5 Inhibitors of Hepatitis C Virus NS3•4A Protease: An Overdue Line of Therapy

Progress in Medicinal Chemistry - Vol. 39, Edited by F.D. King and A.W. Oxford 0 2002 Elsevier Science B.V. All rights reserved.

5 Inhibitors of Hepatitis C Virus NS3.4A Protease: An Overdue Line of Therapy ROBERT B. PERNI and ANN D. KWONG Vertex Pharmaceuticals Inc., 130 Waverly Street, Cambridge, MA 02139, U.S.A.

INTRODUCTION - THE NEED FOR PROTEASE INHIBITORS The disease Current therapies Why target the NS3.4A protease?

216 216 217 218

THE PROBLEM The hepatitis C viral pedigree NS3*4A protease structure and function The nature of the NS3-NS4A interaction

219 219 220 220

THE TOOLS Enzymatic assays Surrogate cells assays Replication assays Animal models

223 223 224 225 225

HEPATITIS C NS304A PROTEASE INHIBITORS HCV subsites and nomenclature General drug design considerations

226 226 226

PEPTIDIC INHIBITORS Non-substrate based inhibitors Substrate-based inhibitors Warheads Aldehydes Boronutes r-Ketoacids. umides and diketones The PI specificity pocket P2 variants and solvent shielding P3, P4 substituents P3 indolinyl derivatives Cvclic derivatives

228 228 229 232 232 234 235 237 239 240 24 1 24 I 215

HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY

216

P’ region based inhibitors

243

NON PEPTIDIC INHIBITORS Screening leads

243 243

SYNTHESIS OF NS3*4A PROTEASE INHIBITORS General comments Boronates Aldehyde synthesis a-Keto acids and derivatives Novel PI carboxylic acids P3 Indolinyl derivatives

245 245 245 245 246 248 249

THE FUTURE: SUMMARY AND OUTLOOK

249

REFERENCES

250

INTRODUCTION: THE NEED AND THE CASE FOR PROTEASE INHIBITORS THE DISEASE

In the world today an essentially unnoticed epidemic rages [ M I . Hepatitis C virus has infected approximately 3% of the world’s population. The majority of these patients are in third world countries though a significant number of infections occur in industrialized nations. Much of the initial spread of the disease was via transfusion of contaminated blood and until 1989 the infection was referred to as non-A, non-B hepatitis as the hepatitis C virus had not yet been identified [ 5 ] . Screening of blood supplies was initiated and the rate of new infections has dropped significantly from 180,000 new cases annually in the United States in the 1980s to approximately 28,000 [6, 71. Unfortunately, while the blood supply has been largely cleared of the hepatitis C virus, in western countries other modes of transmission, mostly parenteral, continue to spread the disease [4, -1. These include intravenous drug use, tatooing, skin-piercing and perinatal transmission. In addition there is a significant fraction of infected individuals where the mode of transmission is unknown [8, 91. Hepatitis C disease progression is exceedingly slow. Typical duration from the time of infection to symptomatic disease runs in the order of 20-30 years. Even with this long duration it is estimated that >50% of infected individuals are asymptomatic and do not seek medical intervention even though the risk of incurring significant liver damage is high. The long-term use of

