Linköping Studies in Science and Technology
Thesis No.
(språkgranska gärna båda titlarna, är lite osäker på ordföljden i den översta titeln)
Division of Chemistry
Department of Physics, Chemistry and Biology
Linköpings Universitet, SE-581 83 Linköping, Sweden
Linköping 2006

© 2006 Marcus Bäck
ISBN 91-85457-57-4
ISSN 0345-7524
Printed in Sweden by UniTryck
Linköping 2006

This thesis covers the design, synthesis and structure activity relationships analysis of potential hepatitis C virus (HCV) NS3 protease inhibitors. Furthermore are discussed the background to the disease in question as well as the class of enzymes called proteases and why we find them suitable as targets when combating diseases in general, and why find the HCV NS3 protease suitable when battling the viral infectious disease HCV in particular. Moreover is briefly considered some strategies used when designing protease inhibitors and what desired properties a potential drug candidates should possess.
The synthesis of linear and macrocyclic NS3 protease inhibitors comprising a designed trisubstituted cyclopentane moiety as an N -acyl-(4 R )-hydroxyproline bioisostere is addressed and several very potent and promising compounds are evaluated.

I.
This thesis is based on the following papers, which are referred to in the text by their
Roman numerals:
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.
II.
Bäck, M.; Johansson, P-O.; Wångsell, F.; Thorstensson, F.; Kvarnström, I.;
Alvarez, S. A.; Maltseva, T.; Vrang, L.; Hamelink, E.; Hallberg, A.;
Rosenquist, Å. and Samuelsson, B.
Potent Macrocyclic Inhbitors of the Hepatitis C Virus NS3 Protease
Incorporating P2 Cyclopentane- and Cyclopentene-derived Scaffolds.

NMR
NS
NS3
Nva
PDB
Pro
RCM
RT
SAR
Ser
TFA
THF
Abu L-

-aminobutyric acid
Asp
Boc
Cat D
Chg
DIAD
DIPEA
DMAP
DMF
EC
50
L-aspartic acid t -butyloxycarbonyl cathepsin D
L-cyclohexylglycine diisopropyl azodicarboxylate
N,N -diisopropylethylamine
4-dimethylaminopyridine
N,N -dimethylformamide inhibitor concentration causing 50% inhibition of replication in a cell culture system
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide EDC
Fmoc
Gly
HATU
HCV
His
HIV
IC
50
K i
9-fluorenylmethoxycarbonyl
L-glycine
O -(7-azabenzotriazol-1-yl)-
N,N,N’,N’
tetramethyluroniumhexafluorophosphate hepatitis C virus
L-histidine human immunodeficiency virus inhibitor concentration causing a 50% decrease in the enzyme activity dissociation constant of an enzyme (E) - inhibitor (I) complex; K i
=
[E][I]/[EI] nuclear magnetic resonance nonstructural nonstructural protein 3
L-norvaline protein data bank
L-proline
Ring-closing metathesis room temperature structure-activity relationship
L-serine trifluoroacetic acid tetrahydrofuran
Val
L
-valine

Publications ................................................................................................. 4
1. Introduction ............................................................................................. 7
1.1 Proteases – Proteins that Cleave Proteins ............................................................ 7
1.1.1 Serine Proteases ..................................................................................................................... 7
Figure 11.1.2 Potential Drug Targets .............................................................................................. 8
1.1.2 Potential Drug Targets ........................................................................................................... 8
1.2 Describing Enzyme/substrate Interactions ........................................................... 8
Figure 21.3 Protease Inhibitors .................................................................................. 8
1.3 Protease Inhibitors ............................................................................................... 9
1.3.1 Inhibitor Design Strategies ..................................................................................................... 9
1.3.2 Desired Inhibitor Properties ................................................................................................... 9
2. Design, Synthesis, and Structure Activity Relationships (SARs) of
Potential Hepatitis C Virus NS3 Protease Inhibitors (Paper I and II) ...... 10
2.1 Background on the Hepatitis C Virus ................................................................ 10
2.2 The Viral Genome and Life Cycle ..................................................................... 11
2.3 Anti-HCV Therapy – Potential Targets ............................................................. 12
2.3.1 The NS5B RNA Polymerase ................................................................................................ 12
2.3.2 The NS3 Protease ................................................................................................................. 12
2.4 HCV NS3 Protease Inhibitors ............................................................................ 12
2.5 Design, Synthesis and SAR Analysis of Linear HCV NS3 Protease Inhibitors
(Paper I) ................................................................................................................... 14
2.5.1 Design of Linear HCV NS3 Protease Inhibitors (Paper I) ................................................... 14
2.5.2 Synthesis of Linear HCV NS3 Protease Inhibitors (Paper I) ............................................... 14
2.5.3 Structure Activity Relationships Analysis of Linear HCV NS3 Protease Inhibitors (Paper I)
...................................................................................................................................................... 16
2.6 Design, Synthesis and SAR Analysis of Macrocyclic HCV NS3 Protease
Inhibitors (Paper II) .................................................................................................. 20
2.6.1 Design of Macrocyclic HCV NS3 Protease Inhibitors (Paper II) ........................................ 20
2.6.2 Synthesis of Macrocyclic HCV NS3 Protease Inhibitors (Paper II) .................................... 20
2.6.3 Structure Activity Relationships Analysis of Macrocyclic HCV NS3 Protease Inhibitors
(Paper II) ....................................................................................................................................... 22
3. Concluding Remarks / Personal Thoughts ........................................... 25
4. Acknowledgments ................................................................................ 26
5. References ............................................................................................. 26

This thesis deals with the design and synthesis of protease inhibitors, targeting the hepatitis C virus NS3 protease. But, before getting into any reaction schemes or SAR analyses, let us first begin with the basics. What is a protease, what is their function, and how can medicinal chemists use their properties when combating various diseases? These are some fundamental questions that I intend to answer before getting more specifically into the research that I have been engaged with.
Except for being the macromolecules that build up living organisms, proteins can also be very dynamic and have specific catalytic properties. Proteins with such qualities, known as enzymes, are responsible for speeding up chemical reactions within biological cells and are thus essential to sustain life. They are divided into different classes depending of what kind of catalytic activity they perform. Proteases define a particular class of enzymes that are characterized by their quality to hydrolyze the kind of bonds that link proteins together, i.e. polypeptide bonds. This property allows them to control synthesis, turnover and function of proteins, and thereby to direct essential physiological processes inside living organisms.
1
Proteases can, based on their catalytic mechanism, be further divided into four major subclasses: i.e. the serine-, aspartic-, cysteine-, and metalloproteases.
2 I will discuss the first one in more detail, since it represents the class of proteases for which synthesis of potential drug candidates will be discussed later in this thesis.
1.1.1 Serine Proteases
Nearly one-third of all proteases are represented by serine proteases, named for possessing a nucleophilic Ser residue at the active.
3
They are according to their substrate specificity, particularly by the type of residue found at P1, classified into at least three categories. The trypsin-like proteases have preference for the positively charged residues Lys/Arg at P1, the chymotrypsin-like proteases favor large hydrophobic P1 residues (e.g. Phe/Tyr/Leu) and the elastase-like proteases prefer small hydrophobic residues (e.g. Ala/Val) in P1 position.
2
The active site of serine proteases possesses a catalytic triad with Ser 195, His 57, and
Asp 102 amino acids (chymotrypsin numbering system). It also contains an oxyanion hole, which stabilizes the tetrahedral intermediate formed during the catalytic cleavage of the substrate.
3,4
Depicted in Figure 1 is the general mechanism for amide bond hydrolysis by serine proteases.

