1. No category

Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Pharmacy 197
Design and Synthesis of Hepatitis
C Virus NS3 Protease Inhibitors
Targeting Different Genotypes and Drug-Resistant
Variants
ANNA KARIN BELFRAGE
ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2015
ISSN 1651-6192
ISBN 978-91-554-9166-6
urn:nbn:se:uu:diva-243317
Dissertation presented at Uppsala University to be publicly examined in B41 BMC,
Husargatan 3, Uppsala, Friday, 27 March 2015 at 09:15 for the degree of Doctor of
Philosophy (Faculty of Pharmacy). The examination will be conducted in Swedish. Faculty
examiner: Ulf Ellervik (Lunds tekniska högskola).
Abstract
Belfrage, A. K. 2015. Design and Synthesis of Hepatitis C Virus NS3 Protease Inhibitors.
Targeting Different Genotypes and Drug-Resistant Variants. Digital Comprehensive
Summaries of Uppsala Dissertations from the Faculty of Pharmacy 197. 108 pp. Uppsala:
Acta Universitatis Upsaliensis. ISBN 978-91-554-9166-6.
Since the first approved hepatitis C virus (HCV) NS3 protease inhibitors in 2011, numerous
direct acting antivirals (DAAs) have reached late stages of clinical trials. Today, several
combination therapies, based on different DAAs, with or without the need of pegylated
interferon-α injection, are available for chronic HCV infections. The chemical foundation of
the approved and late-stage HCV NS3 protease inhibitors is markedly similar. This could partly
explain the cross-resistance that have emerged under the pressure of NS3 protease inhibitors.
The first-generation NS3 protease inhibitors were developed to efficiently inhibit genotype 1 of
the virus and were less potent against other genotypes.
The main focus in this thesis was to design and synthesize a new class of 2(1H)-pyrazinone
based HCV NS3 protease inhibitors, structurally dissimilar to the inhibitors evaluated in clinical
trials or approved, potentially with a unique resistance profile and with a broad genotypic
coverage. Successive modifications were performed around the pyrazinone core structure to
clarify the structure-activity relationship; a P3 urea capping group was found valuable for
inhibitory potency, as were elongated R6 residues possibly directed towards the S2 pocket.
Dissimilar to previously developed inhibitors, the P1’ aryl acyl sulfonamide was not essential
for inhibition as shown by equally good inhibitory potency for P1’ truncated inhibitors. In
vitro pharmacokinetic (PK) evaluations disclosed a marked influence from the R6 moiety on
the overall drug-properties and biochemical evaluation of the inhibitors against drug resistant
enzyme variants showed retained inhibitory potency as compared to the wild-type enzyme.
Initial evaluation against genotype 3a displayed micro-molar potencies. Lead optimization, with
respect to improved PK properties, were also performed on an advanced class of HCV NS3
protease inhibitors, containing a P2 quinazoline substituent in combination with a macro-cyclic
proline urea scaffold with nano-molar cell based activities.
Moreover, an efficient Pd-catalyzed C-N urea arylation protocol, enabling high yielding
introductions of advanced urea substituents to the C3 position of the pyrazinone, and a Pdcatalyzed carbonylation procedure, to obtain acyl sulfinamides, were developed. These methods
can be generally applicable in the synthesis of bioactive compounds containing peptidomimetic
scaffolds and carboxylic acid bioisosteres.
Keywords: hepatitis C virus, HCV, NS3 protease inhibitors, structure-activity relationship,
2(1H)-pyrazinone, quinazoline, resistance, Pd catalysis
Anna Karin Belfrage, Department of Medicinal Chemistry, Organic Pharmaceutical
Chemistry, Box 574, Uppsala University, SE-75123 Uppsala, Sweden.
© Anna Karin Belfrage 2015
ISSN 1651-6192
ISBN 978-91-554-9166-6
urn:nbn:se:uu:diva-243317 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-243317)
Till Johan och Hedda ♥
List of Papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.
I
II
III
IV
V
Nilsson, M., Belfrage, A K., Lindström, S., Wähling, H.,
Lindquist, C., Ayesa, S., Kahnberg, P., Pelcman, M.,
Benkestock, K., Agback, T., Vrang, L., Terelius, Y., Wikström,
K., Hamelink, E., Rydergård, C., Edlund, M., Eneroth, A., Raboisson, P., Lin, T-I., de Kock, H., Wigerinck, P., Simmen, K.,
Samuelsson, B., Rosenquist, Å.* Synthesis and SAR of potent
inhibitors of the hepatitis C virus NS3/4A protease: Exploration
of P2 quinazoline substituents. Bioorg. Med. Chem. Lett., 2010,
20, 4004-4011.
Gising, J.,# Belfrage, A K.,# Alogheli, H., Ehrenberg, A.,
Åkerblom, E., Svensson, R., Artursson, P., Karlén, A., Danielson, U. H., Larhed, M., Sandström, A.* Achiral pyrazinonebased inhibitors of the hepatitis C virus NS3 protease and drugresistant variants with elongated substituents directed toward
the S2 pocket. J. Med. Chem., 2014, 57, 1790-1801.
Belfrage, A K., Abdurakhmanov, E., Åkerblom, E., Oshalim,
A., Gising, J., Skogh, A., Danielson, U. H., Sandström, A. Pyrazinone based hepatitis C virus NS3 protease inhibitors targeting genotypes 1a and 3a, and the drug-resistant enzyme variant
R155K. Manuscript.
Belfrage, A K.,* Gising, J., Svensson, F., Åkerblom, E., Sköld,
C., Sandström, A. Efficient and selective palladium-catalysed
C3-urea couplings to 3,5-dichloro-2(1H)-pyrazinones. EurJOC.,
2015, 978-986.
Belfrage, A K., Wakchaure, P., Larhed, M., Sandström, A. Palladium-catalyzed carbonylation of aryl iodides with sulfinamides. Manuscript.
*Corresponding author.
#
These authors contributed equally.
Reprints were made with permission from the respective publishers.
Contents
1 Introduction .............................................................................................. 11
1.1 Hepatitis C Virus ............................................................................. 11
1.1.1 General Background .............................................................. 11
1.1.2 Course and Outcome of HCV Infection................................. 12
1.1.3 The Viral Life Cycle .............................................................. 13
1.1.4 The Viral Proteins .................................................................. 15
1.2 Treatment Strategies ....................................................................... 15
1.2.1 Current Treatment .................................................................. 16
1.3 Resistance to Direct Acting Antiviral Drugs .................................. 19
1.4 HCV NS3 Protease.......................................................................... 21
1.5 Discovery and Development of HCV NS3 Protease Inhibitors ...... 24
1.5.1 A Historical View of the Discovery of HCV NS3 Protease
Inhibitors ................................................................................ 25
1.6 Model Systems for Analyzing HCV NS3 Protease Inhibitors ........ 28
2 Aims of the Thesis ................................................................................... 32
3 P2 Quinazoline Substituents in HCV NS3 Protease Inhibitors
(Paper I) .................................................................................................... 33
3.1 Pharmacokinetic Aspects in Drug Discovery ................................. 33
3.2 Harmonizing Antiviral Potency with PK Properties in the
Development of HCV NS3 Protease Inhibitors (Paper I) ............... 34
3.3 Synthesis of P2 Quinazoline-Containing HCV NS3 Protease
Inhibitors ......................................................................................... 39
4 2(1H)-Pyrazinone-Based HCV NS3 Protease Inhibitors
(Papers II and III) ..................................................................................... 45
4.1 Pyrazinones in Medicinal Chemistry .............................................. 45
4.2 From Tripeptides to Pyrazinone-Based HCV NS3 Protease
Inhibitors ......................................................................................... 46
4.3 Development of Achiral Inhibitors (Paper II) ................................. 46
4.4 Exploration of the P1P1’ Substituents (Paper III)........................... 56
4.5 P3P4 Urea Elongations (Paper III) .................................................. 61
4.6 Evaluation Towards the Genotype 3a of HCV NS3 Protease
(Paper III) ........................................................................................ 64
5 Synthesis of Pyrazinone-Based HCV NS3 Protease Inhibitors
(Papers II and III) ..................................................................................... 66
5.1 Formation of the 2(1H)-Pyrazinone Core ....................................... 66
5.2 Introduction of Urea Substituents ................................................... 68
5.3 Synthesis of P1P1’ Building Blocks ............................................... 70
5.4 Amide Coupling of P3 Pyrazinones and P1P1’ Building Blocks ... 73
5.5 Final Decorations and Modifications .............................................. 74
6 Method Development (Papers IV and V) ................................................. 76
6.1 Palladium as a Catalyst in Organic Synthesis ................................. 76
6.2 C-N Urea Arylation ......................................................................... 76
6.3 Palladium Catalyzed Urea Couplings to 3,5-Dichloro-2(1H)Pyrazinones (Paper IV) ................................................................... 77
6.4 Palladium Catalyzed Carbonylations .............................................. 84
6.5 Synthesis of Acyl Sulfinamide, a Versatile Functionality
(Paper V) ........................................................................................ 85
7 Concluding Remarks ................................................................................ 92
8 Acknowledgements .................................................................................. 94
9 References ................................................................................................ 97
Abbreviations
ACCA
ADME
Ala or A
Arg or R
Asp or D
CD
CDI
Clint
CMC
CO
CYP3A
Cys or C
DAA
DBU
DCM
DFT
DMAP
DME
DMSO
dppe
dppp
EC50
ER
FRET
Gln or Q
Gly or G
Gt
HATU
His or H
HPLC
aminocyclopropane carboxylic acid
absorption, distribution, metabolism
and excretion
alanine
arginine
aspartic acid
candidate drug
carbonyl diimidazole
intrinsic clearance
chemistry, manufacturing and control
carbon monoxide
cytochrome P450 Isoform 3A
cysteine
direct-acting antiviral agent
1,8-diazabicyclo[5.4.0]undec-7-ene
dichloromethane
density functional theory
4-dimethylaminopyridine
1,2-dimethoxyethane
dimethylsulfoxide
1,2-bis(diphenylphosphino)ethane
1,3-bis(diphenylphosphino)propane
inhibitor concentration giving 50%
inhibition in a cell-based system
endoplasmic reticulum
fluorescence resonance energy transfer
glutamine
glycine
genotype
N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridine-1-yl-methylene]-Nmethylmethanaminium hexafluorophosphate N-oxide
histidine
high-performance liquid chromatography
HSPG
HTA
HCV
IFN
IRES
LC-MS
log D7.4
Lys or K
Met or M
MW
NMR
NS
Papp
Pd
PEG
Phe
PK
pKa
RBV
RNA
SAR
Ser or S
SOC
SRB
SVR
THF
Thr or T
TMS
Val or V
wt
Xantphos
heparan sulfate proteoglycan
host-targeting antiviral agent
hepatitis C virus
interferon
internal ribosome entry site
liquid chromatography-mass
spectrometry
logarithm of the distribution coefficient
at pH 7.4
lysine
methionine
microwave
nuclear magnetic resonance
non-structural
apparent permeability coefficents
palladium
pegylated
phenyl alanine
pharmacokinetics
the acid dissociation constant at logarithmic scale
ribavirin
ribonucleic acid
structure-activity relationship
serine
standard of care
scavenger receptor class B
sustained virological response
tetrahydrofurane
threonine
trimethylsilyl
valine
wild-type
4,5-Bis(diphenylphosphino)-9,9dimethylxanthene
1 Introduction
1.1 Hepatitis C Virus
Forty years ago, in 1974, it was found that a previously unknown infectious
agent, different from the known hepatitis A and B viruses, caused transfusion-associated hepatitis.1 Fifteen years later, in 1989, the viral agent was
characterized and named hepatitis C virus (HCV).2 Another twenty-two
years later, in 2011, the first direct-acting antiviral agents (DAAs) against
HCV entered the market. This was the beginning of a new era in HCV history.
1.1.1
General Background
Each year, 350 000-500 000 people die from HCV-related liver diseases.3 In
the western world, HCV is the primary indication for liver transplantations,
and the supply of organs remains less than the demand.4 It is estimated that
130-150 million people worldwide are chronically infected, but the majority
of people carrying HCV are still undiagnosed.3,5 HCV is unevenly distributed among the different regions of the world, as well as between age and risk
groups within countries.6 The highest rates of prevalence are found in North
Africa and the Middle East.7 Approximately 15% of the citizens of Egypt are
infected; this high figure is the result of a health campaign running from the
1950s to the 1980s which had the objective of reducing the prevalence of
schistosomiasis, a parasitic worm infection. The campaign was successful,
but HCV was widely spread among the population at the same time, because
of insufficient knowledge about transmittance of the virus. In North America, Australia, Japan and Northern and Western Europe, the prevalence is
lower, with an upper limit of 2%.6
Unsafe drug injections are nowadays the most common method for
transmission of HCV in developed countries, whereas unscreened blood or
poor handling of medical equipment in healthcare are still common reasons
in resource-poor countries.8 The virus can also be transmitted sexually or
from infected mother to her baby, but these causes of infection are less
common.3
Six major genotypes (Gts) and several subtypes of HCV are unevenly distributed over the world (Figure 1). Gt 1 is found worldwide, and is the major
variant in America, Europe, Australia and Japan. Gt 2 is found in the indus11
trialized countries as well as in South America and Asia. Gt 3, a variant that
appears to be increasing in prevalence, is common in Europe, North America, Australia and Southern Asia. Gts 4-6 are isolated to specific countries:
Gts 4-5 in Africa and the Middle East and Gt 6 in Southeast Asia.6
Figure 1. Global distribution of HCV genotypes.9 Reproduced with the permission
of the publisher.
1.1.2 Course and Outcome of HCV Infection
HCV causes both acute and chronic hepatitis. One to two weeks after exposure to the virus, HCV ribonucleic acid (RNA) can be detected in the serum.
Subsequently, the levels of serum alanine aminotransferase elevate, indicative of hepatocyte injury. The symptoms are diffuse, (they include flu-like
symptoms, nausea and jaundice) but are often absent during the early phases
of the infection. Therefore, infected persons seldom seek medical care at this
stage.
About 20% of those infected will recover spontaneously but around 80%
will develop a chronic infection, often asymptomatic for up to 20-30 years.
Progression to cirrhosis is estimated to occur in 15-20% of those with a
chronic infection; cirrhosis can ultimately develop into end-stage liver disease or hepatocellular carcinoma.10 Liver transplantation is the only lifesaving alternative at this stage.4
12
Figure 2. A schematic representation of the HCV life cycle. Attachment and entry
into the hepatocyte is followed by release of (+) RNA into the cytoplasm, translation by the ribosomes on the endoplasmic reticulum, polyprotein processing into ten
mature viral proteins, replication and assembly and release of new virus particles.11
Reproduced with the permission of the owner.
1.1.3 The Viral Life Cycle
HCV belongs to the virus family Flaviviridae; it is a single-stranded (+)
RNA virus.12 The viral RNA genome is protected by a nucleocapsid layer
and the envelope glycoproteins E1 and E2. Replication of HCV mostly occurs in the hepatocytes, which are present in the liver.10,13 The HCV viral life
cycle is illustrated in Figure 2, and described in the text below.
Cell binding and entry: The HCV particles that circulate in the serum of an
infected person can be bound to host lipoproteins or immunoglobulins or be
present as free HCV particles.14 Both the lipoproteins and the envelope glycoproteins E1 and E2 are crucial for the interaction between HCV and the
host cell.15 Several host proteins are involved in the HCV-host interaction
and subsequent cell entry, which takes place by an endocytosis process.11
Some of the host cell proteins bind directly to the envelope glycoproteins,
while others have indirect functions essential for binding and entry.15 The
tetraspanin CD81 binds directly to E2, but also contributes to post-binding
processes which assist in the internalization of the virus particle. The scavenger receptor class B1 (SRB1) is another direct binding partner for E2.
Moreover, SRB1 is a receptor for lipoproteins, which could provide addi13
tional interactions with the viral particle. It has also been suggested that
SRB1 is involved in the entry of HCV into cells. Unlike the other known
host factors, the only identified mission for the heparan sulfate proteoglycans
(HSPGs) is to mediate HCV attachment to target cells via E1 and E2. The
tight-junction protein claudin 1 interacts with CD81 rather than directly with
the E1 or E2, but is nevertheless essential for HCV entry. A second tightjunction protein, occludin, is also important in the HCV entry process, but
whether it has direct or indirect interactions with the HCV envelope is not
yet understood.13,15
Translation and processing: Inside the cell, the RNA genome is released into
the cytoplasm, where translation occurs in ribosomes at the endoplasmic
reticulum (ER). The translation of the HCV genome encodes a polyprotein
of more than 3000 amino acids, which subsequently cleaves into ten mature
structural and non-structural (NS) proteins (Figure 3, further described in
1.1.4). The structural proteins and the p7 protein are processed by the ER
signal peptidase. The NS proteins (NS2/3/4A/4B/5A/5B), on the other hand,
are processed by viral proteases. These proteins are essential for replication,
and for subsequent formation of new virus particles. The NS3 protease is the
drug target in focus of this thesis and will be described in more detail in section 1.4.14,16
RNA replication: After the described cleavages into mature proteins, a replication complex based on several viral proteins is formed on the ER membrane.13 A template, the complementary negative-sense RNA strand, is first
synthesized, and this is followed by replication of the positive-sense RNA.11
Virion assembly and release: The newly formed positive-sense RNA is successively incorporated into new virus particles. The assembly of new virus
particles is not completely understood, but it has been suggested that the
viral envelope is built at the ER membrane. Finally, the virus particles are
thought to be released through the secretory pathway.11,13
14
Figure 3. Schematic representation of the organization of the HCV polyprotein. The
host cell enzymes, NS2 protease and NS3 protease cleave the protein into structural
and non-structural proteins.
1.1.4 The Viral Proteins
The structural HCV proteins C, E1 and E2 make up the outer structure of the
virion (Figure 3). The core protein (C) forms the nucleocapsid, which surrounds the genetic material. E1 and E2 are glycosylated proteins which form
a non-covalent complex, integrated in the viral envelope. The membrane
protein p7 is thought to form ion channels, and could be important in the
maturation and release of the viral particles.14
The zinc-dependent metalloprotease NS2, together with the N-terminal of
NS3 protease cleave at the NS2/NS3 junction. In complex with the cofactor
NS4A, NS3 protease cleaves at four junctions of the polyprotein (i.e. the
junctions between NS3/4A, NS4A/4B, NS4B/5A and NS5A/5B).17
The membrane-bound NS4B protein induces the formation of the membranous web, which works as a scaffold for the HCV replication complex.11
Although not fully understood, the phosphoprotein NS5A has multiple functions that are essential in the HCV replication process, viral assembly and
viral release.11,18 The RNA-dependent RNA polymerase NS5B catalyzes the
replication of viral RNA.11
1.2 Treatment Strategies
The purpose of therapy is to eliminate HCV from the body (i.e. to achieve a
sustained virological response, SVR), and thus to avoid HCV-related liver
and extra-hepatic diseases (e.g. fibrosis, cirrhosis and death).19 HCV does
not incorporate into the host cellular DNA, meaning that an HCV infection
can be cured, in contrast to for example an HIV infection.20,21 There is still
no preventative vaccine available, although progress has been made in this
research field.22
15
All stages of the HCV life cycle could be targets for DAAs. Indeed, most
of the viral proteins described above have been evaluated as drug targets,
with varying levels of success.23
NS3 protease has been widely investigated in recent decades and these efforts have yielded several inhibitors, both the so-called serine-trapping electrophilic inhibitors (Figure 4) and product-based inhibitors (Figure 5), which
are currently in varying stages of development. NS5A protein inhibitors and
NS5B polymerase inhibitors are also now available as anti-HCV drugs (Figure 6). In addition, so-called host-targeting antivirals (HTAs), which focus
on the host factors required for viral replication, are being considered.20,23
Before 2011, the standard of care (SOC) for HCV infection consisted of a
weekly injection of the immunomodulatory agent interferon (IFN)-α attached to polyethylene glycol (PEG) in combination with the broad-spectrum
antiviral ribavirin (RBV). This therapy continued for 24 to 48 weeks, with
varying success depending on the genotype of the virus.24 For Gt 1 infection,
the SVR ranges from 42% to 46%. For Gts 2 and 3, the SVR is somewhat
higher, between 76% and 80%.25 Factors like sex, age, body weight and coinfections also influence the outcome of this treatment regimen. In some
cases the side-effects, such as influenza-like symptoms, depression and hematological abnormalities, cause patients to abandon the treatment.24
Figure 4. The electrophilic HCV NS3 protease inhibitors telaprevir26 and boceprevir27.
1.2.1 Current Treatment
In 2011, the “first-wave-first-generation” HCV NS3/4A protease inhibitors,
telaprevir28
(IncivekTM,
Vertex)
and
boceprevir27
(VictrelisTM,
Merck/Schering) were approved for use in combination with PEG-IFN-α and
RBV against Gt 1 infection (Figure 4). The launching of these drugs was the
beginning of a new era, demonstrating that DAAs for HCV could reach the
market. The cure rate increased to 75% with the addition of telaprevir to
SOC and to 70% for boceprevir. Also, a factor of significant importance, the
treatment was reduced to 24-28 weeks, compared to 48 weeks with the previous SOC.7 Despite these treatments having similar potency, efficacy, side
effects and cost, sales for the third quarter of 2011 to the fourth quarter of
16
2012 were $ 2.11 billion for telaprevir and $ 0.114 billion for boceprevir;
telaprevir became the fastest drug in history to reach a billion dollars in
sales.5
Figure 5. HCV NS3 protease inhibitors in clinical trials or approved. Simeprevir,29
paritaprevir,30 danoprevir,31 vaniprevir,32 grazoprevir,33 asunaprevir,34 faldaprevir,35
vedroprevir,36 sovaprevir37 and ciluprevir.38
This appeared to be a tremendous step forward in the struggle to cure HCVinfected patients. However, these drugs have disadvantages that have impeded broad, lasting success. For instance, both drugs need to be taken in high
doses and both are associated with side effects, such as anemia and severe
17
rashes, in addition to the adverse effects associated with PEG-IFN-α and
RBV.7 In some patient populations the efficacy is low,5 and the risk of resistance development has been detected both in vitro and in vivo for these
inhibitors.39–41
Figure 6. Approved DAAs used in anti-HCV therapeutic regimens: sofosbuvir
(NS5B nucleoside inhibitor),42 ledipasvir43 and ombitasvir44 (NS5A inhibitors) and
dasabuvir (NS5B non-nucleoside inhibitor).45
In 2013 the “second-wave-first-generation” NS3 protease inhibitor simeprevir29 (OlysioTM) (Figure 5) and the NS5B polymerase inhibitor sofosbuvir46 (SovaldiTM) (Figure 6) were approved; these drugs have more beneficial dosage regimens, fewer side effects and better pharmacokinetic (PK)
properties than previous therapies.47,48 Due to this competition from new
anti-HCV drugs, the sale of telaprevir was discontinued in 2014, three years
after it entered the market as a successful drug.49 In January 2015 it was announced that Merck planned to stop selling boceprevir by the end of 2015.
The biggest weakness in the current treatment regimens is the reliance on
PEG-IFN-α and RBV, as required for preventing the emergence of resistant
variants.21,23 Recently, the first all-oral combination therapy, HarvoniTM,
reached the market. This contains a fixed-dose combination pill of ledipasvir
(NS5A inhibitor)43 (Figure 6) and sofosbuvir42 and is the first approved regimen that does not require concurrent administration of PEG-IFN-α or RBV.
Soon after, a combination therapy containing simeprevir and sofosbuvir,
without PEG-IFN-α or RBV, was also approved.
In December 2014, a so-called “3D” (three DAAs) therapy, Viekira
PakTM, developed by AbbVie (Abbott/Enanta), was approved. This new
treatment regimen consists of a combination pill, ViekiraxTM, containing the
antiviral drugs paritaprevir (NS3 protease inhibitor)30 (Figure 5) and om18
bitasvir (NS5A inhibitor)44 (Figure 6) plus ritonavir; it is intended to be added to dasabuvir (ExvieraTM; a non-nucleoside NS5B polymerase inhibitor)45
(Figure 6) with or without RBV, for HCV Gt1 infections. The NS3 protease
inhibitor in this therapy, paritaprevir, is highly metabolized by the enzyme
cytochrome P450 isoform 3A (CYP3A). As a consequence, ritonavir, which
is a strong inhibitor of CYP3A, is needed to prolong the half-life and increase the plasma concentrations of paritaprevir. This calls for careful planning and monitoring in order to avoid drug-drug interactions, since CYP3A
metabolizes many other drugs. Another concern is the relatively short halflife of the polymerase inhibitor dasabuvir, since it needs to be administered
twice daily, which complicates the overall dosage regimen.30,44 However, the
advantage of this 3D therapy is that PEG-IFN-α is not needed and the sideeffects are relatively mild (e.g. fatigue, itching and nausea).30,45
These drugs will most likely be followed by new therapies as several
DAAs (e.g. HCV NS3 protease inhibitors, Figure 5) are currently being
evaluated, in various combinations, in clinical trials.23 However, at the same
time, the recent approvals have forced the withdrawal of several advanced
DAAs in late clinical trials because of the changes in the accessability of
HCV drugs and a few additional benefits associated with new DAAs.
1.3 Resistance to Direct Acting Antiviral Drugs
Because of fast turnover rate and lack of a proofreading function of the
NS5B polymerase, HCV rapidly produces mutated variants which results in
a wide genetic diversity of different genotypes (Gts 1 to 6) and subtypes (a,
b, c…). In an infected individual an array of slightly different virus strains,
so-called quasispecies, can be present.50 Under the pressure of selective antiviral drugs, resistance-associated amino acid substitutions emerge in the
drug target, which is a major challenge in anti-HCV drug discovery.51 The
striking similarities in structure among advanced HCV NS3 protease inhibitors (Figure 5) could partly explain the cross-resistance that has been observed during evaluation of this class of DAAs.18
The strategy in the IFN-free treatments that are now available is to combine DAAs of different targets and dissimilar resistance profiles to create a
high enough resistance barrier to inhibit the development of resistant variants.21,23 Table 1 summarizes the level of the barrier to resistance, the extent
of genotype coverage and the extent of antiviral activity of the different classes of DAA.
19
Table 1. Barrier to resistance, genotype coverage and antiviral activity18
Barrier to
resistance
Genotype
coverage
Antiviral
activity
NS3
protease
inhibitors
NS5A
inhibitors
NS5B
nucleoside
inhibitors
NS5B
non-nucleoside
inhibitors
Low
Low
High
Low
Narrow/Broad
Broad
Broad
Narrow
High
High
Intermediate/
High
Low/
Intermediate
NS3/4A protease inhibitors: Both in vitro and in vivo studies have shown
selection of drug-resistant variants under the pressure of NS3/4A protease
inhibitors. Several inhibitors in this class show overlapping resistance profiles. Common positions for amino acid substitutions caused by mutations
that lead to resistance are R155, A156 and D168. The R155K/T/Q substitution confers resistance to most NS3 protease inhibitors, both approved and in
clinical trials (Table 2).18
Table 2. Substitutions at amino acid positions within the NS3/4A protease associated with resistance 18,52
V36A/M
Telaprevir
Boceprevir
Simeprevir
Paritaprevir
Danoprevir
Vaniprevir
Grazoprevir
Asunaprevir
Faldaprevir
Vedroprevir
Sovaprevir
Ciluprevir
T54S/A
V55A
Q80R/K
R155K/T/Q
A156S/T/V
D168A/V/T/H
*
*
*
*
*
*
D170A/
T
*
In vitro data.