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interferodribavirin as front-line therapy and the associated side-effects (persistent flu-like symptoms, depression) also discourages patient compliance. The prognosis for infected individuals varies greatly with age, duration of infection, viral genotype and alcohol use among other factors [lo]. Hepatitis C virus is an infectious agent related to thejaviridae family of viruses. A small percentage of infected patients spontaneously clear the virus (<15%) while the remaining population progresses to a chronic infected state [ l 11. The physical manifestations of this chronic state vary widely from being completely asymptomatic to fulminant liver disease, though deaths from fulminant HCV disease are rare. Fatalities occur from HCV associated chronic liver disease, e.g., cirrhosis, hepatocellular carcinoma since the HCV virus does not appear to be directly cytopathic to the liver [12, 131. There are an estimated 8,00&10,000 deaths annually from HCV derived disease, about 0.2% of the estimated 4 million infected individuals, and this figure is expected to triple over the next twenty years [14]. As a public health problem, the healthcare costs are high. HCV induced cirrhosis and carcinoma are responsible for most of the liver transplants currently performed [4, 151. Though a study has recently shown that the current standard of care, the interferon/ribavirin combination therapy, is cost effective relative to interferon alone, the costs are still high [16, 171. The major reservoir of the virus is the liver but several recent reports have shown evidence for the presence of replicating virus in the bone marrow [ 181 and peripheral mononuclear blood cells, however the observed levels are very low [ 191. Unchecked, hepatitis C viral particles are turned over at a rapid rate. Up to 10I2 particles are produced per day and the half-life of these virions is approximately 2.7 h. Clearance of the virus varies significantly because the rate of infected cell death varies widely among individuals. Cell death tl/2 range from less than 2 days to 70 days [2&22]. Regardless of extra-hepatic pools of virus, the evidence indicates that the overwhelming predominance is in the liver. CURRENT THERAPIES

Current therapies revolve around immunological manipulation by interferon-a (IFN-a) often in tandem with a nucleoside antiviral, ribavirin [23-261. While this type of therapy has improved somewhat with the introduction of polyethyleneglycolated (PEG)- interferon [27-291, the treatment is still arduous for the patient and often not successful in producing a long-term sustained response. The response rate has also been shown to vary with virus genotype and race. An additional issue for interferon therapy is the high cost of treatment [3]. Indeed while PEG-interferon has recently been given regulatory approval, the standard interferon-ribivarin combination therapy produced a sustained

218

HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY

response in less than 50% of patients. A definition of response has been slow to be adopted [30]. A recent preliminary report describes the possible benefits of using a combination of interferon-cr plus zinc [3 11. Research is ongoing in many groups on a number of non-immunological approaches to the eradication of the hepatitis virus which have been reviewed [32, 331. For example, amantadine, an established antiviral agent, has been studied but has been shown to be relatively ineffective with significant sideeffects [34]. Ribozyme therapy is another option being investigated. Ribozymes (catalytic RNA molecules) that target HCV RNA may be introduced via gene introduction and expression [35] or delivered as an ordinary drug. A manufactured ribozyme, heptazyme, is currently in clinical trials [36]. Heptazyme is designed to selectively cleave viral RNA and consequently inhibit replication. The inhibition of the internal ribosomal entry site (IRES) has been receiving significant attention. The IRES is required for translation and consequently may present a useful target. So far, IRES inhibitors that have been reported appear to function as intercalators [37]. This research is in the early preclinical stages. Simultaneous, multiple lines of attack may be more likely to be successful since the hepatitis C virus exists in at least six known genotypes (I-VI) and an increasing number of subtypes. This situation is the result of a high mutation rate and the constant production of quasi-species. Even within a single individual the virus mutates rapidly and evolves into a large number of viral variants. A potential anti-viral agent must have the ability to interfere with replication regardless of genotype or subtype. The clinical situation and outlook for hepatitis C patients has been recently summarized [38, 391. WHY TARGET THE NS3*4A PROTEASE?

In a patient population where the majority of patients are chronically infected and asymptomatic and the prognoses are unknown, an effective drug must possess significantly fewer side-effects than currently available treatments. Since therapy is more likely to be more effective before the onset of symptoms of hepatitis and liver damage the patient treatment burden must be very low. Unlike IFN-u, protease inhibitors have the potential to fulfill this need. The hepatitis C non-structural protein3 (NS3) is a proteolytic enzyme required for processing of the viral polyprotein and consequently viral replication. Fortunately, despite the huge number of viral variants associated with HCV infection, the active site of the NS3 protease remains highly conserved thus making its inhibition an attractive mode of intervention. Recent successes in the treatment of HIV with protease inhibitors support the concept that the inhibition of the NS3 is a key target in the battle against HCV.