Figure 1
1.1.2 Potential Drug Targets
As mentioned above, proteases are, by virtue of being able to control folding, turnover and function of proteins, able to regulate many essential physiological functions such as digestion, growth, aging, fertilization, immunological defense and wound healing.
Being able to control mammalian, viral, bacterial or parasitic proteases associated with such qualities are of great interest when trying to come up with therapies against various diseases.
1,2,4
Examples of proteases that have been targeted for drug development are many. Here follow some: renin (human blood pressure regulating protease), thrombin (human protease that facilitates blood clotting), HIV-1 protease, plasmepsins I and II (found in the most dangerous malarial parasite), β-secretase
( assumed to be involved in the neurodegenerative cascade leading to Alzheimer’s disease) and the HCV NS3 protease.
Most proteases have active sites that are sequence-specific, i.e. the size and hydrophobicity/hydrophilicity of the enzyme sites allow certain substrate residues, and exclude others, for proper enzyme/substrate interactions.
1,2
Schechter and Berger
5 introduced the nomenclature that today is standard when describing interactions between substrate/inhibitor (peptide) and enzyme. The amide bond of the peptide, normally cleaved by a protease, is referred to as the scissile bond. The part of the substrate to the right of the scissile bond is called the prime side, and the part to the left is called the non-prime side. The amino acids are designated P (or P’) for pocket, and the corresponding sites with which the amino acids interact on the enzyme are called S (or S’) for sub site. The positions are numbered according to their relative positions away from the scissile bond (Figure 2).
Figure 2

What medicinal chemists want to achieve, when utilizing proteases as targets against a particular disease, is to prevent/inhibit the enzyme from catalyzing the reactions of its natural substrate. Drugs/inhibitors that possess properties to interact in an efficient way with a particular protease consequently have the potential to slow or even halt the progression of the selected disease.
1.3.1 Inhibitor Design Strategies
Traditional ways of developing protease inhibitors employed screenings of natural products or large libraries of compounds. Subsequent refining of promising compounds found in such screenings would then hopefully lead to a drug candidate.
A more modern approach used when designing protease inhibitors is to optimize the part of the natural substrate that interacts with the active site during cleavage
(substrate analogs). Moreover, proteases are often prone to inhibition by cleavage products from their natural substrates (product analogs). The initial optimization strategy is often to start from a rather defined part (less than 10 amino acids) of a substrate or cleavage product, and replace the scissile amide bond with a noncleavable isostere. At this point, random structural modifications, basically through trial and error, were earlier the common way to optimize the structure. This procedure has substantially improved, however, in recent years. Nowadays the aid from threedimensional structural information, acquired from X-ray studies and computer models of inhibitors docked in the active sites of proteases, makes a much more qualified and rational drug design possible.
2,6
1.3.2 Desired Inhibitor Properties
When designing a protease inhibitor, there are some desired properties that have to be taken into consideration. Besides possessing activity against the protease of interest, a drug candidate should also show selectivity toward other proteases. Low toxicity, a relatively long half-life and a high therapeutic index,
7
are also qualities that need to be fulfilled. Moreover, good bioavailability is necessary when administering a protease inhibitor orally to a patient. A few guidelines have been given
8
to ease the prediction of whether a drug will show the last-mentioned criterion or not (i.e. molecular weight
< 500, hydrogen bond acceptors < 10, hydrogen bond donors < 5, and Log P < 5).
Furthermore according to a recent study,
9
the drug should not possess more than 10 rotatable bonds or have a polar surface area that is more than 140 Å 2
for good oral bioavailability. Another feature, that has to do with the criteria mentioned above, is for the inhibitor not to be too peptide-like. Peptides tend to be unstable and show poor biopharmaceutical qualities.
2

For many years a serious chronic form of hepatitis was spread unknowingly all over the world, mainly through blood transfusions.
10
The development of new tests for hepatitis A (HAV) and hepatitis B (HBV) in the mid-1970s led to the discovery that a large proportion of cases were caused by neither of these two viruses.
11
With scientists now being aware of a new pathogen, experts thought that it would soon be identified. As a matter of fact it took 15 years before Houghton and co-workers,
12 with the use of serum from chimpanzees, were able to clone and identify the genome of the causative agent.
11
The new form, previously referred to as non-A non-B hepatitis, was named hepatitis
C virus (HCV).
13
The main reason why HCV remained such a subtle pathogen for decades is its silent onset and asymptomatic evolution into a chronic form of hepatitis.
14
Moreover, HCV proved to be difficult to grow reliably in cell-culture, and chimpanzees seem to be the only animal, except for humans, that can be reliably infected.
15
These are facts that brought about high costs as well as ethical issues.
Hepatitis C today afflicts more than 170 million people or 3% of the global human population.
16
It thus represents a human epidemic that is nearly five times more prevalent than that of the human immunodeficiency virus (HIV).
17
The large amount infected people in the world are primarily due to the uncontrolled spreading that began in the early 1960s and lasted till the end of 1980s;
10
and even though blood screenings have been implemented the amount of infected people in the world continues to grow mainly due to inadequate detection and injection drug abuse.
14
What sets HCV apart from most other viruses, is its possessed nature to cause a chronic disease. Unlike infection with HAV, that usually only lasts for a couple of weeks, HCV infection may last for decades.
11
The virus primarily infects the hepatocytes, i.e. the cells of the liver. In accordance with other viruses, HCV does not kill the infected cells itself, but triggers a self- destroying response or mechanism from the immune system of its infected host.
18
The disease is associated with a slow progressive inflammation and fibrosis of the liver, which in time result in liver cirrhosis and eventually hepatic failure or hepatocellular carcinoma.
19
Accordingly, HCV infection is today the leading cause for liver transplantation in western countries,
10
and since infected persons may be carriers of the virus for a decade or more before the symptoms manifest, the population requiring medicinal treatment with possible need for liver transplants may increase dramatically the next 10-20 years.
17