NS5A inhibitors: Although the barrier to resistance is low (Table 1), no
cross-resistance with DAAs from other classes has been observed. Thus,
NS5A inhibitors may be useful in combination therapies. Indeed, as described above, the NS5A inhibitor ledipasvir was recently approved in fixed
combination with sofosbuvir, without the need for IFN-α and ribavirin18 and
the NS5A inhibitor ombitasvir was later approved in combination therapy
containing three DAAs.41
NS5B polymerase inhibitors: Since NS5B polymerase is essential for HCV
replication, it has been a well-studied target for DAAs.18 The DAAs that are
20
active against NS5B polymerase are divided into two classes: the nucleoside
and non-nucleoside polymerase inhibitors.
The nucleoside polymerase inhibitors are associated with a high genetic
barrier to the development of resistance (Table 1). Although a single substitution close to the active site of the enzyme, i.e. S282T, impaired the potency
of this class of inhibitors in vitro, this mutation has shown limited relevance
clinically. Moreover, they show a broad genotype coverage.18,53
The non-nucleoside inhibitors bind to allosteric sites distant from the active site of NS5B polymerase. As a consequence, the barrier to resistance is
low, since mutations at this site do not affect the function of NS5B polymerase (Table 1).53
1.4 HCV NS3 Protease
The HCV NS3 protein consists of a protease and a helicase/nucleoside triphosphatase.11 The NS3 serine protease located in the N-terminal, makes up
one-third of the NS3 protein. In complex with its cofactor NS4A,17 it plays
an important role in the HCV life cycle since it is essential for the formation
of infectious viral particles.16,54,55 Cleavage of the NS3/4A site occurs intramolecularly (i.e. cis), while the other cleavages occur intermolecularly (i.e.
trans)54 (Figure 3). In general, Schechter-Berger nomenclature is used when
describing interactions between a protease and its substrates or inhibitors
(Figure 7).56
Figure 7. A schematic outline of protease-substrate interactions. Schechter-Berger
nomenclature56 is used to describe protease-substrate interactions. The sidechains
(P) are numbered according to their positions relative to the cleavage site (scissile
bond). The corresponding subsites of the protease are denoted S.
The amide bond that is cleaved by the protease is called the scissile bond.
The amino acid residues on the N-terminal side are denoted P1, P2, P3, etc.,
starting from the scissile bond, while the C-terminal residues are named P1’,
P2’, P3’, etc., going in the other direction from the scissile bond. The corresponding subsites of the protease are termed S1, S2, S3 and S1’, S2’, S3’.
The P1 residue in the NS3 protease substrates is essential for substrate
recognition, showing specificity for small hydrophobic residues, preferably a
21
cystein, which stack to the aromatic side chain of the phenylalanine
(Phe154) positioned in the S1 pocket.57,58
In addition to cleaving the HCV polyprotein, the NS3 protease suppresses
the host’s immune response by limiting IFN production.59 Thus, inhibition of
the NS3 protease both blocks replication of viral particles and restores the
innate immune response. As described above, four NS3 protease inhibitors
have been approved as agents in anti-HCV therapies (Figures 4 and 5).
Structure of the NS3 protein
In 1996, the protease domain of the NS3 protein was structurally solved by
X-ray crystallography, which established a classification into a chymotrypsin-like serine protease stabilized by coordination to a Zn2+ ion. Unlike other
chymotrypsin family proteases, the binding site of the NS3 protein is flat and
featureless without specific pockets or cavities.60,61
Figure 8. The full-length NS3 protein and the N-terminal part of NS4A (pdb code
1CU1).62 NS3 protein: grey; NS4A: blue; catalytic triad: magenta; NS3 helicase:
green; P6-P1 cleavage product from the NS3/4A cleavage site: orange.
In 1999, the crystal structure of the full-length NS3 protein (i.e. the bifunctional protease-helicase) was determined (Figure 8). As a result of this it was
found that the protease and the helicase domains are covalently linked by a
short solvent-exposed strand. The catalytic site is located in the interface
between the protease and the helicase (Figure 8). It is considered possible
that the bifunctional protease-helicase makes up for the lack of specific cavities in the protease, resulting in strong interactions with the substrate.62
22
Catalytic mechanism
The serine protease, HCV NS3, follows the catalytic mechanism outlined in
Figure 9. The catalytic triad comprises Ser139, His57 and Asp81. Ser139
and Gly137 compose the oxyanion hole.
Asp 81
Asp 81
a)
b)
O
O
O-
OH
P1
N term.
N
H
O
H
N
N
C term.
P 1'
O
P1
N term.
His 57
N
H
H
N
N
H
N
H
O
Gly 137
H
N
H
N
O-
Asp 81
P1'
H
N
Ser 139
H
N
d)
O
O
P1
N
H O-
N
N
H
N
Gly 137
H
H O
H
N
Ser 139
N
H O-
His 57
O
P1
N term.
O
H
C term.
C term.
P1'
O-
His 57
Asp 81
H 2N
N term.
H
N+
O
Gly 137
c)
O
Gly 137
H
OH
N
H
N
Ser 139
O-
O
H
N
H
N+
His 57
Ser 139
Asp 81
e)
O
P1
N term.
OH
OH
N
H
N
O
N
H
N
Gly 137
His 57
OH
H
N
Ser 139
Figure 9. General mechanism for serine protease substrate cleavage. a) The substrate, positioned in the active site, is attacked at the P1 carbonyl by the activated
serine. b) Proton transfer from His57 enables the collapse of the tetrahedral intermediate, yielding the covalent acyl complex and releasing the C-terminal fragment.
c) Water attacks the complex, which produces a second tetrahedral intermediate. d)
Protonation of Ser139 by His57 forms (e) the N terminal cleavage product.63
23
1.5 Discovery and Development of HCV NS3 Protease
Inhibitors
The fact that HCV NS3 protease inhibitors are available on the market
means that these inhibitors have successfully passed the entire process of
drug discovery and development. The goal of drug discovery is to develop
and identify candidate drugs (CDs) that have properties favorable for success
in the subsequent processes of drug development.
In general, the drug discovery process starts with validation of the drug
target and identification of a hit compound (screening libraries of synthesized compounds or starting from the natural ligand of the target, e.g. the
natural substrates of enzymes). Structure-activity relationship (SAR) studies
locate crucial moieties important for biological activity. Lead optimization to
improve binding to the target and the in vitro and in vivo PK properties will
subsequently support the selection of a CD.64,65
Prior to evaluation in humans, animal models are used to establish the
dosage regimen and determine potential toxicities in heart, lungs, brain, kidney, liver and digestive system. Chemistry, manufacturing and control
(CMC) includes establishment of the physicochemical properties of the
compound and the synthetic process for generating it on a kilogram to ton
scale. The best formulation is also agreed on. If pre-clinical testings and
CMC provide information that supports approval by the regulatory authorities, the compound will enter clinical trials.64
Clinical trials comprise three or four stages which can take 5-7 years to
complete. In Phase I trials, the safety and dosage are determined in 100-200
healthy volunteers. In Phase II trials, a small group of sick patients is exposed to the drug to evaluate efficacy and safety. In the following Phase III
trials, a large group of patients is included in order to determine efficacy and
safety more reliably. In some cases, a Phase IV study is also carried out; this
is called post-market surveillance, and is used to detect any unexpected side
effects.64 With a verified desired effect and an established acceptable toxicity
and safety profile, the drug can be submitted for marketing approval.
From peptides to peptidomimetics
As mentioned above, the starting point in the development of a new drug
could be the natural ligand of the target; this is a relevant strategy in HCV
drug discovery since NS3 protease inhibitors are often derived from the natural peptide substrates of the protease (see section 1.5.1). Nevertheless, due
to their often very flexible and unstable structures, peptides have poor oral
bioavailability, are rapidly degraded by proteases, permeate cell membranes
poorly, are associated with nonselective receptor binding, and their preparation is often time-consuming.66
A proven strategy for circumventing these difficulties is to develop peptide-like (peptidomimetic) compounds designed to exert the same biological
24
effects as the natural substrates but with modified properties such as improved stability, bioavailability and selectivity. SAR studies, which identify
the most important amino acids in the peptide, are often used in this process.
The peptide is systematically truncated to find the shortest sequence needed
for affinity. Thereafter, crucial properties like stereochemistry, charge and
hydrophobicity can be elucidated by exchanging the amino acids in the peptide sequence.67 The peptide backbone can be replaced with building blocks
that is less prone to protease cleavages, e.g. the replacement of amide bonds
with azapeptides, ureas, sulfonamides or extensions of the peptide chain by
introducing a methylene linker or a heteroatom.67,68 The side chains, i.e. the
individual amino acids that make up the peptide, are often responsible for
strong, specific interactions between the protein and the peptide.67 Thus, a
peptidomimetic scaffold needs to allow the substituents to remain in the
correct 3D-position in order to achieve the highest potency for the compound. Conformational restrictions in the structure, e.g. macrocyclizations or
introduction of rigid components into the backbone, enable pre-organization
of the bioactive conformation, which decreases the entropic penalty upon
binding to the active site. This strategy can therefore improve the potency,
and also increase the metabolic stability and permeation characteristics of
the compound.
The discovery of the highly potent HCV NS3 protease inhibitor ciluprevir
(Figure 5, section 1.2.1) is an example of successful drug design which started from the peptide substrate. The process will be summarized in the following section.
1.5.1 A Historical View of the Discovery of HCV NS3 Protease
Inhibitors
Independently of each other, Istituto di Ricerche di Biologica Molecolare
(IRBM) in Italy and Boehringer Ingelheim in Canada found that NS3 protease was inhibited by its own cleavage products.58,69 This higher affinity for
the cleavage product than for the corresponding substrate is an unusual property.69 This pioneering discovery initiated the design and evaluation of peptide-based inhibitors that have engaged numerous researchers in both academia and the industry (Figure 10).
Initial modifications
Initial optimization of the peptides (Figure 10) revealed that a C-terminal
carboxylic acid was crucial for potency.57,58 An acetyl capping group on the
N-terminal also increased the potency to some extent.57,70 Replacement of the
cysteine in P2 with a proline, which rigidified the backbone, gave hexapeptide B. Furthermore, the P1 substituent influenced on the potency signifi-
25
cantly, in agreement with other peptide-based inhibitors of serine
proteases.58,71
Figure 10. Hexapeptides derived from cleavage products from the NS4A/4B (A)69
and NS5A/5B (B)58 cleavage sites.
The P1 cysteine appeared to be important for substrate specificity, which
possibly relied on interactions between the sulfhydryl group and the aromatic side chain of Phe154 positioned in the S1 pocket.57,58 In order to replace the metabolically unstable cysteine, a norvaline was identified as a
promising alternative (C, Figure 11).70
Figure 11. Replacement of the cysteine in B with norvaline yielded inhibitor C.
In other serine protease inhibitors, the placement of electrophilic derivatives
such as α-ketoamides, α-ketoesters, α-ketoacids, etc. in the C-terminal has
produced potent inhibitors due to efficient serine trapping.72 Early investigations showed that electrophilic functionalities in this series also gave promising inhibitors. However, inhibition by these molecules was only slightly
better, and sometimes even worse, than that by the product-based analogs.70
This finding enabled the development of inhibitors with reduced specificity
problems since the electrophilic inhibitors, in theory, could react covalently
with numerous nucleophiles present in the host.63 The first approved NS3
protease inhibitors contained α-ketoamides (Figure 4, section 1.2.1). Howev26
er, the many side effects associated with these inhibitors were partly responsible for the subsequent focus on highly potent product-based inhibitors
(Figure 5, section 1.2.1).
Key improvements
Introduction of aromatic substituents on the P2 proline in combination with
further optimization of the P1 side chain, i.e. introduction of 1aminocyclopropane carboxylic acid (ACCA), allowed N-terminal truncations
(D, Figure 12). One of the key improvements was achieved by placing
(1R,2S)-1-amino-2-vinylcyclopropane carboxylic acid (vinyl ACCA) at P1,
which impressively improved the potency of compounds in this series of
inhibitors (E, Figure 12).73,74
Figure 12. Lead optimization process leading to the clinical candidate, ciluprevir.
Macrocyclization
Nuclear magnetic resonance (NMR) experiments indicated that peptides in
this series were extended in a well-defined configuration (P1-P4), and that
the P3 and P1 side chains lay in proximity to each other.75 It was hypothesized that the entropic penalty upon binding to the protease could be reduced
by pre-organizing the bioactive conformation. Hence the P3 and P1 side
chains were connected, yielding a macrocyclic inhibitor.76 The clinical CD,
ciluprevir was generated by further lead optimization (Figure 12).77,78
27
Ciluprevir was the first of the HCV NS3 protease inhibitors to enter clinical trials.78 Proof-of-concept was established as a result of significant reduction in HCV RNA plasma levels in HCV-infected patients who received
ciluprevir.77
Unfortunately, further evaluation of this inhibitor was halted in phase II
trials because of cardiotoxicity in rhesus monkeys that received high doses
of the agent for four weeks.79 However, the research process, from the initial
peptides to the highly potent peptidomimetic clinical candidate, was groundbreaking and motivated the development of the HCV NS3 protease inhibitors currently available on the market as anti-HCV therapies (Figure 5, section 1.2.1).
Further development of product-based inhibitors
Simeprevir, developed in a research collaboration between Medivir and
Janssen, was the first product-based inhibitor to reach the market (Figure 5,
section 1.2.1).29 The characteristic moiety in this compound is the unhydrolyzable proline mimic cyclopentane, which inverts the back-bone amide.80
The product-based inhibitors, which bind non-covalently to the active site
of NS3 protease, all contain a carboxylic acid/acyl sulfonamide at P1; this is
involved in hydrogen bonding to the oxyanion cavity.70 Explorations previously undertaken by our group revealed that the replacement of the Cterminal carboxylic acid (pKa ~4.5) with a phenyl acyl sulfonamide (pKa ~5)
gave more potent inhibitors.81,82 Furthermore, these inhibitors proved to be
highly selective for HCV NS3 protease.83 In parallel, Bristol-Myers Squibb
submitted a patent application for HCV NS3 protease inhibitors containing
an acyl sulfonamide.84 The two approved inhibitors in this class to date
(simeprevir and paritaprevir) contain an acyl sulfonamide, as do the majority
of the advanced NS3 protease inhibitors in clinical trials, as shown in Figure
5, which demonstrates the importance of this discovery.
1.6 Model Systems for Analyzing HCV NS3 Protease
Inhibitors
Improved understanding of the HCV life cycle, via the development of cell
culture models and elucidation of the structures of HCV proteins, has been
crucial for the discovery of novel targets and the development of DAAs. The
continuing evaluation of drugs with regard to, for example, drug resistance,
relies on these techniques.13,85 Various model systems are used in parallel to
study the specific functions of a potential anti-HCV drug. These are complementary to each other, and yield both detailed and general information
regarding the evaluated substance.
28
Enzyme inhibition analysis
The NS3 protein, in vivo, comprises a protease and a helicase. However, the
truncated protein (isolated NS3 protease) is often used for analysis since the
full-length NS3 can be difficult to express and purify, resulting in low yields
of the final protein.86 Studies have suggested that the NS3 protease alone is
sufficient for proteolytic activity,87 while other investigations have proposed
an impact of the helicase on the activity of the protease domain.88,89
The inhibitors presented in this thesis were evaluated in a fluorescence
resonance energy transfer (FRET) assay (Figure 13), using the full-length
NS3 protein together with part of its co-factor NS4A on both the wild-type
enzyme and drug-resistant enzyme variants.
Figure 13. Illustration of a protease activity assay utilizing Fluorescence Resonance
Energy Transfer (FRET). The fluorescent signal is detectable upon substrate cleavage. F = Fluorescent donor; Q = Quenching acceptor.
The enzyme´s capacity to hydrolyze a depsipeptide, in the presence of the
evaluated inhibitor, was measured. The depsipeptide, which resembles the
natural enzyme substrate, contains a fluorescent group (EDANS) near the Nterminal as well as a quenching group (DABCYL) in the C-terminal part.
The fluorescence is quenched in the intact substrate, since these groups are
in close proximity to each other. However, when the substrate has been hydrolyzed by the NS3 protease, the distance between the groups changes, and
a fluorescent signal is detected. In the presence of an inhibitor, the velocity
of the process changes, resulting in an altered signal. The enzyme-catalyzed
conversion rate of substrate into product is described by the MichaelisMenten equation:
V=
[ ]
+[ ]
29
The inhibition is measured at six inhibitor concentrations, in triplicate. Introducing a competitive inhibitor modifies the equation:
V=
[
[
× (1 +
[
]
]
)+[ ]
]
This allows the inhibition constant, Ki to be estimated.86 Ki is the dissociation
constant for the enzyme-inhibitor complex, defined as: Ki = KD = [E][I]/[EI];
E+I
EI
The HCV replicon model
In the HCV research area, the main challenge for many years was to establish a trustworthy, cell-based HCV replication system. From the first molecular cloning of an HCV genome it took another ten years before a reliable
cell culture system could be developed.2,90 It was found that human hepatoma cells can be transfected by replicon RNA by modifying the viral genome,
which enabled monitoring the HCV RNA replication.
The HCV subgenomic replicon RNA uses the segment of the genome
coding for the non-structural proteins, controlled by the encephalomyocarditis virus internal ribosome entry site (IRES), and a gene coding for resistance, driven by the HCV IRES. This allows the selection of transfected
cells encompassing the replication machinery.90
In contrast to in vitro assays using isolated NS3 protein, the replicon assay provides information on how an inhibitor performs in a complex cellular
system. The cell-based activities are measured in Huh7 replicon cell lines
containing a luciferase readout. The concentration of inhibitor needed to
decrease the luciferase signal by 50% is determined (EC50).91 In this thesis,
advanced HCV NS3 protease inhibitors (in late lead optimization stage)
were evaluated for their inhibitory potencies in a cell-based replicon assay.
Pharmacokinetic (PK) profiling
Different approaches are followed in order to evaluate the compounds with
respect to their absorption, distribution, metabolism and excretion (ADME)
properties. In vivo PK models resemble the real life environment of a drug,
which clearly needs to be evaluated at some point in the drug discovery process. However, for a medicinal chemist, the isolated in vitro assays provide
early information on specific properties, such as solubility, permeability
characteristics and metabolic stability, which are useful for the iterative drug
discovery process in guiding the optimization of the lead compound.65
In this thesis, a few compounds at the stage of lead selection were evaluated for in vitro physicochemical and PK properties, i.e. solubility, metabolic
stability (Clint) and permeability coefficient (Papp). The most promising compounds in a series of advanced HCV NS3 protease inhibitors, in late lead
30
optimization, were also evaluated for their in vivo liver-to-plasma ratios and
PK properties in a rat model.
Molecular modeling
Molecular modeling could support the design of novel bioactive molecules.
The molecular modeling studies in this project used the HCV NS3/4A protease-helicase crystal structure in complex with a macrocyclic protease inhibitor (PDB code 4A92).92 The inhibitors were modeled in Maestro (Schrödinger 2011) and induced-fit docking was performed by FLO (QXP
200605).93 Four hydrogen bond constraints, which mimic the hydrogen pattern found between the bound cleavage product and the protein, were used to
restrict the inhibitor within the active site: between the NH of alanine
(Ala157) and the carbonyl of P3, between the backbone carbonyl oxygen of
arginine (Arg155) and the NH of P1, and between the NH of glycine
(Gly137) and Ser139 and the carbonyl oxygen of P1. Ten unique binding
modes were generated using 6000 Monte Carlo perturbation cycles
(mcldock) for each inhibitor, and the most plausible binding mode was chosen by visual inspection.
In this thesis, molecular modeling was carried out for inhibitors in the pyrazinone series in order to clarify the SAR as well as to guide the design of
inhibitors for further evaluation.
31
2 Aims of the Thesis
Numerous HCV NS3 protease inhibitors have entered the market and clinical trials during the time covered by this thesis. However, at the same time,
drug-resistant enzyme variants have appeared which can complicate future
treatment of HCV infection.
The overall aim of this thesis was:
•
to design and synthesize a new lead class of HCV NS3 protease
inhibitors based on a pyrazinone scaffold, structurally different
from the advanced agents in clinical trials and on the market, possessing a unique resistance profile and potentially with a broad
genotypic coverage.
The specific objectives were:
32
•
to synthesize and evaluate the PK properties (in vitro and in vivo)
of late stage, macrocyclic HCV NS3 protease inhibitors, containing a P2 quinazoline, in order to increase awareness of how structural features can affect permeability and stability and in the long
run influence the bioavailability.
•
to investigate the structure-activity relationships (SARs) regarding
the inhibition of the wild-type NS3 protease, Gt 1a and Gt 3a, and
drug-resistant enzyme variants of pyrazinone based inhibitors.
•
to evaluate in vitro PK properties of the pyrazinone based inhibitors to identify how various substituents influence solubility, permeability and metabolic stability.
•
to evaluate procedures for synthesizing the designed compounds,
including establishment of generally applicable Pd catalyzed protocols useful in the synthesis of bioactive molecules.
3 P2 Quinazoline Substituents in HCV NS3
Protease Inhibitors (Paper I)
3.1 Pharmacokinetic Aspects in Drug Discovery
A revealing paper published in 1988 presented the reasons for the failure of
drugs in development.94 Alarmingly, 39% of drugs failed due to poor PK
properties and bioavailability. Years of invested money and time were lost,
and the introduction of new drugs on the market was delayed. This ultimately affected the patients in need of new pharmaceuticals.
Contemporary drug discovery and development has a different approach.
At its best, a drug discovery program is a highly iterative process, where
properties such as solubility, permeability and metabolic stability are evaluated in parallel with optimizations in terms of binding to the target. A less
active compound could have advantageous PK properties which enable a
better in vivo therapeutic response and, eventually, might offer more convenient dosage regimens for the patient. A successful research program needs to
consider and attempt to anticipate how the various properties of a drug cooperate at its final destination inside the human body.65
Lipinski’s well known “rule-of-five” has, since it was presented in 1997,95
guided the choice of compounds that will proceed in the discovery process.
While favorable PK properties and solubility can be predicted from the molecular qualities, the emerging area of demanding and novel targets as well
as poor outcomes from big pharma have challenged researchers to think
“outside the box”, and this can be rewarding. In the HCV research field, for
example, the approved HCV NS3 protease anti-HCV drugs violate at least
one of the rules, since they have a molecular weight of >700. One could
consider the drug-like properties as guidelines but should also bear in mind
that the success of a drug depends on how well various properties are balanced with each other. Moreover, oral drug space is likely to expand with
improved formulation techniques.96
33
3.2 Harmonizing Antiviral Potency with PK Properties
in the Development of HCV NS3 Protease
Inhibitors (Paper I)
During the development of the now approved HCV NS3 protease inhibitor
simeprevir, which contains a quinoline P2 substituent (Figure 14), other P2
heterocycles were also evaluated (e.g. pyrimidines97, I, Figure 14). The ether
linkage found in simeprevir, which connects the P2 core and the P2 heterocyclic group, was replaced with a carbamate moiety (II, Figure 14) in another series.98 However, neither the pyrimidine- nor the carbamate-linked P2
aromatic substituents yielded optimal properties for the inhibitors.29
During these explorations, a novel P2 quinazoline substituent was identified (III, Figure 14); this was combined with a cyclopentane core (as in
simeprevir) and a proline urea core in further optimizations. The quinazoline
substituent was modified with the goal of balancing antiviral potency with
the PK properties.
Figure 14. Pyrimidine, phenyl carbamate and quinazoline as P2 substituents.
Initial modifications
The initial lead in this series (1, Figure 15), which contained a 2phenylquinazoline on the cyclopentyl scaffold, showed excellent permeation
through Caco-2 cells, moderate stability in human liver microsomes, high
potency in the enzyme assay and moderate potency in the cell-based assay.
Introduction of a methoxy moiety in position 7 of the quinazoline (2) improved the cell-based potency. The metabolic stability, on the other hand,
decreased. This property was adjusted by a fluoro-moiety on the para position of the phenyl substituent in the quinazoline (3).
34
Figure 15. Initial modifications on the quinazoline substituent. Ki (NS3fl1a): inhibition constant. EC50 (NS31b): cell-based activity. The cut-off values for stability in
human liver microsomes (HLM), intrinsic clearance (µL/min/mg): Clint< 30: no
risk; 30 < Clint < 92: moderate risk; Clint > 92: high risk. The cut-off values for
Caco-2 permeability (cm/s): Papp < 2×10-6: low; 2×10-6 < Papp < 20×10-6: moderate;
Papp > 20 × 10-6: high.65
Interestingly, the introduction of a thiazolyl substituent reduced the enzyme- and the cell-based activity drastically (4 and 5), in contrast to the outcomes found in the quinoline series, where such a moiety improved the potency.29
Figure 16. Left: quinoline-based P2 substituent containing a thiazolyl moiety. Right:
quinazoline-based P2 substituent containing a thiazolyl moiety, indicating a possible
repulsion between one nitrogen on the quinazoline and the nitrogen in the thiazolyl
moiety.
35
A likely reason for the lower potency is that repulsion between the hetero
atoms in the thiazolyl moiety and the nitrogens in the quinazoline impeded
the bioactive co-planar conformation of the thiazolyl substituent, leading to
reduced interactions with the enzyme (Figure 16). A similar reason could
possibly explain the drastic decrease in both enzyme and cell-based assays
for compounds 6 and 7, i.e. that the non-aromatic rings did not adopt a bioactive conformation, leading to reduced potencies.
The main improvements for the initial optimizations were the addition of
a methoxy group in position 7 of the quinazoline, which improved the cellbased potency (2), and the introduction of a fluoro moiety on the phenyl
group, which increased the metabolic stability (3).
Table 3. SAR and in vitro PK for the P2 cyclopentane series
a
a
Clint
Papp
(µL/min/mg) (cm/s×10-6)
NS3fl 1a
Ki (nM)
NS3 1b
EC50 (nM)
3
0.2
11
32
20
8
0.1
5
<6
13
9
0.4
10
<6
3
10
0.4
20
10
5
11
0.6
8
11
9
12
>1000
>1000
N.d.
N.d.
Cmpd
R1
R2
N.d.: not determined; a See Figure 15.
36
SAR and PK properties for the cyclopentane series
Rewardingly, an 8-methyl on the quinazoline drastically improved the metabolic stability (8-11, Table 3) as well as preserving the potency. However,
the Caco-2 permeability decreased, especially for the analogs 9 and 10
which contained a fluoro moiety. This could be explained by lowered solubility due to increased lipophilicity of the compounds, which possibly provided less substance in solution.
Importantly, in the absence of an aromatic moiety on the quinazoline (12),
the potency radically decreased, which emphasizes the importance of an
aromatic moiety for crucial interactions with the S2 pocket of the NS3 enzyme. Besides good potency and metabolic stability, there was still room for
improvement regarding the Caco-2 permeability to these compounds.
