my own investigation, except where otherwise stated. Other

sources are acknowledged by explicit references.

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AcknowledgmentsI wish to express my gratitude to my supervisor, Dr. Andrea Brancale, whose knowledge,

assistance and encouragement have contributed greatly to my experience in a project I have

truly enjoyed. I would like to extend my thanks to all of Andrea’s colleagues in the Medicinal

Chemistry Department of The Welsh School of Pharmacy, not least Marcella Bassetto and Dr.

Antonio Ricci. Their kindness, and enthusiasm for chemistry is highly admirable, and I can

only hope this is reflected in my work. Special thanks of course go to my family, without

whom none of this would have been possible. Their love and belief is constant, and has

supported me both through life and my time at university(s). Finally, I must acknowledge

everyone at The Welsh School of Pharmacy for providing me not only with an opportunity to

learn, but also to do so amongst the best of friends. It has been a pleasure and a privilege to

share these memorable years with a group of great people; a time I will look back on forever

with happiness.

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AbstractThe hepatitis C virus (HCV) is a small, enveloped positive-sense RNA virus responsible for the

infection of up to 170 million people worldwide. Current standard of care combination

therapy with pegylated interferon alfa and ribavirin is effective in less than 50 per cent of

cases, and has a severe associated side effect profile. For these reasons, there is an urgent

interest in the development of more potent and better-tolerated targeted therapies for

hepatitis C. The HCV NS3 helicase, which has an essential role in viral replication through the

unwinding of duplex nucleic acids, has been proposed as a promising orphan target, with no

drug candidates currently in clinical trials. Described herein are the structure-based design,

molecular docking, and synthesis of a novel series of inhibitors of the NS3 helicase.

A compound library of 112 potential inhibitors was designed based around the structural

modification of the symmetrical lead compound 1, which demonstrated modest antiviral

activity against subgenomic HCV replicon cell lines (EC50 = 8 μM). With the aim of improving

activity, the compounds were docked into the RNA binding site of the 3KQH crystallised NS3

helicase structure using GLIDE software, and the best ranked poses were screened by visual

inspection for interactions with key surface residues. Eight outstanding candidates were

selected for further development, and all were synthesised by a simple two-step reaction

scheme in good overall yields.

Of the proposed compounds, 13, 16, and 22 showed the most extensive interactions with the

RNA binding site, although they did not appear to associate with the crucial Trp501 residue,

which is understood to stack against the 3’-terminal base of the bound nucleic acid.

Compound 14 demonstrated clear potential, with binding both to Trp501 and Glu493, a key

residue of similar mechanistic importance. Despite a relatively low docking score, the

asymmetrical compound 17 was chosen for synthesis both due to its evident association with

the nearby Glu493 residue, and its clear superiority over other candidates in terms of

molecular weight.

Currently, the study is awaiting the results of HCV replicon and helicase enzymatic assays for

4

all eight synthesised compounds, which will determine the next stage of investigation. The

main priority will be the rational optimisation of the compound(s) showing greatest activity

to ultimately obtain a novel class of potent anti-HCV inhibitors.

Abbreviations and Definitions

ATP Adenosine triphosphate

GLIDE Grid-based ligand docking with energetics

HCV Hepatitis C virus

HIV Human immunodeficiency virus

HOBt Hydroxybenzotriazole

IRES Internal ribosome entry site

NMR Nuclear magnetic resonance

NS Non-structural protein

NTR Non-translated region

Peg-IFN-α Pegylated-interferon-alfa

RdRp RNA-dependent RNA polymerase

RNA Ribonucleic acid

SVR Sustained virological response

TBTU O-(Benzotriazol-1-yl)-N,N,N´,N´-tetramethyluronium tetrafluoroborate

TLC Thin layer chromatography

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ContentsAbstract...................................................................................................................... 4

Abbreviations and Definitions..................................................................................... 5

1 Introduction............................................................................................................. 7

1.1 The Hepatitis C Virus (HCV)…………………………………………………………………………. 7

1.1.1 HCV Background……………………………………………………………………….... 7

1.1.2 Current Treatments…………………………………………………………………….. 7

1.1.3 HCV Genome, Life Cycle, and Strategies for Inhibition………………... 8

1.2 An Orphan Drug Target……………………………………………………………………………….. 12

1.2.1 HCV NS3 Helicase……………………………………………………………………….. 12

1.2.2 Structure of HCV NS3 Helicase……………………………………………………. 12

1.2.3 Mechanism of HCV NS3 Helicase Action……………………………………… 14

1.2.4 HCV NS3 Helicase as a Drug Target……………………………………………… 15

1.3 Molecular Docking…………………………………………………………………………………...... 17

1.4 Research Objectives……………………………………………………………………………………. 18

2 Results and Discussion.............................................................................................. 19

2.1 Structure-based Design: A Novel Compound Library…………………………………... 19

2.2 Compounds Selected for Chemical Synthesis…………………………………………...... 23

2.3 Docking Studies…………………………………………………………………………………………… 25

2.4 Chemical Synthesis……………………………………………………………………………………….34

2.5 Conclusions and Future Work……………………………………………………………………... 41

4 Experimental Section................................................................................................ 42

4.1 Molecular Modelling........................................................................................ 42

4.2 General Synthetic Procedures.......................................................................... 42

4.3 Synthesis of N-(bromoalkyl)-arenesulfonamides............................................. 43

4.4 Synthesis of N,N'-(alkylarenesulfonamide)-piperazines...................................44

4.5 Synthesis of N-(propyl-2-naphthalenesulfonamide)-piperidine.......................45

4.6 Synthesis of 3-(arenesulfonamide)-propanoic acids........................................ 45

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4.7 Synthesis of N,N'-(3,3'-(piperazine-1,4-diyl)bis(3-oxopropane-3,1-diyl))-

diarenesulfonamides.............................................................................................. 46

5 References................................................................................................................ 48

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1. Introduction1.1 The Hepatitis C Virus (HCV)

1.1.1 HCV Background

The Hepatitis C Virus (HCV), a small, enveloped RNA virus belonging to the Hepacivirus genus

of the Flaviviridae family,1,2 was first identified in 1989 as the agent responsible for the

majority of post-transfusion non-A, non-B hepatitis cases.3 It is estimated that HCV is

prevalent amongst up to 3% of the global population, corresponding to 170 million infected

people worldwide.4 Few patients show early signs of acute infection, such as malaise or

jaundice, but the majority – up to 80% – will progress to chronic disease beyond six months.5

This persistence is partly due to the virus's unique ability to evade the host immune

response. In around 40% of cases, chronic hepatitis C leads to cirrhosis,6 which remains the

primary reason for liver transplantation in the developed world.7 A significant proportion of

sufferers will go on to develop hepatocellular carcinoma, and overall prognosis is poor.

There are six known genotypes of HCV, which share up to 70% nucleotide sequence similarity

and may be further divided into more closely related subtypes. Genotypes 1a, 1b and 3a,

which have spread widely as a result of infected blood transfusions, transplantation or

needle-sharing, now contribute the most infections to the Western world.8 The virus

circulates in affected individuals as a series of variants, often referred to as quasispecies. The

existence of quasispecies appears to be an overriding factor in viral persistence, as chronic

hepatitis C patients tend to show greater genetic complexity than those with self-limiting

infection.9

1.1.2 Current Treatments

Historically, treatment of chronic hepatitis C has been restricted to the use of an

immunomodulatory interferon alongside the broad-spectrum antiviral ribavirin. Interferons

work by increasing the expression of major histocompatibility complex proteins to allow the

immune system to more efficiently recognise presenting viral antigens, as well as enhancing

the function of natural killer cells and macrophages.10 Ribavirin, a synthetic guanosine

nucleoside analogue, exerts its antiviral activity by several mechanisms, including depletion

of the intracellular pool of GTP, and direct incorporation of its metabolites into viral RNA to

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interrupt mRNA synthesis.11,12 The non-specific combination of interferon-alpha (IFN-α) with

ribavirin proved to be relatively ineffective, with barely 40% of patients showing signs of any

lasting benefit.13 Studies showed that by increasing the interferon dosage, and limiting its

clearance rate, it would be possible to maximise its therapeutic effectiveness. This soon led

to the introduction of pegylated interferon-alpha (peg-IFN-α),14,15 which offered a modest

improvement due to its prolonged half-life.