There are at least six existing genotypes of HCV today (1-6); with genotype 1 being predominant.
20
The many genotypes, which are a result of the high mutation rate of the virus, 10 have made the development of efficient drugs against HCV very challenging. At present there is no vaccine against HCV and the current therapy administered to infected persons, pegylated interferon-α in combination with the nucleoside analogue ribavirin, 21 is poorly tolerated and effective in less than 50% of patients infected with HCV genotype 1.
16
Consequently, there is an urgent need to come up with new and improved drugs to treat HCV infection.
HCV is a relatively small positive single strand RNA virus belonging to the
Flaviviridae viral family. The viral genome comprises an open reading frame (ORF) of approximately 9600 nucleotides that, once inside the host cell, codes for a large polyprotein. This polyprotein undergoes proteolytic cleavage into smaller proteins, three structural and six non-structural (NS) proteins.
22
The structural proteins include the nucleocapsid core (C) protein and two envelope glycoproteins E1 and E2. The core protein forms the nucleocapsid, encapsulating the viral RNA, and E1 and E2 are heavily glycosylated envelope proteins that help the virus to interact with membranes of hepatocytes and other cells.
10
The structural proteins are separated from the NS proteins by a small membrane peptide designated p7, suggested to have ion channel properties. The NS proteins, NS2, NS3, NS4A, NS4B, NS5A and NS5B, are essential for polyprotein processing and for directing the viral translation and replication.
Autocatalytic cleavage of the NS2-NS3 metalloprotease produces NS2 and NS3. The
NS3 protein possesses an N-terminal serine protease domain and a C-terminal RNA helicase/NTPase domain, where the protease in complex with the cofactor, NS4A, is responsible for cleavages of the remaning NS proteins. The function of the small hydrophobic protein NS4B is unknown, whereas the NS5A and NS5B proteins are responsible for the replication machinery (Figure 3).
23
Figure 3
Figure 4 presents a rather simplified HCV life cycle. Attachment and endocytosis (cell entry) is promoted by the interaction of the viral-envelope proteins with specific surface receptors of the cell. Within the cell, low pH fusion mediates the release of the single-stranded RNA which then serves three main roles: for translation of the polyprotein; as a template for replication; and as the genome to be packaged into new virus particles (virions).
23
Figure 4
Due to a very high replication rate and the lack of proofreading function by the NS5B polymerase the genetic variability found in the HCV RNA genome is very high. As mentioned above, this has resulted in six main genotypes and several subtypes of
HCV. The predominant genotype 1 is further divided into subtypes 1a and 1b. Both subtypes are commonly found in the United States and Europe, whereas 1b is the predominant subtype in Asia.
10

Of the NS proteins coded for by HCV, all are in theory equally suitable as targets for anti-HCV therapy. Nonetheless two enzymes have gained a little more interest: The
HCV NS3 protease, which represents the HCV target that this thesis is focusing on, and the NS5B RNA polymerase. I will only discuss the last mentioned briefly.
2.3.1 The NS5B RNA Polymerase
The HCV NS5B RNA-dependent RNA polymerase (RdRp) is an essential enzyme for the viral replication. It facilitates the synthesis of positive and negative stranded viral
RNA. There are no human enzymes with similar biochemical activity, making it possible to identify very selective inhibitors of this enzyme.
15
The crystal structure of the polymerase has been resolved and several nucleoside analogues as well as nonnucleoside inhibitors have been identified. Moreover, active RdRp inhibitors have been shown to reduce the viral load in vivo.
17
Altogether, the HCV NS5B polymerase makes a very promising anti-HCV target.
2.3.2 The NS3 Protease
NS3 is the most intensively studied and best characterized antiviral target of the NS proteins and emerged at an early stage as the most popular Anti-HCV target. It is a bifunctional enzyme of 631 amino acids where the first 180 amino acids are defined by a serine protease, whereas the remainder of the protein encompasses an RNA helicase.
16
Structurally the protease is a member of the chymotrypsin serine protease family 24 but is yet unique in demanding a noncatalytic structural zinc atom and a small peptide co-factor, NS4A, for catalysis.
21
The NS3 protease in complex with the cofactor is responsible for cleavages of the entire downstream region of the polyprotein,
25
thereby making it essential for viral replication and a potential pharmaceutical target.
26
Moreover, with the success of using protease inhibitors in the treatment of HIV,
25
the concept of inhibiting key proteases in the battle against viral diseases was proven. Furthermore, the efficacy of inhibiting the HCV NS3 protease was validated in recent proof of concept studies.
27
The HCV NS3 protease has a shallow and solvent exposed active site and requires a long peptide substrate with many weak interactions distributed along an extended surface area. The protease also shows an established preference for a cysteine in the
P1 position,
28
which initially rendered medicinal chemists substantial problems (se below). These facts, along with the high mutation rate of the virus, show to some extent the difficulties associated with HCV NS3 protease inhibitor design.
Nonetheless, great efforts from many research groups around the world have led to the identification of several potent inhibitors in a rather short period of time.
Among the first inhibitors to emerge were decamer substrate analogs, spanning from
P6 to P4’ position of the enzyme active site. The activity of these peptides was gained

through the incorporation of cyclic residues like proline or pipecolinic acid in P1’ position, making the peptides uncleavable, and thereby given the potential to inhibit the enzyme in a competitive manner.
28,29
During the substrate specificity studies of the NS3 protease, the enzyme was found to be prone to feed-back inhibition from hexapeptidic substrate cleavage products.
30,31
Based on these observations two research groups set out to optimize these hexapeptides. Compounds A
32
and B
33
(Figure 5) represent such early stage product analog inhibitors, which were shown to require a two acid “anchor” in P5-P6 position, as well as a cysteine P1 residue with a terminal carboxylic acid, for optimum activity.
Since cysteine is not a preferred building block in drug synthesis, major efforts were done with the goal to come up with acceptable P1 replacements. This resulted in ( S )-
4,4-difluoro-2-aminobutyric acid inhibitor C
28
and 1-aminocyclopropyl carboxylic acid inhibitor D , 33 exhibiting equipotent activities compared to the corresponding cysteine inhibitors.
However, it is a well-known fact that polypeptides and multiple carboxylate functionalities is not a favorable combination when it comes to stability and bioavailability of a potential drug. Therefore truncation of the P5-P6 positions of inhibitor D , optimization of a P2 aromatic moiety and insertion of an exceptionally good P1 residue led to the extremely potent tetrapeptide inhibitor E .
34
The inhibitor was the result of years of systematic research and optimization led primarily by
Llinàs-Brunet and co-workers 30,34-38
at Boehringer Ingelheim, Canada. The use of
(1 R ,2 S )-1-amino-2-vinylcyclopropane carboxylic acid as an excellent cysteine mimic, along with large P* aromatic systems extending from P2 position, was a major breakthrough making it possible to produce small peptide inhibitors without substantial loss in activity. Although very potent, compound E suffered from poor biopharmaceutical properties mainly due to its higly peptidic nature. The analysis of crystallographic- and NMR data had however revealed that the P1 and P3 side chains of bound inhibitors were positioned close to each other in space, pointing in the same direction.
39
Thus further refinements of inhibitor E , such as capping the P3 position with a cyclopentyl moiety, 34 rigidification of the inhibitor into its bound conformation and replacement of the phenyl moiety of the qinolinol with an aminothiazole derivative resulted in the less peptidic macrocyclic inhibitor F ( BILN 2061)
27,40-43
(Figure 5), the first HCV protease inhibitor to enter clinical trials. Early testing proved
BILN 2061 to be very effective displaying a rapid decline in virus concentration in all treated patients infected with HCV genotype 1.
15
Unfortunately, cardiac toxicity was observed in laboratory animals administered with high doses for four weeks, and the clinical trials have as a consequence been put on hold.
44
Another very promising compound to enter clinical trials is α-ketoamide G ( VX-
950 ).
16,45-47
Whereas all other compounds in figure 5 belong to the non-covalent product analog class of inhibitors, VX-950 represents another category known as transition state analogs or serine trap inhibitors. These inhibitors use electrophilic groups to form a covalent adduct with the catalytic serine of the protease, a covalent bond that is slowly reversible in the case of VX-950 . The initial clinical trials have shown very promising results but more detailed evaluation is required and is currently underway.
16
An important feature to be added is that experiments have revealed BILN 2061 resistant mutants still to be sensitive to VX-950 and vice versa. This indicates the use of combination therapy to be a possible strategy to treat HCV infected patients in the future.
48