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THE PROBLEM THE HEPATITIS C VIRAL PEDIGREE

The hepatitis C virus is aflaviridae type RNA virus and was not identified until 1989 [40]. The hepatitis C viral genome is enveloped and contains a single strand RNA molecule composed of circa 9600 base pairs. The RNA encodes a polypeptide comprised of approximately 30 10 aminoacids. Significant efforts in the study of the molecular virology of the hepatitis C virus have resulted in some understanding of the viral replicative process. This area has been thoroughly reviewed [41-43]. The HCV polyprotein is processed by viral and host peptidases into 10 discreet peptides (Figure 5.1) which serve a variety of functions [44]. There are three structural proteins, C , El and E2. The P7 protein is of unknown function and is comprised of a highly variable sequence. There are six nonstructural proteins. NS2 is a zinc-dependent metalloproteinase that functions in conjunction with a portion of the NS3 protein. NS3 incorporates two cataytic functions (separate from its association with NS2): a serine protease at the N-terminal end which requires NS4A as a cofactor [45] and an ATP-ase dependent helicase function at the carboxy terminus. NS4A is a tightly associated but non-covalent cofactor of the serine protease [4&49]. The function of NS4B is currently unknown. NSSA may be involved in immunomodulation and NSSB functions as the viral RNA dependent RNA polymerase (RdRP). The NS3a4A protease is responsible for cleaving four sites on the viral polyprotein [SO]. The sequence location of the protease activity was determined in 1995 [Sl]. The NS3-NS4A cleavage is autocatalytic, occurring in cis. The remaining three hydrolyses, NS4A-NS4B, NS4B-NSSA and NSSA-NSSB all occur in trans (Figure 5.2).

- - --

Structural proteins

HzN-

NS3-cofactor

Protease

-

C-EI-EZ-P7-NSZ-NS3-NS4A-NS4B-NS5A-NSSB-COOH

n

H2N-[Protease-181-AA]-[Helicase]-COOH

Figure 5.1. HCV polyprotein structure.

Polymerase

220

HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY

IIII cis

H2N-C-El

-E2-P7-NS2-NS3 NS4A NS4B NSBA NSBBtrans trans trans

Figure 5.2. HCV NS3 mediated polyprotein cleavage sites.

NS3-4A PROTEASE STRUCTURE AND FUNCTION

NS3 is a serine protease which is structurally classified as a chymotrypsin like protease. The distinguishing features as determined by X-ray analysis are two structural domains containing a twisted /?-sheet and a characteristic chymotrypsin-like fold (Figure 5.3) [52-541. Various forms of the enzyme have been studied including full length as well as truncated forms which exclude the carboxy-terminus helicase region of NS3. Truncated enzyme usually consists of amino acids 1-181. Recently an active single chain N S 3 4 A construct was described [55,56]. NMR structural analyses have also been carried out [57]. The NS3 protein is thought to be localized at least partially in the cellular nuclei although it also appears to be difisely distributed in the cytoplasm [58, 591. THE NATURE OF THE NS3-NS4A INTERACTION

While the NS3 serine protease possesses proteolytic activity by itself, the HCV protease is not an efficient enzyme in terms of catalyzing polyprotein cleavage [60]. It has been shown that a central hydrophobic region of the NS4A protein is required for this enhancement [61]. For example, in the presence of NS4A the cleavage of NS5A/5B is increased by over 20-fold (Table 5. I). The NS4A protein is most likely membrane associated and anchors the NS3*4A complex [62]. Evidence suggests that the NS4A protein serves to make the NS3 active site more rigid [63, 641. Indicative of the growth of this topic an increasing number of detailed reports and reviews on the structure and function of the NS3*4A complex are appearing [65-67]. NS4A peptides have been prepared synthetically and have been engineered into a soluble form while maintaining activity and presumably the secondary structure [62]. In addition, mutated versions of an NS4A peptide have been investigated and activity has been restored by biotinylation [68]. The function and sequence of NS4A is well conserved among HCV genotypes Structurally this implies that the NS3-4A binding motif is also conserved ~91.