SAR and PK properties for the proline urea series
Gratifyingly, when switching to the proline urea scaffold (Table 4), enhanced Caco-2 permeabilities were obtained for compounds 13-15 compared
with the corresponding cyclopentane-based inhibitors 8, 9 and 11. The potencies and metabolic stabilities were preserved.
The pyridine-containing inhibitors, 16 and 17, on the other hand, were
less potent. Compound 16, with a much lower cell-based activity than the
other inhibitors in the series, might be repulsed from the nitrogens on the
quinazoline with the ortho nitrogen on pyridine. The Caco-2 cells, however,
became totally impermeable to the potent pyridine-containing inhibitor 18,
which could be explained by the partially protonated pyridine nitrogen, and
the possibly zwitterionic character of the compound.
Two compounds containing an expanded macrocyclic ring (15membered, 19 and 20) were evaluated. Besides potencies and Caco-2 permeabilities similar to those of the 14-membered derivatives 13 and 14, they
exhibited decreased metabolic stability.
In vivo PK evaluation
The evaluated inhibitors exhibited excellent potencies and in vitro drug metabolism and PK properties, which endorsed an extended in vivo PK evaluation on selected compounds. Since promising properties were detected in all
three series, i.e. the cyclopentane series and in both the 14- and 15membered proline urea series, compounds 8, 13, 14 and 20 were chosen for
evaluation in vivo (Table 5).
The data presented in Table 5 include the PK properties after intravenous
(iv) injection and after oral administration in Sprague-Dawley rats. A high
drug concentration in the liver is essential for this type of compound, since
the main location for replication of HCV is the hepatocytes. The urea-based
compounds 13, 14 and 20 were all well distributed to the liver, with liver-toplasma ratios of 32-100. Interestingly, for the cyclopentane derivative 8, the
level of compound concentration was not detectable.
37
Table 4. SAR and in vitro PK for the P2 proline series
NS3 1b
a
a
Clint
Papp
EC50
(µL/min/mg) (cm/s×10-6)
(nM)
n
NS3fl 1a
Ki
(nM)
13
1
0.2
11
17
26
14
1
0.3
16
<6
32
15
1
0.6
3
8
33
16
1
0.9
130
N.d.
N.d.
17
1
2.8
13
N.d.
N.d.
18
1
0.2
7
14
0.4
19
2
0.3
23
26
38
20
2
0.2
16
20
32
Cmpd
R1
R2
N.d.: not determined. a See Figure 15.
The volume of distribution (0.56-2 L/kg) indicated that the compounds were
evenly distributed to blood and tissues. The proline compound, 13, was absorbed more slowly (Tmax, 5 h) and had a higher Cmax (3.4 µM) than the cyclopentane 8 (Tmax, 2 h; Cmax, 0.66 µM), which resulted in greater exposure
to the drug (AUC: 15 µM h, approx. 10500 h·ng/mL vs 3.3 µM h, approx.
38
2400 h·ng/mL). Interestingly, the properties of the 14-membered urea 14
were distinctly different from those of the 15-membered urea 20. Compound
20 had a higher clearance rate, and was associated with a lower Cmax and
AUC than compound 14. This correlates with the predicted outcomes from
the in vitro evaluations; although the in vitro clearance of compound 20 was
not high, it was higher than that of the 14-membered derivative (14). However, the oral bioavailabilities (F) were good for the evaluated compounds,
especially for compound 13 (73%).
Table 5. Mean plasma levels and PK parameters after single intravenous and
oral administrations in male Sprague-Dawley rats.
iv (2 mg/kg, n = 2)
Cl (L/h/kg)
Vdss (L/kg)
AUC (µM h)
Liver/plasma ratio (6 h)
8
13
14
20
1
0.56
N.d.
N.d.a
0.69
1.1
8.4
38
0.56
2
N.d.
N.d.
1.6
1.2
N.d.
2.5
Oral (10 mg/kg, n = 2)
AUC (µM h)
3.3
15
11
1.7
0.66 3.4
2.6
0.39
Cmax (µM)
2
5
5
2
Tmax (h)
t½ (h)
3.8
N.d.
N.d. N.d.
F (%)
40
73
N.d. 38
42
100
N.d.a 32
Liver/plasma ratio (6 h)
Cl, clearance; Vdss, volume of distribution at steady state; AUC, area under the plasma concentration-time curve; Cmax, maximum plasma concentration; Tmax, time to maximum plasma
concentration; t1/2, plasma half-life; F, percentage of dose reaching systemic circulation. N.d.:
not determined. Cut-off values: Low: Cl < 0.6 (L/h/kg); Vd < 1 L/kg; AUC < 500h·ng/mL;
Tmax < 1 h; T1/2 < 1 h; F < 20%. High: Cl > 2.7 (L/h/kg); Vd > 10 L/kg; AUC > 2000h·ng/mL;
Tmax > 3 h; T1/2 > 3 h; F > 50%; a Levels were not detectable.
3.3 Synthesis of P2 Quinazoline-Containing HCV NS3
Protease Inhibitors
The synthesis of the investigated inhibitors is outlined in the following sections. The P2 quinazoline building blocks were separately prepared
(Schemes 1 and 2) and then incorporated into the cyclopentane (Scheme 3)
and proline (Scheme 4) scaffolds under Mitsunobu conditions.
Synthesis of the quinazolinol building blocks
Two approaches were followed to prepare the quinazolinol building blocks,
as outlined in Scheme 1 (25a-c and 28) and Scheme 2 (35a-h).
Starting with the carboxylic acid 21 (Scheme 1), the acyl chloride formed
in situ was displaced with ammonia, yielding the primary amide 22. Reduc39
tion of the nitro moiety gave the corresponding amine 23. Coupling of 23
and 26 with carboxylic acids generated the amide-containing intermediates
24a-c and 27, in which the rings were subsequently closed to form the
quinazolinol building blocks 25a-c and 28, by refluxing in ethanol and water
in the presence of sodium carbonate.
Scheme 1. Reagents and conditions: (a) oxalyl chloride, DMF cat., DCM, then NH3,
71%. (b) Raney-Ni, EtOH, H2, 3.5 bar, 95%. (c) R2-COOH, HOBt, EDAC, Et3N,
DMF, 55-90%. (d) Na2CO3, EtOH/H2O 1:1, reflux, 77-90%.
The 8-methyl-containing quinazolinols were prepared as summarized in
Scheme 2. The hydroxyl on 29 was initially methylated, generating the methyl ether 30. The nitrile moiety was subsequently hydrolyzed under basic
conditions. This approach gave a mixture of the carboxylic acid 31 (30%)
and the primary amide 36 (58%). Both could be utilized, but the carboxylic
acid needed protection, which was established via esterification, to obtain
32. Thereafter the coupling of carboxylic acids and hydrolysis of the methylester provided the intermediates 34a-c which were treated under elevated
temperatures in formamide to obtain the quinazolinol building blocks 35a-c.
The intermediate 36 was coupled with acyl chlorides to generate the amidecontaining intermediates 34d-g which were subsequently dissolved in
EtOH/water and refluxed in the presence of Na2CO3 to produce the quinazolinols 35d-h.
40
Scheme 2. Reagents and conditions: (a) MeI, K2CO3, DMF. (b) 2 M NaOH, EtOH.
(c) R2-COOH, HOBt, EDAC, Et3N, DMF, 50-56%. (d) 1 M LiOH, EtOH, 60°C. (e)
formamide, 150°C, 71-89% over two steps. (f) R2-COCl, pyridine, THF. (g)
Na2CO3, EtOH/H2O 1:1, reflux, 60-95% over two steps. 35h was formed as a byproduct when (g) was performed in DMF rather than in EtOH.
Synthesis of the cyclopentane-based inhibitors
The final cyclopentane-based inhibitors were prepared via two cyclopentane
scaffolds. The key operation for generating the final compounds 1, 6 and 7
was a nucleophilic aromatic substitution in order to introduce the quinazoline moiety.99 However, herein the focus will be on the main pathway, namely the route using a Mitsunobu reaction for introduction of the quinazoline
building blocks (Scheme 3).
41
Scheme 3. Reagents and conditions: (a) quinazolinol (25a-c, 28, 35a,b,d,e,h,
Scheme 1 and 2), PPh3, DIAD, THF, 0°C to rt, overnight, (68-99%). (b) 2:1
DCM/TFA, Et3SiH, 0°C to rt. (c) N-methylhex-5-en-amine, HATU, DIPEA, DMF,
0°C to rt,1-3 h, (60-85%) over two steps. (d) Hoveyda-Grubbs catalyst, 1st or 2nd
generation, 1,2-dichloroethane, reflux, 4-12 h (30-80%). (e) 2:1:1 THF/MeOH/H2O,
aq 1 M LiOH. (f) EDAC, DCM, rt, 3-12 h, then cyclopropylsulfonamide, DBU, rt,
6-12 h, (10-40%). R1 substituents are included in Table 3.
The building block 3780 was coupled with the quinazolinols 25a-c, 28,
and 35a,b,d,e,h (Schemes 1 and 2) under Mitsunobu conditions, which generated 38a-i in good to excellent yields (68-99%). Selective hydrolysis of the
42
tert-butyl ester was accomplished under acidic conditions, using Et3SiH as a
carbocation scavenger, which gave the carboxylic acid intermediates 39a-i.
Coupling of the N-methylhex-5-enamine using N-[(dimethylamino)-1H1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium
hexafluorophosphate N-oxide (HATU) as coupling reagent produced 40a-i
in 60-85% yield. The macrocyclic, 14-membered ring was well formed in
30-80% yield employing Hoveyda-Grubbs catalyst (1st or 2nd). Finally, hydrolysis of the ethyl ester under basic conditions and successive coupling of
the cyclopropylsulfonamide, via the formation of oxazolidinone, yielded the
final inhibitors 2-5 and 8-12 in moderate yields (10-40%) after preparative
high-performance liquid chromatography (HPLC) purification.
Synthesis of the proline urea-based inhibitors
The generation of proline urea-based final inhibitors was accomplished as
outlined in Scheme 4. The successful introduction of the quinazolinol moiety
late in the procedure was a rewarding synthetic strategy; this enabled fast
production of target compounds containing various quinazoline substituents.
N-deprotection of 42100 and further treatment with phosgene delivered the
acid chloride 44 in situ which thereafter was coupled with N-methylhex-5en-1-amine or hept-6-enyl-(p-methoxybenzyl)-amine with a satisfying yield
of 85-90% over three steps. Successful macrocyclization (74% yield of 46)
followed by selective hydrolysis of the 4-nitrobenzyl ester revealed the alcohol (80% yield of 47) in 55-65% yield over two steps.
The quinazolinol building blocks 35a-c, e-g were introduced to the 14membered intermediate 47 under Mitsunobu conditions (30-85%). Finally,
ester hydrolysis and coupling to the cyclopropylsulfonamide, as described
before, provided the final inhibitors 13-18 with 40-85% yield over two steps
after preparative HPLC purification.
In parallel, the 15-membered compounds 53a and 53b were prepared with
a total yield of 20% and 12%, respectively, from intermediate 44. The pmethoxybenzyl protecting group was removed under acidic conditions, using
DCM/TFA, yielding the target compounds 19 and 20 with 62% and 38%
yields, after preparative HPLC purification.
43
Scheme 4. Reagents and conditions: (a) 2:1 DCM/TFA, 0°C to rt, 1.5 h. (b) phosgene in toluene, THF, NaHCO3, rt, 1 h. (c) DCM, NaHCO3, N-methylhex-5-en-1amine, or hept-6-enyl-(p-methoxybenzyl)-amine, rt, overnight, 85-90% over three
steps. (d) Hoveyda-Grubbs catalyst 2nd generation, 1,2-dichloroethane, reflux, 4-12
h. (e) aq 1 M LiOH, 2:1:1 THF/MeOH/H2O, 0°C, 4 h, 55-65% over two steps. (f)
quinazolinol (35a-c, e-g, Scheme 2), PPh3, DIAD, THF, 0°C to rt, overnight, 3085%. (g) 2:1:1 THF/MeOH/H2O, aq 1 M LiOH, 40-50 °C, overnight. (h) EDAC,
DCM, rt, 5h, then cyclopropylsulfonamide, DBU, rt, overnight, 45-80%. (i) 2:1
DCM/TFA, rt, 30 min, 38-62%. R1 substituents are included in Table 4.
44
4 2(1H)-Pyrazinone-Based HCV NS3
Protease Inhibitors (Papers II and III)
4.1 Pyrazinones in Medicinal Chemistry
Pyrazinone is a versatile scaffold which has been evaluated as a core structure in protease inhibitors of thrombin, tryptase, tissue factor VIIa, caspase-3
and HCV NS3.101–105 This heterocyclic template has also provided the basis
for other types of bioactive compounds such as kinase inhibitors intended for
anticancer drugs, µ-opioid receptor agonists with analgesic effects and natural product analogs.106–108
The advantages of the use of the pyrazinone structure in the protease inhibitors discussed above are correlated with its ability to induce β-strand
conformations, which is an important aspect of the recognition and subsequent binding to an active site in the proteases.109,110 The pyrazinone ring
contributes to the rigid structure, forming the hydrogen bond interactions
normally found between the natural substrate and the protease (Figure 17).
Figure 17. Hydrogen bond interactions from the peptide, i.e. natural substrate (left)
and from the corresponding P3 pyrazinone scaffold (right) to the protease. HBA:
hydrogen bond acceptor; HBD: hydrogen bond donor.
Another important aspect is the opportunity for decorating the core structure with various functional groups.111,112 This allows efficient and informative SAR studies to be accomplished using straightforward synthetic procedures. Various synthetic approaches, e.g. Pd-catalyzed cross-coupling reactions, such as C-N arylations and Suzuki couplings, and amide coupling procedures yielding peptidomimetic inhibitors are presented in chapters 5 and 6.
45
4.2 From Tripeptides to Pyrazinone-Based HCV NS3
Protease Inhibitors
As discussed in section 1.5.1, inhibitors of HCV NS3 protease have often
been developed from the cleavage product of its natural substrate. With the
aim to develop HCV NS3 protease inhibitors, structurally distinct from the
inhibitors in clinical trials, our group shifted focus to alternative tripeptidic
HCV NS3 protease inhibitors e.g. 54113 in Figure 18, containing a phenylglycine P2 substituent instead of the commonly used proline (i.e. inhibitors
in Figure 5, section 1.2.1), and similar P3 and P1P1’ substituents to those
found in potent proline-based inhibitors. Subsequently, introduction of a P3
pyrazinone rigidified the core structure and yielded 55 which was considered
a promising starting point for development of novel peptidomimetic inhibitors.114
Figure 18. Replacing a P3 t-Leucin 54 (left) with a P3 pyrazinone 55 (right).
The P1P1’ substituent was then investigated and an aromatic acyl sulfonamide (56, Table 6) previously found to be useful in other HCV NS3 protease
inhibitors, was equipotent to the P1P1’ found in 55.114 The aromatic P1P1’
enabled further substituents around the P1 and P1’ substituents to be introduced.
4.3 Development of Achiral Inhibitors (Paper II)
Optimization of this class of compounds was initiated after the above modifications. Molecular modeling suggested that the proposed P2 aryl could
possibly be moved to the R6 position of the pyrazinone while retaining the
same or similar interactions with the S2 pocket. Preliminary results supported this idea and inspired further optimizations.114
A carbamate in the 3 position had previously been identified as a promising P3 capping group of the pyrazinone (56, Table 6) and better than other
46
attempts to introduce alkyl and aryl amines and primary amides. However,
the chemical stability of the tert-butyl carbamate was unpredictably low,
which prompted replacement with a more stable derivative.
Introduction of a P3 urea capping group and relocation of the P2
substituent
The 3-tert-butyl carbamate was replaced with a tert-butyl urea moiety, which
to our surprise increased the potency of the inhibitor by a factor of ten (Table
6, 57 compared with 56) while also increasing the stability. Compound 57
contained an additional hydrogen which could possibly be involved in interor intramolecular bonding.
Subsequent relocation of the aromatic P2 group (R2) to the R6 position of
the pyrazinone to yield achiral inhibitor 58 was allowed with only a minor
drop in inhibitory potency. Combining a phenylglycine with the R6 benzyl
did not further improve the inhibitory potency of compound 59.
Table 6. Influence on inhibitory potency by carbamate-to-urea exchange and the
relocation of the R2 group to the R6 position on the pyrazinone.
a
NS3fl 1a; SD, standard deviation.
47
To compare the acyl sulfonamide in the C-terminal with a serine-trapping
electrophilic group, an α-ketoamide (the same as that in boceprevir, Figure 4,
section 1.2.1) was evaluated. As presented in Table 7, this produced the inhibitors 60 and 61 which were less potent than the acyl sulfonamide derivatives 57 and 58 in Table 6.
Accordingly, the achiral compound 58 (Table 6) which contained an aromatic acyl sulfonamide in P1P1’, was selected for further investigations, as
presented in Table 8.
Table 7. Electrophilic serine trap inhibitors containing an α-ketoamide in P1.
a
NS3fl 1a; SD, standard deviation
Elongation of the R6 substituent
Various substituents, e.g. alkyl, elongated phenyl, naphthyl and bromo- and
pyridine-substituted aryls, were introduced and evaluated with respect to
their inhibitory constants in order to develop a SAR around the R6 position
of the pyrazinone (58, 62-81, Table 8).
The potency of 62, which lacks a substituent in R6 was decreased (1.9 µM
vs 0.66 µM for 58). The potency was then increased by adding larger, more
lipophilic substituents, as found in 63, 64, 68 and 69. A slight decrease in
potency was however found for the naphthyl derivatives 66 and 67. Compound 65 which contained an oxygen spacer, also appeared to be less efficient. In order to study the preferred orientation of a second aromatic substituent, 70-75 were prepared, biochemically evaluated and subsequently
used as precursors for compounds 76-81. Slightly improved potencies were
obtained for the bromo-containing phenyl and benzyl derivatives, 70-75,
compared with the unsubstituted benzyl analog 58. More interestingly, the
corresponding pyridyl substituents revealed limitations for the orthoderivatives 78 and 81 with a significant drop in inhibitory potency.
48
Table 8. Pyrazinone based HCV NS3 protease inhibitors containing elongated R6
substituents and their Ki values.
a
NS3fl 1a; SD, standard deviation
49
Molecular modeling of a pyrazinone-based inhibitor
A representative compound from the series (77) was modeled into the protease active site (Figure 19). The binding mode showed backbone interactions
with Ala157 and Cys159 and possible edge-to-face interactions between the
aromatic P1 group and Phe154. Furthermore, the P2 substituent seemed to be
directed towards the S2 pocket, which supported our hypothesis for elongated R6 groups.
An internal hydrogen bond between the urea nitrogen on the P3 capping
group and the 4-nitrogen in the pyrazinone (Figure 19) possibly stabilizes the
bioactive conformation and could partly explain the gain in inhibitory potency observed by the replacement of a P3 carbamate with a urea moiety.
Therefore, the urea functionality was preserved and further evaluated in SAR
studies (sections 4.4 and 4.5).
Figure 19. Compound 77 modeled into the boundary between the NS3 protease
active site (grey surface) and the helicase domain (green) of the 4A92 crystal structure.92 Hydrogen bond interactions are shown in red dashed lines.
Additionally, for the ortho pyridyl compounds 78 and 81, modeling suggested poses where the P2 group was directed out from the S2 pocket, instead forming hydrophobic interactions with the pyrazinone core. The backbone is somewhat distorted in these modes which could explain the loss of
inhibitory potency for 78 and 81.
50
The substrate envelope theory and amino acid substitutions in the NS3
protease
The resistant HCV mutations that have emerged during the development and
evaluation of advanced HCV NS3 protease inhibitors have limited the efficacy of these compounds. As previously mentioned (section 1.3) the most
common amino acid substitutions that lead to resistance occur at positions
R155, A156 and D168 (Figure 20).
Figure 20. Left: Drug resistant amino acid substitutions R155K, A156T and D168V.
Right: The electrostatic network in the S2 pocket between R123, D168, R155. The
hydrogen bond interctions between the P1 residue and the enzyme, as suggested by
the full-length crystal structure.62
Schiffer and co-workers have explained the emergence of drug resistance
against HCV NS3 protease inhibitors using the so-called substrate envelope
theory115 which they previously have utilized in the design of HIV protease
inhibitors showing reduced problems with resistance.116,117 Crystal structures
of the NS3 protease domain with its N-terminal products of the different
cleavage sites (P7-P1) were analyzed and the consensus volume (substrate
envelope) inside the NS3 active site was defined.115 Comparisons between
the substrate envelope and the crystal structures of the NS3 protease in complex with boceprevir, danoprevir and simeprevir (the structures of these inhibitors can be found in Figures 4 and 5, section 1.2.1) revealed that these
drugs protrude outside the substrate envelope at positions associated with
drug-resistant mutations, e.g. R155, D168, etc. These mutations do not affect
the viral fit, but selectively disrupt important interactions with the inhibitors.118 The large aromatic P2 groups found in danoprevir and simeprevir
stack with the guanidinium group of R155, which changes the electrostatic
51
network in the S2 pocket between R123, D168 and R155 (Figure 20). The
extensive packing of these big P2 groups will hence be altered by mutations
at R155 and D168, with a possibly reduced affinity as a consequence. This
could explain why mutations at R155, and especially D168, confer resistance
to the inhibitors containing extended P2 groups.
Furthermore, it was found that A156 and R155 not only interacted with
the P2 groups of simeprevir and danoprevir, but also had apparent interactions with the less bulky P2 group found in boceprevir at places outside the
substrate envelope. Accordingly, substitutions at A156 and R155 would alter
the interactions with the P2 moieties in all three inhibitors. More recent studies on crystal structures of these inhibitors with the drug-resistant enzyme
variants R155K, A156T and D168A supported these suggestions.118 Interestingly, grazoprevir (Figure 5, section 1.2.1), an inhibitor that has shown a
lower level of resistance to R155K than other protease inhibitors, had a different binding conformation with fewer interactions from the P2 group with
R155 and D168. These findings could help in the design of novel inhibitors
of the NS3 protease encompassing a unique resistance profile.
Evaluation of pyrazinone-based inhibitors against drug resistant
enzyme variants
One of the driving forces for our development of coming generation HCV
NS3 protease inhibitors is the possibility to maintain potency against drugresistant enzyme variants. Consequently, three inhibitors in the pyrazinone
series were evaluated against R155K, A156T and D168V. The selected
compounds contained one phenyl glycine inhibitor 57 from Table 6 as well
as two compounds from the achiral pyrazinone series, containing one alkyl
(69) and one aryl (76) R6 substituent (Table 8). Two advanced HCV NS3
protease inhibitors were also included, namely the electrophilic NS3 protease inhibitor telaprevir (Table 4, section 1.2.1) and the first-in-class, productbased inhibitor ciluprevir (Table 5, section 1.2.1). Vitality values,119 which
normalize for the different catalytic abilities of the enzyme variants, were
calculated and presented along with the Ki values (Table 9). If the vitality
value < 1, the enzyme variant is disfavored in the presence of inhibitor compared with the wild-type enzyme. If the value > 1 the amino acid substitution
provides a less vulnerable enzyme to the evaluated inhibitor compared with
the wild-type enzyme.
Both telaprevir and ciluprevir showed a decrease in potency on the
R155K substituted enzyme. A crystal structure of telaprevir in complex with
the R155K substituted protease has previously been published by Schiffer
and co-workers; this demonstrates disrupted interactions by P2 and P4 of the
inhibitor with the protease.118 However, for inhibitors in the pyrazinone series this amino acid substitution was well accepted, with vitality values <1.
The Ki values displayed a preference for the aliphatic R6 group in 69 compared with 57 and 76.
52
Table 9. Inhibition of the protease activity of the wild-type (wt) and the drug resistant enzyme variants A156T, D168V and R155K for the full length NS3 protease,
and corresponding vitality values (V).
The inhibition potencies for telaprevir and ciluprevir of wt, A156T and D168V have previously been reported in Dahl et al.119
The amino acid substitution A156T results in increased bulk, which influences interactions with the P2 and P4 substituents of HCV NS3 inhibitors.
Both ciluprevir and telaprevir lost potency against this mutated form of the
enzyme. The drop in potency was especially distinct for telaprevir, which
displayed a Ki value in a micro molar range. A published crystal structure of
telaprevir in complex with the A156T substituted protease has later clarified
the large drop in potency;118 the A156T substitution resulted in a steric clash
with the P2 moiety of telaprevir which led to a changed position of the inhibitor in the active site and reduced interactions with R155.118 This alteration
also resulted in an extended distance between the ketoamide warhead and the
53
catalytic serine, which reduced the capacity for a covalent bond formation.
Although more potent than telaprevir against the A156T protease, the phenyl
glycine inhibitor 57 showed a slightly larger drop in inhibition potency compared with 69 and 76, which could be because of the altered location of the
P2 substituent. Again, the cyclohexyl-containing compound 69 showed the
most promising Ki value of the three inhibitors 57, 69 and 76; this was close
to the Ki in the presence of the wild-type enzyme.
As previously discussed, the D168V mutation primarily affects inhibitors
with large P2 substituents. Ciluprevir, which contains an extended quinoline
P2 substituent, dramatically lost inhibitory potency against this mutation.
However, telaprevir, which lacks an extended P2 substituent, showed improved inhibitory potency and a vitality value less than 1, implying that this
compound inhibits the D168V mutated form of the enzyme more effectively
than it inhibits the wild-type enzyme. The crystal structure of telaprevir with
a D168A substituted protease (note: not D168V) showed a tighter interaction
with the D168A mutant than with the wild-type, which also resulted in increased interactions with R155 and A156.118 The higher potency against
D168V could possibly be explained by similar interactions. The Ki values for
the pyrazinone-based inhibitors were about three times larger against D168V
mutant than against the wild-type. The vitality values indicated only minor
influence on the inhibition of this mutated form of the enzyme. Among the
pyrazinones, compound 76, which was less flexible with respect to its P2
substituent than 69, seemed to be slightly less potent against the D168V
mutation.
In vitro PK properties for pyrazinone-based inhibitors
Although the stage of this drug discovery project could be considered to be
between hit and lead selection, the awareness of less suitable PK properties
in a prospective candidate drug is of importance for further investigations.
Initially, in silico predictions were performed to estimate pKa and log D7.4
(Table 10). Based on the acyl sulfonamide group, these inhibitors possess
acidic properties, with small differences in pKa (4.7-5.1). The higher value
was seen in 76, which contains the slightly basic moiety pyridyl, which possibly affected the overall pKa value.
The ideal value for the lipophilicity, estimated at pH 7.4, i.e. the log D7.4,
should be between 1 and 3 for well balanced solubility and permeability, and
minimal metabolism.65 For this set of compounds, the log D7.4 ranged between 3.8 and 5.0, with the lowest values found for 65 and 76, which contained heteroatoms in the R6 substituent. Overall, these log D7.4 values indicated low solubility, which could negatively affect the level of absorption.