The somewhat unique feature for non-covalent product-based inhibitors to require a
C-terminal carboxylic acid, led to the search for bioisosteric replacements of this critical group. This exploration resulted in the identification of an array of P1 cysteine replacements where especially acylsulfonamide derivatives, reaching into the S1’ and
S2’ sites, proved to give inhibitors with increased potency in both enzymatic and cellbased assays.
16,49-51 Compound I 52 is an illustrative example of the benefits gained from incorporation of sulfonamide carboxylic acid bioisosteres and should be compared with compound H
34
(Figure 5).
Figure 5
2.5.1 Design of Linear HCV NS3 Protease Inhibitors (Paper I)
The amino acid L -proline is a frequently employed building block in inhibitor drug design. It has been incorporated in numerous molecules targeting various key proteases and diseases.
36,53-55
A substantial amount of effort has therefore been devoted to mimic this motif. Previous work from our laboratories has revealed that N acylproline I (Figure 6), can be replaced with five-membered carbocyclic ring isosteres like II
56
and III
57
producing in the case of proline mimic III moderately potent thrombin inhibitors.
Pioneering work from Boehringer Ingelheim has resulted in the discovery of novel and very potent HCV NS3 protease inhibitors incorporating N -acyl-(4 R )hydroxyproline IV in P2 position (compounds E and F in Figure 5). Inspired by those inhibitors we chose the trisubstituted cylopentane structure V , based on modeling, to be synthesized and incorporated in HCV NS3 inhibitors.
2.5.2 Synthesis of Linear HCV NS3 Protease Inhibitors (Paper I)
Depicted in figure 7 is a generic structure of the linear HCV NS3 inhibitors synthesized in Paper I, all encompassing the trisubstituted cyclopentane scaffold V .
Also included are the different R
1
, R
2
and R
3
substituents used in order to optimize these inhibitors. A majority of the substituents in Figure 7 were commercially available or readily synthesized from commercially available amino acids or precursors. A detailed description of the chemistry employed will therefore not be given here. For clarity some comments should however be made. R
1
amino acid derivatives A1 , A2 and A4 were all commercially available or easily synthesized from suitably protected and commercially available precursors. Vinylcyclopropane amino acid derivative A3 was synthesized according to literature protocol from Llinas-
Brunet et al.
41,58 Synthesis of the R
2
2-phenyl-7-methoxy-4-quinolinol B1 moiety was also performed according to published procedure.
34,59
R
3 dipeptide, or capped amino acid, substituents C1-C8 and C10-C11 were synthesized employing standard

coupling-, protection- and deprotection procedures (se appendix) using commercially available amino acids and amines. In order to obtain N -methylated dipeptide C9 ,
Fmoc protected cyclohexylglycine was first subjected to treatment with paraformaldehyde and p -toluenesulfonic acid in refluxing toluene, followed by treatment with trietylsilane (Et
3
SiH) and trifluoroacetic acid (TFA) in chloroform.
60
This N -methylated amino acid was coupled using standard peptide synthesis procedure to afford dipeptide C9 . The same procedure was used to obtain the corresponding dipeptide, in which the nitrogen on tert -butyl glycine had been methylated instead of the nitrogen on cyclohexylglycine. However, in spite of several attempts using a vast number of coupling reagents and conditions, the dipeptide could not be coupled to the scaffold. As a consequence, this dipeptide, which we intended to include in an N -methylation study of the P3 and P4 substituents together with C8 and
C9 , was never to be evaluated as an R
3
substituent. The low coupling reactivity is likely to be caused by steric hindrance between this particularly bulky amine and the scaffold during coupling.
Figure 7
In order to reach the goal of incorporating a P2 trisubstituted cylopentane as 4hydroxyproline mimic into our potential HCV NS3 inhibitors, bicyclic lactone 3
(Scheme 1) was synthesized. Starting from enantiomerically pure trans -(3 R ,4 R )bis(methoxycarbonyl)cyclopentanone ( (-)-1 ),
61
prepared according to procedure described by Rosenquist et al ., 62 alcohol 2 63 was prepared in 76% yield using sodium borohydride in MeOH. Both methyl esters of 2 were then hydrolyzed using NaOH in
MeOH. Next treatment with acetic anhydride in pyridine
64
effected lactonization to give 3 63 in a total yield of 88%. It then turned out to be necessary to protect lactone 3 in two different ways in order to have orthogonal protecting groups when coupling to differently protected R
1
substituents. Using methyl iodide and silver (I) oxide in acetone produced methyl ester protected scaffold 4 in 81% yield, whereas reaction with ditert -butyl dicarbonate (Boc
2
O) and 4-dimethylaminopyridine (DMAP) in dichloromethane (DCM) yielded the corresponding tert -butylester protected scaffold
5 in 52%. An initial approach to promote tert -butyl ester protection involved the use of tert -butanol, EDC and DMAP in DCM.
65 Unfortunately this procedure constantly produced less product and more byproducts compared to the Boc
2
O protocol.
Scheme 1
Depending on which of the two scaffolds and which R
1
substituent that were used, two slightly different methods were employed when synthesizing the target compounds (Table 1). Scheme 2 depicts the synthesis of target molecule 9 , according to Method I, also employed in the synthesis of target molecules 14-20 (Table 1).
Methyl ester protected lactone 4 was initially opened using H-Nva-O t Bu, diisopropylethylamine (DIPEA) and 2-hydroxypyridine in refluxing THF to yield amide 6 in 96%. 2-hydroxypyridine is a bifunctional catalyst that is known to catalyze the amide formation between amines and different kind of esters;
66
an accelerating effect that proved to be very important, in order to shorten the reaction time and to obtain high yields, when opening this lactone.
Next, Mitsunobu-like conditions using R
2
substituent 2-methyl-7-methoxy-4quinolinol ( B1 ), triphenylphosphine (PPh
3
) and diisopropyl azodicarboxylate (DIAD) in dry THF 41 gave compound 7 in 78% yield. Treating methyl ester 7 with LiOH in