R.B. PERNI AND A.D. KWONG

Figure 5.3. Structure

22 1

the HCV NS3 protease comple.xed with NS4A (light stick structure) and the ED WCCSMSY substrate (dark stick structure).

of

Since the NS4A peptide affects the prime-side and non-prime side portions of the enzyme differently, the enzymatic interactions with inhibitors differ according to their binding location. A model is shown in Figure 5.4 [60]. The enzyme kinetics have been studied in detail [60]. The NS4A cofactor improves the intrinsic catalytic efficiency to the point of producing viable viral replication but relative to similar viral proteases, the competency is still low. The cleavage rates of a number of natural sequences were determined in the absence or presence of NS4A. The results are shown in Table 5.1. The formation of the three component (NS3 NS4A cofactor inhibitor) complex, is strongly supported by the determination of active site occupancy by measurement of fluorescence energy transfer [70]. An X-ray and CD study of the ternary complexes demonstrated that even for inhibitors that bind with varying potencies, the protein undergoes a

+

+

222

HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY

Table 5.1. KINETIC PARAMETERS FOR THE HYDROLYSIS OF SYNTHETIC PEPTIDE SUBSTRATES BY t-NS3 IN THE PRESENCEIABSENCE OF 4A PEPTIDE Substrate

kcu,

*

0.6 0.007 0.18f0.01 0 . 2 6 i 0.005 0.05 f 0.0002 ND ND 0.2 f 0.005 0.012 f0.002

5A/5B H-EDVV(Abu)C*SMSY-OH 4A/4B H-DEMEEC*SQHLPYI-OH 4B/5A H-ECTTPC*SGSWLRD-OH 5A-pNA

H-EDVV(Abu)C*(p-Nitroanilide)

KWI

kmtlKrn

32i2 270 f 38 16Oi11 805 f 73 ND ND 1010i 157 1080f 167

20,000 700 1,600 60 110 4 200 10

The asterisks denote the site of hydrolysis; Abu denotes L-aminobutyric acid.

I

4A

Ki,

Ea4A.I

-

I

J

I

€-4A

-

I€*4A*J

Kj(with 4A)

Ki(with 4A)

€*4A Figure 5.4. Kinetic model for the HCV NS3 and its interaction with NS4A. non-prime-side inhibitors (I) and prime-side inhibitors (4.

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rearrangement of tertiary structure to accommodate the inhibitor molecules in an identical fashion. In other words the CD spectra for all the compounds studied were superimposable [7 1, 721. This conformational change implies that an induced fit mechanism is operative in inhibitor binding. The binding of the NS4A to the NS3 also occurs via an induced fit. This results in a partial reorganization of the NS3 to a conformation suitable for efficient binding to substrate. Despite detailed experiments and an ever-increasing understanding of the subtleties of the enzyme fimction, published values for kinetic parameters vary significantly. There are several reasons for this. Several forms of the enzyme have been employed (i.e., NS3, NS31431, native and protease domain). The enzyme itself is very sensitive to a number of experimental variables including pH, salt concentration, and even the specific detergent used. The active site of the HCV NS3 protease is very shallow by comparison to other proteases [52]. For example, the NS3 lacks the high flaps surrounding the active site which exist in thrombin and that serve to immobilize the substrate as well as exclude solvent from the active site.

THE TOOLS ENZYMATIC ASSAYS

The p-nitroaniline (pNA) derived assay has been the mainstay of protease compound screening and has been shown to be useful for the NS3*4A protease as well [60]. The substrate probe is usually an NS5A derived protein incorporating the non-prime side of the NS5 with a p-nitroaniline terminus at the cleavage site. The pNA assay can utilize a 181-amino acid version of the NS3 that lacks the helicase domain or, alternatively, the full-length protein, although the full-length NS3 poses handling problems. Excess exogenous NS4A is added to the reaction containing NS3 alone. This is a high throughput assay that is usually run in 96 well plates. Another spectrophotometric method uses an 0-4-phenylazaphenylester (PAP) coupled peptide, AcDTEDVVP(Nva)-OPAP. This system allows for continuous monitoring of the cleavage reaction. Similar to the pNA assay, this system can be formatted in 96-well plates [73]. Product formation from the polyprotein processing can be monitored directly using HPLC methodology. An assay has been described that uses a synthetic 20-residue peptide to mimic the NS5A/5B cleavage site [74]. A closely related alternative uses the NSSA-NS5B substrate itself with a similar detection method [75]. This methodology is also suited for time course experiments.