One could expect good permeability, which is associated with high lipophilicity, but at the same time, an increased risk of metabolism due to binding to metabolic enzymes could be expected.120
54
Table 10. In vitro PK properties, in silico pKa values and log D7.4 values.
pKa
logD7.4
Solubility
(µM)
Papp a-b
(10-6 cm/s)
In vitro
Clint
(µL/min/mg)
In silico
t½ (min)
57
63
65
68
69
76
4.7
4.2
4.7
4.4
4.7
3.8
4.7
4.3
4.8
5.0
5.1
4.1
38
23
22
21
31
53
1.1
± 0.4
0.3
± 0.2
4.0
± 1.1
0.04
± 0.02
0.12
± 0.09
7.0
± 1.5
26 ± 2
<13
17 ± 4
33 ± 13
52 ± 3
17 ± 3
54 ± 3
>100
81 ± 19
42 ± 16
27 ± 2
82 ± 13
Papp = apparent permeability coefficient; a-b = apical to basolateral; Clint = in vitro clearance;
t1/2 = in vitro half-life; Predicted risk of oxidative first-pass metabolism in vivo based on in
vitro intrinsic clearance (µL/min/mg), cut-off values: Clint< 30: no risk; 30 < Clint < 92: moderate risk; Clint > 92: high risk; Caco-2 permeability, apparent permeability coefficent,
Papp (cm/s):121 Papp < 0.2 × 10-6 : low; 0.2 × 10-6 < Papp < 1.6 × 10-6 : moderate; Papp > 1.6 ×
10-6 : high.
The in vitro solubility, Caco-2 permeability and metabolic stability were
determined, as presented in Table 10. The solubilities were overall classified
as moderate, indicating that solubility is not a major issue for this class of
compounds despite the predictions made by in silico calculations. The pyridine containing inhibitor 76 was the most soluble in the series and gives
important insights for further optimizations. Interestingly, when evaluating
the permeability, the pyridine compound 76 and the ether compound 65,
showed the most promising Papp values. In contrast, the alkyl-containing R6
moieties in 68 and 69 were associated with low Caco-2 permeabilities.
The in vitro intrinsic clearance studies, performed in human liver microsomes, indicated low or moderate risk of high oxidative first-pass metabolism in vivo for this class of compounds, in contrast to the predicted in silico
calculations. It was found that the compounds containing alkyls in R6 (57, 68
and 69) were less stable than the aromatic derivatives 63, 65 and 76.
It is interesting to observe the big variation in PK properties for the different R6 substituents situated on an otherwise unchanged large scaffold. The
predicted in silico data supported to some extent the in vitro PK properties,
indicating that a lower log D7.4 could relate to balanced PK properties, as
exemplified by 65 and 76 compared with 68 and 69.
55
4.4 Exploration of the P1P1’ Substituents (Paper III)
The SAR investigation around the R6 position improved understanding of the
function of the substituents at this site in the pyrazinone. The importance of
a urea as a P3 capping group was also noticed. Further, an aromatic P1P1’
block was identified as a better substituent than a classical serine-trapping,
α-keto amide (57 and 58, Table 6, compared to 60 and 61, Table 7) or amino
acid based carboxylic acid or acyl sulfonamide.
Preceding these investigations it had been found that the classical acyl
sulfonamide (as in 55) gave more promising Ki values than the corresponding carboxylic acid.114 The acyl sulfonamide is a widely used carboxylic acid
bioisostere, found in most advanced HCV NS3 protease inhibitors (Figure 5,
Section 1.2.1).
Based on those previous findings, further investigations were focused on
the interactions with the protease from P1 and P1’ substituents and the aromatic acyl sufonamide building block was considered to be a suitable scaffold. The template could be modified on both the P1 and P1’ aromatic moieties, which is valuable in SAR studies.
Regioisomers
The investigation was started by varying the regioisomer in order to study if
there were any preferences for specific isomers (Table 11). Initially, we explored the inhibitory potencies against full length NS3, Gt 1a. Surprisingly,
going from lead 69, with the acyl sulfonamide in the ortho position to the
meta (82) or para (83) positions did not affect the inhibitory potencies much.
The electron-withdrawing trifluoromethyl moiety can have two functionalities in this type of compound: affecting the acidity of the acyl sulfonamide
hydrogen or providing hydrophobic interactions with the protease. However,
the trifluoromethyl group was varied between the ortho (84), meta (85) and
para (69) positions with similar inhibitory potencies. Even replacement with
a methyl group (86) yielded similar Ki values. These modifications indicated
that the P1’ moiety did not have any specific interactions with the enzyme.
Moreover, the increased acidity of the acyl sulfonamide hydrogen was not
beneficial in terms of stronger interactions with the oxyanion cavity, in contrast to previous findings for other HCV NS3 protease inhibitors.122
56
Table 11. Pyrazinone based HCV NS3 protease inhibitors containing various P1P1’
aromatic acyl sulfonamides.
NS3fl 1a
Ki ±SD (µM)
NS3fl 1a
R155K
Ki ±SD (µM)
NS3fl 3a
Ki ±SD (µM)
69
0.3 ± 0.1a
0.13 ± 0.04
N.d.
82
0.36 ± 0.16
N.d.
N.d.
83
0.40 ± 0.28
0.23 ± 0.1
1.0 ± 0.38
84
0.27 ± 0.17
N.d.
N.d.
85
0.55 ± 0.27
N.d.
N.d.
86
0.26 ± 0.11
N.d.
N.d.
87
0.40 ± 0.1
N.d.
N.d.
Cmpd
P1P1’
57
NS3fl 1a
Ki ±SD (µM)
NS3fl 1a
R155K
Ki ±SD (µM)
NS3fl 3a
Ki ±SD (µM)
88
0.36 ± 0.11
N.d.
N.d.
89
0.21 ± 0.08
0.17 ± 0.04
3.44 ± 1.0
90
0.22 ± 0.11
0.14 ± 0.05
0.93 ± 0.26
91
0.30 ± 0.07
N.d.
N.d.
92
0.53 ± 0.2
N.d.
N.d.
93
94
0.22 ± 0.06
0.20 ± 0.08
0.05 ± 0.01
0.02 ± 0.01
N.d.
N.d.
95
0.17 ± 0.1
0.09 ± 0.05
0.80 ± 0.57
96
>10
N.d.
N.d.
Cmpd
P1P1’
N.d.: not determined. aThe Ki value was determined on the same occasion as the other inhibitors in this series. (Ki = 0.11 µM in a previous measurement, Table 8).
58
Next, we focused on the P1 aryl, and examined an aryl bromine (87)
which was subsequently replaced with vinyl (88) and pyrimidyl (89) moieties. A fluorine in the same position was also examined (90), as well as a CF3
group with the acyl sulfonamide in the meta position (91). Although there
was a big difference in size and electronic properties, the influence on inhibitory potencies was small. Of interest, the bulky pyrimidine (89) was well
tolerated but showed no additional interactions with the protease, based on
similar Ki values for 89 and 69. These modifications to the P1 aryl, which
has potentially more acidic acyl sulfonamides, questioned the importance of
an acidic functionality. The alkylated acyl sulfonamide 92 which lacks an
acidic hydrogen, supported this assumption, showing a maintained inhibitory
potency compared with 69.
Truncation
The surprisingly flat SAR obtained from the modifications performed on the
P1 and P1’ aromatic moieties resulted in further analysis of truncated derivatives. Thus, 93, 94 and the carboxylic acid 96, which lacks the entire P1P1’
aromatic building block, were evaluated (Table 11). Remarkably, similar
potencies were retained for the P1 carboxylic acid (93) and for the P1 methyl
ester derivative (94). However, removal of the P1 aromatic functionality (96)
resulted in a considerable loss of inhibitory potency, verifying the need for
the P1 moiety in combination with the P2P3 pyrazinone scaffold. A slightly
improved inhibitory potency was achieved for the more flexible inhibitor 95.
Reversed acyl sulfonamides
One advantage of the aryl acyl sulfonamides, compared with alkyl analogs,
is the possibility to easily reverse the acyl sulfonamide functionality, which
could alter the opportunity of reaching interactions in the oxyanion cavity.
Accordingly, the inhibitors in Table 12 were evaluated. Again, it was found
that even the reversed acyl sulfonamides were allowed. No well defined
favorable influence from this rearrangement was found, although slightly
lower Ki values were obtained for compounds 97-99 than for 67. However,
in combination with the better fitting phenethyl P2 group, as previously
found, the potencies were similar for the original acyl sulfonamide (63) as
for the reversed analog (100).
The results shown in Tables 11 and 12 indicated that the P1 aryl improved
potency. However, the P1’ aromatic moiety did not show any additional
benefits. The acidic acyl sulfonamide was not specifically needed for good
binding to the protease for these pyrazinone based inhibitors, in contrary to
most HCV NS3 protease inhibitors known. Further investigations are needed
to confirm and shed light on this observation.
59
Table 12. Pyrazinone based HCV NS3 protease inhibitors containing reversed
P1P1’ aromatic acyl sulfonamides.
Cmpd
67
97
98
99
63
100
P1P1’
R6
A
A
A
A
B
B
NS3fl 1a
Ki ±SD
(µM)
NS3fl 1a
NS3fl 3a
R155K
Ki ±SD (µM)
Ki ±SD (µM)
0.81 ± 0.23a
0.15 ± 0.07
1.3 ± 0.4
0.51 ± 0.2
0.08 ± 0.04
0.78 ± 0.14
0.38 ± 0.15
0.12 ± 0.08
0.87 ± 0.18
0.61 ± 0.14
N.d.
N.d.
0.30 ± 0.13b
N.d.
N.d.
0.35 ± 0.1
0.10 ± 0.02
N.d.
N.d.: not determined. a,bThe Ki value was determined on the same occasion as the other inhibitors in this series. (aKi = 0.29 µM, bKi = 0.12 µM in a previous measurement, Table 8).
Cyclobutyl
A high aromatic content could be a problem in terms of the bioavailability of
a potential candidate drug.123 A cyclobutyl moiety was therefore evaluated as
an alternative to the aromatic P1 substituent. Spirocyclobutyls have previously been evaluated in electrophilic serine-trap HCV NS3 protease inhibitors.124 If promising, the inherent opportunity for incorporating substituents
60
into the spirocyclobutyl group, via a 3-hydroxyl moiety, would be useful in
forthcoming SAR studies.
Table 13. Pyraonzine based HCV NS3 protease inhibitors containing P1 cyclobutyl
acyl sulfonamides.
Cmpd
R1
R2
R6
NS3fl 1a
Ki ±SD (µM)
NS3fl 1a R155K
Ki ±SD (µM)
101
Cl
A
B
0.11 ± 0.14
0.11 ± 0.04
102
Cl
A
A
Isomer a: 0.40 ± 0.13
Isomer b: 0.50 ± 0.17
N.d.
H
A
>10
N.d.
103
H
N.d.: not determined.
As presented in Table 13, this modification improved the inhibitory potency about three-fold (101 vs 69 found in Table 11, and 102 vs 58 found in
Table 14). The compound containing the free hydroxyl group (103) also
lacked the chlorine in position 5 of the pyrazinone (synthesis in section 5.35.5) which hampered a truly reliable comparison of the compound. The absence of the benzyl and the C5 chlorine decreased the inhibitory potency
substantially. Efforts to further evaluate this spirocyclobutyl moiety would
be of interest. Possibly, the P1’ aromatic part could be removed, and the
substituent on the ring might enable novel interactions with the protease.
4.5 P3P4 Urea Elongations (Paper III)
In 2011, Schiering et al. published the crystal structure of a macrocyclic
HCV NS3 protease inhibitor in the presence of the full-length NS3 protein,
i.e. the protease and the helicase. This showed interactions between the P4
capping group and the helicase,92 which supported suggestions from our
group emphasizing the importance of having the full-length protein for evaluation of inhibitors and resistance profiling.119 Because of our interest in
developing inhibitors that are also potent against known drug-resistant enzyme variants, the hypothesis of designing inhibitors to fit inside the socalled substrate envelope was appealing. When applied to the pyrazinonebased inhibitors, elongation of the inhibitors to span the P4P5 region, in
combination with a relatively small P2 group, could be a possible strategy. A
61
cyclic imide (found in 107 and 108, Table 14) that previously had been evaluated in an electrophilic inhibitor of the HCV NS3 protease125 was examined
as a P4P5 substituent in a pyrazinone based inhibitor by molecular modeling
(Figure 21).
Figure 21. A close derivative to compound 108 modeled into the boundary between
the NS3 protease active site (grey surface) and the helicase domain (green) of the
4A92 crystal structure. Hydrogen bond interactions are shown in red dashed lines.
The molecular modeling of a close derivative of compound 108 (i.e. lacking a fluorine on the P1 aryl) showed hydrogen bond interactions between
His528, positioned in the helicase, and one of the oxygens on the P4 capping
4,4-dimethylpiperidine-2,6-dione. The backbone interactions with Lys136,
Ala157 and Cys159 and an internal hydrogen bond between the urea hydrogen and the pyrazinone were suggested.
These initial indications of possible interactions with the helicase supported the evaluation of a small series of diverse P3 and P4(P5) capping
groups (Table 14). 104 and 105 which contained short P3 capping groups
and lacked possible internal hydrogen bond interactions with the pyrazinone,
gave lower inhibitory potencies than the reference compound containing the
tert-butyl urea (58). Gratifyingly, compounds 106 and 107 which contained
elongated P4P5 substituents, showed improved inhibitory potencies compared to 58. In combination with a better P2 moiety, the potencies were simi-
62
lar to the tert-butyl urea (108 vs 69 found in Table 11), but were well accepted.
Table 14. Pyrazinone based HCV NS3 protease inhibitors containing various ureas
in the 3-position of the pyrazinone.
R
NS3fl 1a
Ki ±SD (µM)
NS3fl 1a
R155K
Ki ±SD
(µM)
NS3fl 3a
Ki ±SD
(µM)
H
A
1.28 ± 0.38 a
0.58 ± 0.1
N.d.
104
H
A
2.82 ± 0.42
N.d.
N.d.
105
H
A
3.8 ± 1.4
N.d.
N.d.
106
F
A
0.40 ± 0.18
0.14 ± 0.02
1.9 ± 0.6
107
F
A
0.27 ± 0.14
0.13 ± 0.03
1.3 ± 0.6
108
F
B
0.30 ± 0.16
0.10 ± 0.01 0.61 ± 0.21
2
R
58
Cmpd
1
R
6
N.d.: not determined. aThe Ki value was determined on the same occasion as the other inhibitors in this series. (Ki = 0.66 µM in a previous measurement, Table 6).
Evaluation against the R155K enzyme variant
Selected inhibitors in Tables 11-14 were evaluated against the most prevalent drug-resistant enzyme variant R155K, which is resistant to most of the
HCV NS3 protease inhibitors under clinical evaluation or available on the
market, as discussed in section 4.3.52 Satisfyingly, in general, this series
showed more promising Ki values against the R155K mutant than against the
63
wild-type. To some extent, this could be explained by the relatively small P2
substituents compared with the extended P2 blocks found in the advanced
inhibitors (Figure 5, section 1.2.1). The inhibitors displayed Ki values ranging from 0.02 µM to 0.58 µM which could aid in the future design of optimized inhibitors directed against the R155K variant of the virus. Notably,
the P1’ truncated inhibitors 93 and 94 were the most active compounds in
this series, which accentuates the potential of this class of inhibitors.
4.6 Evaluation Towards the Genotype 3a of HCV NS3
Protease (Paper III)
Gt 3a of HCV is currently considered the most difficult genotype to treat.126
Moreover, there are indications that the prevalence of this genotype is increasing and that an infection with Gt 3a is connected with a greater risk of
severe liver inflammation, faster progression of fibroses and an increased
risk of hepatocellular carcinoma.6
The HCV NS3 protease inhibitors available on the market are not effective against Gt 3a of the virus. The so-called second generation NS3 protease
inhibitors (e.g. grazoprevir, Figure 5, section 1.2.1) have addressed this
weakness; they have promising SVRs against Gt 3a.33,127
As previously discussed for Gt 1b, NS3 protease inhibitors containing
large P2 substituents force R155 into an extended conformation which favors the salt bridge between R123, D168 and R155 in the S2 pocket (Figure
20). Gallo and co-workers have used NMR spectroscopy to structurally
characterize Gt 3a.128 This study showed that the previously described salt
bridge is lacking in Gt 3a, due to a glutamine (Q) in position 168 and a threonine (T) in position 123. This partly explains the lower inhibitory potencies
for inhibitors with extended P2 substituents. In presence of inhibitors interacting with R155 and Q168 (i.e. containing P2 substituents) the otherwise
solvated, charged residue (Arg) will have to compensate for desolvation.
Moreover, the flexible side chains of R155 and Q168 become more rigid in
presence of inhibitors leading to loss of entropy.128 Recently, it was suggested from molecular dynamics simulation that part of the reduced inihibitory
potency of HCV NS3 protease inhibitors against Gt 3a was due to reduced
catalytic activity of the enzyme. This was explained by a different distance
between the amino acids in the catalytic triad, which results in a less efficient acid-base support from His57 (Figure 9, section 1.4).129
Pyrazinone-based inhibitors against genotype 3a
The increased understanding of the Gt 3a, i.e. the different properties in the
S2 pocket compared with the Gt 1a, partly supported the previously discussed strategy for dealing with drug resistance against R155K and D168V,
64
i.e. to design inhibitors with smaller P2 substituents and elongations in the
P4P5 region.
A few inhibitors in the pyrazinone series (Tables 11, 12 and 14) have so
far been selected for evaluation against this enzyme variant. Notably, although relatively high Ki values, they differed five- to six-fold, ranging from
0.61 µM to 3.44 µM. The highest Ki value was found for the bulky pyrimidine containing inhibitor 89 which fitted well in Gt 1a (0.2 µM) but was less
suitable for Gt 3a (3.44 µM). In comparison, inhibitor 108, which encompassed an elongated urea as a P4 capping group, showed a promising inhibitory potency also for Gt 3a (0.61 µM; Gt 1a, 0.30 µM).
65
5 Synthesis of Pyrazinone-Based HCV NS3
Protease Inhibitors (Papers II and III)
The P3 pyrazinone-based inhibitors, containing P1P1’ substituents, P4(P5)
ureas and varied R6 substituents (Figure 22) can be synthesized in a five- to
seven-step procedure starting from aldehyde or alcohol, via the formation of
α-aminonitriles and subsequent ringclosure (Scheme 5). Displacement of the
C3-chlorine with a urea (Scheme 9) followed by introduction of the P1P1’
building blocks via amide coupling (Scheme 13) provide target compounds.
Further decoration of the aromatic R6 group can be achieved in a final step
using Suzuki-Miyaura couplings (Scheme 14).
In this chapter, synthetic strategies are outlined and discussed. Optimization of a C-N urea protocol is further evaluated in chapter 6.2.
Figure 22. A schematic overview of the synthetic strategies for the preparation of
pyrazinone based HCV NS3 protease inhibitors.
5.1 Formation of the 2(1H)-Pyrazinone Core
The 2(1H)-pyrazinone core structures 113-120 were constructed via a procedure previously established by our group.130 The aldehydes 110a-h comprising the R6 moiety, which in the pyrazinone is located in position 6, reacted
with the glycine benzyl ester (109), forming the imin. Addition of trimethylsilyl cyanide and microwave irradiation yielded the intermediate
α-aminonitrile, which was purified by flushing the crude material through a
plug of silica. In order to avoid a symmetrical oxamide in the cyclization
step, the α-aminonitrile was first enriched with HCl gas with a subsequent
cyclization using oxalyl chloride and microwave heating at 145-170 °C for
66
10-25 min, or by reflux over-night, to obtain 113-120 in 28-66% yield over
two steps.
The desired R6 groups found in 121-127 were not available as aldehydes.
Thus, the corresponding alcohols (112a-g) were first oxidized by sulfur trioxide-pyridine complex, dimethylsulfoxide (DMSO) and Et3N in DCM at
room temperature. For the naphthyl derivatives (111a and b), the corresponding carboxylic acids were first reduced to the alcohols (112a and b)
followed by oxidation to aldehydes and a subsequent Strecker-type reaction
by in situ addition of TMS-cyanide and 109, under reflux for one hour. The
ring closure procedure described previously gave 121-127 in 25-43% yield
over three steps.
Scheme 5. Reaction conditions: (a) TMSCN, DIPEA, DME, MW, 110-170 °C,
10 min or reflux, 30 min; (b) LiAlH4, THF, -78 °C to rt; (c) SO3-Py, Et3N, DMSO,
DCM, rt, 1 h; (d) Dess-Martin periodinane, DCM, rt, 30 min; (e) 109, TMSCN,
DIPEA, reflux, (f) HCl gas, Et2O, then oxalyl chloride, DME, MW, 145 °C for 25
min or reflux over-night (25-66% from 109).
67
5.2 Introduction of Urea Substituents
The urea moieties in position 3 of the pyrazinone were introduced in a palladium (Pd)-catalyzed C-N urea arylation protocol, which was evaluated and
established for various urea derivatives as described in section 6.2. However,
before the protocol was thoroughly investigated, another strategy was tried,
as described in the following section.
Introduction of P4P5 urea via isocyanate
In the first attempt to incorporate a sophisticated urea moiety, the C3chlorine on the pyrazinone was replaced with an amino functionality by addition of 25% NH3 in H2O to 127 at 110 °C for 6 h which gave 128 in 55%
yield (Scheme 6). The amino group in 128 was weakly nucleophilic and after
unsuccessful trials using pyridine as the base, NaH was applied, which enabled the amino functionality to react with the isocyanate 129.125,131 However,
the 4,4-dimethylpiperidine-2,6-dione moiety on 130 hydrolyzed to some
extent in THF and 1,2-dimethoxyethane (DME) at 100 °C for 15-30 min.
Additionally, the benzylester present in 128 partly hydrolyzed resulting in a
complicated reaction mixture. After several attempts it was, however, possible to obtain 43% of 130.
Scheme 6. Reaction conditions: (a) 25% NH3 in H2O, 110°C, 55%; (b) 60% NaH,
DME, rt, 129, MW 100°C, 15 min, 43%.
Formation of urea building blocks
Clearly, the above procedure was unreliable and difficult to master. Thus, a
protocol was optimized and evaluated to facilitate the introduction of complicated urea building blocks to position 3 of the pyrazinone (section 6.2).
The ureas 136 and 137 were prepared as outlined in Scheme 7.
First, the phthalimido group of 131132,133 was deprotected with 40% methylamine in H2O. Generation of the 3-pyridine sulfonamide 133 was followed by alkylation of the nitrogen using methyl iodide and cesium carbonate (134). Along with the isocyanate 135133 in the following step, the
corresponding symmetrical urea was formed, which explained the low yield
68
(25%). In the literature it was found that symmetrical ureas can be formed
under the described conditions in the presence of a tertiary amine (i.e. the
pyridine in this reaction).134 In the absence of pyridine (or any tertiary
amine) when synthesizing 137, no byproduct was detected. The desired ureas were finally obtained by addition of 0.5 M ammonia in dioxane (136,
quant. and 137, 93% yield).
Scheme 7. Reaction conditions: (a) 40% CH3NH2 in H2O, rt, 88%; (b) 3-pyridine
sulfonylchloride, TEA, DCM, 0°C/rt, 66%; (c) Cs2CO3, CH3I, DMF, 0°C/rt, 76%;
(d) i. 4 M HCl in dioxane, rt, ii. sat. NaHCO3 aq., DCM, triphosgene, 0°C/rt; (e) 0.5
M NH3 in dioxane, 0°C/rt.
Interestingly, when treating 133 with 4 M HCl in dioxane in order to remove the Boc group, followed by treatment with triphosgene to obtain 138,
the ring-closed compound 139 was formed in high yields (Scheme 8). Therefore, it was not possible to manufacture the urea via this protocol.
Scheme 8. Reaction conditions: (a) 4 M HCl in dioxane, rt, sat. NaHCO3, DCM,
triphosgene, 0°C/rt.
Palladium-catalyzed C-N urea arylation
The urea/urea derivatives tert-butyl urea, benzohydrazide, 1-(tertbutyl)imidazolidin-2-one, 136 or 137 were installed via chemoselective Pdcatalyzed C-N arylation of the C3-chlorine on pyrazinones 113-127 (Scheme
69
9) to yield 140-158 in 34-89%. It was found that the benzyl ester in some
entries was partly or fully converted into the methyl ester due to residues of
methanol in the DME. Exchange of the solvent to THF in the preparation of
155 conserved the benzyl ester.
As found previously111,112 the chlorine in position 3 was much more reactive than the chlorine in position 5, because of low electron density on the
C3. More surprisingly, complete C3 selectivity was also noticed when the
reaction conditions were applied to the pyrazinones 118-120 and 124-126,
which contained R6 aryl bromide moieties. For similar selectivity patterns
under Pd-catalyzed cross coupling, see section 6.2 and density functional
theory (DFT) calculations.
Scheme 9. Reaction conditions: (a) tert-butylurea, Benzohydrazide, 1-(tertbutyl)imidazolidin-2-one, 136 or 137, Pd(OAc)2, Xantphos, Cs2CO3, DME or THF,
MW 100-110 °C, 15-20 min or reflux, 0.5-1 h.
5.3 Synthesis of P1P1’ Building Blocks
The P1P1’ building blocks were prepared as described in Schemes 10-12 to
furnish an amino functionality successively acting as nucleophile in the amide coupling reactions found in Scheme 13.
Aromatic acyl sulfonamides
The various P1P1’ building blocks 161-171 and 174 containing different
regioisomers and P1 substituents were prepared as outlined in Scheme 10,
utilizing a carbonyl diimidazole (CDI)-promoted coupling of the sulfonamides with the carboxylic acids (160a-e and 172). In general this reaction
worked well (47-80%) but in two reactions the yields were surprisingly low
(163, 19% and 173, 26%). The lower yield of 163 can probably be explained
by steric hindrance from the trifluoro methyl group situated in the ortho position of the aryl sulfonamide.
70
The bromo derivative 166 was further modified by displacing the halogen
with a vinyl (169) and a pyrimidyl (170) moiety via microwave-assisted
Suzuki coupling (66% and 48% yield, respectively). The sulfonamide nitrogen on 161 was methylated to 171. Finally, in order to uncover the free
amine, the Boc group was removed using 4 M HCl in dioxane, which gave
the hydrochloride salt of the amines. The nitro group in 173 was reduced to
the amine by Pd catalyzed hydrogenation to give 174 in 92% yield.
Scheme 10. Reaction conditions: (a) Boc2O, NaOH, dioxane; (b) CDI, THF, 66-68
°C, 2-CF3-benzene sulfonamide, 3-CF3-benzene sulfonamide, 4-CF3-benzene sulfonamide or 4-(CH3)-benzene sulfonamide, DBU, rt; (c) 2,4,6-trivinyl cycloboroxane pyridine complex or pyrimidine boronic acid, Pd(OAc)2, HP(tBu)3BF4, K2CO3,
H2O, DME, MW 100 °C, 15 min; (d) Cs2CO3, MeI, DMF, 65 °C; (e) 4 M HCl in
dioxane, rt, quant.; (f) Pd/C (10%), H2, EtOAc, 1 atm, rt, 92%.
P1 cyclobutyl-containing building blocks
The spirocyclobutyl intermediate 179 (Scheme 11) was obtained via a fourstep procedure previously described,135,136 starting from a 2(chloromethyl)oxirane, and including epoxide opening (176), cyclization to a
cyclobutyl (177),135 selective hydrolysis (178) and Curtius rearrangement
(179).136 The ethylester was thereafter hydrolyzed, and coupled with the
sulfonamide, in a similar manner to that described above, which gave 181 in
an excellent yield (89%) with subsequent Boc deprotection. From 178 and
onwards the cyclobutyl moiety contained two isomers (cis and trans).