Currently, the only recommended treatment is combination therapy with peg-IFN-α and

ribavirin, usually lasting for six to twelve months.16,17 The primary aim is a 'sustained

virological response', or SVR, which is defined as an undetectable serum HCV RNA level

twenty-four weeks after the discontinuation of therapy.18 Despite recent progress, and the

promise of improved forms of ribavirin, treatment efficacy remains strictly limited, and varies

considerably with genotype. Clinical studies with both forms of peg-IFN-α (2a and 2b) have

consistently shown an SVR in just 40-50% of patients with genotype 1 infection, and 70-80%

of those with genotypes 2 and 3.18-20 Current treatments are also limited by a relatively

severe side effect profile, particularly in those affected by other diseases, such as HIV.18-20,22

Influenza-like symptoms, including fatigue, headache, pyrexia and insomnia, are especially

common, while more severe psychiatric disorders – mainly depression-related events – can

also be a problem. Furthermore, there have been several reports of neutropenia, haemolytic

anaemia, and thrombocytopenia, which have necessitated the immediate dose reduction or

complete withdrawal of therapy. For these reasons, there is an urgent interest in the

development of more effective and better-tolerated targeted therapies for hepatitis C.23

1.1.3 HCV Genome, Life Cycle, and Strategies for Inhibition

The cloning of the HCV genome in 1989,3 combined with recent advances in replicon

models,24 has helped us to characterise the viral life cycle, and identify opportunities for drug

development. The HCV genome is a single-stranded, positive-sense RNA molecule of ~ 9600

bp in length, which includes a large open reading frame (ORF) encoding for a 3000 amino

acid polyprotein.24 Translation of the HCV codon is initiated by a 5' non-translated region

(NTR), which acts as an internal ribosome entry site (IRES) to allow the 40S ribosomal subunit

to bind directly at the start of the ORF. The function of the 3' NTR is less well understood, but

is thought to be involved in minus-strand priming essential to viral replication. The precursor

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polyprotein is post-translationally cleaved by host and viral proteases to form three structural

proteins (the core nucleocapsid protein, and the two envelope proteins, E1 and E2), a

putative ion channel-forming protein (p7), and six non-structural proteins (NS2, NS3, NS4A,

NS4B, NS5A, and NS5B).18,25,26

Figure 1. The HCV genome and its encoded proteins

Although the exact functions of all the non-structural proteins are yet to be fully elucidated,

researchers are aware that each step in the HCV life cycle represents a potential target for

anti-viral activity. Rapid progress in the treatment of HIV infection is seemingly leading the

way, as therapies target the inhibition of key viral enzymes. Protease inhibitors, for example,

have been of proven benefit in the treatment of HIV infection, contributing to a more than

80% reduction in AIDS-related mortality.27 Pharmaceutical companies are now focusing on

replicating this success in the treatment of hepatitis C.

HCV possesses two proteolytic enzymes: the zinc-dependent NS2/3 metalloprotease, which

catalyses the cleavage of NS2 from the polyprotein, and the NS3/4A serine protease, which

sequentially cleaves the remaining four junctions. To date, NS2/3 has been largely

overlooked as an antiviral target due to obscurities in the cleavage process,28 though novel

models for precise identification of protein function, and isolation of the enzyme catalytic

domain, have led to increasing optimism for the future. The NS3/4A protease is a somewhat

more attractive target,29 and as such several inhibitors have reached clinical trials. The first

was BILN-2061, or ciluprevir, which demonstrated a transient but significant and rapid

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response in viral decline amongst patients infected with HCV genotype 1.30 Unfortunately,

trials were ended prematurely due to its significant cardiotoxicity.31 Side effects have also

contradicted the benefits of other similar inhibitors, such as boceprevir (SCH503034) and

telaprevir (VX-950).32,33

Figure 2. Structures of the NS3/4A protease inhibitors boceprevir and telaprevir

The NS5B RNA-dependent RNA polymerase (RdRp), which is key to viral replication, is

another good anti-HCV target. It is a viral-specific enzyme with no corresponding host-cell

homologues. Several novel inhibitors have progressed to clinical development including

IDX184, a once daily, oral HCV nucleotide polymerase inhibitor based on Idenix’s proprietary

liver targeting technology.35 Furthermore, recent work at The Welsh School of Pharmacy by

McGuigan et al. has identified a novel ‘Protide’ nucleoside analogue, INX-189,36 which has

shown great promise in early phase clinical trials. The results of a phase 1b trial in Genotype

1 naive HCV patients are eagerly anticipated,37 with U.S. company Inhibitex seeking to gain

marketing authorisation for INX-189 within the foreseeable future. This would be an

important milestone in the introduction of specific anti-HCV therapies.

Figure 3. Structure of the NS5B RNA-dependent RNA polymerase inhibitor INX-189

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Boceprevir (Schering) Telaprevir (Vertex)

The first HCV protein to be crystallised was the portion of NS3 that acts as a helicase.38

However, thus far no drug candidates have progressed to clinical development, perhaps due

to uncertainties in its mechanism of action. Although its exact physiochemical role is still

unclear, NS3 helicase is clearly essential to viral replication and as such represents a

promising orphan target for the development of novel inhibitors of HCV.

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1.2 An Orphan Drug Target

1.2.1 HCV NS3 Helicase

The HCV non-structural protein 3 (NS3) fulfils two distinct enzymatic functions. The N-

terminal hosts a serine protease, responsible for post-translational polyprotein processing,

while the 450 C-terminal amino acid residues constitute an RNA helicase domain.39 This NS3

helicase was the first HCV protein to be crystallised,38 and was shown almost 20 years ago to

hydrolyse ATP to catalyse the unwinding of duplex RNA or DNA. Although its precise role is as

yet unclear, helicase activity is understood to be essential for HCV replication. As such, HCV

RNA with mutations inactivating the NS3 helicase is unable to replicate in both in vivo

chimpanzee models and in subgenomic replicons.40

1.2.2 Structure of HCV NS3 Helicase

Sequence-based classification has placed the C-terminal domain of HCV NS3 into helicase

superfamily 2 (SF2), as described by Gorbalenya and Koonin,41 with a structure consisting of

three separate domains. Crystallised structures have shown that the most N-terminal domain

(domain 1) and the middle domain (domain 2) lie above the C-terminal domain (domain 3).

ATP binds at the cleft formed at the interface between domains 1 and 2, and RNA or DNA

binds between domain 3 and domains 1 and 2.42 Studies have identified a flexibly-linked

rotation of domain 2 relative to domains 1 and 3,43 which allows the NS3 helicase to exist in

two distinct conformations. In some structures, domain 2 faces away from the other

domains, confirming its existence in an “open” conformation, while some show domain 2

closer to its neighbours in a “closed” conformation. The enzyme must be in its closed state

for ATP hydrolysis to occur, and RNA binding can only take place in the open conformation.