dioxane/H
2
O 1:1 gave the corresponding acid, which was subsequently coupled to amine C6 using the coupling reagent HATU and DIPEA in DMF to provide 8 in 81% yield. Final treatment with TFA and Et
3
SiH in DCM removed the tert -butyl ester and produced target compound 9 in quantitative yield.
Scheme 2
The synthesis of target molecule 13 , according to Method II, is outlined in Scheme 3.
The same method was also used for preparation of target compounds 21-25 . Initial attempts to use vinylcyclopropyl amino acid A3 , to open lactone 5 according to
Method I, were unsuccessful in our hands. Instead a different method was applied where lactone 5 was firstly opened by careful treatment with LiOH in dioxane/H
2
O
1:1. Subsequent coupling of the obtained acid to amine A3 using HATU and DIPEA in DMF afforded compound 10 in 89% yield. Mitsunobu-like procedure, according to
Method I (Scheme 2), was used to attach quinoline moiety B1 with inversion of configuration, providing 11 in 68% yield. Tert-butyl ester removal and coupling to amine C6 , using TFA and Et
3
SiH in DCM and HATU and DIPEA in DMF, respectively, gave compound 12 in 74% yield. Final, ethyl ester hydrolysis employing
LiOH in THF/MeOH/H
2
O 2:1:1, produced the desired target compound, 13 , in 67% yield.
Scheme 3
2.5.3 Structure Activity Relationships Analysis of Linear HCV NS3 Protease
Inhibitors (Paper I)
In Table 1 are presented structures of all target compounds, along with synthesis methods employed, total yields over the last five or six steps and biological data. The inhibitors were screened against the HCV NS3 1a protease and the percent inhibition was determined at three different concentrations: 10, 1 and 0.1 μM. K i
values were also determined for the most promising inhibitors in the initial screenings. For the inhibitors in Table 1 where K i
values were not determined, instead percent inhibition at 10 μM was used as a comparison of potency.
Table 1
All inhibitors included in Table 1 contain a P2 2-phenyl-7-methoxy-4-quinolinol substituent, which has been frequently employed in potent HCV NS3 protease inhibitors,
16,39
e.g. compounds E
34
and I
27,40-43
(Figure 5). Such aromatic P2 elongations have been reported to play an important role in stabilizing the catalytic machinery in the right geometry by shielding that part of the protease from exposure to solvent.
35,67
Furthermore, the P2 aryl substituent is suggested to interact in a favorable manner with the helicase domain of the NS3 protein.
68-70
The incorporation of P2 substituents, along with optimizations of the critical P1 position, have largely revolutionized the synthesis of HCV NS3 protease inhibitors and are the main reasons for medicinal chemists being able to produce a new generation of small, less peptidic

and drug-like inhibitors with equal or even better inhibitory activities than the previous generation of hexapeptide inhibitors.
Let us now analyze the properties of the inhibitors in Table 1 in more detail. Using the cyclopentane central core, we were initially concerned about which stereochemistry to use at the P3 and P4 positions in our novel inhibitors. When comparing compounds incorporating our new cyclopentane-derived template with compounds based on the
4-hydroxyproline (e.g. E in Figure 5), you notice some striking differences: Our scaffold protrudes one atom farther than the 4-hydroxyproline scaffold, and the P3-P4 substituents have to be turned away from the N-C direction (used in 4-hydroxyproline compounds) towards the C-N direction when coupling to the carboxylic acid functionality of our template. In addition the 1-position in our P2 template is sp
3 hybridized, whereas proline with its corresponding nitrogen in this ring position is planar. Considering these differing P2 template properties it was not certain that the
L
-
L
stereochemistry of the P3-P4 substituents, producing the most potent 4hydroxyproline-based inhibitors, would yield the most potent inhibitors when using our scaffold.
A modeling study was set with the purpose to predict the stereochemical requirements of the substituents at the P3-P4 positions. As starting point for this modeling the X-ray crystal structure of a bound product of the NS3-mediated cleavage
70
(the C-terminal of the full length single strand NS3 construct) was used (Figure 8). Compond 14 ,
(Table 1) having the
D
-configuration at the P3 and P4 positions, was then aligned with the NS3 product. The result, depicted in Figure 9, shows that no good alignment could be obtained between the P3-P4 side chains in compound 14 and the side chains of the product.
In contrast, a very good overlay is given from the alignment of compound 15 , (Table
1) having the
L
-amino acids at P3 and P4, and the bound cleavage product (Figure 10).
When taking a closer look at Figure 10 you notice that: the acyl cyclopentyl moiety of compound 15 adopts the same position as the P3-carbonyl of the product, the P3-NH of 15 shows the same interaction as the P2-NH of the product and that the P3 side chains of the two compounds are equally positioned in space. The same pattern of binding and positioning in space is valid for the side chain and the amide of the P4 substituent. In view of this modeling study the
L
-configuration was thus anticipated to be preferred for the P3 and P4 amino acids.
Although molecular modeling is this way can be helpful when trying to explain similarities and differences regarding the adopted positions and directions of substituents in an active site, it is still rather hypothetical. Consequently, in order to verify the modeling experimentally compounds 14 and 15 , which both incorporate 2aminobutyric acid (Abu) as a cysteine mimic in P1 position, were synthesized.
Neither of these compounds are very active, even though compound 15 , having the
L
configured P3 valine and P4 cyclohexylglycine moieties, displays the better percent inhibition at 10 μM, 65%, compared to 21% for compound 14 with the corresponding
D
-configured moieties. The results of these compounds suggest that in accordance with the 4-hydroxyproline-based inhibitors the
L
-configuration of the P3 and P4 residues also gives our novel inhibitors, incorporating the cyclopentane-based template, the best fit in the active site; an observation that is in agreement with the modeling predictions. The somewhat weak potencies of these compounds can be explained by the use of the rather poor Abu cysteine mimic in the P1 position. When introducing the reportedly better norvaline cysteine mimic, is obtained compound 16, with a measurable K i
value of 2.3 μM and 100% inhibition at a concentration of 10