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HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY

NS3e4A enzyme inhibition can also be measured using a scintillation proximity assay [76]. The cleavage protein is biotinylated and tritiated to give ~~o~~~-DEMEECASHLPYK[~~O The ~ ~assay O~~ monitors ~ - ~ Hthe ] -cleaNH~. vage of substrate and shows a decreasing signal as the hydrolysis proceeds and the amount of tritium in proximity to the biotin (i.e., in the same molecule) decreases. This is an automatable high-throughput assay. SURROGATE CELL ASSAYS

The development of secondary in vivo bioassays to test whether the inhibitor and its derivatives can function in a cellular environment is a critical step in the drug development process. Ideally, one would test whether a potential NS3/4A protease inhibitor inhibits HCV replication in cultured cells infected with HCV. Unfortunately, the testing of candidate antiviral molecules has been limited by the lack of a robust in vitro system for growing HCV in cultured cells. Surrogate cell assays with readouts such as luciferase or alkaline phosphatase activity have been developed which are amenable for screening compounds in 96-well format on a large scale and provide meaningful SAR information. Several cell-based reporter systems have been developed for testing the activity of NS3 protease inhibitors. Hirowatari and co-workers [77] have developed a system in mammalian cells in which NS3 protease cleavage is required for detection of chloroamphenicol acetyltransferase (CAT) activity. Conversely, Song and co-workers developed a system in yeast in which inhibition of NS3 protease cleavage increases B-galactosidase activity [78]. Cho et al. describe an assay that incorporates a NS3.4A-SEAP (secreted alkaline phosphatase) chimeric gene which has been shown to be relatively simple and fast [79]. A drawback for this assay is that it is carried out in yeast cells and not in mammalian cells. Such differences in cellular penetration and transport may provide misleading results. Researchers at Vertex have described a novel method for determining activity of inhibitory drug candidates against a protease [go]. This method uses a multi-domain fusion protein comprised of a protease cleavage site which is used to monitor protease activity in a cell via a reporter (such as luciferase) gene expression system. In the presence of the hepatitis C NS304A serine protease, the expression of a reporter gene is significantly reduced. Because of the low fidelity of RNA-dependent RNA polymerases, the development of resistance to antiviral drugs could become a major hurdle in small-molecule antiviral therapy for HCV as has been the case for anti-HIV therapy. In the absence of a robust HCV viral replication system, several groups have developed recombinant HCV viruses with other RNA viruses such that the

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replication of the chimeric or surrogate virus is dependent on the activity of the HCV NS3 protease domain. Several different RNA virus backbones have been used resulting in the development of chimeric sindbis virus [79, 811, chimeric polio virus [82], and chimeric bovine viral diarrhoea virus [83]. Another surrogate system that is being developed for in vitro cell culture and as an animal model is the virus which is most closely related to HCV, namely GB Virus-B (GBV-B), which can infect tamarins [84]. REPLICATION ASSAYS

Over the last ten years there have been numerous descriptions of HCV replication systems in cell culture. These have been summarized and reviewed [MI. Both peripheral blood neutrophils and hepatocytes are thought to be replication sites of the virus, with the majority of the virus coming from the liver [19]. The source of HCV for these experiments has been sera or plasma collected from infected people. The type of cells which have been reported in use ranges from animal cell lines to human hepatic and haemopoietic cell lines and primary liver cells to chimpanzee primary liver cells. Some of the cell lines reported to support HCV replication include MT-2 cells [86, 871, HPB-Ma cells [88-901, Daudi cells [88, 911, HepG2 cells [92], and Huh7 cells [93]. In most cases, the replication rate is low and sporadic. HCV signal has been shown to persist in some cases for greater than a year. In addition to immortalized cell lines, researchers have investigated the use of in vitro infection with HCV of primary adult human hepatocytes [94, 951, and primary fetal human hepatocytes [96, 971. The recent publication of a HCV replicon construct [98, 991 has initiated a flurry of work to characterize the system [loo, 1011, to format the replicon into a useable assay [102], and subsequently to evolve the replicon into a true replication assay. To date no truly reproducible infectious virus replication assay in human cells has been described which is suitable for drug discovery. ANIMAL MODELS