Figure 4. Stereo ribbon diagram illustrating the

overall fold of the HCV NS3 helicase with bound

ssDNA. Domain 1 is coloured blue, domain 2 red

and domain 3 green. DNA is coloured yellow.38

The topologically similar domains 1 and 2

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contain the seven conserved sequence motifs common to all related helicase enzymes.44

They lie in close proximity to each other around the bound ATP, suggesting an essential

involvement in its binding and hydrolysis, or the transfer of ATP-dependent energy to nucleic

acid unwinding. Site-directed mutagenesis studies have shown that mutations in motifs I to

VI impact the ability of the helicase to both hydrolyse ATP and unwind duplex nucleic

acids.45,46.

Domains 1 and 2 host two motifs that are conserved in all HCV isolates but not other similar

helicases. The first, as described by Lam et al.,47 is centred around an arginine residue

(Arg393), which is understood to clamp the helicase to the nucleic acid strand. The second

motif, or Phe-loop, connects two anti-parallel sheets between SF2 motifs 5 and 6.

The role of domain 3 is less well characterised, though it is clearly essential for NS3 helicase

activity as deletion of 97 amino acids from the C-terminus of NS3 inactivates the enzyme.

Site-directed mutagenesis has identified two critical residues: Trp501, which stacks against

the 3'-terminal base of the RNA strand,48 and Glu493, which repels nucleic acids from the

binding cleft whilst ATP is bound in the closed conformation.49

Figure 5. Key residues in the RNA binding cleft of the HCV NS3 helicase

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1.2.3 Mechanism of HCV NS3 Helicase Action

The cycle of helicase activity begins as ATP is bound in the cleft between the two adjacent

domains 1 and 2.42 The subsequent break down (hydrolysis) of ATP into ADP and inorganic

phosphate releases energy to allow domain 2 to rotate away from domain 1.

The exact mechanism through which NS3 helicase unwinds double-stranded RNA is still the

subject of intense debate. Most theories consider the conformational switch to correlate

directly to movement of the enzyme along the nucleic acid. As domains 1 and 2 move along

the tracking strand, one nucleotide at a time, ATP binding and ADP release induce closing

and opening of the two domains, respectively. In its closed conformation, NS3 helicase has a

low affinity for RNA, compared with a high affinity in its open conformation. This means that,

as the enzyme cycles between these two states, it is able to sequentially grab and release the

RNA tracking strand in order to translocate and unwind the polynucleotide.

These changes can be visualised in the interactions between two highly conserved threonine

residues, Thr269 and Thr411, and RNA.50,51 In a crystal structure of NS3 helicase, isolated in

the absence of ATP, these particular residues bind to two RNA phosphates that are 3

nucleotides apart. Structures of similar SF2 helicases show that the closure of domains 1 and

2 upon ATP binding brings the equivalent threonines one nucleotide closer, evidencing the

bulk movement of the protein in a 3'-to-5' direction.

The “ratcheting inchworm” model proposed by Kim et al.38 considers the Trp501 residue of

domain 3 to act as a 3'-bookend, remaining stacked against the 3'-terminal base to maintain

the relative position of the nucleic acid fixed while domains 1 and 2 translocate. This

prediction is validated by site-directed mutagenesis studies, which confirm that HCV helicase

is unable to unwind RNA without a bulky aromatic amino acid at position 501.48 The residue

that acts as the analogous 5'-bookend is believed to be Val432.38,42 The suggested mechanism

for translocation and unwinding of a single base-pair is illustrated in Figure 6.

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Figure 6. A schematic diagram illustrating the mechanism of translocation and unwinding of

a single base-pair by HCV NS3 helicase

Electrostatic analysis of the RNA binding site has identified a key ionisable residue, Glu493,

which must be protonated for the helicase to be optically active.49 This observation led to the

development of the “propulsion-by-repulsion” model for NS3 helicase activity, in which an

acidic patch on the protein surface provides the driving force for its translocation along the

nucleic acid strand.42 Negatively-charged RNA binds in the negatively-charged cleft and is

unable to exit the enzyme as it remains fixed in position by Trp501 and the Arg-clamp of

domain 2. This leads to the build-up of potential energy. As domain 2 rotates, it brings with it

the positively-charged Arg-clamp, thus attracting the negatively-charged phosphodiester

backbone of RNA, which moves free of the Trp501 bookend. The binding cleft then repels the

similarly-charged polynucleotide, which propels through the protein as the potential energy

is released.

1.2.4 HCV NS3 Helicase as a Drug Target

The HCV helicase represents an exciting prospect for the development of novel anti-viral

agents, but has often been overshadowed by work on the more prominent non-structural

proteins, such as the NS5B polymerase or NS3/4A protease.23 As we begin to understand its

mechanism of action, NS3 helicase is increasingly recognised as a crucial target for

inhibition,52 with the potential not only to directly halt viral replication but also to stimulate a

cellular antiviral response brought about by the expected build-up of non-associated duplex

RNA. Despite the wealth of structural information available for well over a decade, only a

small number of NS3 helicase inhibitors have thus far been reported.The possible strategies

of inhibition are highlighted in Figure 7 and summarised below.23

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Inhibition of ATPase activity

Competitive inhibitionof RNA binding

Inhibition of unwinding through intercalation of

the RNA duplex

Figure 7. Strategies for Inhibition of HCV NS3 Helicase

i. Inhibition of ATPase Activity

As previously discussed, the ATPase cycle is required to provide the energy that drives

the protein along the length of polynucleotide.42 Examples of competitive ATPase

inhibitors to be tested include ribavirin 5’-triphosphate (RTP) and ribavirin 5’-

disphosphate (RDP). However, although these compounds showed good ATPase

activity in the low μM range, they failed to elicit a significant reduction in helicase

unwinding rate.23 This unfortunate phenomenon was mimicked by paclitaxel. More

recently, a number of halogenated benzimidazoles and benzotriazoles have shown

improved inhibition,53 while a new class of ring-expanded nucleoside (REN) analogues

have also limited helicase unwinding.

Figure 8. ATPase inhibitors: Structures of benzimidazole and benzotriazole derivatives53

To overcome the problem of partial inhibition, several allosteric mechanisms have

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DRB (R = Cl)DRBB (R = Br)

DRBT (R = Cl) α-DMRB (R = Me) TRBT (R = Cl)TBBT (R = Br)

been investigated, whereby antagonists such as trifluoroperazine block accessibility to

the ATP-binding site in a non-competitive manner.54 Cytotoxicity remains an issue, as

compounds that inhibit ATPase activity may also disrupt analogous host cell

mechanisms.

ii. Inhibition of Unwinding through Intercalation of the RNA Duplex

Nucleic acid duplexes are more stable when bound with an intercalating agent,

requiring a greater amount of energy to unwind. Progress into the development of

RNA-modulators has been slow, however, with effective inhibitors such as epirubicin

and nogalamycin proving to be highly toxic and non-selective.55

iii. Competitive Inhibition of RNA Binding

A more selective mechanism for anti-HCV activity appears to be the competitive

inhibition of RNA binding, which directly correlates to a decrease in unwinding

activity. There are several important leads in this field including QU663, which was

discovered by Maga et al. through optimisation of a series of compounds known to

bind HIV reverse transcriptase at the non-nucleoside binding site.56 QU663

competitively inhibits HCV helicase-catalysed DNA unwinding independent of its

associated ATPase activity, with a Ki of 750 nm. Molecular docking studies suggest

this compound binds at the RNA binding site in place of the polynucleotide, with key

interactions with both Trp501 and Arg393.