μM, as compared to >10 μM and 65% inhibition, respectively, for the corresponding
Abu compound, 15 (Table 1). In order to make sure not combinations of
D
-
L
or
L
-
D configurations of the P3-P4 substituents could provide inhibitors with a better fit, compounds 17 (
D
-
L
) and 18 (
L
-
D
) were prepared. The activities displayed by these inhibitors, 35% and 37% inhibition at a concentration of 10 μM for 17 and 18 , respectively, further confirms the importance of
L
-configuration of both the P3 and P4 residue.
So far all compounds discussed incorporate a P4 methyl ester capped amino acid.
Since the methyl amide is believed to be of greater metabolic stability compared to the more easily hydrolyzed methyl ester, compound 19 was prepared. Unfortunately the introduction of the methyl amide resulted in a slight decrease in potency, with a K i value of 6.6 μM for compound 19 , compared to the K i
value of 2.3 μM displayed by the corresponding methyl ester compound 16 . Previous reports have revealed that replacing valine for tert -butyl glycine in the P3 position produce more potent NS3 inhibitors. Accordingly, this modification yielded compound 9 with a K i
value of 1.7
μM, which makes it almost four times more potent than the corresponding valine compound 19 . The corresponding methyl ester capped compound 20 , exhibiting a K i value of 1.2 μM, is essentially equipotent to compound 19 . It thus appears that the methyl ester can be replaced for the methyl amide with preserved activity in more optimized compounds.
Llinas-Brunet et al. have shown the (1 R ,2 S )-1-amino-2-vinylcyclopropane carboxylic acid to be an excellent cysteine replacement and P1 substituent with an outstanding fit in the hydrophobic S1 pocket;
38,41
and it has been incorporated into several highly potent inhibitors, e.g. compound E and I (Figure 5).
The incorporation of this P1 substituent into our cyclopentane series felt like a natural move at this stage, since the S1 site interaction for optimum activity cannot be underestimated. Hence, introduction of the vinyl cyclopropane P1 residue yielded compound 13 with a very promising K i
value of 0.022 μM, making it almost 80 times more potent than the corresponding norvaline substituted compound 9 .
At this point a methylation study was conducted with the purpose to further examine the binding modes, and with that especially the hydrogen bond interactions of the P3-
P4 portion, of these inhibitors. The compound where the amide nitrogen closest to the cyclopentane ring had been methylated could never be evaluated due to lack of success, presumably because of steric hindrance, during the coupling step. The inhibitor with an added methyl group on the nitrogen of the capping group, compound
21 , exhibits a K i
value of 0.016 μM, which makes it almost equipotent to compound
13 ; suggesting the hydrogen of the capping methyl amide not to take part in any hydrogen bond interactions. In contradiction to compound 21 with the methylated amide capping group, compound 22 with the methylated amide nitrogen of the cyclohexylglycine moiety, displays a K i
value of >10 μM, emphasizing very important hydrogen bond interaction in this area of the active site.
In attempts to truncate the P3-P4 portion of inhibitor 13 we synthesized compounds
23 and 24 where the P4 substituent was replaced by simple amines, in this case cyclopentylamine and tert-butyl amine, respectively. Notably this resulted in a significant activity loss with 23 displaying a K i
value of 2.7 μM and 24 a K i
of 6.9
μM, indicating that inhibitors based on the cyclopentane scaffold, although optimized, are very sensitive to modifications in the P3-P4 region.
Another very good cysteine mimic in the P1 position is the ( S )-2-amino-4,4difluorobutyric acid (compound C in Figure 5) previously reported by Narjes et al.
71

When introducing this P1 substituent an increase in activity is gained compared to the corresponding molecules comprising Abu or norvaline. Nonetheless the activity exhibited by compound 25 , 0.56
μM, still makes it significantly less potent, approximately 25 times, compared to the corresponding (1 R ,2 S )-1-amino-2vinylcyclopropane carboxylic acid incorporated inhibitor ( 13 ).
It should finally be mentioned that the enantiomeric scaffold ( VI ) and the scaffold with the acyl substituents in cis configuration ( VII ) were also synthesized and incorporated in target molecules for proper evaluation. The target compounds based on these isomeric scaffolds (Figure 11) turned out to be totally inactive against the
NS3 protease. This is proof that the scaffold we chose ( V ), based on molecular modeling, also was the scaffold that gave the best fit in NS3 active site.
Figure 11
When summarizing the results in Table 1 we can see that incorporation of systematically optimized and evaluated P1 and P3-P4 substituents, into our novel trisubstituted cyclopentane-based template, have resulted in several very promising inhibitors in the nanomolar range, e.g. 13 , 21 and 25 , displaying K i
values of 22, 16 and 560 nM, respectively. The substituents found to give the best fit were the (1 R ,2 S )-
1-amino-2-vinylcyclopropane carboxylic acid in P1 position and
L
tert -butyl glycine and
L
-cyclohexylglycine in P3 and P4 position, respectively. A striking feature seen when comparing the inhibitors of our cyclopentane-based series with inhibitors utilizing the 4-hydroxyproline central core is that our inhibitors seem to be very sensitive to modifications in the P3-P4 portion, e.g. N -methylation in certain position or truncation of the P4 substituent resulting in dramatic loss in activity. Furthermore, the use of the right P1 substituent was found to be crucial for obtaining potent compounds.
Nonetheless we have shown that a trisubstituted cyclopentane dicarboxylic acid can be readily synthesized and successfully used as a 4-hydroxyproline mimic to produce very promising HCV NS3 protease inhibitors; and as you are about to see (below) further refinements have rendered even more potent and drug-like compounds.

2.6.1 Design of Macrocyclic HCV NS3 Protease Inhibitors (Paper II)
In paper I we were able to show that incorporation of a novel trisubstituted cyclopentane dicarboxylic acid in P2 position, as a replacement for the much more frequently used N -acyl-(4 R )-hydroxyproline, produced very promising linear HCV
NS3 protease inhibitors. Encouraged by these results and inspired by results from previous reports, especially those concerning BILN 2061 (figure 5) (Llinas-Brunet et al. at Boehringer Ingelheim, Canada), we were now interested to investigate if macrocyclization could provide our inhibitors with more desirable properties.
Previous analyses have revealed the P1 and P3 substituents of bound 4hydroxyproline-based inhibitors to be in close proximity.
39 Thus, if connecting these positions in an appropriate way you may be able to rigidify the structure, and by that reduce the entropic penalty paid upon binding as well as obtain a less peptidic and drug-like inhibitor.
43
Moreover, the introduction of carboxylic acid bioisosteres, like acyl sulfonamides, has been reported to improve inhibitory activities both in enzymatic- and cell-based assays.
16,49,51,52
Since we in this paper were aiming for synthesizing compounds with better biopharmaceutical properties, replacing the cterminal carboxylic acid in our inhibitors with such cell-activity enhancing isosteres, was another interesting modification we were interested to explore. A recent report from our group has also proved a trisubstituted cyclopentene dicarboxylic acid to be an effective N -acyl-(4 R )-hydroxyproline mimic in the synthesis of novel linear NS3 inhibitors.
72
With this in mind we set out to synthesize P2 cyclopentane- and cyclopentene incorporated hydrazine-functionalized macrocyclic HCV NS3 inhibitors of different ring sizes, utilizing olefin ring-closing metathesis (RCM).
2.6.2 Synthesis of Macrocyclic HCV NS3 Protease Inhibitors (Paper II)
Ring-closing metathesis is a modern and very convenient approach to create rings intramolecularly from molecules incorporating two olefin functionalities. Depicted in
Scheme 4 is the synthesis of four P3 Boc hydrazine olefin linkers used in the coupling steps generating diolefins 31a-d (Scheme 5).
Scheme 4
The hydrazine linkers were obtained according to two different protocols. Direct alkylation of commercially available tert -butyl carbazate with 5-bromo-1-pentene
( 26a ) and 6-bromo-1-hexene ( 26b ) at 100 °C in DMF 73 produced hydrazine olefin linkers 27a and 27b in 72% and 75%, respectively. The second approach employed a two-reaction reductive amination protocol, where the commercially available alcohols
6-heptenol ( 28a) and 7-octenol ( 28b) were initially oxidized using a catalytic amount of tetrapropylammonium perruthenate (TPAP) and 4Å molecular sieves in DCM with
N -methylmorpholine N -oxide as reoxidizing agent,
74
producing the crude aldehydes
29a and 29b in 74% and 98%, respectively. Due to the extreme volatility of the obtained aldehydes concentration of the reaction mixture and evaporation after purification were performed without heating, and no further drying under reduced