71
Scheme 11. Reaction conditions: (a) BnBr, HgCl2, 160 °C, 55%; (b) 60% NaH,
diethyl malonate, dioxane, 0 °C to reflux, 53%; (c) NaOH, H2O/EtOH reflux, 54%;
(d) DPPA, EtN3 toluene, t-butoxide, reflux, 40%; (e) LiOH, H2O, THF, rt, quant.; (f)
CDI, THF, 66 °C, 4-CF3-benzene sulfonamide, DBU, rt; (g) 4 M HCl in dioxane, rt,
quant.
Reversed aromatic acyl sulfonamides
For the preparation of the reversed acyl sulfonamides 184-186 we again
utilized a nitro moiety as a masked amine (182a-c). Coupling with 4trifluoromethyl benzoyl chloride gave the intermediates 183a-c in moderate
yields (45-58%) as depicted in Scheme 12. Reduction of the nitro group
produced the building blocks 184-186 in 65-87% yield. It is known from the
literature that acyl sulfonamides containing an amino group in the ortho
position are precursors in the preparation of benzothiadiazine analogs by
exposure to heat under acidic conditions.137,138 It was indeed noticed that
during hydrogenation of the ortho derivative 183a, a byproduct with the
actual molecular weight of the ring-closed/dehydrated compound was
formed. The amount of byproduct seemed to increase during the prolonged
reaction time. Since the desired reduction of the nitro moiety proceeded efficiently in only two hours, it was possible to suppress the quantity of the byproduct (<5%) and the free amine was immediately coupled with the pyrazinone scaffold as described in Scheme 13.
Scheme 12. Reaction conditions: (a) 4-pyrrolidino pyridine, pyridine, 4-(CF3)benzoyl chloride, toluene, rt, N2-atm; (b) Pd/C (10%), H2, EtOAc, 1 atm, rt.
72
5.4 Amide Coupling of P3 Pyrazinones and P1P1’
Building Blocks
After urea N-arylation (Scheme 9), the methyl/benzyl esters were hydrolyzed, followed by peptide coupling between the formed carboxylic acid and
the building blocks presented in Schemes 10-12 (Scheme 13).
Scheme 13. Reaction conditions: (a) (i) K2CO3, CH3CN/H2O, MW 100-120 °C, 1520 min (except for 130), (ii) P1P1’ building block, POCl3, pyridine, -15 °C/rt, 489% over two steps, or P1P1’ building block, HATU, DIEA, DCM, rt to 45 °C, 1168% over two steps.
Initially, we used HATU to promote the coupling. However, the reaction
appeared slow and low yielding, probably because of the poor nucleophilicity of the P1P1’ building block 161, and much of the starting material remained intact in the HATU-promoted coupling (58, 22% yield). Hence, we
decided to investigate alternative coupling procedures and found that phosphoryl chloride in pyridine at low temperatures provided the final inhibitors
62-75, 82, 83, 86, 87, 90-92, 95, 97-100 in 14-89% yield. The low yields of
compounds 84 (10%), 85 (4%) and 105 (11%) were partly the result of several purifications on a silica column, followed by preparative HPLC, which
resulted in very small amounts of the pure compounds. These yields could
therefore possibly be improved. However, compound 104 (8%) which contained the hydrazide moiety as a P3 capping group, was partly unstable and
present in at least two tautomeres. This complicated both the introduction of
the hydrazide moiety (155, Scheme 9) and purification of the final target
compound 104.
During the synthesis of compound 88 we needed to reconsider HATU as
a coupling reagent since the vinyl moiety was unstable in the presence of
phosphoryl chloride. Interestingly, HATU worked very well, in just 2.5 h
reaction time (67%). Compounds 89 (54%), 94 (57%), 102 (68%), 106
(52%), 107 (61%) and 108 (71%) were also successfully coupled in the pres-
73
ence of HATU. The low yield of 101 (11%) was due to repeated purification
attempts, providing small amounts of the pure target compound.
5.5 Final Decorations and Modifications
For some of the inhibitors (76-81, 93, 96 and 103) a final conversion was
needed to achieve the desired inhibitors, as described in the two following
sections.
Suzuki coupling to the R6 arylbromide
Decoration of the aryl bromides 70-75 was achieved under microwaveassisted Suzuki-Miyaura coupling conditions, using 3-pyridyl boronic acid
(Scheme 14). The yields ranged between 5% and 69%, with poor outcomes
for the sterically hindered ortho derivatives 78 (20%) and 81 (5%) and higher yields for the meta and para analogs (76, 77, 79 and 80, 41-69%). Although five equivalents of boronic acid were used, no competing coupling
with the C5 chlorine was observed.
Scheme 14. Reaction conditions: (a) 3-Pyridyl boronic acid, Pd(PPh3)2Cl2, Na2CO3,
H2O, EtOH, DME, MW, 120 °C, 15 min (5-69%).
Removal of protecting groups
The inhibitors 93, 96 and 103 were finally synthesized as described in
Scheme 15. The methyl ester in 94 and the benzyl ester in 141 were hydrolyzed in good to excellent yields (75% and 98%, respectively). The benzylprotecting group positioned on the spirocyclobutyl hydroxyl in compound
102 was removed via Pd-catalyzed hydrogenation under mild conditions (rt,
1 atm). However, the chlorine in position 5 was also removed, and a shorter
reaction time did not selectively remove the benzyl group. Although undesirable in this case, from a synthetic point of view this observation was interesting since the C5 chlorine is often considered to be more or less unreactive.
74
Scheme 15. Reaction conditions: (a) K2CO3, CH3CN/H2O, MW 100 °C, 15 min. (b)
Pd/C (10%), H2, MeOH, 1 atm, rt, 59%.
75
6 Method Development (Papers IV and V)
6.1 Palladium as a Catalyst in Organic Synthesis
In 1959, the so-called Wacker oxidation process emphasized the d-block
transition metal Pd as a valuable catalyst. Nowadays, Pd is one of the most
frequently used transition metals in preparative organic synthesis, as a result
of its ability to catalyze C-C and C-heteroatom bond formations with high
chemo- and regio-selectivity.139 The importance of the Pd(0)-catalyzed
cross-coupling reactions was accentuated in 2010, when the Nobel Prize in
Chemistry was awarded to Heck, Negishi and Suzuki for their contributions
in this research area.140
The usefulness of Pd can be explained by its inherent fundamental properties. The moderately large atomic size provides a stable, but also wideranging, reactive metal. Pd is primarily found in oxidation states 0 or +2,
with a tetrahedral (d10) or a square planar (d8) geometry and a low barrier for
moving between these states (oxidation/reduction). The relatively electronegative Pd makes the C-Pd bond rather nonpolar which explains the low
reactivity towards polar groups, such as ketones, esters, amides and aldehydes, etc. These properties combined with a low risk for radical or oneelectron reactions endorse Pd as a useful catalyst with relatively few unwanted side reactions.139,141
Indeed, since the beginning of the 1970s when the C-C bond formation
was demonstrated under Pd catalysis, this type of reaction has been widely
used and further developed by researchers all over the world, both in academia and the industry. The influence of the steric and electronic properties
of ligands led to the design and preparation of numerous ligands which subsequently enabled cross couplings, to generate bonds like C-N, C-O and C-S,
etc.140
6.2 C-N Urea Arylation
In 1995, Buchwald and Hartwig, independently of each other, discovered
Pd-catalyzed amination of aryl halides.142,143 The scope of this reaction subsequently also covered arylation of less reactive nucleophiles, e.g. amides,144
carbamates145 and sulfonamides.146,147 Six years later, in 2001, Beletskaya
and co-workers managed to arylate ureas under Pd-catalyzed conditions.148
76
This was an important alternative to the conventional synthesis of ureas using toxic isocyanate or phosgene. The catalytic cycle for Pd-catalyzed amidation (Figure 23) includes oxidative addition of aryl halide to the Pd0 species, creating a PdII complex, followed by the introduction of the nucleophile
(e.g. a urea moiety). Subsequently, the catalytic cycle is closed by a reductive elimination, forming the desired C-N bond and regenerating the Pd0
species. Mechanistic studies suggest that the reductive elimination is the
rate-determining step for the ureidation reaction.149,150 The development and
evaluation of various ligands in Pd-catalyzed amidation reactions indicated
that the rate of reductive elimination is increased by steric and electrondeficient ligands on Pd, electron-deficient aryl halides and electron-rich nucleophiles.148–152
Several modified protocols have been developed for the arylation of various ureas, including both mono- and bidentate ligands as well as a variety of
aryl halides and substrates.149,150,153–155 Recently, a few heteroaryl halides
were investigated as components of the catalytic cycle.156,157 However studies on heterocyclic structures that are interesting as templates in medicinal
chemistry are still limited.
Figure 23. The proposed catalytic cycle for the palladium-catalyzed amidation (i.e.
ureidation).149 Ar = Aryl, X = Leaving group, L = Ligand.
6.3 Palladium Catalyzed Urea Couplings to 3,5Dichloro-2(1H)-Pyrazinones (Paper IV)
As discussed in a previous section (4.1) the 2(1H)-pyrazinone molecule has
found its application in various medicinal chemistry projects. We found that
a tert-butyl urea as a P3-capping group improved the inhibitory potency of
pyrazinone-based HCV NS3 protease inhibitors (section 4.3.1). However,
the introduction of urea to the C3 position of a pyrazinone is not a well77
established procedure. In a few patent applications, urea moieties have been
installed using a pyrazinone amine and an isocyanate158 or an activated carbamate.159
A literature survey revealed a considerable number of biologically active
N-aryl and N-heteroaryl ureas, which are able to improve the biological stability of peptidomimetic inhibitors.160–162 Given our increased knowledge of
C-N arylations, and the potential to exploit the pyrazinone scaffold in various peptidomimetic compounds, we felt encouraged to explore scope and
limitations of a C-N urea arylation to the 3,5-dichloro-2(1H)-pyrazinone
(Figure 24).
Figure 24. Pd-catalyzed C-N arylation of urea analogs to the 3,5-dichloro-2(1H)pyrazinone.
Ligand screen
The investigation of urea coupling to pyrazinone was initiated with a ligand
screen, in order to analyze the impact of employing mono- or bidentate ligands to the protocol (Figure 25). 1-Benzyl-3,5-dichloro-6-methyl-2(1H)pyrazinone (187a) was selected as a model substrate and tert-butylurea was
used as nucleophile. Pd(OAc)2 was chosen as the catalyst, Cs2CO3 was used
as base and DME as solvent. All reactions were performed in a dedicated
microwave reactor (MW), at 100 °C for 20 min (Table 15).
Figure 25. Ligands evaluated in the optimization study.
78
Monodentate ligands (188a,b) and the nitrogen-based bidentate ligand
(188c) gave poor yields of 189a (6-11%). Along with the formation of product, a major byproduct was also detected in entries 1-3, which explained the
low yield of product in combination with small amounts of recovered starting material. Analysis of the byproduct by NMR and liquid chromatographymass spectrometry (LC-MS) suggested structure 190a (Figure 26), which
originated from two coupled pyrazinones. A possible explanation for the
formation of 190a is that the azomethine nitrogen of a second 2(1H)pyrazinone coordinated to Pd, making the actual C-N formation feasible
during the reductive elimination step.
Table 15. Optimization study of C-N urea arylation between the 3,5-dichloro-2(1H)pyrazinone and tert-butyl urea.
Entry
Ligand
Base
189a (%)a
187a (%)b
1
2
3
4
5
6
7e
8f
9
10
11
12
13
14g
188a
188b
188c
188d
188e
188f
188f
188f
188f
188f
188f
188f
-
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
K2CO3
K3PO4
Et3N
Cs2CO3
Cs2CO3
11
7
6
73c
63d
91
92
91
82
86
45
22
2
0
3
6
13
0
0
0
0
0
0
0
43
52
23
n.d.
Reaction conditions: 187a (0.5 mmol, 1.0 equiv.), tert-butylurea (1.5 mmol, 3.0 equiv.),
Pd(OAc)2 (0.025 mmol, 0.05 equiv.), 188a-f (0.04 mmol, 0.08 equiv.) and Cs2CO3 (1.0 mmol,
2.0 equiv.) in 3.0 mL of DME, 100 °C, 20 min. aIsolated yield. bRecovered starting material
187a. c10% isolated yield of 190b (Figure 26). d4% isolated yield of 190b (Figure 26). e80 °C,
20 min. f100 °C, 10 min. gWithout Pd(OAc)2.
79
Figure 26. Byproducts 190a and 190b.
Gratifyingly, an improved outcome, with no formation of this byproduct,
was achieved by changing to phosphorous-based bidentate ligands (entries
4-6). However, 1,2-bis(diphenylphosphino)ethane (dppe) (188d, entry 4,
73%) and 1,3-bis(diphenylphosphino)propane (dppp) (188e, entry 5, 63%)
caused partial cleavage of the urea moiety, yielding some of the amino compound 190b (Figure 26); this was not found when employing 4,5bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos) (188f, entry 6).
Moreover, absence of ligand (entry 13) or removal of the Pd catalyst (entry
14), resulted in low or no formation of product. These outcomes further emphasized the importance of the ligand for the reaction to proceed successfully. Consequently, Xantphos was identified as the most suitable ligand in this
study and was selected for further optimization studies.
Influence of base and reaction conditions
Although a satisfying outcome was achieved via the introduction of
Xantphos as ligand, we decided to evaluate a few different bases, as well as
the reaction time and temperature, in order to determine the robustness of the
protocol. It was found that K2CO3 and K3PO4 gave good yields (82% and
86%, respectively) which were close to the yield found for Cs2CO3 (91%).
Et3N, on the other hand, hampered the reaction and resulted in incomplete
conversion of starting material with a low yield of 189a (45%). Removal of
base gave only partial conversion of starting material and low product yield
(22%). Thus, the presence and choice of base were essential for an efficient
reaction.
The reaction was performed with Xantphos and Cs2CO3 at 80 °C for 20
min as well as at 100 °C for 10 min (entries 7 and 8). The excellent yields
indicated that the protocol was reliable with fast conversion also at decreased
temperatures. The milder conditions were examined on the somewhat more
complex urea derivatives 1-(tert-butyl)imidazolidin-2-one and 1-(pyridine-3yl)urea (80 °C, 20 min and 100 °C, 10 min, respectively) which resulted in
incomplete conversion of 187a. Bearing in mind that the protocol should
allow installation of a wide variety of ureas, these control experiments sup-
80
ported the choice of conditions found in entry 6 (100 °C, 20 min) for the
extended study.
Scope and limitations
A broad investigation to cover a wide variety of ureas was carried out next,
including aliphatic, aromatic and sterically hindered ureas (Table 16). Overall, the protocol performed well with most of the ureas. The aliphatic methyl,
dimethyl and ethyl ureas were arylated in good to excellent yields (82-94%,
entries 2-4) as was the sterically hindered cyclic urea (96% yield, entry 5).
The reaction protocol also handled the morpholino urea well (189f, 82%
yield).
Table 16. Palladium-catalyzed C3 coupling of various ureas to 3,5-dichloro2(1H)pyrazinone.
Product
Yield (%)a
1
189a
91
2
189b
94
3
189c
90
4
189d
82
5
189e
96
6
189f
82
7
189g
191
45
41
Entry
Urea
81
8
189h
50
9
189i
67
10
189j
85b
11
189k
84
12
189l
71
13
189m
96c
14
189n
56
67c
15
189o
Tracesc
Reaction conditions: The reaction was performed under microwave irradiation in a sealed
vial: 187a (0.5 mmol, 1.0 equiv.), urea analog (entries 1-15) (0.75 - 1.5 mmol, 1.5 – 3.0
equiv.), Pd(OAc)2 (0.025 mmol, 0.05 equiv.), Xantphos (0.04 mmol, 0.08 equiv.) and Cs2CO3
(1.0 mmol, 2.0 equiv.) in 3.0 mL of DME, 100 °C, 20 - 25 min. aIsolated yield. b25 min reaction time. c1.5 equiv. urea.
When the reaction conditions were applied to the allyl urea (entry 7) the
byproduct 191 (41%, Figure 27) negatively affected the formation of 189g
(45%). At least two pathways could possibly give the byproduct 191. Either
the more hindered nitrogen of the allylurea was coupled to the pyrazinone
followed by hydrolysis of the urea, or the free amine of allylurea hydrolyzed
in situ competed as a component in the catalytic cycle. Despite a low yield,
these results presented a reasonable approach to the introduction of this
chemical handle which can be utilized in further modifications, including
macrocyclizations,163 epoxidations164 and aziridinations,164 etc., which are
reactions widely performed to prepare bioactive compounds.163,164
82
Figure 27. Byproduct 191 formed in entry 7, Table 16.
The hydroxyethylurea, with potentially two nucleophilic positions available, produced only the desired product (189h). However, during purification
on a silica column, the product decomposed and provided a lower amount of
189h (50%) than expected. The biuret (entry 9) provided 189i in 67% yield,
despite some di-arylation (<10%) of the biuret.
In general, the protocol handled the aromatic ureas very well (entries 1012, 71-85% yield). In order to reach full conversion of the basic pyridyl urea
(entry 10), a 25 min reaction time was required (189j, 85%). The fairly acidic hydroxyl group (entry 11) was well tolerated in the protocol, giving 189k
in 84% yield. Gratifyingly, 5H-dibenzo[b,f]azepine-5-carboxamide (entry
12) gave 189l in 71% yield despite the sterically congested urea.
One important investigation within this set of reactions was to examine
the selectivity pattern between the 3,5-dichloro-2(1H)-pyrazinone and the
chloro-, bromo- and iodophenyl ureas (entries 13-15). The purification of the
bromo compound (189n, 56%) was complicated due to the presence of byproducts which most likely originated from the excess urea reactant. In an
attempt to reduce the amount of byproducts formed, the reaction was performed using 1.5 equiv. of 4-bromophenyl urea. This was a fruitful modification, which indeed reduced the amount of byproducts and facilitated the
purification (189n, 67% yield). The same strategy was followed in entries 13
and 15, using the chloro- and iodophenyl ureas, respectively. Interestingly,
the outcomes of these two reactions were entirely different. The chlorophenyl derivative (entry 13) gave an excellent yield (189m, 96%) while the
4-iodophenyl urea (entry 15) only provided traces of product 189o. The potentially higher reactivity of the 4-iodophenyl urea compared to the C3 chlorine on the 2(1H)-pyrazinone 187a and polymerization of the iodophenyl
urea could explain the low amount of product formed. We decided to scrutinize the selectivity pattern by performing DFT calculations.
DFT calculations have previously been used to investigate the oxidative
addition of aryl halides to Pd complexes.165,166 However, the oxidative addition of the 3,5-dichloro-2(1H)-pyrazinone, which has two potentially reactive positions for this type of reaction, has not been investigated. Initially,
the DFT calculations showed a free energy requirement of 30 kJ mol-1 for
oxidative addition to the C3 position, compared with 104 kJ mol-1 for the C5
chlorine. The expected trend in free energy requirement between the various
aryl halides was verified, i.e. Cl > Br > I with energies of 107 kJ mol-1, 72 kJ
83
mol-1 and 60 kJ mol-1, respectively. Thus, it is suggested that the chlorine in
the C3 position is more reactive than the iodophenyl urea with respect to
oxidative addition. The initial hypothesis suggesting that polymerization of
the iodo phenyl urea could account for the poor formation of product was
therefore contradictory to the DFT calculations. For that reason, the reaction
was further examined by a few control experiments.
First, we ran a reaction following the conditions in entry 1, Table 16, with
the addition of 3 equiv. of 3-iodotoluene. Minor amounts (<10%) of 189a
were formed, in contrast to the outcome for entry 6 (91%). This indicated
that the presence of an aryl iodide somehow impeded the reaction. It was
further noted that 3-iodotoluene did not react with tert-butylurea. Moreover,
the byproduct 190a (Figure 26) was observed, implying a suboptimal catalytic system for the desired reaction to occur. Subsequently, a control reaction was performed in the absence of the pyrazinone 187a. Minor amounts
(<10%) of 3-iodotoluene coupled to tert-butylurea, which also supported the
idea that aryl iodides prohibited the C-N urea formation. Taken together,
these experiments verified the DFT calculations suggesting the C3 chlorine
on the pyrazinone was more prone to oxidative addition to Pd than aryl iodides.
Although limited to urea containing aryl bromides and chlorides, the results presented in entries 13 and 14 allow for selective couplings to the C3
chlorine on the pyrazinone, which permit further elaborations on the installed aryl, e.g. Pd cross-coupling reactions.140
6.4 Palladium Catalyzed Carbonylations
The initial efforts to provide amides and esters via amino- and alkoxycarbonylations of aryl or vinyl halides by the use of a Pd catalyst and carbon
monoxide (CO) gas were presented by Heck and co-workers in 1974.167,168 A
proposed mechanism of a typical carbonylation reaction is outlined in Figure
28.139 The oxidative addition of aryl halide generates the PdII complex, followed by insertion of CO. The nucleophile then attacks the electron poor PdII
complex, which expels the formed carbonyl compound. Finally, a reductive
elimination step follows, which regenerates the Pd0 catalyst.169
84
Figure 28. Proposed catalytic cycle for the carbonylation of aryl halides.L = Ligand,
Ar = Aryl, X = Leaving group.
An interesting feature regarding the carbonylation chemistry is the possibility of delivering a wide variety of compounds by simply changing the
nucleophile. This makes it a suitable reaction in the development of compound libraries of biologically active analogs. A major drawback, however,
is the use of gaseous CO, which requires special equipment that can endure
high pressure.170 Moreover, the CO gas is toxic and flammable, which calls
for caution when performing these experiments. Gas-free alternatives are
therefore highly valuable. As a matter of fact, several crystalline and liquid
CO sources are frequently used in small-scale synthetic procedures, where
CO is released in situ by an activator or heat and is subsequently incorporated into the catalytic cycle.171–174
Mo(CO)6, a gas-free CO source, has been widely explored by researchers
at our department.174 Recently, Skydstrup and co-workers developed a twochamber reaction system172 which enabled ex situ generation of CO. This
system has been further evaluated using Mo(CO)6 as the CO source.175
6.5 Synthesis of Acyl Sulfinamide, a Versatile
Functionality (Paper V)
In line with our overarching goal to investigate bioactive molecules and bioisosteres that are useful to drug discovery, we identified acyl sulfinamide
(I) (Figure 29) as an interesting functionality found in different bioactive
molecules.176–178 Acyl sulfinamide has been considered as a carboxylic acid
bioisostere due to its slightly acidic proton (ca. pKa 6)179 in, for example,
HCV NS3 protease inhibitors.180 Besides its direct application in drug discovery, acyl sulfinamide also works as a precursor in the preparation of sulfonimidamides (II)181–187 and acyl sulfonimidamides (III and IV)188,189 (Figure 29).
85
Figure 29. Acyl sulfinamide (I), sulfonimidamide (II), acyl sulfonimidamides (III),
(IV).188,189
These acyl sulfinamide derivatives have recently been evaluated in drug
discovery-related research. The sulfonimidamide (II) has been recognized as
a sulfonamide bioisostere in γ-secretase inhibitors.186,190 PK evaluation revealed increased solubility, decreased lipophilicity, and decreased plasma
protein binding compared with derivatives containing a sulfonamide.186 Cyclic sulfonimidamides have also been evaluated as novel carboxylic acid
bioisosteres, since they are associated with higher Caco-2 permeability and
lower efflux than the carboxylic acid isostere, tetrazole.191 Moreover, the
acyl sulfonimidamides (III and IV) have been identified as potential carboxylic acid bioisosteres based on their in vitro PK properties.192
Preparation of acyl sulfinamide
Acyl sulfinamide has typically been constructed from sulfinamide and carboxyl acid anhydride, by exposure to n-BuLi or NaH at low temperatures.
179,193
With an interest in further evaluating acyl sulfinamides and derivatives
thereof as carboxylic acid bioisosteres, we took the opportunity to facilitate
the synthesis of this functionality to make it more accessible.
The capacity of Pd-catalyzed carbonylation is expansive, which is demonstrated by the number of reported functionalities installed by various carbonylation protocols, e.g. amides, carboxylic acids, esters, ketones and alkynes.170,194 However, to the best of our knowledge, sulfinamide has not yet
been evaluated as a nucleophile in this type of reaction. If a satisfying protocol could be attained, varied aromatic moieties could be introduced next to
the carbonyl functionality, which should be of interest in diverse library syntheses for SAR studies as well as for adjustment of other properties important to drug design (Figure 30).
Figure 30. A schematic description of a Pd catalyzed carbonylation reaction.
Initially, the reaction conditions previously developed for Mo(CO)6-based
carbonylative transformations for the functionalization of sulfonimidamides
(III) (Figure 29) were applied.188,189 A two-vial system was used (Table 17)
and chamber 1 (C1) was charged with sulfinamide (192a), 4-iodoanisole,
Pd(OAc)2 and Et3N in 1,4-dioxane. Thereafter, Mo(CO)6 together with DBU,
86
which has been suggested to facilitate the release of CO from Mo(CO)6,174 in
1,4-dioxane were added to chamber 2 (C2). The two vials were capped, and
the reaction system was heated to 80 °C for 6 h (entry 1, Table 17). The substrate 192a was fully consumed but purification only provided 20% yield of
the desired product (193a). It was found that a byproduct, the thioester 194
(Figure 31)195 was generated along with the product, which explained the
relatively low yield.
Table 17. Optimization of reaction conditions between sulfinamide and 4iodoanisol.
Entry
Base
Temp
Time
Yield (%)a
1
Et3N
80
6
20
2b
Et3N
80
3
17
Et3N
80
2
12
3b
4c
Et3N
60
1
0
5c
Et3N
70
18
22
70
2
71
K2CO3
6
7
80
2
79
K2CO3
8d
80
2
32
K2CO3
80
1
16
K2CO3
9e
Reagents and conditions: C1: sulfinamide (0.354 mmol), 4-iodoanisol (0.885 mmol), base
(0.885 mmol), Pd(OAc)2 (0.018 mmol) and 1,4-dioxane (2 mL). C2: Mo(CO)6 (0.885 mmol),
DBU (1.563 mmol) and 1,4-dioxane (2 mL). aIsolated yields. b0.1 equiv. Xantphos. c2 equiv.
DMAP. dCO (75 psi). eCO (50 psi).
Figure 31. Byproduct (194) formed using Et3N as base in the carbonylation reaction (Table
17).
These disappointing, yet challenging, results needed to be addressed before an acceptable yield could be obtained. We planned to successively vary
the reaction time, investigate the impact of ligand, and stimulate the nucleophilic attack of the sulfinamide on the catalytic system.
We speculated that a prolonged reaction time could support the formation
of 194 at the expense of 193a by reducing unreacted starting material to
thiol. Therefore, the reaction time was reduced to 3 h and Xantphos, a ligand
that potentially enhances the progress of the reaction, was added (entry 2).196
However, a similar yield to that in entry 1 was obtained with no obvious
87
advantage from the ligand. Shorter reaction time (entry 3) further lowered
the yield of 193a.