Figure 9. Structure of the NS3 helicase RNA

binding inhibitor QU663

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1.3 Molecular Docking

Since its introduction in the early 1980s, molecular docking has fast become established as

an important tool for drug lead discovery and optimisation.57 A common computational

strategy involves the virtual screening of large compound databases, where a docking

program is able to predict all possible complex structures for different ligands bound at the

target protein active-site. These complexes are then scored and ranked by binding energy,

and the best candidates (hits) are selected for further development.

Early approaches to docking, such as the original DOCK algorithm,58 were based simply on a

rigid shape-complementarity between ligand and receptor. It is now clear that rigid docking

is of limited practical use, as the vast majority of ligands tend to exist in a variety of flexible

conformations according to the orientation of peripheral groups and internal bond angles. To

overcome this issue, several incremental construction algorithms including FlexX have been

introduced to pre-generate a variety of low-energy ligand conformations prior to docking and

scoring.59-61 Further improvements, as seen in the docking program GLIDE,62 have allowed the

shape and properties of the receptor to be mapped onto a grid of fields with progressively

more accurate scoring of each docked ligand pose.

All molecular docking studies in this project are performed on GLIDE (grid-based ligand

docking with energetics) software,63 which is chosen for its proven robustness in binding-

mode prediction. It is able to perform a near exhaustive search of the positional,

orientational and conformational space available to the ligand, whilst retaining sufficient

computational speed to cover large compound libraries.62 Initial ligand conformations are

screened across the entire phase space to predict a number of low-energy poses, which are

further minimised by a standard molecular mechanics energy function in conjunction with a

distance-dependent dielectric model. The predicted poses are then ranked by binding affinity

by a composite scoring function (Emodel) based on the 'GlideScore' combined with the

ligand-receptor molecular mechanics interaction energy and ligand strain energy.

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1.4 Research Objectives

RNA binding agents remain at the forefront of investigation into novel HCV NS3h inhibitors.

Previous screening studies at the Welsh School of Pharmacy have identified 1 as a compound

with modest antiviral activity.

Figure 10. Structure of Compound 1

Table 1. Inhibitory activity (μM) of 1 against a cell-based HCV replicon replication and b isolated HCV NS3 helicase enzyme

EC50 (μM) a IC50 (μM) b

Compound 1 8 50

These data provide proof-of-concept in that 1 clearly has an inhibitory effect on viral

replication, although effective concentrations in the high micromolar range suggests

relatively low potency compared with other anti-HCV agents.

This report proposes the design and synthesis of a novel series of potential HCV NS3 helicase

inhibitors, based around the systematic variation of the structure of 1. With the aim of

enhancing potency, and increasing selectivity for the helicase enzyme, a small library of

newly designed compounds will be evaluated by docking simulation using GLIDE.63 The best

ranked poses will then be subject to visual inspection, with the consideration of specific

interactions with the active-site, focusing on the key residues of NS3 helicase action

previously described. The most promising compounds will be selected for synthesis and all

synthesised compounds will be tested in HCV replicon tissue culture and NS3 helicase

enzymatic assays.

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2 Results and Discussion2.1 Structure-based Design: A Novel Compound Library

A series of potential NS3 helicase inhibitors is proposed based around the structural

modification of the symmetrical lead compound 1 at three positions, as shown in Figure 11.

Figure 11. Sites of Lead Compound Modification

The majority of substituents at A retain the aromatic ring (with the exception of a simple

methyl control group), as this is thought to interact with the key Trp501

residue of the

helicase enzyme. Direct replacement of the chlorine atom provides the para-substituted

methyl-, trifluoromethyl-, cyano-, nitro- and methoxybenzene derivatives, while a number of

dimethoxy- variations are also considered. The naphthalene, quinoline, and quinazoline

products are speculated to further enhance the aromaticity of the molecule.

At B, the linker chain is both shortened and extended (to 2C and 4C respectively) to

investigate its effects in coordinating the functional groups at the receptor site. For instance,

it would be desirable to find the most appropriate chain length to bring the aromatic group

into close proximity with the interacting Trp501

residue. Whilst a simple alkyl linker remains

flexible, allowing a multitude of conformations in the active-site, it is also possible to

maintain chain rigidity, with the use of a peptide bond adjacent to the core piperazine. It

21

Modification of linker chain

Variation of end-chain substituent

B

remains to be seen what effect this will have on the binding affinity.

It is important to note that modifications at A and B are consistent on either side of the

molecule, which retains its symmetry as a result. Molecules with dissimilar groups bonded to

each piperazine nitrogen atom would be too difficult to synthesise, and therefore do not

represent realistic candidates for drug development. However, it can be possible to replace

the piperazine group at C with a piperidine group, thus yielding a mono-substituted

asymmetrical product.

The various structural modifications that determine a library of 112 possible novel

compounds are detailed in Table 2. These compounds were docked into the 3KQH HCV

protein structure using GLIDE 63 in its standard precision mode, and the best ranked results

were visually inspected for signs of promising interactions with the RNA binding site.

Table 2. Structural Modifications at Variable Positions A, B and C

A B C

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2.2 Compounds Selected for Chemical Synthesis

Following molecular docking, eight outstanding candidates were selected from the

compound library for synthesis and further development. They were chosen according to a

system that combined both the GLIDE 63 docking score and a visual inspection of interactions

with certain key binding site residues. Ease of synthesis was also an important consideration.

The highly conserved aromatic residue, Trp501, is a prime target in the search for novel NS3

helicase inhibitors. Several reported compounds are known to interact with the RNA binding

cleft in the region of Trp501, possibly interfering with its role in stacking against the 3'-

terminal base of the nucleic acid strand.38,42 It is hypothesised that such interactions will

interrupt the cycle of helicase activity by rendering the tryptophan residue inert, allowing

RNA to move free independent of enzyme conformation. Consideration of the docking

results showed that several compounds formed clear associations with Trp501, particularly

through aromatic naphthalene and dimethoxybenzene groups at the terminal ends of the

symmetrical molecule. Along with those that bind to Val432, which is speculated to act as

the analogous 5'-terminal bookend,38 these compounds represent promising choices to

advance to synthesis.

Interactions with the key ionisable Glu493 residue are exploited by six of the eight selected

compounds, particularly through hydrogen bonds donated by the core heterocyclic ring.

Glu493 is thought to have a highly significant role in the helicase mechanism of RNA

unwinding, acting to repel the negatively-charged nucleic acid upon ATP binding.42,49 Potential

24

inhibitors aim to bind and deionise this glutamic acid residue, thus disintegrating the

proposed cycle of propulsion-by-repulsion along the RNA strand. A similar case is observed

for Arg393, the focal residue of the Arg-clamp in domain 2,47 which associates by hydrogen

bonding to a sulfonyl oxygen atom in several proposed compounds.

Associations with other proximal groups will act to further stabilise the positioning of the

compounds at the RNA binding site, but are of incomparable significance in terms of

mechanistic effects. A good proportion of the chosen compounds demonstrated interactions

with the RNA-binding Thr411 residue, which is considered to be a good target for anti-

helicase activity due to its high sequence conservation between isolates.50,51 This should

confer a good degree of selectivity for the chosen NS3 helicase active-site.