pressure was allowed. Following treatment of aldehydes 29a and 29b with tert -butyl carbazate in MeOH with 3Å molecular sieves gave the hydrazones, which were subsequently reduced to their corresponding hydrazine cyanoborane adducts using sodium cyanoborohydride in acetic acid/THF 1:1. Final hydrolysis of the borane adducts with NaOH in MeOH
75
rendered the desired hydrazines 30a and 30b in 45% and 38% total yield, respectively, over three steps.
The direct alkylation using a large excess of tert -butyl carbazate was found to work surprisingly well in our hands producing the monoalkylated hydrazine linkers 27a and
27b in good yields. Initially we were concerned with the possibility of dialkylation; hence us employing the reductive amination protocol when synthesizing analogues
30a and 30b . The inconvenient synthesis of shorter aldehydes than 29a , due to their extreme volatility, together with the longer reaction sequence and the poor total yields obtained using the reductive amination protocol made us try the direct alkylation approach when synthesizing analogues 27a and 27b .
Scheme 5 shows the synthesis of the macrocyclic target compounds containing a P2 cyclopentane proline mimic. Building block 11 , synthesized according to Scheme 1 and 3, was employed as a starting template when obtaining the cyclopentane-based inhibitors in Table 2. Initial tert-butyl ester hydrolysis using TFA and Et
3
SiH in DCM produced the corresponding carboxylic acid, which was subsequently coupled to amines 27a , 27b , 30a and 30b employing HATU and DIPEA in DMF to give dienes
31a-d in yields ranging from 64-85%. The pivotal macrocyclization step was effected by letting dienes 31a-d react with a catalytic amount of 2 nd
generation Hoveyda-
Grubbs ruthenium catalyst in refluxing DCM,
41
which gave 13-, 14-, 15- and 16membered macrocyclic compounds 32a-d in 10-79% yield (Scheme 5). The poor yield afforded for the 13-membered ring 32a , is probably due to unfavorable ring strain as well as the increased difficulty with which the olefins are able to interact during the metathesis step when using the short olefin linker, resulting in more byproducts. Ethyl esters 32a-d were hydrolyzed with lithium hydroxide in refluxing
THF/MeOH/H
2
O 2:1:1 yielding the first target compounds 33a-d in 32-100%.
Treating compounds 32a-d with TFA and Et
3
SiH in DCM gave hydrazines 34b-d in yields ranging from 63-74%. Subsequent hydrolysis of the ethyl esters according to the synthesis of 33a-d rendered target molecules 35b-d in 46-71% yield.
For the introduction of an acyl sulfonamide, as a c-terminal carboxylic acid bioisostere, a slightly different coupling procedure was employed. Since sulfonamides are rather deactivated compared to ordinary amines, compound 33b was first subjected to a pre-activation using N -(3-Dimethylaminopropyl)N
′-ethylcarbodiimide
(EDC) hydrochloride in DCM. Subsequent reaction of pre-activated compound 33b with cyclopropanesulfonic acid amide and DBU in DCM gave target compound 36 in
80% yield. Final Boc-removal according to the synthesis of compounds 34b-d furnished target compound 37 in 95% yield (Scheme 5).
Scheme 5
Colleagues of mine performed the synthesis of the target compounds employing a cyclopentene moiety as a proline mimic. Diastereomeric building block 38 (Scheme
6) was used as a precursor; for which the synthesis was previously described.
62,72,76,77
Coupling of carboxylic acid 38 to amines 2b and 5a according to the synthesis of 31ad furnished dienes 39a and 39b in 81% and 79% yield, respectively. Ring-closing

metathesis using 2 nd
generation Hoveyda-Grubbs catalyst produced macrocycles 40a and 40b in 81% and 53% yield, respectively. Final hydrolysis of the tert -butyl ester as well as the Boc group of 40a and 40b with TFA and triethylsilane in DCM generated target compounds 41a and 41b (diastereomeric mixtures) in 38% and 47% yield, respectively (Scheme 6).
Scheme 6
2.6.3 Structure Activity Relationships Analysis of Macrocyclic HCV NS3
Protease Inhibitors (Paper II)
All target compounds were screened against the HCV NS3 1a protease and K i
values and EC
50
values were determined. The entire structures of the inhibitors, along with inhibition constants against the HCV NS3 protease, are summarized in Table 2. Also presented in Table 2 is selectivity data for selected compounds against the human serine proteases cathepsin B, chymotrypsin and elastase. All inhibitors discussed below have Z -configured double bonds, according to NMR-analyzes. They also contain a P2 2-phenyl-7-methoxy-4-quinoline substituent, a motif frequently employed as P2 extension in potent HCV NS3 protease inhibitors e.g. compounds E and I in Figure 5 and compound 13 from our linear inhibitor series in paper I (Table
1). As mentioned above when discussing the linear inhibitors, the P2 aryl substituent is besides being crucial for shielding of the catalytic machinery from exposure to solvent,
35,67
also likely to interact in a favorable manner with the helicase domain of the NS3 protein.
68-70
The benefits gained upon connecting the P1 and P3 positions of our inhibitors were anticipated to be several. Fixing the inhibitor into its bound conformation is suggested to reduce the entropy penalty paid upon binding and increase the overall binding energy of the inhibitor. Furthermore, the transformation of a linear inhibitor, like 13 , into a macrocycle decreases the peptidic nature and is thus likely to improve the biopharmaceutical properties of the potential drug.
43
Table 2
We were initially interested in establishing the ring size that would provide our cyclopentane-based inhibitors with the best fit in the S1-S3 pocket of the active site.
When analyzing the structure of BILN 2061 in more detail you find it to be a 15membered macrocycle with a plane bond protruding from the proline nitrogen.
Moreover you notice that the “linker” protrudes from the sp 3 -configured P3 α-carbon.
In contrast our inhibitors possess an sp
3
-hybridized carbon in the corresponding P2 ring position and the linker protrudes from a plane nitrogen bond (Figure 12).
With these differences in mind the 15-membered ring size used in BILN 2061 was not certain to be the optimal size of ring when using our cyclopentane-based template.
Figure 12
The first compound in Table 2, the 13-membered macrocycle 33a , exhibits a K i
value of 130 nM. Although very promising, the activity of 33a still indicates that the S1-S3