Next we considered an approach for investigating whether the introduction of the sulfinamide was the problematic stage. 4-Dimethylaminopyridine
(DMAP) has previously been applied for enhancing the carbonylation of
poor or sterically hindered nucleophiles by acting as an acylating agent.197
However, in this protocol, no improvement was obtained (entries 4 and 5). A
temperature of 60 °C (entry 4) was not sufficient for the reaction to commence, since neither product nor byproduct were detected after 1 h of heating. At 70 °C with an extended reaction time (entry 5), it was noted that after
about 2-3 h of reaction the amount of 193a did not increase, indicating that
the catalytic system at that point was no longer active.
To summarize these initial investigations, it was found that the byproduct
194 was formed in entries 1-3 and 5, regardless of varying reaction conditions and the presence of ligand and DMAP. A likely explanation is that the
reaction conditions in these entries enabled the reduction of sulfinamide to
the corresponding thiol which subsequently competed with 192a in the nucleophilic attack on the Pd complex. As mentioned, after 2-3 h of reaction,
the formation of product seemed to halt. This could be explained by poisoning of the Pd catalyst, due to the presence of thiol.198,199
We considered that the choice of base could have affected the outcome of
the reaction, so the organic base Et3N in chamber 1 was exchanged to K2CO3
which dramatically increased the formation of product (entry 6). Most importantly, the byproduct 194 was not detected and full consumption of 192a
after 2 h at 70 °C was realized, with a slightly increased yield at 80 °C (entry
8).
It has previously been confirmed that reduction-sensitive moieties, like
the nitro group, are viable under carbonylation conditions, provided that a
two-chamber system is used.175 A nitro group could be considered as a
masked amine, useful for further modifications, so this equipment widens
the application of carbonylations. When employing the sulfinamide as a
component in the Pd-catalyzed carbonylation protocol, our results indicated
that the two-vial system was crucial for successful formation of product. In a
single vial reaction several byproducts were formed, most likely due to the
sensitive sulfinamide group. Moreover, under molybdenum-free conditions
using CO-gas, a pressurized reactor was employed. Lower yields were obtained (entries 8 and 9, 32% and 16%, respectively) which emphasized the
benefits of a two-compartment system for this reaction.
Scope and limitations
To evaluate the protocol, electron-deficient, -neutral and -rich aryl and heteroaryl iodides were investigated, using aryl and alkyl sulfinamides following the conditions in entry 7 (Table 17). For all the presented entries, full
88
consumption of the sulfinamide was reached. Yields in the range of 34% to
79% were obtained (Table 18).
Table 18. Scope of aryl iodides in the carbonylative synthesis of acyl sulfinamides.
Entry R-SO-NH2
Ar-I
Yield
(%)a
Product
1
192a
193a
79
2
’’
193b
76
3
’’
193c
46
4
’’
193d
68
5
’’
193e
58
6
’’
193f
65
7
’’
193g
51
8
’’
193h
38
9
’’
193i
66
10
’’
193j
41
89
11
’’
193k
38
12
’’
193l
71b
13
192b
193m
69
14
’’
193n
76
15
’’
193o
62
16
’’
193p
34
17
192c
193q
49
18
’’
193r
53
Reagents and conditions: C1: sulfinamide (0.354 mmol), aryl iodide (0.885 mmol), K2CO3
(0.885 mmol), Pd(OAc)2 (0.018 mmol) and 1,4-dioxane (2 mL). C2: Mo(CO)6 (0.885 mmol),
DBU (1.563 mmol) and 1,4-dioxane (2 mL), 80 °C, 2 h; aIsolated yields; bReaction time, 3 h.
The aryl iodides with para and meta methoxy substituents (193a, 193b
and 193m) and the electron-neutral aryl iodides (193d-f, 193n, 193o) gave
the highest yields.200 For the ortho iodoanisole (entry 3), steric hindrance
probably affected the outcome of the reaction, since this has been observed
previously.188
Among the aryl iodides containing electron-withdrawing substituents in
the para position, the cyano derivative (entry 9) gave a markedly higher
yield (193i, 66%) than 193g 51%, 193h 38%, 193p 34% or 193r 53%. The
nitro moiety was conserved, with no reduction to the corresponding aniline.
The heteroaryl derivatives (entries 10-12) were relatively well achieved.
Notably, the trityl-protected imidazole (193k) was stable under the present
reaction conditions with no deprotection detected (38%). A comparable high
yield was obtained for the possible PdII-coordinating pyridyl derivative
90
(193l, 71%). However, the 2-iodopyridyl only provided traces of product
(not included in the table). The achievable introduction of heteroaryl iodides
broadens the scope of this protocol, providing means for elaboration with PK
properties in for example the design of bioactive molecules.
Surprisingly, for the benzene sulfinamide (entries 17 and 18), the difference in yield between the electron-rich 4-iodoanisole and the electron-poor
para-nitro aryliodide was small (193q, 49% and 193r, 53%), in contrast to
previous entries. It is important to mention that purification in some entries
was challenging. The compound adhered to the silica, possibly due to the
slightly acidic proton on the acyl sulfinamide nitrogen. For the compounds
containing electron-withdrawing aryls, and thus an increased acidic character
of the acyl sulfinamide functionality, this phenomenon appeared to be more
distinct. This could be a reason for the generally lower isolated yields of
compounds containing electron-withdrawing aryl substituents. Unfortunately, when the reaction conditions were applied to the chiral sulfinamides,
192a and 192b, partial racemization seemed to occur as suggested by optical
rotation.
Arylbromides did not work under these relatively mild conditions. A few
attempts were made to identify a suitable catalytic system by adding a ligand
or changing the Pd source (e.g. Xantphos, Palladacycle/Fu salt, Pd(PPh3)4,
Pd(dppf)Cl2/DCM) and by raising the temperature to 120 °C. However, so
far a protocol that works smoothly also with aryl bromide has not yet been
established.
To summarize chapter 6, a chemoselective and robust palladiumcatalyzed N-arylation protocol to install various ureas at the 3-position of the
3,5-dichloro-2(1H)-pyrazinone scaffold was developed. Bidentate ligands,
Xantphos in particular, were superior to monodentate ligands and in presence of aryl chloride and aryl bromide, the C-3 chlorine was selectively displaced. Subsequently, a novel palladium-catalyzed protocol for the carbonylative synthesis of acyl sulfinamides, was developed. The two-chamber
system, using Mo(CO)6 as CO-source, and the choice of base, K2CO3, were
crucial for the successful carbonylation of various aryl/heteroaryl iodides
with the sensitive sulfinamide functionality.
91
7 Concluding Remarks
The work presented in this thesis was based on two drug discovery projects
within the HCV area, both aiming to inhibit the drug target, NS3 protease.
The criteria differed with respect to the various stages of discovery they represented. In the P2 quinazoline macrocyclic series, the lead structure was
optimized for improved PK properties along with sub-nanomolar Ki and
nanomolar EC50 values. The pyrazinone series, on the other hand, represents an early stage of drug discovery aiming for new lead compounds,
which could be further optimized into coming generation of HCV NS3 protease inhibitors. The main findings are summarized below:
•
92
Improved Caco-2 permeability and promising in vivo PK properties for
inhibitors containing the optimized P2 quinazoline were obtained by replacing the cyclopentane scaffold with the proline urea analog.
For the P3 pyrazinone-based inhibitors, the following main results were
obtained during the evaluation and optimization of the substituents around
the core structure:
•
A urea moiety in the C3 position improved both stability and inhibitory
potency compared with the carbamate analog. Inhibitors containing
P4P5-ureas were prepared and evaluated and indicated allowance for
substituents in this area.
•
Relocation of the P2 group to the R6 position was well accepted and
resulted in achiral inhibitors with improved inhibitory potencies for
elongated R6 moieties. Moreover, the R6 substituents influenced the PK,
with favorable properties for a pyridyl moiety.
•
The resistance profile for this class of inhibitors showed retained inhibitory potencies against known drug-resistant variants of the virus, i.e.
R155K, A156T and D168V. Initial evaluation against genotype 3a displayed promising inhibitory potencies for a set of inhibitors with Ki values 0.6-3.4 µM.
•
Based on evaluation of several P1P1’ building blocks, preliminary results suggested that the acyl sulfonamide did not improve the inhibitory
potency. The P1’ aryl did not appear to have any specific interactions
with the S1’ pocket, as supported by comparable inhibitory potencies for
truncated derivatives. It was found that the P1 aryl in combination with
the P3 pyrazinone and a C3 urea were important for sub-micromolar Ki
values, suggesting that this could be the new lead structure.
•
An efficient Pd-catalyzed C-N urea arylation to the C3 position of the
pyrazinone was developed and successively applied to inhibitors with
elongated P4P5 urea substituents. In line with our interest in identifying
carboxylic acid bioisosteres, a novel Pd-catalyzed carbonylation protocol for sulfinamides yielding acyl sulfinamides was developed.
93
8 Acknowledgements
I have had the privilege to work with many skillful and nice researchers at the Department of Medicinal Chemistry. You have made these years so enjoyable and
rewarding. Thank you all! I would like to express my sincere gratitude to the following people:
My excellent supervisor, Docent Anja Sandström, for your enthusiasm and confidence in the projects and in me. You are a true inspiration both in research and in
teaching and you have gently guided me during these years.
Docent Eva Åkerblom, your experience and caring have been invaluable when
supporting me, regardless of good or bad results. I will miss your engagement in the
HCV project and our always fruitful and pleasant discussions over a cup of tea.
My assistant supervisor, Prof. Mats Larhed, for sharing your experience and showing me what is possible to accomplish with limited time.
Former and present members of the HCV team: Anja, Eva, Anna L, Pernilla, Johan
and Krzysztof for sharing ideas and experiences during the group meetings and in the
lab. Angelica, Eldar, Sofia and Prof. Helena Danielson, for the biochemical evaluation and for discussions regarding assays. Dr. Richard Svensson and Prof. Per Artursson and for providing me with PK data and new insights into the properties of
these molecules. Hiba and Prof. Anders K for molecular modeling and for the beautiful pictures in this thesis. The Pd group for contributing to my C-N urea arylationproject. Fredrik and Docent Christian for the DFT calculations. Anna S and Anna O,
for skillful laboratory work during your master thesis work and SOFOSKO project.
Dr. Prasad, for your contribution and positive attitude in my “last but not least”project. Dr. Sanyaj, for your enthusiasm and chemistry suggestions. Antona Wagstaff, for excellent linguistic revision of this thesis. Linda Å, Drs Patrik, Rebecca,
Eva and Anja for constructive criticism and comments on my thesis.
Rebecca and Anna L, your open arms when I moved to Uppsala made me feel like
home. You shared my “first-grey-hair”-moment and I will never forget our trip to
San Diego; we are probably better scientists than surfers! Charlotta W, Linda A
and Pernilla, for early on sharing your experience what PhD studies is about. Ulrika
for good support and valuable insights. Per and Patrik, for your company in Boston
and in New York and for crossing Manhattan carrying heavy back-packs in the dark.
94
Gunnar, for making the equipment working with us and not against us. Your support
is appreciated and the lab is empty on Wednesdays! My room-mates: Vivek, Suresh
and Johanna for a nice atmosphere and for chats about chemistry and life. Karin, for
kindly serving me coffee and cake during weekends of thesis-writing. Anna S and
Linda Å for your open office door and for inspiring talks about life, society, and
matters outside the life of chemistry. Hiba, for lunches sharing our thoughts. Sara
and Rebecka, for all fun and nice lunch and coffee breaks! Bobo, for knocking on
my closed office door just to make sure I was still alive during the last months of
hard work. Gunilla, for taking such good care of OFK and for supportive company
during late nights. Prof. Anders H, for spreading your genuine passion for medicinal
chemistry. My mentor Dr Jenny Ekegren, for valuable conversations during the first
part of my PhD studies.
Former colleagues at Medivir who inspired me to take this journey! Some of you
have remained good friends. Bertil Samuelsson, Åsa Rosenquist and Xiao-Xiong
Zhou for accepting me at Medivir and guiding me in industrial medicinal chemistry
but also supporting my inherent wish to complete a PhD. Nils-Gunnar Johansson for
introducing me to the challenging world of nucleoside analogs. Per-Ola Johansson
for excellent supervision during my master thesis work at Linköping University and
for letting me synthesize my first protease inhibitors.
Members of the Pharmaceutical PhD Student Council (FDR) 2010-2011, the Doctoral Board 2010-2011 and the Academic Senate 2011-2012, for inspiring and interesting discussions and good work.
The Swedish Academy of Pharmaceutical Sciences and Anna-Maria Lundins stipendiefond for making it possible to attend international conferences. Thanks to:
Stiftelsen Hanna Roos af Hjelmsäters testamentsfond, stiftelsen Hierta af Petersens
fond, Farmaceutiska fakultetens jubelfeststipendium, Cronhielms fond, Stiftelsen von
Krusenstiernas fond, Pauli OG stiftelse and Belfrageska släktföreningen for recognition and support during these years.
Annika, vi delade underbara, roliga, galna och ibland slitsamma år i Stockholm. Alla
våra samtal har varit och är ovärderliga! Robert, så fint att du kom in i Annikas liv
och att vi har fått lära känna varandra. Ert paradis på Rindö har blivit ett härligt
ställa att umgås på.
Sofie, tänk att vi till slut hamnade i samma stad igen! Du är en så fin vän som tror på
mig och min förmåga när jag själv tvivlar. Jag hoppas vi snart kan spela in vår nästa
skiva; den senaste inspelningen har några år på nacken… David, vi tar en lunchdejt snart för vi befinner ju oss löjligt nära varandra om dagarna!
Ylva, jag önskar ofta att Uppsala låg på västkusten. Det var underbart att vara med
dig och din fina familj i somras!
95
Pia, Ulrika och Lotta med familjer, jag missade julgransplundringen i år, men framöver kan ni lita på min närvaro!
Lotta, det är så härligt att få hänga med dig och din Felix. Vi har följts åt på ett speciellt sätt och har sett hur mirakel kan ske. Än är de inte över!
Jan, Jörgen, Per och Anna, förra året blev det två maffiga 50-årskalas, och jag ser
fram emot fler stora som små händelser att fira. Alltid fint att vara med er, vänner!
Kerstin, vilken lyx att ha dig så nära! Du har en förmåga att förgylla med en bukett
tulpaner, eller något litet ”smått och gott” och stöttar på så många sätt i vardagen!
Ingvar, jag är så glad att vi hann lära känna varandra och jag hoppas att du hade
gillat avhandlingen! Annika, Pelle, Jesper, Emrik, Astor och Mira, jag flyttade solo
till Uppsala, och fick inom ett halvår en stor, extra-familj. Jag ser fram emot lite mer
umgänge efter en lång ”upptagen” tid.
Mina älskade bröder, Erik och Hans, vi får se till att träffas oftare för jag saknar er!
Ni är så kloka på många sätt, och jag behöver lite syskonhäng snart! Jennyh, för att
du finns. Din kämparanda är inspirerande! Mina fina, goa brorsbarn Emil och Jennifer, hoppas ni väljer att plugga i Uppsala om sådär 10-15 år, för jag vill se er oftare!
Farmor, det känns lite ledsamt att du inte fick vara med in i mål! Du som inte hade
möjlighet till studier som ung tyckte att en doktorsexamen var ett alldeles självklart
val för mig!
Mamma och Pappa, för att ni alltid har trott på mig och ställt upp när det har behövts, inte minst med all flytthjälp mellan olika städer och lägenheter genom åren.
Det var fint att få fira en tidig jul med er här i Uppsala när den senaste julledigheten
blev väldigt begränsad. Ser fram emot Strömstad i sommar med salta bad och fixande med huset.
Johan♥! Du är en fantastisk person och en superfin pappa med enorm kapacitet. Du
stöttar och tror på mig, och du är den förste jag frågar om råd – vad det än gäller!
Hoppas jag kan fixa det där som kallas hemarbete snart för nu är det i jämställdhetens namn din tur att tänka på annat!
Hedda☼! Du är en väldigt klok ”snart 3-åring” som börjat fundera över vad det är
för spännande jag håller på med på jobbet eftersom ”det aldrig är stängt”! Kanske du
en dag vill läsa den här boken och finna svaren på egen hand?... Älskar dig!
Anna Karin, 8 February 2015
96
9 References
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
Feinstone, S. M.; Kapikian, A. Z.; Purcell, R. H.; Alter, H. J.; Holland, P. V.
Transfusion-Associated Hepatitis Not Due to Viral Hepatitis Type A or B. N. Engl. J.
Med. 1975, 292, 767–770.
Choo, Q.; Kuo, G.; Weiner, A. M. Y. J.; Overby, L. R.; Bradley, D. W.; Houghton, M.
Isolation of a cDNA Clone Derived from a Blood- Borne Non-A , Non-B Viral
Hepatitis Genome. Science (80-. ). 1989, 244, 359–362.
World Health Organization. Hepatitis C: Fact Sheet No 164. 2014, 1–6.
Brown, R. S. Hepatitis C and Liver Transplantation. Nature 2005, 436, 973–978.
Kwong, A. D. The HCV Revolution Did Not Happen Overnight. ACS Med. Chem.
Lett. 2014, 5, 214–220.
Ansaldi, F.; Orsi, A.; Sticchi, L.; Bruzzone, B.; Icardi, G. Hepatitis C Virus in the
New Era: Perspectives in Epidemiology, Prevention, Diagnostics and Predictors of
Response to Therapy. World J. Gastroenterol. 2014, 20, 9633–9652.
Nature Outlook Hepatitis C. A Smouldering Public- Health Crisis. Nature 2011, 474,
S1–S21.
Shepard, C. W.; Finelli, L.; Alter, M. J. Global Epidemiology of Hepatitis C Virus
Infection. Lancet Infect. Dis. 2005, 5, 558–567.
WHO. Guidelines for the Screening, Care and Treatment of Persons with Hepatitis C
Infection. 2014.
Hoofnagle, J. H. Course and Outcome. Hepatology 2002, 36, S21–S29.
Moradpour, D.; Penin, F.; Rice, C. M. Replication of Hepatitis C Virus. Nat. Rev.
Microbiol. 2007, 5, 453–463.
Miller, R. H.; Purcell, R. H. Hepatitis C Virus Shares Amino Acid Sequence
Similarity with Pestiviruses and Flaviviruses as Well as Members of Two Plant Virus
Supergroups. Proc. Natl. Acad. Sci. U. S. A. 1990, 87, 2057–2061.
Bartenschlager, R.; Lohmann, V.; Penin, F. The Molecular and Structural Basis of
Advanced Antiviral Therapy for Hepatitis C Virus Infection. Nat. Rev. Microbiol.
2013, 11, 482–496.
Penin, F.; Dubuisson, J.; Rey, F. a; Moradpour, D.; Pawlotsky, J.-M. Structural
Biology of Hepatitis C Virus. Hepatology 2004, 39, 5–19.
Zeisel, M. B.; Felmlee, D. J.; Baumert, T. F. Hepatitis C Virus: From Molecular
Virology to Antiviral Therapy. 2013, 369, 87–112.
Bartenschlager, R.; Ahlborn-Laake, L.; Mous, J.; Jacobsen, H. Nonstructural Protein 3
of the Hepatitis C Virus Encodes Serine-Type Proteinase Required for Cleavage at the
NS3/4 and NS4/5 Junctions. J. Virol. 1993, 67, 3835–3844.
Failla, C.; Tomei, L.; Francesco, R. De. Both NS3 and NS4A Are Required for
Proteolytic Processing of Hepatitis C Virus Nonstructural Proteins. J. Virol. 1994, 68,
3753–3760.
Vermehren, J.; Sarrazin, C. The Role of Resistance in HCV Treatment. Best Pract.
Res. Clin. Gastroenterol. 2012, 26, 487–503.
EASL Recommendations on Treatment of Hepatitis C 2014. EASL Recomm. Treat.
Hepat. C 2014 2014.
Yang, P.; Gao, M.; Lin, K.; Liu, Q.; Villareal, V. A. Anti-HCV Drugs in the Pipeline.
Curr. Opin. Virol. 2011, 1, 607–616.
Wyles, D. L. Antiviral Resistance and the Future Landscape of Hepatitis C Virus
Infection Therapy. J. Infect. Dis. 2013, 207 Suppl , S33–S39.
97
(22)
(23)
(24)
(25)
(26)
(27)
(28)
(29)
(30)
(31)
(32)
(33)
98
Swadling, L.; Capone, S.; Antrobus, R. D.; Brown, A.; Richardson, R.; Newell, E. W.;
Halliday, J.; Kelly, C.; Bowen, D.; Fergusson, J.; Kurioka, A.; Ammendola, V.;
Sorbo, M. Del; Grazioli, F.; Esposito, M. L.; Siani, L.; Traboni, C.; Hill, A.; Colloca,
S.; Davis, M.; Nicosia, A.; Cortese, R.; Folgori, A.; Klenerman, P.; Barnes, E. A
Human Vaccine Strategy Based on Chimpanzee Adenoviral and MVA Vectors That
Primes, Boosts, and Sustains Functional HCV-Specific T Cell Memory. Sci. Transl.
Med. 2014, 6, 1–16.
Lange, C. M.; Jacobson, I. M.; Rice, C. M.; Zeuzem, S. Emerging Therapies for the
Treatment of Hepatitis C. EMBO Mol. Med. 2014, 6, 4–15.
Pearlman, B. L. Hepatitis C Treatment Update. Am. J. Med. 2004, 117, 344–352.
Feld, J. J.; Hoofnagle, J. H. Mechanism of Action of Interferon and Ribavirin in
Treatment of Hepatitis C. Nature 2005, 436, 967–972.
Lin, C.; Kwong, A. D.; Perni, R. B. Discovery and Development of VX-950, a Novel,
Covalent, and Reversible Inhibitor of Hepatitis C Virus NS3/4A Serine Protease.
Infect. Disord. - Drug Targets 2006, 6, 3–16.
Malcolm, B. A.; Liu, R.; Lahser, F.; Agrawal, S.; Belanger, B.; Chase, R.; Gheyas, F.;
Hart, A.; Hesk, D.; Jiang, C.; Kong, R.; Lu, J.; Pichardo, J.; Prongay, A.; Skelton, A.;
Tong, X.; Venkatraman, S.; Xia, E.; Njoroge, F. G.; Butkiewicz, N.; Ingravallo, P.;
Girijavallabhan, V. SCH 503034, a Mechanism-Based Inhibitor of Hepatitis C Virus
NS3 Protease , Suppresses Polyprotein Maturation and Enhances the Antiviral Activity
of Alpha Interferon in Replicon Cells. Antimicrob. Agents Chemother. 2006, 50, 1013–
1020.
Perni, R. B.; Almquist, S. J.; Byrn, R. A.; Chandorkar, G.; Chaturvedi, P. R.;
Courtney, L. F.; Decker, C. J.; Dinehart, K.; Gates, C. A.; Harbeson, S. L.; Heiser, A.;
Kalkeri, G.; Kolaczkowski, E.; Lin, K.; Luong, Y.; Rao, B. G.; Taylor, W. P.;
Thomson, J. A.; Tung, R. D.; Wei, Y.; Kwong, A. D.; Lin, C. Preclinical Profile of
VX-950 , a Potent , Selective , and Orally Bioavailable Inhibitor of Hepatitis C Virus
NS3-4A Serine Protease. Antimicrob. Agents Chemother. 2006, 50, 899–909.
Rosenquist, Å.; Samuelsson, B.; Johansson, P.-O.; Cummings, M. D.; Lenz, O.;
Raboisson, P.; Simmen, K.; Vendeville, S.; de Kock, H.; Nilsson, M.; Horvath, A.;
Kalmeijer, R.; de la Rosa, G.; Beumont-Mauviel, M. Discovery and Development of
Simeprevir (TMC435), a HCV NS3/4A Protease Inhibitor. J. Med. Chem. 2014, 57,
1673–1693.
Carrion, A. F.; Gutierrez, J.; Martin, P. New Antiviral Agents for the Treatment of
Hepatitis C: ABT-450. Expert Opin. Pharmacother. 2014, 15, 711–716.
Jiang, Y.; Andrews, S. W.; Condroski, K. R.; Buckman, B.; Serebryany, V.;
Wenglowsky, S.; Kennedy, A. L.; Madduru, M. R.; Wang, B.; Lyon, M.; Doherty, G.
A.; Woodard, B. T.; Lemieux, C.; Do, M. G.; Zhang, H.; Ballard, J.; Vigers, G.;
Brandhuber, B. J.; Stengel, P.; Josey, J. A.; Beigelman, L.; Blatt, L.; Seiwert, S. D.
Discovery of Danoprevir (ITMN-191/R7227), a Highly Selective and Potent Inhibitor
of Hepatitis C Virus (HCV) NS3/4A Protease. J. Med. Chem. 2014, 57, 1753–1769.
McCauley, J. A.; McIntyre, C. J.; Rudd, M. T.; Nguyen, K. T.; Romano, J. J.;
Butcher, J. W.; Gilbert, K. F.; Bush, K. J.; Holloway, M. K.; Swestock, J.; Wan, B.L.; Carroll, S. S.; DiMuzio, J. M.; Graham, D. J.; Ludmerer, S. W.; Mao, S.-S.;
Stahlhut, M. W.; Fandozzi, C. M.; Trainor, N.; Olsen, D. B.; Vacca, J. P.; Liverton, N.
J. Discovery of Vaniprevir (MK-7009), a Macrocyclic Hepatitis C Virus NS3/4a
Protease Inhibitor. J. Med. Chem. 2010, 53, 2443–2463.
Summa, V.; Ludmerer, S. W.; McCauley, J. a; Fandozzi, C.; Burlein, C.; Claudio, G.;
Coleman, P. J.; Dimuzio, J. M.; Ferrara, M.; Di Filippo, M.; Gates, A. T.; Graham, D.
J.; Harper, S.; Hazuda, D. J.; McHale, C.; Monteagudo, E.; Pucci, V.; Rowley, M.;
Rudd, M. T.; Soriano, A.; Stahlhut, M. W.; Vacca, J. P.; Olsen, D. B.; Liverton, N. J.;
Carroll, S. S. MK-5172, a Selective Inhibitor of Hepatitis C Virus NS3/4a Protease
with Broad Activity across Genotypes and Resistant Variants. Antimicrob. Agents
Chemother. 2012, 56, 4161–4167.
(34)
(35)
(36)
(37)
(38)
(39)
(40)
(41)
(42)
(43)
Scola, P. M.; Sun, L.; Wang, A. X.; Chen, J.; Sin, N.; Venables, B. L.; Sit, S.; Chen,
Y.; Cocuzza, A.; Bilder, D. M.; D’Andrea, S. V; Zheng, B.; Hewawasam, P.; Tu, Y.;
Friborg, J.; Falk, P.; Hernandez, D.; Levine, S.; Chen, C.; Yu, F.; Sheaffer, A. K.;
Zhai, G.; Barry, D.; Knipe, J. O.; Han, Y.; Schartman, R.; Donoso, M.; Mosure, K.;
Sinz, M. W.; Zvyaga, T.; Good, A. C.; Rajamani, R.; Kish, K.; Tredup, J.; Klei, H. E.;
Gao, Q.; Mueller, L.; Colonno, R. J.; Grasela, D. M.; Adams, S. P.; Loy, J.; Levesque,
P. C.; Sun, H.; Shi, H.; Sun, L.; Warner, W.; Li, D.; Zhu, J.; Meanwell, N. A.;
McPhee, F. The Discovery of Asunaprevir (BMS-650032), An Orally Efficacious
NS3 Protease Inhibitor for the Treatment of Hepatitis C Virus Infection. J. Med.