The selected compounds have been numbered according to synthetic reaction schemes 1

and 2. They comprise a logical SAR investigation, in that the end-chain substituent is varied

only between three structures – naphthalene, 2,5-dimethoxybenzene, and 3,4-

dimethoxybenzene – linked to a central piperazine ring by an alkyl chain consisting of two or

three carbon atoms. Note that no compounds with a four-carbon chain are included, since

they were far surpassed by the shorter chain analogues in terms of docking score and key

interactions. Whilst most compounds tend to exceed a molecular weight of 500, they

generally fulfil the Lipinski criteria for good pharmacokinetics. Dimethoxybenzene-

substituted compounds, for instance, demonstrate good aqueous solubility. Log P is

calculated based on the unprotonated product form; hence the true values may fall as the

amide nitrogen is protonated in its natural state. Compounds 22 and 23 remain

unprotonated due to the stabilisation effects of an electron-withdrawing carbonyl group

adjacent to the piperazine ring. 23 has a relatively low docking score of -5.65, but the

presence of two key interactions with Thr411 and Arg393 merits its further investigation. It

will be interesting to consider how peptide rigidity and loss of charge on the amide nitrogen

affects inhibitory activity.

Of the proposed compounds, 13, 16 and 22 showed the most extensive interactions with the

RNA binding site, and thus can be speculated to offer the most promise for in vitro

investigation. Conversely, these compounds did not seem to interact with the crucial Trp501

25

residue, which must surely be weighted in terms of importance to activity. For this reason,

compound 14 demonstrated clear potential, with binding both to Trp501 and the key Glu493

residue. 14 also has a relatively low log P, which is highly significant in terms of delivery to its

target site.

The inclusion of a mono-substituted piperidine ring appeared to be detrimental to

interactions at the RNA binding site. However, compound 17 has been chosen for synthesis

both due to its evident association with the nearby Glu493 residue and its clear superiority

over other candidates in terms of molecular weight.

2.3 Docking Studies

Tables 3 – 10 summarise the results of molecular docking studies in GLIDE,63 with the

protein-ligand interactions displayed alongside the important structural and chemical

properties of the relevant compounds.

It is important to note that docking is merely a predictive interpretation of the most likely

interactions at the binding site, hence visual inspection of ligand orientation amidst

surrounding residues remains integral to investigation. For this reason, the proximal Trp501

residue has been identified by the author as a possible site of interaction with compounds 15

– 17 (shown in brackets), despite not being reflected in the displayed results.

26

Table 3. Key properties of proposed compound (12)

Name N,N'-(diethyl-2-naphthalenesulfonamide)-piperazine

Structure

Molecular Weight 552.72

Log P 2.87

Docking Score -6.61

27

Protein-ligand Interactions

Key Interactions Trp501


Name N,N'-(dipropyl-2-naphthalenesulfonamide)-piperazine

Structure


28

Log P 3.65

Docking Score -7.03


Key Interactions Glu493 Val432 Thr411 Arg393


Name N,N'-(diethyl-2,5-dimethoxybenzenesulfonamide)-piperazine

Structure

29


Log P 0.60

Docking Score -6.63


Key Interactions Trp501 Glu493


Name N,N'-(dipropyl-2,5-dimethoxybenzenesulfonamide)-piperazine

Structure


30

Log P 1.38

Docking Score -6.25


Key Interactions Glu493 Val432 (Trp501)


Name N,N'-(diethyl-3,4-dimethoxybenzenesulfonamide)-piperazine

31

Structure


Log P 0.60

Docking Score -6.77


32

Key Interactions Glu493 Val432 Thr411 (Trp501)


Name N-(propyl-2-naphthalenesulfonamide)-piperidine

Structure


Log P 2.99

Docking Score -5.02

33


Key Interactions Glu493 (Trp501)


Name N,N'-(3,3'-(piperazine-1,4-diyl)bis(3-oxopropane-3,1-diyl))-dinaphthalene-2-sulfonamide

34

Structure


Log P 2.70

Docking Score -7.32


Key Interactions Glu493 Thr411 Arg393


Name N,N'-(3,3'-(piperazine-1,4-diyl)bis(3-oxopropane-3,1-diyl))-di-2,5-dimethoxybenzenesulfonamide

35

Structure


Log P 0.43

Docking Score -5.65


Key Interactions Thr411 Arg393

2.4 Chemical Synthesis

Compounds 12 – 17 were synthesised by reaction scheme 1, involving two distinct SN2

mechanistic steps. All reactions were successful at the first attempt, with good product yields

36

in the range of 66 – 89 per cent. Since most products were isolated in sub-gram quantities,

apparatus error in calculation of mass may render these figures as underestimates.

Dimethoxybenzene-substituted products were simplest to purify, as they were fully soluble in

the solvent and hence were added in solution to the column. In contrast, naphthalene-

substituted products were relatively insoluble and had to be suspended in the solvent.

Compound 13 presented the most difficulty in the work-up, as the large particles formed

from the reaction were not suspended fully for the column. In retrospect, the acceptable

yield of 71 per cent could be improved drastically by refining the particle size prior to

purification. All products eluted well in low percentage methanol in dichloromethane,

although separation was less distinct when the process was attempted with ethyl acetate as

the organic solvent. Intermediate compounds were verified by 1H NMR spectroscopy alone,

while the purity of final products was confirmed by both 1H and 13C NMR. Replicon and

enzymatic assays required a minimum purity of 99 per cent, which was achieved in all cases.

Reaction Scheme 1a

a Reagents and conditions: (i) NEt3, Ch2Cl2, 10 min; (ii) piperazine, NaHCO3, EtOH, reflux, 24 h;

(iii) piperidine, NaHCO3, EtOH, reflux, 24 h.

Step (i): 64 Two equivalents of triethylamine were used: one to release the primary amine

from the ammonium salt to react by nucleophilic substitution (SN2), as shown

by the mechanism in Figure 12, and the second to neutralise the HCl formed

from the reaction.

37

Figure 12. SN2 Mechanism for Reaction Step (i)

Step (ii): 65 Two equivalents of sodium bicarbonate were used: one to deprotonate both

the monosubstituted and disubstituted products formed in the two-stage SN2

mechanism shown in Figure 13. The reactions were successful in that the

monosubstituted species could not be detected by 1H or 13C NMR, since the

reaction had been forced to completion by an excess of the sulfonamide.

Figure 13. Two-stage SN2 Mechanism for Reaction Step (ii)

Step (iii): 65 The monosubstituted piperidine product, formed from a similar SN2

mechanism, was deprotonated by one equivalent of sodium bicarbonate.

38

Figure 14. SN2 Mechanism for Reaction Step (iii)

Compounds 22 and 23 were synthesised by reaction scheme 2. Both carboxylic acid

intermediates were obtained successfully by step (iv) at the first attempt, with good yields

despite some difficulties in maintaining a consistent basic pH. Indeed, proton NMR confirmed

the presence of the product with minimal starting material remaining, allowing 20 and 21 to

be used as reactants for step (v). This proved to be the most challenging step of all the

chemical synthesis, with two failed attempts before achieving a pure product. The reasons

for this are detailed below. Ultimately, however, good product yields of 77 and 80 per cent

were obtained for 22 and 23 respectively.

Reaction Scheme 2a

a Reagents and conditions: (iv) β-alanine, NaOH, H2O, 20˚C, until pH 9 then rt, 1 h; (v)

piperazine, TBTU, N,N-Diisopropylethylamine, Ch2Cl2, rt, 5 h.

Step (iv): 66 One equivalent of 2M NaOH was used to deprotonate the carboxylic acid

39

group of β-alanine, freeing it into solution to react by the SN2 mechanism

indicated in Figure 15. The pH was monitored and 1M NaOH added to

maintain basic conditions at pH 9 to retain the product in solution.