pocket may accommodate larger rings. Compound 33b , containing a 14-membered ring, is more than four times better than the corresponding 13-membered ring, with an even more promising K i
value of 31 nM. The 15-membered macrocyclic compound in our series, 33c , exhibits a K i
value of 710 nM, a result indicating 14-membered rings to possess a better fit in the S1-S3 pocket for cyclopentane derived type of inhibitors.
When increasing the ring size further a total loss of activity is seen with a K i
value of
>10 μM for the 16-membered macrocycle 33d .
At this point we decided to explore the effect of removing the Boc groups of compounds 33b-d . This modification provided a more than five-fold increase in potency for compounds 35b and 35c, compared to the corresponding Boc protected derivatives, with K i
values of 6 nM and 120 nM, respectively. Boc removal had no effect on the 16-membered ring 35d , exhibiting a K i value of >10 μM, further indicating this large ring to be rejected from the S1-S3 pocket. Due to lack of material the corresponding free hydrazine, derived from the 13-membered ring, was not synthesized. Nontheless, the activity data obtained for compounds 33a-d , along with the potency trend seen for compounds 35b-d , indicates the 14-membered ring be the optimal ring size when using our trisubstituted cyclopentane central core.
Compounds 33b and 35b are rather small and very specific NS3 protease inhibitors, displaying excellent selectivity over selected human serine proteases (Table 1).
However, due to their zwitter-ionic nature, they suffer from poor biopharmaceutical properties. Previous reports have shown that replacement of the carboxylic acid of product-based inhibitors for bioisosteres, like acyl sulfonamides, not only improves the pharmacokinetic properties, but also substantially increases the potency of HCV
NS3 inhibitors.
16,49,51,52
With this knowledge we were intrigued to introduce a cyclopropyl acyl sulfonamide as a carboxylic acid bioisostere into the most promising compounds, 33b and 35b .
This modification furnished compounds 36 and 37 , exhibiting impressive K i
values of
0.07 nM and 0.19 nM, respectively. In addition, as a result of the replacement, the compounds gained promising properties on cell, with EC
50
values of 530 nM for 36 and 33 nM for 37 , and to our delight the selectivity was preserved.
Diastereomeric cylopentene derivatives 41a and 41b share, with the proline nitrogen, the same planar configuration at the P2 ring position, but differ just like the cyclopentane derivatives from BILN 2061 with the linkers protruding from a plane nitrogen bond. Still the 14-membered macrocyclic inhibitor 41a displays better activity compared to the 15-membered macrocycle 41b , an observation that is in accordance with their corresponding cyclopentane derivatives 35b and 35c . Inhibitor
41a is slightly less potent compared to 35b whereas 41b and 35c are almost equipotent; displaying K i
values of 15 nM vs . 6 nM and 110 nM vs . 120 nM, respectively. Accordingly cyclopentane compounds 35b and 35c exhibit equal or better activities compared to cyclopentene compounds 41a and 41b .
These observations, along with the more inconvenient synthesis route for the cyclopentene target compounds, providing diastereomers, and the fact that these inhibitors are potential Michael acceptors rendering them unsuitable as drug candidates , were the main reasons for us not exploring the cyclopentene derivatives further.

When summarizing the results obtained from the macrocyclic compound series in
Table 2, it is evident that very promising and selective HCV NS3 protease inhibitors were produced from the macrocyclization of P2 cyclopentane- and cyclopentenebased linear inhibitors. When introducing a cyclopropylsulfonamide as carboxylic acid bioisostere into the most promising compounds, i.e. the 14-membered macrocyclic compounds 33b and 35b , inhibitors with impressive activities on the enzymatic assay and very promising activities on the cell-based assay were obtained; i.e. compounds 36 and 37 with K i
values of 0.07 and 0.019 and EC
50
values of 530 and 33 nM, respectively.

The aim of the medicinal chemistry studies presented in this thesis was to explore the potential of incorporating a trisubstituted cyclopentane dicarboxylic acid in the P2 position of HCV NS3 protease inhibitors. (As mentioned previously, colleagues of mine performed the synthesis of the macrocyclic cyclopentene-derived compounds incorporated in Paper II). This gave rise to several novel and very promising compounds. But before summarizing the results in paper I and II it may be appropriate with a little sense of humility and perspective.
The active site of an enzyme is very dynamic and complex; making the exact prediction of the way a molecule will interact with it almost impossible. The human body is a masterpiece that has evolved during a very long period of time. The slow evolution unfortunately made it sensitive to sudden changes of the outer and inner environment. The sensitivity towards sudden changes of the last-mentioned makes the selectivity and distribution of a potential drug very critical. To be able to come up with a molecule with the quality of possessing all the properties needed to fulfill all the criteria of a suitable drug is in other words an extremely difficult, time consuming and expensive task. I guess what I am trying to say is that, although I was fortunate enough to take part of a project that almost from the start gave potent compounds, the road from potency to a commercial drug is very, very long….
Paper I:

A novel P2 cyclopentane-derived scaffold was synthesized with the aim to function as an N -acyl-(4 R )-hydroxyproline bioisostere in novel HCV NS3 protease inhibitors.

Two synthetic routes were developed in order to have orthogonal protecting groups when coupling to different substituents. These routes readily gave several target compounds in good yields.

Systematic variation of P1 and P3-P4 substituents produced promising inhibitors, some of which display activities in the nanomolar range; i.e. 13 , 21 , and 25 with
K i
values of 22, 16 and 560 nM, respectively.

The most potent inhibitors were obtained by incorporation of (1 R ,2 S )-1-amino-2vinylcyclopropane carboxylic acid at the P1 position and
L
tert -butyl glycine and
L
-cyclohexylglycine at the P3 and P4 positions, respectively. All inhibitors contain a 2-phenyl-7-methoxy-4-quinolinol substituent elongating from the P2 position.
Paper II:

Very potent and selective macrocyclic hydrazine functionalized P2 cyclopentane- and cyclopentene-derived HCV NS3 protease inhibitors were synthesized from two previously developed series of linear inhibitors.

Utilizing ring-closing metathesis 13-, 14-, 15- and 16-membered rings were obtained, of which the 14-membered macrocycles gave the best active site fit; i.e.
33b , 35b and 41a with K i
values of 31, 6 and 15 nM, respectively.


The introduction of a cyclopropylsulfonamide as carboxylic acid bioisostere into compounds 33b and 35b , gave inhibitors with even better activity on enzymatic assay, and very promising activity on cell-based assay; i.e. 36 and 37 with K i values of 0.07 and 0.019 and EC
50
values of 530 and 33 nM, respectively.
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