Chem. 2014, 57, 1730–1725.
White, P. W.; Llinàs-Brunet, M.; Amad, M.; Bethell, R. C.; Bolger, G.; Cordingley,
M. G.; Duan, J.; Garneau, M.; Lagacé, L.; Thibeault, D.; Kukolj, G. Preclinical
Characterization of BI 201335, a C-Terminal Carboxylic Acid Inhibitor of the
Hepatitis C Virus NS3-NS4A Protease. Antimicrob. Agents Chemother. 2010, 54,
4611–4618.
Yang, H.; Robinson, M.; Corsa, A. C.; Peng, B.; Cheng, G.; Tian, Y.; Wang, Y.;
Pakdaman, R.; Shen, M.; Qi, X.; Mo, H.; Tay, C.; Krawczyk, S.; Sheng, X. C.; Kim,
C. U.; Yang, C.; Delaney, W. E. Preclinical Characterization of the Novel Hepatitis C
Virus NS3 Protease Inhibitor GS-9451. Antimicrob. Agents Chemother. 2014, 58,
647–653.
Agarwal, A.; Zhang, B.; Olek, E.; Robison, H.; Robarge, L.; Deshpande, M. Rapid
and Sharp Decline in HCV upon Monotherapy with NS3 Protease Inhibitor, ACH1625. Antivir. Ther. 2012, 17, 1533–1539.
Llinas-Brunet, M.; Bailey, M. D.; Bolger, G.; Brochu, C.; Faucher, A.; Ferland, J. M.;
Garneau, M.; Ghiro, E.; Gorys, V.; Grand-maıtre, C.; Halmos, T.; Lapeyre-Paquette,
N.; Liard, F.; Poirier, M.; Rheaume, M.; Tsantrizos, Y. S.; Lamarre, D. StructureActivity Study on a Novel Series of Macrocyclic Inhibitors of the Hepatitis C Virus
NS3 Protease Leading to the Discovery of BILN 2061. J. Med. Chem. 2004, 47,
1605–1608.
Lin, C.; Lin, K.; Luong, Y.-P.; Rao, B. G.; Wei, Y.-Y.; Brennan, D. L.; Fulghum, J.
R.; Hsiao, H.-M.; Ma, S.; Maxwell, J. P.; Cottrell, K. M.; Perni, R. B.; Gates, C. a;
Kwong, A. D. In Vitro Resistance Studies of Hepatitis C Virus Serine Protease
Inhibitors, VX-950 and BILN 2061: Structural Analysis Indicates Different
Resistance Mechanisms. J. Biol. Chem. 2004, 279, 17508–17514.
Sarrazin, C.; Kieffer, T. L.; Bartels, D.; Hanzelka, B.; Müh, U.; Welker, M.;
Wincheringer, D.; Zhou, Y.; Chu, H.-M.; Lin, C.; Weegink, C.; Reesink, H.; Zeuzem,
S.; Kwong, A. D. Dynamic Hepatitis C Virus Genotypic and Phenotypic Changes in
Patients Treated with the Protease Inhibitor Telaprevir. Gastroenterology 2007, 132,
1767–1777.
Susser, S.; Welsch, C.; Wang, Y.; Zettler, M.; Domingues, F. S.; Karey, U.; Hughes,
E.; Ralston, R.; Tong, X.; Herrmann, E.; Zeuzem, S.; Sarrazin, C. Characterization of
Resistance to the Protease Inhibitor Boceprevir in Hepatitis C Virus-Infected Patients.
Hepatology 2009, 50, 1709–1718.
Afdhal, N.; Reddy, K. R.; Nelson, D. R.; Lawitz, E.; Gordon, S. C.; Schiff, E.;
Nahass, R.; Ghalib, R.; Gitlin, N.; Herring, R.; Lalezari, J.; Younes, Z. H.; Pockros, P.
J.; Di Bisceglie, A. M.; Arora, S.; Subramanian, G. M.; Zhu, Y.; Dvory-Sobol, H.;
Yang, J. C.; Pang, P. S.; Symonds, W. T.; McHutchison, J. G.; Muir, A. J.; Sulkowski,
M.; Kwo, P. Ledipasvir and Sofosbuvir for Previously Treated HCV Genotype 1
Infection. N. Engl. J. Med. 2014, 370, 1483–1493.
Link, J. O.; Taylor, J. G.; Xu, L.; Mitchell, M.; Guo, H.; Liu, H.; Kato, D.;
Kirschberg, T.; Sun, J.; Squires, N.; Parrish, J.; Keller, T.; Yang, Z.; Yang, C.; Matles,
M.; Wang, Y.; Wang, K.; Cheng, G.; Tian, Y.; Mogalian, E.; Mondou, E.; Cornpropst,
M.; Perry, J.; Desai, M. C. Discovery of Ledipasvir (GS-5885): A Potent, Once-Daily
Oral NS5A Inhibitor for the Treatment of Hepatitis C Virus Infection. J. Med. Chem.
2014, 57, 2033–2046.
99
(44)
(45)
(46)
(47)
(48)
(49)
(50)
(51)
(52)
(53)
(54)
(55)
(56)
(57)
(58)
(59)
(60)
100
Stirnimann, G. Ombitasvir (ABT-267), a Novel NS5A Inhibitor for the Treatment of
Hepatitis C. Expert Opin. Pharmacother. 2014, 15, 2609–2622.
Kati, W.; Koev, G.; Irvin, M.; Beyer, J.; Liu, Y.; Krishnan, P.; Reisch, T.; Mondal, R.;
Wagner, R.; Molla, A.; Maring, C.; Collins, C. In Vitro Activity and Resistance
Profile of Dasabuvir, a Non-Nucleoside HCV Polymerase Inhibitor. Antimicrob.
Agents Chemother. 2014.
Sofia, M. J.; Bao, D.; Chang, W.; Du, J.; Nagarathnam, D.; Rachakonda, S.; Reddy, P.
G.; Ross, B. S.; Wang, P.; Zhang, H.-R.; Bansal, S.; Espiritu, C.; Keilman, M.; Lam,
A. M.; Steuer, H. M. M.; Niu, C.; Otto, M. J.; Furman, P. A. Discovery of a β-D-2’Deoxy-2'-α-Fluoro-2'-β-C-Methyluridine Nucleotide Prodrug (PSI-7977) for the
Treatment of Hepatitis C Virus. J. Med. Chem. 2010, 53, 7202–7218.
Vaidya, A.; Perry, C. M. Simeprevir: First Global Approval. Drugs 2013, 73, 2093–
2106.
Lawitz, E.; Mangia, A.; Wyles, D.; Rodriguez-Torres, M.; Hassanein, T.; Gordon, S.
C.; Schultz, M.; Davis, M. N.; Kayali, Z.; Reddy, K. R.; Jacobson, I. M.; Kowdley, K.
V; Nyberg, L.; Subramanian, G. M.; Hyland, R. H.; Arterburn, S.; Jiang, D.; McNally,
J.; Brainard, D.; Symonds, W. T.; McHutchison, J. G.; Sheikh, A. M.; Younossi, Z.;
Gane, E. J. Sofosbuvir for Previously Untreated Chronic Hepatitis C Infection. N.
Engl. J. Med. 2013, 368, 1878–1887.
Johnson, C. Important Safety Information : Discontinuation of INCIVEK (telaprevir)
in the United States. 2014.
De Francesco, R.; Migliaccio, G. Challenges and Successes in Developing New
Therapies for Hepatitis C. Nature 2005, 436, 953–960.
Schneider, M. D.; Sarrazin, C. Antiviral Therapy of Hepatitis C in 2014: Do We Need
Resistance Testing? Antiviral Res. 2014, 105, 64–71.
Sarrazin, C.; Zeuzem, S. Resistance to Direct Antiviral Agents in Patients with
Hepatitis C Virus Infection. Gastroenterology 2010, 138, 447–462.
Mani, N. Clinically Relevant HCV Drug Resistance Mutations Figure and Tables.
Ann. Forum Collab. HIV Res. 2012, 14, 1–8.
Tomei, L.; Failla, C.; Santolini, E.; Francesco, R. D. E.; Molecolare, B. NS3 Is a
Serine Protease Required for Processing of Hepatitis C Virus Polyprotein. J. Virol.
1993, 67, 4017–4026.
Grakoui, A.; Mccourt, D. W.; Wychowski, C.; Feinstone, S. M.; Rice, C. M.
Characterization of the Hepatitis C Virus-Encoded Serine Proteinase : Determination
of Proteinase-Dependent Polyprotein Cleavage Sites. J. Virol. 1993, 67, 2832–2843.
Schechter, I.; Berger, A. On the Size of the Active Site in Proteases. Biochem.
Biophys. Res. Commun. 1967, 2, 157–162.
Ingallinella, P.; Altamura, S.; Bianchi, E.; Taliani, M.; Ingenito, R.; Cortese, R.;
Francesco, R. De; Steinku, C.; Pessi, A.; Molecolare, B.; Irbm, P. A.; Km, V. P.;
February, R. V; Re, V.; Recei, M.; April, V. Potent Peptide Inhibitors of Human
Hepatitis C Virus NS3 Protease Are Obtained by Optimizing the Cleavage Products.
Biochemistry 1998, 2960, 8906–8914.
Llinas-Brunet, M.; Bailey, M.; Fazal, G.; Goulet, S.; Halmos, T.; Laplante, S.;
Maurice, R.; Poirier, M.; Poupart, M.; Wernic, D.; Lamarre, D. Peptide-Based
Inhibitors of the Hepatitis C Virus Serine Protease. Bioorg. Med. Chem. Lett. 1998, 8,
1713–1718.
Gale, M.; Foy, E. M. Evasion of Intracellular Host Defence by Hepatitis C Virus.
Nature 2005, 436, 939–945.
Kim, J. L.; Morgenstern, K. A.; Lin, C.; Fox, T.; Dwyer, M. D.; Landro, J. A.;
Chambers, S. P.; Markland, W.; Lepre, C. A.; Malley, E. T. O.; Harbeson, S. L.; Rice,
C. M.; Murcko, M. A.; Caron, P. R.; Thomson, J. A.; Street, W. Crystal Structure of
the Hepatitis C Virus NS3 Protease Domain Complexed with a Synthetic NS4A
Cofactor Peptide. Cell 1996, 87, 343–355.
(61)
(62)
(63)
(64)
(65)
(66)
(67)
(68)
(69)
(70)
(71)
(72)
(73)
(74)
(75)
(76)
(77)
Love, R. A.; Parge, H. E.; Wickersham, J. A.; Hostomsky, Z.; Habuka, N.; Moomaw,
E. W.; Adachi, T.; Hostomska, Z. The Crystal Structure of Hepatitis C Virus NS3
Proteinase Reveals a Trypsin-like Fold and a Structural Zinc Binding Site. Cell 1996,
87, 331–342.
Yao, N.; Reichert, P.; Taremi, S. S.; Prosise, W. W.; Weber, P. C. Molecular Views of
Viral Polyprotein Processing Revealed by the Crystal Structure of the Hepatitis C
Virus Bifunctional Protease – Helicase. Structure 1999, 7, 1353–1363.
Leung, D.; Abbenante, G.; Fairlie, D. P. Protease Inhibitors : Current Status and
Future Prospects. J. Med. Chem. 2000, 43, 305–341.
Patrick, G. L. An Introduction to Medicinal Chemistry; Oxford University Press Inc.:
New York, 2005.
Kerns, E. H.; Di, L. Drug-like Properties; Concepts, Structure Design and Methods;
Elsevier Inc.: London,UK, 2008.
Vlieghe, P.; Lisowski, V.; Martinez, J.; Khrestchatisky, M. Synthetic Therapeutic
Peptides. Drug Discov. Today 2010, 15, 40–56.
Grauer, A.; König, B. Peptidomimetics - A Versatile Route to Biologically Active
Compounds. European J. Org. Chem. 2009, 2009, 5099–5111.
Avan, I.; Hall, C. D.; Katritzky, A. R. Peptidomimetics via Modifications of Amino
Acids and Peptide Bonds. Chem. Soc. Rev. 2014, 43, 3575–3594.
Steinkuhler, C.; Biasiol, G.; Brunetti, M.; Urbani, A.; Koch, U.; Cortese, R.; Pessi, A.;
Francesco, R. De; Re, V.; Recei, M.; April, V. Product Inhibition of the Hepatitis C
Virus NS3 Protease. Biochemistry 1998, 37, 8899–8905.
Llinas-Brunet, M.; Bailey, M.; Deziel, R.; Fazal, G.; Gorys, V.; Goulet, S.; Halmos,
T.; Maurice, R.; Poirier, M.; Poupart, M.-A.; Rancourt, J.; Thibeault, D.; Wernic, D.;
Lamarre, D. Studies on the C-Terminal of Hexapeptide Inhibitors of the Hepatitis C
Virus Serine Protease. Bioorg. Med. Chem. Lett. 1998, 8, 2719–2724.
Demuth, H. Recent Developments in Inhibiting Cysteine and Serine Proteases. J.
Enzyme Inhib. 1990, 3, 249–278.
Mehdi, S. Synthetic and Naturally Occuring Protease Inhibitors Containing an
Electrophilic Carbonyl Group. Bioorg. Chem. 1993, 21, 249–259.
Rancourt, J.; Cameron, D. R.; Gorys, V.; Lamarre, D.; Poirier, M.; Thibeault, D.;
Llinas-Brunet, M. Peptide-Based Inhibitors of the Hepatitis C Virus NS3 Protease :
Structure-Activity Relationship at the C -Terminal Position. J. Med. Chem. 2004, 47,
2511–2522.
Llinas-Brunet, M.; Bailey, M. D.; Ghiro, E.; Gorys, V.; Halmos, T.; Poirier, M.;
Rancourt, J.; Goudreau, N. A Systematic Approach to the Optimization of SubstrateBased Inhibitors of the Hepatitis C Virus NS3 Protease: Discovery of Potent and
Specific Tripeptide. J. Med. Chem. 2004, 47, 6584–6594.
LaPlante, S. R.; Cameron, D. R.; Aubry, N.; Lefebvre, S.; Kukolj, G.; Maurice, R.;
Thibeault, D.; Lamarre, D.; Llinas-Brunet, M. Solution Structure of Substrate-Based
Ligands When Bound to Hepatitis C Virus NS3 Protease Domain. J. Biol. Chem.
1999, 274, 18618–18624.
Tsantrizos, Y. S.; Bolger, G.; Bonneau, P.; Cameron, D. R.; Goudreau, N.; Kukolj, G.;
Laplante, S. R.; Llinàs-brunet, M.; Nar, H.; Lamarre, D. Macrocyclic Inhibitors of the
NS3 Protease as Potential Therapeutic Agents of Hepatitis C Virus Infection. Angew.
Chem. Int. Ed. Engl. 2003, 115, 1393–1398.
Lamarre, D.; Anderson, P. A.; Bailey, M.; Beaulieu, P.; Bolger, G.; Bonneau, P.; Bös,
M.; Cameron, D. R.; Cartier, M.; Cordingley, M. G.; Faucher, A.-M.; Goudreau, N.;
Kawai, S. H.; Kukolj, G.; Lagacé, L.; LaPlante, S.; Narjes, H.; Poupart, M.-A.;
Rancourt, J.; Sentjens, R. E.; George, R. S.; Simoneau, B.; Steinmann, G.; Thibeault,
D.; Tsantrizos, Y. S.; Weldon, S. M.; Yong, C.-L.; Llinàs-brunet, M. An NS3 Protease
Inhibitor with Antiviral Effects in Humans Infected with Hepatitis C Virus. Nature
2003, 426, 1–5.
101
(78)
(79)
(80)
(81)
(82)
(83)
(84)
(85)
(86)
(87)
(88)
(89)
(90)
(91)
(92)
(93)
(94)
(95)
102
Tsantrizos, Y. S. The Design of a Potent Inhibitor of the Hepatitis C Virus NS3
Protease: BILN 2061-from the NMR Tube to the Clinic. Biopolymers 2004, 76, 309–
323.
Reiser, M.; Hinrichsen, H.; Benhamou, Y.; Reesink, H. W.; Wedemeyer, H.;
Avendano, C.; Riba, N.; Yong, C.-L.; Nehmiz, G.; Steinmann, G. G. Antiviral
Efficacy of NS3-Serine Protease Inhibitor BILN-2061 in Patients with Chronic
Genotype 2 and 3 Hepatitis C. Hepatology 2005, 41, 832–835.
Johansson, P.-O.; Bäck, M.; Kvarnström, I.; Jansson, K.; Vrang, L.; Hamelink, E.;
Hallberg, A.; Rosenquist, Å.; Samuelsson, B. Potent Inhibitors of the Hepatitis C
Virus NS3 Protease: Use of a Novel P2 Cyclopentane-Derived Template. Bioorg.
Med. Chem. 2006, 14, 5136–5151.
Johansson, A.; Poliakov, A.; Åkerblom, E.; Wiklund, K.; Lindeberg, G.; Winiwarter,
S.; Danielson, U. H.; Samuelsson, B.; Hallberg, A. Acyl Sulfonamides as Potent
Protease Inhibitors of the Hepatitis C Virus Full-Length NS3 (ProteaseHelicase/NTPase): A Comparative Study of Different C-Terminals. Bioorg. Med.
Chem. 2003, 11, 2551–2568.
Johansson, A.; Åkerblom, E.; Lindeberg, G.; Poliakov, A.; Danielson, U. H.;
Hallberg, A. Drugs Fut; 2002; Vol. 27, p. 100.
Poliakov, A.; Johansson, A.; Akerblom, E.; Oscarsson, K.; Samuelsson, B.; Hallberg,
A.; Danielson, U. H. Structure-Activity Relationships for the Selectivity of Hepatitis
C Virus NS3 Protease Inhibitors. Biochim. Biophys. Acta 2004, 1672, 51–59.
Campbell, J. A.; Good, A. Hepatitis C Tripeptide Inhibitors. WO 02/060926 A2 2002.
Lohmann, V.; Bartenschlager, R. On the History of Hepatitis C Virus Cell Culture
Systems. J. Med. Chem. 2014, 57, 1627–1642.
Poliakov, A.; Hubatsch, I.; Shuman, C. F.; Stenberg, G.; Danielson, U. H. Expression
and Purification of Recombinant Full-Length NS3 Protease-Helicase from a New
Variant of Hepatitis C Virus. Protein Expr. Purif. 2002, 25, 363–371.
Thibeault, D.; Massariol, M.-J.; Zhao, S.; Welchner, E.; Goudreau, N.; Gingras, R.;
Llinàs-brunet, M.; White, P. W. Use of the Fused NS4A Peptide - NS3 Protease
Domain To Study the Importance of the Helicase Domain for Protease Inhibitor
Binding to Hepatitis C Virus NS3-NS4A. Biochemistry 2009, 48, 744–753.
Johansson, A.; Hubatsch, I.; Åkerblom, E.; Lindeberg, G.; Winiwarter, S.; Danielson,
H. U.; Hallberg, A. Inhibition of Hepatitis C Virus NS3 Protease Activity by ProductBased Peptides Is Dependent on Helicase Domain. Bioorg. Med. Chem. Lett. 2001,
11, 203–206.
Beran, R. K. F.; Pyle, A. M. Hepatitis C Viral NS3-4A Protease Activity Is Enhanced
by the NS3 Helicase. J. Biol. Chem. 2008, 283, 29929–29937.
Lohmann, V.; Körner, F.; Koch, J.-O.; Herian, U.; Theilmann, L.; Bartenschlager, R.
Replication of Subgenomic Hepatitis C Virus RNAs in a Hepatoma Cell Line. Science
(80-. ). 1999, 285, 110–114.
Lohmann, V.; Hoffmann, S.; Herian, U.; Penin, F.; Bartenschlager, R. Viral and
Cellular Determinants of Hepatitis C Virus RNA Replication in Cell Culture. J. Virol.
2003, 283, 3007–3019.
Schiering, N.; Arcy, A. D.; Villard, F.; Simi, O.; Kamke, M.; Monnet, G.; Hassiepen,
U. A Macrocyclic HCV NS3 / 4A Protease Inhibitor Interacts with Protease and
Helicase Residues in the Complex with Its Full-Length Target. PNAS 2011, 108,
21052–21056.
McMartin, C.; Bohacek, R. S. QXP : Powerful, Rapid Computer Algorithms for
Structure-Based Drug Design. J. Comput. Aided. Mol. Des. 1997, 11, 333–344.
Prentis, R. A.; Lis, Y.; Walker, S. R. Pharmaceutical Innovation by the Seven UKOwned Pharmaceutical Companies (1964-1985). Brittish J. Clin. Pharmacol. 1988,
25, 387–396.
Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Experimental and
Computational Approaches to Estimate Solubility and Permeability in Drug
Discovery and Development Settings. Adv. Drug Deliv. Rev. 1997, 23, 3–25.
(96)
(97)
(98)
(99)
(100)
(101)
(102)
(103)
(104)
(105)
(106)
(107)
(108)
(109)
Zhao, H. Lead Optimization in the Nondrug-like Space. Drug Discov. Today 2011,
16, 158–163.
Belfrage, A. K. G. L.; Classon, B. O.; Lindquist, K. C.; Nilsson, K. M.; Raboisson, P.
J.-M. B.; Rosenquist, Å. A. K.; Samuelsson, B. B.; Wähling, H. J. Pyrimidine
Substituted Macrocyclic HCV Inhibitors. US20100113440 2010.
Antonov, D.; Ayesa Alvarez, S.; Belfrage, A. K. G. L.; Jönsson, C. E. D.; Mc Gowan,
D. C.; Nilsson, K. M.; Raboisson, P. J.-M. B.; Rosenquist, Å. A. K.; Samuelsson, B.
B. HCV Inhibiting Macrocyclic Phenylcarbamates. US20100041728 2010.
Nilsson, M.; Belfrage, A. K.; Lindström, S.; Wähling, H.; Lindquist, C.; Ayesa, S.;
Kahnberg, P.; Pelcman, M.; Benkestock, K.; Agback, T.; Vrang, L.; Terelius, Y.;
Wikström, K.; Hamelink, E.; Rydergård, C.; Edlund, M.; Eneroth, A.; Raboisson, P.;
Lin, T. I.; de Kock, H.; Wigerinck, P.; Simmen, K.; Samuelsson, B.; Rosenquist, Å.
Synthesis and SAR of Potent Inhibitors of the Hepatitis C Virus NS3/4A Protease:
Exploration of P2 Quinazoline Substituents. Bioorganic Med. Chem. Lett. 2010, 20,
4004–4011.
Vendeville, S.; Nilsson, M.; de Kock, H.; Lin, T.-I.; Antonov, D.; Classon, B.; Ayesa,
S.; Ivanov, V.; Johansson, P.-O.; Kahnberg, P.; Eneroth, A.; Wikström, K.; Vrang, L.;
Edlund, M.; Lindström, S.; Van de Vreken, W.; McGowan, D.; Tahri, A.; Hu, L.;
Lenz, O.; Delouvroy, F.; Van Dooren, M.; Kindermans, N.; Surleraux, D.; Wigerinck,
P.; Rosenquist, Å.; Samuelsson, B.; Simmen, K.; Raboisson, P. Discovery of Novel,
Potent and Bioavailable Proline-Urea Based Macrocyclic HCV NS3/4A Protease
Inhibitors. Bioorg. Med. Chem. Lett. 2008, 18, 6189–6193.
Burgey, C. S.; Robinson, K. A.; Lyle, T. A.; Sanderson, P. E. J.; Lewis, S. D.; Lucas,
B. J.; Krueger, J. A.; Singh, R.; Miller-stein, C.; White, R. B.; Wong, B.; Lyle, E. A.;
Williams, P. D.; Coburn, C. A.; Dorsey, B. D.; Barrow, J. C.; Stranieri, M. T.;
Holahan, M. A.; Sitko, G. R.; Cook, J. J.; Mcmasters, D. R.; Mcdonough, C. M.;
Sanders, W. M.; Wallace, A. A.; Clayton, F. C.; Bohn, D.; Leonard, Y. M.; Detwiler,
T. J.; Lynch, J. J.; Yan, Y.; Chen, X. Z.; Kuo, L.; Gardell, X. S. J.; Shafer, J. A.;
Vacca, J. P. Metabolism-Directed Optimization of 3-Aminopyrazinone Acetamide
Thrombin Inhibitors. Development of an Orally Bioavailable Series Containing P1
and P3 Pyridines. J. Med. Chem. 2003, 46, 461–473.
Hopkins, C.; Neuenschwander, K.; Scotese, A.; Jackson, S.; Nieduzak, T.; Pauls, H.;
Liang, G.; Sides, K.; Cramer, D.; Cairns, J.; Maignan, S.; Mathieu, M. Novel
Pyrazinone Inhibitors of Mast Cell Tryptase: Synthesis and SAR Evaluation. Bioorg.
Med. Chem. Lett. 2004, 14, 4819–4823.
Jones, D. E.; South, M. S. Synthesis of a Versatile 2(1H)-Pyrazinone Core for the
Preparation of Tissue Factor-Factor VIIa Inhibitors. Tetrahedron 2010, 66, 2570–2581.
Han, Y.; Giroux, A.; Colucci, J.; Bayly, C. I.; Mckay, D. J.; Roy, S.; Xanthoudakis,
S.; Vaillancourt, J.; Rasper, D. M.; Tam, J.; Tawa, P.; Nicholson, D. W.; Zamboni, R.
J. Novel Pyrazinone Mono-Amides as Potent and Reversible Caspase-3 Inhibitors.
Bioorg. Med. Chem. Lett. 2005, 15, 1173–1180.
Zhang, X.; Schmitt, A. C.; Jiang, W.; Wasserman, Z.; Decicco, C. P. Design and
Synthesis of Potent, Non-Peptide Inhibitors of HCV NS3 Protease. Bioorg. Med.
Chem. Lett. 2003, 13, 1157–1160.
Caldwell, J. J.; Veillard, N.; Collins, I. Design and Synthesis of 2(1H)-Pyrazinones as
Inhibitors of Protein Kinases. Tetrahedron 2012, 68, 9713–9728.
Jinsmaa, Y.; Miyazaki, A.; Fujita, Y.; Li, T.; Fujisawa, Y.; Shiotani, K.; Tsuda, Y.;
Yokoi, T.; Ambo, A.; Sasaki, Y.; Bryant, S. D.; Lazarus, L. H.; Okada, Y. Oral
Bioavailability of a New Class of Micro-Opioid Receptor Agonists Containing 3,6bis[Dmt-NH(CH(2))(n)]-2(1H)-Pyrazinone with Central-Mediated Analgesia. J. Med.
Chem. 2004, 47, 2599–2610.
Mai, A. H.; Pawar, S.; De Borggraeve, W. M. Synthesis of 1-Benzyloxypyrazin2(1H)-One Derivatives. Tetrahedron Lett. 2014, 55, 4664–4666.
Tyndall, J. D.; Fairlie, D. P. Conformational Homogeneity in Molecular Recognition
by Proteolytic Enzymes. J. Mol. Recognit. 1999, 12, 363–370.