Figure 15. SN2 Mechanism for Reaction Step (iv)

Step (v): 67 The reaction was initially trialled with dimethylformadide (DMF) as a solvent,

but problems in purification of the final compound necessitated a change in

procedure. The product tended to elute from the packed column stacked

alongside N-Hydroxybenzotriazole (HOBt). Better purification was achieved

with dichloromethane in place of DMF, and HOBt was removed from the

reaction to leave TBTU as the solitary peptide coupling reagent.

TBTU is understood to exist in two different isomeric forms, of which the

guanidinium isomer predominates.68 This equilibrium is shown in Figure 16.

Figure 16. Equilibrium Showing the Formation of the Reactive Guanidinium Coupling Agent

N,N-Diisopropylethylamine was used as a base to deprotonate the carboxylic

acid group prior to reacting. TBTU was then used as a coupling agent to form

peptide bonds between two equivalent carboxylate ions and a core piperazine

ring. Figure 17 shows the mechanism for the formation of the

40

monosubstituted species, while Figure 18 shows the addition of a second

carboxylate ion to form the final product.

Figure 17. Mechanism for the Formation of the Monosubstituted Species in Reaction Step (v)

Figure 18. Mechanism for the Formation of the Final Product in Reaction Step (v)

1H NMR Comment: It is interesting to note that, for both dimethoxybenzene- and

41

naphthalene-substituted products, the amino proton presents furthest

downfield with a three-carbon linker chain, and furthest upfield with

two carbons. The peptide-linked compounds 22 and 23 give an

intermediate amino proton shift.

It is also worth commenting that the 1H NMR spectrum for compound

16 does not fully resolve many of the characteristic peak splits, which

instead present as multiplets. However, since the peaks occur in a shift

pattern similar to other compounds, 16 has clearly been isolated in its

purified form.

Overall, NMR spectra are clear and unquestionable, and act to confirm

the purity of the eight final compounds.

42

Table 11. Structures of Compounds 2 - 17

Compound R Y Product Yield (%)

2 - -

3 - -

4 - -

5 - CH2 -

6 - C2H4 -

7 CH2 73

8 C2H4 88

9 CH2 86

10 C2H4 70

11 CH2 89

12 CH2 89

13 C2H4 71

14 CH2 67

15 C2H4 66

16 CH2 66

17 C2H4 75

Table 12. Structures of Compounds 18 - 23

Compound R1 Product Yield (%)

18 -

19 -

20 71

21 72

22 77

23 80

43

2.5 Conclusions and Future Work

Poor efficacy and tolerability of current treatments has necessitated the development of novel

specific therapies for Hepatitis C.23 The Hepatitis C Virus (HCV) NS3 Helicase enzyme was identified

as a promising orphan target for investigation, with no drug candidates currently in clinical trials.

Development of a lead compound with modest antiviral activity yielded a library of 112 potential

inhibitors, which were screened by molecular docking studies for interactions with key RNA

binding site interactions.

Based on the docking results, eight compounds were selected for synthesis and further

development. All proposed compounds were successfully synthesised in good overall yields, and

the study is currently awaiting the results of HCV replicon tissue culture and NS3 helicase

enzymatic assays, which will determine the next stage of investigation. Selectivity may need to be

confirmed through identification of mutation of the helicase enzyme in a resistant viral strain.

Nonetheless, the main priority will be the rational optimisation of the compound(s) showing

greatest activity to ultimately obtain a novel class of potent anti-HCV inhibitors.

44

3 Experimental Section3.1 Molecular Modelling

All molecular modelling studies were performed on a MacPro running Ubuntu using Molecular

Operating Environment (MOE) 69 2009.10 and GLIDE 63 as docking suite molecular modelling

software.

All the minimisations were performed with MOE until RMSD gradient of 0.05 Kcal mol-1 Å-1 with the

MMFF94x forcefield and the partial charges were automatically calculated.

Docking experiments were carried out using GLIDE in Standard Precision (SP) with the default

options. The output of GLIDE docking was visualised in MOE.

3.2 General Synthetic Procedures

General Information

All chemicals, reagents and solvents were purchased from Aldrich or purified by standard

techniques.

Thin Layer Chromatography

Silica gel plates (Merck Kieselgel 60F254) were used and were developed by the ascending method.

After solvent evaporation, compounds were visualised by irradiation with UV light at 254nm and

366nm.

Column Chromatography

Glass columns were slurry packed in the appropriate eluent under gravity, with Woelm silica (32-

63mm). Samples were applied as a concentrated solution in the same eluent. Fractions containing

the product were identified by TLC, combined and the solvent removed in vacuo.

The purified products were obtained by gradient elution beginning with a wash of 200 mL 100%

dichloromethane. Compounds 12, 14, 15, 16 and 17 eluted with 1% methanol, 22 with 2%

methanol, 13 with 3% methanol, and 23 with 5% methanol.

NMR Spectroscopy1H, 13C, DEPT NMR spectra were recorded on a Bruker AVANCE 500 spectrometer (500MHz and

75MHz respectively) and auto calibrated to the deuterated solvent reference peak. Chemical shifts

are given in relative to tetramethylsilane (TMS); the coupling constants (J) are given in Hertz. The

spectra were recorded in CDCl3 or DMSO at room temperature; TMS served as an internal standard

(δ = 0 ppm) for 1H NMR and CDCl3 was used as an internal standard (δ = 77.0 ppm) for 13C NMR.

45

3.3 Synthesis of N-(bromoalkyl)-arenesulfonamides 64

N-(2-bromoethyl)-2-naphthalenesulfonamide (7). 2-naphthalenesulfonyl chloride (2, 1000 mg,

4.42 mmol, 0.99 equiv) and 2-bromoethylammonium bromide (5, 1006 mg, 4.91 mmol, 1.1 equiv)

were dissolved in 8 mL dry dichloromethane under a nitrogen atmosphere. The mixture was

treated dropwise with 1.43 mL triethylamine under ice-cooling and then stirred for 5 min under

cooling. The reaction mixture was washed twice with 15 mL 2M hydrochloric acid and 15 mL

saturated sodium chloride solution. Evaporation of the organic solvent after drying over

magnesium sulphate gave the crude product, as a white powder (73% yield), which was used for

the following step without further purification. 1H NMR (CDCl3, 500 MHz): δ 8.48 (s, 1H), 8.00 (m,

2H), 7.95 (d, J = 7.9 Hz, 1H), 7.87 (dd, J1 = 8.6 Hz, J2 = 1.8 Hz, 1H), 7.67 (m, 2H), 5.16 (s, br, 1H), 3.44

(m, 4H).

N-(3-bromopropyl)-2-naphthalenesulfonamide (8). The procedure was similar to the procedure

for 7 except that 3-bromopropylammonium bromide (6, 1075 mg, 4.91 mmol, 1.1 equiv) was used.

Compound 8 was isolated as a white powder (88% yield). 1H NMR (CDCl3, 500 MHz): δ 8.48 (s, 1H),

8.00 (m, 2H), 7.95 (d, J = 7.9 Hz, 1H), 7.87 (dd, J1 = 8.6 Hz, J2 = 1.8 Hz, 1H), 7.67 (m, 2H), 4.85 (t, J =

6.4 Hz, 1H), 3.45 (t, J = 6.3 Hz, 2H), 3.20 (q, J = 6.4 Hz, 2H), 2.07 (m, 2H).

N-(2-bromoethyl)-2,5-dimethoxybenzenesulfonamide (9). The procedure was similar to the

procedure for 7 except that 2,5-dimethoxybenzenesulfonyl chloride (3, 700 mg, 2.96 mmol, 0.99

equiv) and 2-bromoethylammonium bromide (5, 673 mg, 3.29 mmol, 1.1 equiv) were dissolved in

5.6 mL dry dichloromethane. The mixture was treated dropwise with 0.96 mL triethylamine.