There is currently no validated system to mimic human infection in animals though a number of possible models are in development. There is a chimpanzee infection model [ 103-1 051 but this model is expensive and impractical for use with the large number of animals required to obtain statistically meaningful results. The Trimera mouse is a SCID mouse derived model wherein a section of human liver tissue is implanted in the mouse kidney capsule after the animal has been irradiated [106]. The model suffers from the drawback that the

226

HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY

infection is relatively transient and that if a drug has a delayed effect this model may not give a positive result. The extensive homology the GB virus B shares with HCV has prompted its development as a chimeric HCV model in tamarins [107]. Infectivity has been demonstrated in the tamarin and the model awaits testing with therapeutic agents. HEPATITIS C NS3*4A PROTEASE INHIBITORS HCV SUBSITES AND NOMENCLATURE

The nomenclature for protease sub-sites has been standardized [ 1081. Residues on the N-terminal side of the catalytic site of the protease are counted sequentially from the catalytic site, S1, S2, etc. Similarly, residues on the Cterminus side of the catalytic site are referred to sequentially beginning at the catalytic site as Sl’, S2’ etc. Residues on substrates or inhibitors are given the complementary designations P1, P2, P1’ P2’ etc, analogously as described above for the enzyme. A summary of subsites and properties for HCV protease is shown in Figure 5.5. GENERAL DRUG DESIGN CONSIDERATIONS

Though there are a number of therapeutic molecular targets being investigated as previously discussed, the vast majority of the effort appears to be focused on the HCV protease. Inhibition of proteases has become a fashionable research area. This may be due at least in part to the successful application of this strategy against the human immunodeficiency virus coupled with the omnipresence of proteolytic enzymes in a vast number of biological processes [109].

Salt Bridge

Solvent

Lipophillc

.

Solvent

Llpophilic

Small Llpophillc

Figure 5.5. HCV NS3 non-prime side sub-site characteristics

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221

Table 5.2. ACTIVE-SITE ANALYSIS OF VARIOUS PROTEASES Enzyme

Inhibitor

MW

K,

Buried Surface

NS3-4A Elastase ICE Thrombin S. Grieseus Protease HIV-1 Protease

Hexapeptide Aldehyde Tetrapeptide CMK Tetrapeptide Aldehyde NAPAP Tetrapeptide Aldehyde Amprenavir (VX-478)

725 521 493 510 412 406

1 pM 20 n M 9 nM 40nM 15 nM 0.6 nM

849 867 868 968 769 1092

1.2 I .7 1.8 1.9 1.9 2.2

One measure of the effectiveness of protease binding can be derived from a calculation of the buried surface area of an inhibitor. That is the surface area of a bound inhibitor covered by the protein and not accessible to solvent. The calculation can be normalized to area per unit molecular weight. One can easily see from Table 5.2 that it is extremely difficult for the NS3 to cover much of a bound inhibitor unlike other common proteases such as thrombin or HIV protease [ 1 101. The shallow binding groove of the NS3 protease poses particular challenges to the design of peptidyl and peptidomimetic inhibitors. The natural recognition length of ten amino acid residues is a starting point but not a practical end. In addition, the hydrophobic nature of most of the protein groove does not provide tight binding handles for an inhibitor to latch onto. High-energy covalent and/or ionic interactions occur only at the catalytic site or at the terminii of the recognition groove [65, 11 11. A drug designer is left with essentially two obvious possibilities: a covalent inhibitor that reacts reversibly or irreversibly with the catalytic serine or a noncovalent inhibitor that incorporates a strong electrostatic interaction with the active site. In both cases the inhibitor would be truncated to a minimum size that includes hydrophobic groups that are complementary to the hydrophobic sub-sites near the catalytic triad of the NS3. The shallow binding trough creates a situation where a small molecule does not have sufficient contact with the enzyme for strong binding. As mentioned, the hydrophobic nature of the groove with the exceptions of the termini gives few opportunities for energetically strong interactions. The net effect is that the resultant inhibitor binding interaction is weak. While acidic groups at the termini provide potent inhibitors from electrostatic attraction, the presence of multiple, spatially distant, carboxylates, makes these compounds unlikely to penetrate cells and are, therefore, unlikely to be effective drugs. Detailed structural studies primarily by NMR have shown subtle shielding effects wherein the highly solvent exposed active site is shielded from solvent by the catalytic histidine. This arrangement allows for better binding equilibria - i.e., slower off rates. Unfortunately, exploiting this finding has been difficult.