103
(110) Fairlie, D. P.; Tyndall, J. D.A.; Reid, R. C.; Wong, A K.; Abbenante, G.; Scanlon, M.
J.; March, D. R.; Bergman, D. A; Chai, C. L.; Burkett, B. A. Conformational
Selection of Inhibitors and Substrates by Proteolytic Enzymes: Implications for Drug
Design and Polypeptide Processing. J. Med. Chem. 2000, 43, 1271–1281.
(111) Kaval, N.; Appukkuttan, P.; Eycken, E. Van der. The Chemistry of 2-(1H)Pyrazinones in Solution and on Solid Support. Microwave-Assisted Synth. 2006, 1,
267–304.
(112) Pawar, V.; De Borggraeve, W. 3,5-Dihalo-2(1H)-Pyrazinones: Versatile Scaffolds in
Organic Synthesis. Synthesis (Stuttg). 2006, 2006, 2799–2814.
(113) Örtqvist, P.; Peterson, S. D.; Åkerblom, E.; Gossas, T.; Sabnis, Y. A.; Fransson, R.;
Lindeberg, G.; Helena Danielson, U.; Karlén, A.; Sandström, A. Phenylglycine as a
Novel P2 Scaffold in Hepatitis C Virus NS3 Protease Inhibitors. Bioorg. Med. Chem.
2007, 15, 1448–1474.
(114) Örtqvist, P.; Gising, J.; Ehrenberg, A. E.; Vema, A.; Borg, A.; Karlén, A.; Larhed, M.;
Danielson, U. H.; Sandström, A. Discovery of Achiral Inhibitors of the Hepatitis C
Virus NS3 Protease Based on 2(1H)-Pyrazinones. Bioorg. Med. Chem. 2010, 18,
6512–6525.
(115) Romano, K. P.; Ali, A.; Royer, W. E.; Schiffer, C. A. Drug Resistance against HCV
NS3/4A Inhibitors Is Defined by the Balance of Substrate Recognition versus
Inhibitor Binding. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 20986–20991.
(116) Chellappan, S.; Kairys, V.; Fernandes, M. X.; Schiffer, C.; Gilson, M. K. Evaluation
of the Substrate Envelope Hypothesis for Inhibitors of HIV-1 Protease. Proteins 2007,
567, 561–567.
(117) Altman, M. D.; Ali, O. A.; Reddy, G. S. K. K.; Nalam, M. N. L.; Anjum, S. G.; Cao,
H.; Chellappan, S.; Kairys, V.; Fernandes, M. X.; Gilson, M. K.; Schiffer, C. A.;
Rana, T. M.; Tidor, B. HIV-1 Protease Inhibitors from Inverse Design in the Substrate
Envelope Exhibit Subnanomolar Binding to Drug-Resistant Variants. J. Am. Chem.
Soc. 2008, 130, 6099–6113.
(118) Romano, K. P.; Ali, A.; Aydin, C.; Soumana, D.; Ozen, A.; Deveau, L. M.; Silver, C.;
Cao, H.; Newton, A.; Petropoulos, C. J.; Huang, W.; Schiffer, C. A. The Molecular
Basis of Drug Resistance against Hepatitis C Virus NS3/4A Protease Inhibitors. PLoS
Pathog. 2012, 8, 1–15.
(119) Dahl, G.; Sandström, A.; Åkerblom, E.; Danielson, U. H. Resistance Profiling of
Hepatitis C Virus Protease Inhibitors Using Full-Length NS3. Antivir. Ther. 2007, 12,
733–740.
(120) Artursson, P.; Lennernäs, H.; v. d. Waterbeemd, H. Methods and Principles in
Medicinal Chemistry; Comer, J. E. A., Ed.; Wiley, Weinheim, Germany, 2003; pp.
21–45.
(121) Bergström, C. A. S.; Strafford, M.; Lazorova, L.; Avdeef, A.; Luthman, K.; Artursson,
P. Absorption Classification of Oral Drugs Based on Molecular Surface Properties. J.
Med. Chem. 2003, 46, 558–570.
(122) Rönn, R.; Lampa, A.; Peterson, S. D.; Gossas, T.; Åkerblom, E.; Danielson, U. H.;
Karlén, A.; Sandström, A. Hepatitis C Virus NS3 Protease Inhibitors Comprising a
Novel Aromatic P1 Moiety. Bioorg. Med. Chem. 2008, 16, 2955–2967.
(123) Lovering, F.; Bikker, J.; Humblet, C. Escape from Flatland: Increasing Saturation as
an Approach to Improving Clinical Success. J. Med. Chem. 2009, 52, 6752–6756.
(124) Venkatraman, S.; Velazquez, F.; Wu, W.; Blackman, M.; Madison, V.; Njoroge, F. G.
Potent Ketoamide Inhibitors of HCV NS3 Protease Derived from Quaternized P1
Groups. Bioorg. Med. Chem. Lett. 2010, 20, 2151–2155.
(125) Chen, K. X.; Nair, L.; Vibulbhan, B.; Yang, W.; Arasappan, A.; Bogen, S. L.;
Venkatraman, S.; Bennett, F.; Pan, W.; Blackman, M. L.; Padilla, A. I.; Prongay, A.;
Cheng, K.-C.; Tong, X.; Shih, N.-Y.; Njoroge, F. G. Second-Generation Highly
Potent and Selective Inhibitors of the Hepatitis C Virus NS3 Serine Protease. J. Med.
Chem. 2009, 52, 1370–1379.
104
(126) Tapper, E. B.; Afdhal, N. H. Is 3 the New 1: Perspectives on Virology, Natural
History and Treatment for Hepatitis C Genotype 3. J. Viral Hepat. 2013, 20, 669–677.
(127) Rudd, M. T.; McIntyre, C. J.; Romano, J. J.; Butcher, J. W.; Holloway, M. K.; Bush,
K.; Nguyen, K. T.; Gilbert, K. F.; Lyle, T. a; Liverton, N. J.; Wan, B.-L.; Summa, V.;
Harper, S.; Rowley, M.; Vacca, J. P.; Carroll, S. S.; Burlein, C.; DiMuzio, J. M.;
Gates, A.; Graham, D. J.; Huang, Q.; Ludmerer, S. W.; McClain, S.; McHale, C.;
Stahlhut, M.; Fandozzi, C.; Taylor, A.; Trainor, N.; Olsen, D. B.; McCauley, J. a.
Development of Macrocyclic Inhibitors of HCV NS3/4A Protease with Cyclic
Constrained P2-P4 Linkers. Bioorg. Med. Chem. Lett. 2012, 22, 7207–7213.
(128) Gallo, M.; Bottomley, M. J.; Pennestri, M.; Eliseo, T.; Paci, M.; Koch, U.; Bazzo, R.;
Summa, V.; Carfì, A.; Cicero, D. O. Structural Characterization of the Hepatitis C
Virus NS3 Protease from Genotype 3a: The Basis of the Genotype 1b vs. 3a Inhibitor
Potency Shift. Virology 2010, 405, 424–438.
(129) Kramer, M.; Halleran, D.; Rahman, M.; Iqbal, M.; Anwar, M. I.; Sabet, S.; Ackad, E.;
Yousef, M. Comparative Molecular Dynamics Simulation of Hepatitis C Virus
NS3/4A Protease (Genotypes 1b, 3a and 4b) Predicts Conformational Instability of
the Catalytic Triad in Drug Resistant Strains. PLoS One 2014, 9, e104425.
(130) Gising, J.; Örtqvist, P.; Sandström, A.; Larhed, M. A Straightforward Microwave
Method for Rapid Synthesis of N-1, C-6 Functionalized 3,5-Dichloro-2(1H)Pyrazinones. Org. Biomol. Chem. 2009, 7, 2809–2815.
(131) Venkatraman, S.; Njoroge, F. G.; Wu, W.; Girijavallabhan, V.; McKittrick, B.; Su, J.;
Velazquez, F.; Pinto, P. A. WO 2009/058856 A1, 2009.
(132) Busacca, C. A.; Grossbach, D.; Spinelli, E. A Convenient Synthesis of (1S)-tertButyl-1,2-Ethylenediamine. Tetrahedron:Asymmetry 2000, 11, 1907–1910.
(133) Venkatraman, S.; Blackman, M.; Wu, W.; Nair, L.; Arasappan, A.; Padilla, A.;
Bogen, S.; Bennett, F.; Chen, K.; Pichardo, J.; Tong, X.; Prongay, A.; Cheng, K.-C.;
Girijavallabhan, V.; George Njoroge, F. Discovery of Novel P3 Sulfonamide-Capped
Inhibitors of HCV NS3 Protease. Inhibitors with Improved Cellular Potencies. Bioorg.
Med. Chem. 2009, 17, 4486–4495.
(134) Perveen, S.; Abdul Hai, S. M.; Khan, R. A.; Khan, K. M.; Afza, N.; Sarfaraz, T. B.
Expeditious Method for Synthesis of Symmetrical 1,3‐Disubstituted Ureas and
Thioureas. Synth. Commun. 2005, 35, 1663–1674.
(135) Michejda, C. J.; Comnick, R. W. Acetolysis of 3,3-Disubstituted Cyclobutyl
Tosylates. J. Org. Chem. 1975, 40, 1046–1050.
(136) Allan, R. D.; Hanrahan, J. R.; Hambley, T. W.; Johnston, G. A. R.; Mewett, K. N.;
Mitrovic, A. D. Synthesis and Activity of a Potent N-Methyl-D-Aspartic Acid
Agonist, Trans-1-Aminocyclobutane-1,3-Dicarboxylic Acid, and Related Phosphonic
and Carboxylic Acids. J. Med. Chem. 1990, 33, 2905–2915.
(137) Bubenyák, M.; Takács, M.; Blazics, B.; Rácz, Á.; Noszál, B.; Püski, L. Synthesis of
Bioisosteric 5-Sulfa-Rutaecarpine Derivatives. ARKIVOC 2010, 2010, 291–302.
(138) Chernykh, V. P.; Petyunin, P. A. Synthesis and Properties of 3-Substituted 2H-1,2,4Benzothiadiazine 1,1-Dioxides. Chem. Heterocycl. Compd. 1976, 12, 402–406.
(139) Negishi, E. Handbook of Organopalladium Chemistry for Organic Synthesis,Volume
1; Wiley-Interscience: New York. 2002.
(140) Johansson Seechurn, C. C. C.; Kitching, M. O.; Colacot, T. J.; Snieckus, V.
Palladium-Catalyzed Cross-Coupling: A Historical Contextual Perspective to the
2010 Nobel Prize. Angew. Chem. Int. Ed. Engl. 2012, 51, 5062–5085.
(141) Negishi, E. Handbook of Organopalladium Chemistry for Organic Synthesis, Volume
2; Wiley-Interscience: New York. 2002.
(142) Guram, S.; Rennels, R. A.; Buchwald, S. L. A Simple Catalytic Method for the
Conversion of Aryl Bromides to Arylamines. Angew. Chem. Int. Ed. Engl. 1995, 34,
1348–1350.
(143) Louie, J.; Hartwig, J. F. Palladium-Catalyzed Synthesis of Arylamines from Aryl
Halides . Mechanistic Studies Lead to Coupling in the Absence of Tin Reagents.
Tetrahedron Lett. 1995, 36, 3609–3612.
105
(144) Wolfe, J. P.; Rennels, R. A.; Buchwald, S. L. Intramolecular Palladium-Catalyzed
Aryl Amination and Aryl Amidation. Tetrahedron 1996, 52, 7525–7546.
(145) Hartwig, J. F.; Kawatsura, M.; Hauck, S. I.; Shaughnessy, K. H.; Alcazar-Roman, L.
M. Room-Temperature Palladium-Catalyzed Amination of Aryl Bromides and
Chlorides and Extended Scope of Aromatic C-N Bond Formation with a Commercial
Ligand. J. Org. Chem. 1999, 64, 5575–5580.
(146) Yin, J.; Buchwald, S. L. Palladium-Catalyzed Intermolecular Coupling of Aryl
Halides and Amides. Org. Lett. 2000, 2, 1101–1104.
(147) Yin, J.; Buchwald, S. L. Pd-Catalyzed Intermolecular Amidation of Aryl Halides: The
Discovery That Xantphos Can Be Trans-Chelating in a Palladium Complex. J. Am.
Chem. Soc. 2002, 124, 6043–6048.
(148) Artamkina, G. A; Sergeev, A. G.; Beletskaya, I. P. Palladium-Catalyzed Reaction of
Aryl Halides with Ureas. Tetrahedron Lett. 2001, 42, 4381–4384.
(149) Sergeev, A. G.; Artamkina, G. A.; Beletskaya, I. P. Variation of Xanthene-Based
Bidentate Ligands in the Palladium-Catalyzed Arylation of Ureas. Tetrahedron Lett.
2003, 44, 4719–4723.
(150) Abad, A.; Agulló, C.; Cuñat, A. C.; Vilanova, C. Regioselective Preparation of
Pyridin-2-yl Ureas from 2-Chloropyridines Catalyzed by Pd(0). Synthesis (Stuttg).
2005, 915–924.
(151) Hartwig, J. F. Carbon - Heteroatom Bond-Forming Reductive Eliminations of
Amines, Ethers, and Sulfides. Acc. Chem. Res. 1998, 31, 852–860.
(152) Hartwig, J. F.; Richards, S.; Baranano, D.; Paul, F. Influences on the Relative Rates
for C-N Bond-Forming Reductive Elimination and -Hydrogen Elimination of Amides.
A Case Study on the Origins of Competing Reduction in the Palladium-Catalyzed
Amination of Aryl Halides. J. Am. Chem. Soc. 1996, 118, 3626–3633.
(153) McLaughlin, M.; Palucki, M.; Davies, I. W. Efficient Access to Cyclic Ureas via PdCatalyzed Cyclization. Org. Lett. 2006, 8, 3311–3314.
(154) Kotecki, B. J.; Fernando, D. P.; Haight, A. R.; Lukin, K. A. A General Method for the
Synthesis of Unsymmetrically Substituted Ureas via Palladium-Catalyzed Amidation.
Org. Lett. 2009, 11, 947–950.
(155) Breitler, S.; Oldenhuis, N. J.; Fors, B. P.; Buchwald, S. L. Synthesis of
Unsymmetrical Diarylureas via Pd-Catalyzed C-N Cross-Coupling Reactions. Org.
Lett. 2011, 13, 3262–3265.
(156) Soussi, M. A.; Audisio, D.; Messaoudi, S.; Provot, O.; Brion, J.-D.; Alami, M.
Palladium-Catalyzed Coupling of 3-Halo-Substituted Coumarins, Chromenes, and
Quinolones with Various Nitrogen-Containing Nucleophiles. European J. Org. Chem.
2011, 5077–5088.
(157) Yu, S.; Haight, A.; Kotecki, B.; Wang, L.; Lukin, K.; Hill, D. R. Synthesis of a
TRPV1 Receptor Antagonist. J. Org. Chem. 2009, 74, 9539–9542.
(158) South, M. S.; Parlow, J. J.; Case, B. L.; Dice, T.; Lindmark, R.; Hayes, M. J.;
Rueppel, M. L.; Fenton, R.; Franklin, G. W.; Huang, H.-C.; Kusturin, C.; Long, S. A.;
Neumann, W. L.; Reitz, D.; Trujillo, J. I.; Wang, C.-C.; Wood, R.; Zeng, Q.;
Mahoney, M. W. Substituted Polycyclic Aryl and Heteroaryl Pyrazinones Useful for
Selective Inhibition of the Coagulation Cascade. WO 01/87854 A1 2001.
(159) Paone, D. V; Nguyen, D. N.; Shaw, A. W.; Burgey, C. S.; J, T. T.; Graham, S. L.
CGRP Receptor Antagonists. WO 2006/099268 A2 2006.
(160) Gallou, I. Unsymmetrical Ureas. Synthetic Methodologies and Application in Drug
Design. Org. Prep. Proced. Int. 2007, 39, 355–383.
(161) Zhou, G.; Zorn, N.; Ting, P.; Aslanian, R.; Lin, M.; Cook, J.; Lachowicz, J.; Lin, A.;
Smith, M.; Hwa, J.; van Heek, M.; Walker, S. Development of Novel
Benzomorpholine Class of Diacylglycerol Acyltransferase I Inhibitors. ACS Med.
Chem. Lett. 2014, 5, 544–549.
106
(162) Qiao, J. X.; Wang, T. C.; Thibeault, C.; Heureux, A. L.; Schumacher, W. A.; Spronk,
S. A.; Hiebert, S.; Bouthillier, G.; Lloyd, J.; Pi, Z.; Schnur, D. M.; Abell, L. M.; Hua,
J.; Price, L. A.; Liu, E.; Wu, Q.; Steinbacher, T. E.; Bostwick, S.; Chang, M.; Zheng,
J.; Gao, Q.; Ma, B.; Mcdonnell, P. A.; Huang, C. S.; Rehfuss, R.; Wexler, R. R.; Lam,
P. Y. S. Conformationally Constrained Ortho-Anilino Diaryl Ureas: Discovery of
1-(2-(1′-Neopentylspiro[indoline-3,4′-Piperidine]-1-Yl)phenyl)-3- (4(trifluoromethoxy)phenyl)urea, a Potent, Selective, and Bioavailable P2Y1
Antagonist. J. Med. Chem. 2013, 56, 9275–9295.
(163) Grubbs, R. H. Olefin-Metathesis Catalysts for the Preparation of Molecules and
Materials (Nobel Lecture). Angew. Chem. Int. Ed. Engl. 2006, 45, 3760–3765.
(164) Zhu, Y.; Wang, Q.; Cornwall, R. G.; Shi, Y. Organocatalytic Asymmetric
Epoxidation and Aziridination of Olefins and Their Synthetic Applications. Chem.
Rev. 2014, 114, 8199–8256.
(165) Goossen, L. J.; Koley, D.; Hermann, H.; Thiel, W. The Mechanism of the Oxidative
Addition of Aryl Halides to Pd-Catalysts: A DFT Investigation. Chem. Commun.
(Camb). 2004, 2141–2143.
(166) Senn, H. M.; Ziegler, T. Oxidative Addition of Aryl Halides to Palladium(0)
Complexes: A Density-Functional Study Including Solvation. Organometallics 2004,
23, 2980–2988.
(167) Schoenberg, A.; Heck, R. F. Palladium-Catalyzed Amidation of Aryl, Heterocyclic,
and Vinylic Halides. J. Org. Chem. 1974, 39, 3327–3331.
(168) Schoenberg, A.; Bartoletti, I.; Heck, R. F. Palladium-Catalyzed Carboalkoxylation of
Aryl, Benzyl, and Vinylic Halides. J. Org. Chem. 1974, 39, 3318–3326.
(169) Barnard, C. F. J. Palladium-Catalyzed Carbonylation- A Reaction Come of Age.
Organometallics 2008, 27, 5402–5422.
(170) Brennführer, A.; Neumann, H.; Beller, M. Palladium-Catalyzed Carbonylation
Reactions of Aryl Halides and Related Compounds. Angew. Chem. Int. Ed. Engl.
2009, 48, 4114–4133.
(171) Hermange, P.; Gogsig, T. M.; Lindhardt, A. T.; Taaning, R. H.; Skrydstrup, T.
Carbonylative Heck Reactions Using CO Cenerated Ex Situ in a Two-Chamber
System. Org. Lett. 2011, 13, 2444–2447.
(172) Hermange, P.; Lindhardt, A. T.; Taaning, R. H.; Bjerglund, K.; Lupp, D.; Skrydstrup,
T. Ex Situ Generation of Stoichiometric and Substoichiometric 12 CO and 13 CO and
Its Efficient Incorporation in Palladium Catalyzed Aminocarbonylations. J. Am.
Chem. Soc. 2011, 133, 6061–6071.
(173) Morimoto, T.; Kakiuchi, K. Evolution of Carbonylation Catalysis: No Need for
Carbon Monoxide. Angew. Chem. Int. Ed. Engl. 2004, 43, 5580–5588.
(174) Odell, L.; Russo, F.; Larhed, M. Molybdenum Hexacarbonyl Mediated CO Gas-Free
Carbonylative Reactions. Synlett 2012, 23, 685–698.
(175) Nordeman, P.; Odell, L. R.; Larhed, M. Aminocarbonylations Employing Mo(CO)6
and a Bridged Two-Vial System: Allowing the Use of Nitro Group Substituted Aryl
Iodides and Aryl Bromides. J. Org. Chem. 2012, 77, 11393–11398.
(176) Singh, J.; Conzentinot, P.; Cundya, K.; Gainor, J. A.; Gilliam, C. L.; Gordonb, T. D.;
Johnson, J. A.; Morganb, B. A.; Schneiderc, E. D.; Wahl, R. C.; Whipple, D. A.
Relationship between Structure and Bioavailability in a Series of Hydroxamate Based
Metalloprotease Inhibitors. Bioorg. Med. Chem. Lett. 1995, 5, 337–342.
(177) Reddy, P. E.; Reddy, R. M. V. WO2006/025924 A2, 2006.
(178) Bunel, E. E.; Gajewski, R. P.; Jones, C. D.; Lu, J.; Ma, T.; Nagpal, S.; Yee, Y. K.
WO2004/048309 A1, 2004.
(179) Savile, C. K.; Magloire, V. P.; Kazlauskas, R. J. Subtilisin-Catalyzed Resolution of NAcyl Arylsulfinamides. J. Am. Chem. Soc. 2005, 127, 2104–2113.
(180) Rönn, R.; Sabnis, Y. A.; Gossas, T.; Åkerblom, E.; Danielson, U. H.; Hallberg, A.;
Sandström, A. Exploration of Acyl Sulfonamides as Carboxylic Acid Replacements in
Protease Inhibitors of the Hepatitis C Virus Full-Length NS3. Bioorg. Med. Chem.
2006, 14, 544–559.
107
(181) Mancheño, O. G.; Bolm, C. Synthesis of Sulfonimidamides from Sulfinamides by
Oxidation with N-Chlorosuccinimide. Beilstein J. Org. Chem. 2007, 3, 25.
(182) Azzaro, S.; Fensterbank, L.; Lacôte, E.; Malacria, M. Probing the Amino-End
Reactivity of Sulfonimidamides. Synlett 2008, 2008, 2253–2256.
(183) Azzaro, S.; Murr, M.; Fensterbank, L.; Lacôte, E.; Malacria, M. Copper-Catalyzed NArylation of Sulfonimidamides. Synlett 2011, 2011, 849–851.
(184) Worch, C.; Atodiresei, I.; Raabe, G.; Bolm, C. Synthesis of Enantiopure
Sulfonimidamides and Elucidation of Their Absolute Configuration by Comparison of
Measured and Calculated CD Spectra and X-Ray Crystal Structure Determination.
Chemistry 2010, 16, 677–683.
(185) Steurer, M.; Bolm, C. Synthesis of Amino-Functionalized Sulfonimidamides and
Their Application in the Enantioselective Henry Reaction. J. Org. Chem. 2010, 75,
3301–3310.
(186) Sehgelmeble, F.; Janson, J.; Ray, C.; Rosqvist, S.; Gustavsson, S.; Nilsson, L. I.;
Minidis, A.; Holenz, J.; Rotticci, D.; Lundkvist, J.; Arvidsson, P. I. Sulfonimidamides
as Sulfonamides Bioisosteres: Rational Evaluation through Synthetic, in Vitro, and in
Vivo Studies with Γ-Secretase Inhibitors. ChemMedChem 2012, 7, 396–399.
(187) Leca, D.; Toussaint, A.; Mareau, C.; Fensterbank, L.; Lacote, E.; Malacria, M.
Efficient Copper-Mediated Reactions of Nitrenes Derived from Sulfonimidamides.
Org. Lett. 2004, 6, 3573–3575.
(188) Borhade, S. R.; Sandström, A.; Arvidsson, P. I. Synthesis of Novel Aryl and
Heteroaryl Acyl Sulfonimidamides via Pd-Catalyzed Carbonylation Using a
Nongaseous Precursor. Org. Lett. 2013, 15, 1056–1059.
(189) Wakchaure, P. B.; Borhade, S. R.; Sandström, A.; Arvidsson, P. I. Synthesis of Vinyland Aryl-Acyl Sulfonimidamides Through Pd-Catalyzed Carbonylation Using
Mo(CO)6 as Ex Situ CO Source. European J. Org. Chem. 2015, 213–219.
(190) Funes Maldonado, M.; Sehgelmeble, F.; Bjarnemark, F.; Svensson, M.; Åhman, J.;
Arvidsson, P. I. Synthesis and Arylation of Unprotected Sulfonimidamides.
Tetrahedron 2012, 68, 7456–7462.
(191) Pemberton, N.; Graden, H.; Evertsson, E.; Bratt, E.; Lepisto, M.; Johannesson, P.
Synthesis and Functionalization of Cyclic Sulfonimidamides: A Novel Chiral
Heterocyclic Carboxylic Acid Bioisostere. ACS Med. Chem. Lett. 2012, 3, 574–578.
(192) Borhade, S. R.; Svensson, R.; Brandt, P.; Artursson, P.; Arvidsson, P. I.; Sandström,
A. ChemMedChem 2015 10.1002/cmdc.201402497.
(193) Backes, B. J.; Dragoli, D. R.; Ellman, J. A. Chiral N-Acyl-tert-Butanesulfinamides:
The “Safety-Catch” Principle Applied to Diastereoselective Enolate Alkylations. J.
Org. Chem. 1999, 64, 5472–5478.
(194) Grigg, R.; Mutton, S. P. Pd-Catalysed Carbonylations: Versatile Technology for
Discovery and Process Chemists. Tetrahedron 2010, 66, 5515–5548.
(195) Arisawa, M.; Igarashi, Y.; Kobayashi, H.; Yamada, T.; Bando, K.; Ichikawa, T.;
Yamaguchi, M. Equilibrium Shift in the Rhodium-Catalyzed Acyl Transfer Reactions.
Tetrahedron 2011, 67, 7846–7859.
(196) Martinelli, J. R.; Watson, D. A.; Freckmann, D. M. M.; Barder, T. E.; Buchwald, S. L.
Palladium-Catalyzed Carbonylation Reactions of Aryl Bromides at Atmospheric
Pressure : A General System Based on Xantphos. J. Org. Chem. 2008, 73, 7102–7107.
(197) Odell, L. R.; Sävmarker, J.; Larhed, M. Microwave-Promoted Aminocarbonylation of
Aryl Triflates Using Mo(CO)6 as a Solid CO Source. Tetrahedron Lett. 2008, 49,
6115–6118.
(198) Zeysing, B.; Gosch, C.; Terfort, A. Protecting Groups for Thiols Suitable for Suzuki
Conditions. Org. Lett. 2000, 2, 1843–1845.
(199) Cao, H.; McNamee, L.; Alper, H. Palladium-Catalyzed Thiocarbonylation of
Iodoarenes with Thiols in Phosphonium Salt Ionic Liquids. 2008, 5, 3530–3534.
(200) Wan, Y.; Alterman, M.; Larhed, M.; Hallberg, A. Dimethylformamide as a Carbon
Monoxide Source in Fast Palladium-Catalyzed Aminocarbonylations of Aryl
Bromides. 2002, 6232–6235.
108
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