Compound 9 was isolated as a pale yellow oil (86% yield). 1H NMR (CDCl3, 500 MHz): δ 7.44 (d, J =

2.4 Hz, 1H), 7.10 (dd, J1 = 9.1 Hz, J2 = 2.8 Hz, 1H), 6.99 (d, J = 8.9 Hz, 1H), 5.55 (s, 1H), 3.97 (s, 3H),

3.82 (s, 3H), 3.37 (m, 4H).

N-(3-bromopropyl)-2,5-dimethoxybenzenesulfonamide (10). The procedure was similar to the

procedure for 9 except that 3-bromopropylammonium bromide (6, 719mg, 3.29 mmol, 1.1 equiv)

was used. Compound 10 was isolated as a white powder (70% yield). 1H NMR (CDCl3, 500 MHz): δ

7.45 (d, J = 2.9 Hz, 1H), 7.10 (dd, J1 = 9.0 Hz, J2 = 2.9 Hz, 1H), 7.00 (d, J = 8.9 Hz, 1H), 5.54 (s, br, 1H),

3.96 (s, 3H), 3.81 (s, 3H), 3.40 (t, J = 6.2 Hz, 2H), 3.20 (q, J = 6.1 Hz, 2H), 2.10 (m, 2H).

46

N-(2-bromoethyl)-3,4-dimethoxybenzenesulfonamide (11). The procedure was similar to the

procedure for 7 except that 3,4-dimethoxybenzenesulfonyl chloride (4, 900 mg, 3.80 mmol, 0.99

equiv) and 2-bromoethylammonium bromide (5, 866 mg, 4.23 mmol, 1.1 equiv) were dissolved in

7.2 mL dry dichloromethane. The mixture was treated dropwise with 0.96 mL triethylamine.

Compound 11 was isolated as a white powder (89% yield). 1H NMR (CDCl3, 500 MHz): δ 7.52 (dd, J1

= 8.4 Hz, J2 = 2.2 Hz, 1H), 7.36 (d, J = 2.2 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), 4.96 (s, br, 1H), 3.97 (s,

3H), 3.96 (s, 3H), 3.41 (m, 4H).

3.4 Synthesis of N,N'-(alkylarenesulfonamide)-piperazines 65

N,N'-(diethyl-2-naphthalenesulfonamide)-piperazine (12). Compound 7 (766 mg, 2.44 mmol, 2.1

equiv) was dissolved in 4 mL EtOH and added dropwise to a solution of piperazine (100 mg, 1.16

mmol, 1 equiv) and NaHCO3 (205 mg, 2.44 mmol, 2.1 equiv) in 4 mL EtOH. The reaction mixture

was stirred under reflux for 24 h then dried under pressure. The precipitate obtained was

suspended in 50 mL ethyl acetate and washed with 50 mL water and three times with 50 mL

saturated sodium chloride solution. The organic phase was collected and dried under vacuum, and

the white powder (89% yield) purified by acidic gel column chromatography. 1H NMR (CDCl3, 500

MHz): δ 8.45 (s, 1H), 7.97 (t, J1 = 8.2Hz, J2 = 8.8 Hz, 2H), 7.92 (d, J = 8.1 Hz), 7.82 (dd, J1 = 8.7 Hz, J2 =

1.8 Hz), 7.66 (m, 2H), 5.22 (s, br, 1H), 3.01 (s, br, 2H), 2.37 (t, J = 5.8 Hz, 2H), 2.18 (s, br, 4H); 13C

NMR (CDCl3, 75 MHz): δ 137.5, 134.1, 132.1, 129.4, 129.2, 128.8, 128.6, 127.9, 127.7, 122.2, 55.5,

52.3 (x2), 39.2.

N,N'-(dipropyl-2-naphthalenesulfonamide)-piperazine (13). The procedure was similar to the

procedure for 12 except that compound 7 (640 mg, 1.95 mmol, 2.1 equiv), piperazine (80 mg, 0.93

mmol, 1 equiv) and NaHCO3 (164 mg, 1.95 mmol, 2.1 equiv) were used. Compound 13 was isolated

as a white powder (71% yield). 1H NMR (CDCl3, 500 MHz): δ 8.44 (s, 1H), 7.99 (m, 2H), 7.95 (d, J =

7.9 Hz, 1H), 7.83 (dd, J1 = 8.7 Hz, J2 = 1.8 Hz, 1H), 7.66 (m, 2H), 7.32 (s, br, 1H), 3.13 (t, J = 5.5 Hz,

2H), 2.46 (s, br, 6H), 1.66 (m, 2H); 13C NMR (DMSO, 75 MHz): δ 137.4, 134.1, 131.7, 129.3, 129.1,

128.6, 127.8, 127.5, 127.3, 122.2, 54.7, 52.4, 40.9 (x2), 26.0.

N,N'-(diethyl-2,5-dimethoxybenzenesulfonamide)-piperazine (14). The procedure was similar to

the procedure for 12 except that compound 9 (567 mg, 1.75 mmol, 2.1 equiv), piperazine (70 mg,

47

0.83 mmol, 1 equiv) and NaHCO3 (147 mg, 1.75 mmol, 2.1 equiv) were used. Compound 14 was

isolated as a white powder (67% yield). 1H NMR (CDCl3, 500 MHz): δ δ 7.47 (d, J = 3.1 Hz, 1H), 7.08

(dd, J1 = 9.0 Hz, J2 = 3.0 Hz, 1H), 6.96 (d, J = 9.1 Hz, 1H), 5.58 (t, J = 4.9 Hz, 1H), 3.92 (s, 3H), 3.83 (s,

3H), 2.98 (m, 2H), 2.42 (t, J = 5.6 Hz, 2H), 2.31 (s, 4H); 13C NMR (CDCl3, 75 MHz): δ 153.4, 150.3,

127.8, 120.4, 114.9, 113.7, 57.1, 56.1, 56.0, 52.6 (x2), 39.9.

N,N'-(dipropyl-2,5-dimethoxybenzenesulfonamide)-piperazine (15). The procedure was similar to

the procedure for 14 except that compound 10 (592 mg, 1.75 mmol, 2.1 equiv) was used.

Compound 15 was isolated as a white powder (66% yield). 1H NMR (CDCl3, 500 MHz): δ 7.47 (d, J =

3.1 Hz, 1H), 7.08 (dd, J1 = 8.9 Hz, J2 = 3.1 Hz, 1H), 7.00 (d, J = 8.9 Hz, 1H), 6.22 (s, br, 1H), 3.95 (s,

3H), 3.83 (s, 3H), 3.00 (t, J = 6.2 Hz, 2H), 2.55 (s, br, 6H), 1.71 (m, 2H); 13C NMR (CDCl3, 75 MHz): δ

153.4, 150.4, 128.0, 120.2, 114.9, 114.2, 57.3, 56.6, 56.0, 52.8, 40.8 (x2), 25.4.

N,N'-(diethyl-3,4-dimethoxybenzenesulfonamide)-piperazine (16). The procedure was similar to

the procedure for 13 except that compound 11 (632 mg, 1.95 mmol, 2.1 equiv) was used.

Compound 16 was isolated as a white powder (66% yield). 1H NMR (CDCl3, 500 MHz): δ 7.49 (m,

1H), 7.34 (m, 1H), 6.94 (m, 1H), 5.11 (s, br, 1H), 3.96 (m, 3H), 3.94 (m, 3H), 2.99 (s, br, 2H), 2.40 (m,

2H), 2.26 (s, br, 4H); 13C NMR (CDCl3, 75 MHz): δ 152.6, 149.2, 131.3, 121.0, 110.5, 109.7, 56.3,

56.2, 55.6, 52.4 (x2), 39.2.