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HEPATITIS C VIRUS AN OVERDUE LlNE OF THERAPY

An additional complicating factor is the tendency of the protein to undergo surface conformational changes upon binding of inhibitors [62, 112, 1131. This induced fitting phenomenon is particularly true for NS3 in the absence of the NS4A cofactor. These conformational changes are diminished, though not eliminated, by complexation with NS4A. This is further evidence that the NS4A cofactor partially pre-organizes the NS3 conformation to an arrangement more favourable to binding substrates. Even in the presence of NSIA, sub-sites undergo conformational changes to better accommodate differing substituents thus accounting for the relative promiscuity of this enzyme. From the drug design standpoint this ability to adopt conformations specific to an inhibitor implies that inhibitors can be designed which closely resemble the natural substrate. PEPTIDIC INHIBITORS NON-SUBSTRATE BASED INHIBITORS

A number of peptides not based on the natural substrates of the NS3*4A protease have been studied as NS3a4A inhibitors. Eglin C, is a natural peptide isolated from Hirudo medicinalis. It has been found to inhibit a number of serine proteases [ 1141 and was studied as a basis for HCV NS3 inhibitor optimization. Eglin C is comprised of 70 amino acid residues and exhibits several advantageous characteristics. Eglin C is resistant to both acidic and thermal denaturation. Inhibition of the NS3 protease is thought to occur via the interaction of the active site binding loop with the protease catalytic site (residues 3%49). A total of 20 variants of Eglin C were studied. The most potent variant was found to be the ELEMS modification at P5, P4, P3, P2’ and P3’ respectively (Figure 5.6). The optimized peptide exhibited an = 0.06 pM whereas the parent Eglin C was inactive at 180 pM. Another approach to the design of peptidic inhibitor that does not invoke the natural substrate is based on the sequence ofan HCV minibody [ 1 151. A minibody

P6

3.

+

P1

Residue 39 Elain C -E-G-S-P-V-T-L-D-L-R-Y-

Figure 5.6. Eglin C optimization.

P4‘

3.

49

ECYI >180 uM

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Table 5.3. ALANINE SCAN OF MINIBODY INHIBITION AT 1 yM

(26) (27) (28) (29)

(30) (31) (32)

Minihody inhibitor

Mbic H2 loop sequence

96 Inhibition of NS3 activity

Mbic Mbic.2 Mbic.3 Mbic.34 Mbic.4 Mbic.45 Mbic.6

GIEELD GAEELD GIAELD GIAALD GIEALD GIEAAD GIEELA

45 40 30 I 20 22 37

is a minimized version of an antibody hypervariable domain sequence. The minibody is composed of two loops, H1 and H2, that can be modified by mutagenesis and the resulting polypeptides screened for HCV NS3 inhibitory activity. An alanine scan was carried out on the H2 loop and the results are summarized in Table 5.3. Monoclonal antibodies (Mabs) have been studied as potential inhibitors of NS3 [ 1161. A Ki of 39 nM was observed for a Mab designated 8D4. Interestingly, although binding appears to be competitive with respect to substrate, this binding was decreased by the addition of NS4A. In addition, the binding of the Mab to the NS3 only inhibits the in cis processing of the NS3-4A cleavage and does not affect downstream in trans cleavages. SUBSTRATE BASED INHIBITORS