3.5 Synthesis of N-(propyl-2-naphthalenesulfonamide)-piperidine 65

N-(propyl-2-naphthalenesulfonamide)-piperidine (17). The procedure was similar to the

procedure for 12 except that compound 8 (450 mg, 1.37 mmol, 1.1 equiv), piperidine (106 mg,

1.25 mmol, 1 equiv) and NaHCO3 (115mg, 1.37 mmol, 1.1 equiv) were suspended in 6 mL EtOH

before stirring. Compound 17 was isolated as a pale yellow oil (75% yield). 1H NMR (CDCl3, 500

MHz): δ 8.45 (s, 1H), 7.98 (m, 2H), 7.93 (d, J = 7.9 Hz, 1H), 7.86 (dd, J1 = 8.6 Hz, J2 = 1.8 Hz, 1H), 7.64

(m, 2H), 5.31 (s, 1H), 3.11 (t, J = 5.7 Hz, 2H), 2.34 (m, 6H), 1.63 (m, 6H), 1.48 (s, br, 2H); 13C NMR

(CDCl3, 75 MHz): δ 137.2, 134.7, 132.2, 129.3, 129.1, 128.5, 128.2, 127.9, 127.4, 122.4, 72.1, 59.0,

54.5, 53.4, 44.7, 26.0, 24.2, 23.6.

3.6 Synthesis of 3-(arenesulfonamide)-propanoic acids 66

3-(2-naphthalenesulfonamide)-propanoic acid (20). To partially dissolved β-alanine (500 mg, 5.62

48

mmol, 1 equiv) in distilled water (1.5 mL) was added a solution of 2.81 mL 2M NaOH, followed by

the portion-wise addition of 2-naphthalenesulfonyl chloride (2, 1782 mg, 7.87 mmol, 1.4 equiv).

The reaction mixture was vigorously stirred and a solution of 1M NaOH was added portion-wise to

maintain a pH of 9 at 20°C. After complete consumption of alkali, stirring was continued at rt for an

additional 1 h. Unreacted acid chloride was removed by filtration, and the reaction mixture was

acidified with 5M HCl at 0°C to pH 2. The aqueous solution with solid precipitate was stored in the

refrigerator overnight. The crystals were collected by filtration, washed with cold water and dried

to give the crude product as a white powder (71% yield), which was used for the following step

without further purification. 1H NMR (CDCl3, 500 MHz): δ 8.46 (s, br, 1H), 8.00 (m, 2H), 7.93 (m,

1H), 7.85 (m, 1H), 7.65 (m, 2H), 5.83 (s, br, 1H), 3.23 (s, br, 2H), 2.66 (m, 2H). 13C NMR (DMSO, 75

MHz): δ 171.9, 137.2, 134.0, 131.9, 129.4, 129.2, 128.7, 128.0, 127.7, 127.3, 122.2, 38.8, 33.8.

3-(2,5-dimethoxybenzenesulfonamide)-propanoic acid (21). The procedure was similar to the

procedure for 20 except that β-alanine (250 mg, 2.81 mmol, 1 equiv) was dissolved in 1 mL distilled

water, followed by 1.5 mL 2M NaOH and the portion-wise addition of 2,5-

dimethoxybenzenesulfonyl chloride (2, 931 mg, 3.93 mmol, 1.4 equiv). Compound 21 was isolated

as a white powder (72% yield). 1H NMR (CDCl3, 500 MHz): δ 7.47 (d, J = 3.0 Hz, 1H), 7.11 (dd, J1 =

9.0 Hz, J2 = 3.0 Hz, 1H), 7.00 (d, J = 8.9 Hz, 1H), 5.78 (t, J = 6.6 Hz, 1H), 3.95 (s, 3H), 3.84 (s, 3H), 3.21

(q, J = 6.1 Hz, 2H), 2.61 (t, J = 5.9 Hz, 2H). 13C NMR (DMSO, 75 MHz): δ 172.4, 152.3, 150.2, 128.2,

119.5, 114.2 (x2), 56.4, 55.7, 38.7, 33.9.

3.7 Synthesis of N,N'-(3,3'-(piperazine-1,4-diyl)bis(3-oxopropane-3,1-diyl))-

diarenesulfonamides 67

N,N'-(3,3'-(piperazine-1,4-diyl)bis(3-oxopropane-3,1-diyl))-dinaphthalene-2-sulfonamide (22).

Compound 20 (200 mg, 0.72 mmol, 2.2 equiv) and TBTU (241 mg, 0.75 mmol, 2.3 equiv) were

suspended in 3 mL dry dichloromethane at rt. A solution of piperazine (28 mg, 0.33 mmol, 1 equiv)

and N,N-diisopropylethylamine (0.25 mL, 1.50 mmol, 4.6 equiv) in 1 mL dry dichloromethane was

added, and left stirring at rt for 5 h under a nitrogen atmosphere. The reaction mixture was diluted

with 20 mL dry dichloromethane and washed once with 30 mL 5% citric acid solution, once with 30

mL saturated bicarbonate solution, and once with 30 mL saturated sodium chloride solution. The

organic phase was collected and evaporation of the solvent after drying over magnesium sulphate

gave the crude product as a white powder (77% yield), which was purified by acidic gel column

49

chromatography. 1H NMR (CDCl3, 500 MHz): δ 8.45 (s, 1H), 7.99 (m, 2H), 7.94 (d, J = 7.8 Hz, 1H),

7.86 (dd, J1 = 8.7 Hz, J2 = 1.8 Hz, 1H), 7.66 (m, 2H), 5.59 (m, 1H), 3.51 (m, 2H), 3.28 (m, 4H), 2.54 (m,

2H); 13C NMR (DMSO, 75 MHz): δ 169.2, 137.5, 134.1, 131.7, 129.4, 129.1, 128.7, 127.8, 127.6,

127.4, 122.3, 44.6, 44.2, 40.8, 40.6.

N,N'-(3,3'-(piperazine-1,4-diyl)bis(3-oxopropane-3,1-diyl))-di-2,5-dimethoxybenzenesulfonamide

(23). The procedure was similar to the procedure for 22 except that compound 21 (120 mg, 0.42

mmol, 2.2 equiv) and TBTU (139 mg, 0.43 mmol, 2.3 equiv) were suspended in 4mL dry

dichloromethane. A solution of piperazine (16 mg, 0.19 mmol, 1 equiv) and N,N-

diisopropylethylamine (0.14 mL, 0.87 mmol, 4.6 equiv) in 1 mL dry dichloromethane was then

added. Compound 23 was isolated as a white powder (80% yield). 1H NMR (CDCl3, 500 MHz): δ

7.46 (d, J = 3.0 Hz, 1H), 7.09 (dd, J1 = 9.0 Hz, J2 = 3.0 Hz, 1H), 7.00 (d, J = 9.0 Hz, 1H), 5.92 (t, J = 6.1

Hz, 1H), 3.97 (s, 3H), 3.84 (s, 3H), 3.60 (m, 2H), 3.37 (m, 2H), 3.22 (q, J = 6.0 Hz, 2H), 2.55 (t, J = 5.5

Hz, 2H); 13C NMR (DMSO, 75 MHz): δ 168.9, 152.3, 150.2, 128.2, 119.5, 114.3, 114.2, 56.5, 55.8,

44.5, 44.2, 40.8, 40.5.

50

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