Much of the current drug-design effort revolves around evolved peptidic inhibitors derived from the natural occurring substrates. Such an approach has proved fruitful against other targets and there exists a rapidly growing body of patent literature in this area [ 1 17, 1 IS]. Ideally, with increased understanding of the structure-activity relationships, these compounds will evolve into smaller, less peptidic (and more drug-like) analogues [ 1191. For the NS3*4A protease this evolution has been extremely difficult to achieve. Experiments were initially performed, beginning with the natural ten-amino acid sequence, spanning P6 to P4’, to determine the extent to which each substrate residue contributed to the overall affinity of inhibitor for the protein [60]. Using a non-cleavable active-site analogue, incorporating a tetrahydroisoquinoline P2, Landro has quantified the dramatic reduction in potency observed by the truncation of either end of the decamer as shown in Table 5.4. Simple removal of the P4’ tyrosine results in an immediate 80-fold reduction in K,. Continued prime-side truncation of the P3’ serine and P2’ norleucine had

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HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY

Table 5.4. EFFECTS OF PRIME-SIDE AND NON-PRIME SIDE TRUNCATIONS ON K, OF DECAPEPTIDE INHIBITORS K, f&)’

Pepride Sequence

Compound

H-Glu-Asp-Val-Val-Leu-Cys-Tic-Nle-Ser-Tyr-OH H-Glu-Asp-Val-Val-Leu-Cys-Tic-Nle-Ser-OH

0.34 27 17

H-Glu-Asp-Val-Val-Leu-Cys-Tic-OH H-Asp-Val-Val-Leu-Cys-Tic-Nle-Ser-Tyr-OH

14 4.4

H-Glu-Asp-Val-Val-Leu-Cys-Tic-Nle-OH

H-Val-Val-Leu-Cys-Tic-Nle-Ser-Tyr-OH H-Val-Leu-Cys-Tic-Nle-Ser-Tyr-OH H-Leu-Cys-Tic-Nle-Ser-Tyr-OH

79 500 2000

Experiments performed with NS3 in the presence of added kk4A.

less effect. The same trend was observed on the non-prime side. Removal of the glutamic and aspartic acids at P6 and P5, respectively, reduced the Ki more than 200-fold. It has been determined that the products of the natural substrate cleavage reaction are themselves inhibitors of the NS3 protease [ 1201. The data obtained from this study are consistent with the observations by Landro. Potencies for these inhibitors range from ICs0= 1-150 pM for NS4A derived inhibitors (Table 5.5) and 2.1-5.5 pM for NS5A based compounds (Table 5.6). In both NS4A and NS5A based derivatives removal of acidic residues at P6 and P5 results in significant decreases in binding affinities (Table 5.6).

Table 5.5. NS4A PEPTIDES, P5-P6 TRUNCATIONS compound

P6

P5

P4

P3

P2

PI

ICS, (PM)

(9) (10) (1 1)

AcAsp

Glu AcGlu

(12) (13) (14)

SUC

Glu SUC Glut

Met Met AcMet Met Met Met

Glu Glu Glu Glu Glu Glu

Glu Glu Glu Glu Glu Glu

CysOH CysOH CysOH CysOH CysOH CysOH

21 150 1.3 77 69

1

23 1

R.B. PERNI AND A.D. KWONG Table 5.6. NSSA PEPTIDES, P5-P6 REPLACEMENTS Conipound

P6

P5

P4

P3

P2

P1

ICSO (PM)

(15)

AcGlu SUC Glut AcGlu AcAsp

Asp Asp Asp Asp Glu

Val Val Val Val Val

Val Val Val Val Val

Abu Abu Abu Cys Cys

CysOH CysOH CysOH CysOH CysOH

2.8 4.6 5.5 5.3 2.1

(16) (17) (18) (19)

These structures have subsequently been the starting point for optimization studies [ l l l , 1211. The results shown in Table 5.6 also agree well with those from truncation studies and both approaches lead drug designers to the same question. How to design a reversible inhibitor that binds tightly in the catalytic site but does need to extend out to P6 and P4’? From these studies the answer is not clear. Acidic terminal groups coupled with hydrophobic residues capable of efficiently filling surface subsites clearly provides significant binding. The concept of peptidomimetic inhibitor design was validated as a useful methodology in the HIV field. Using the natural substrate as a starting point for inhibitor design greatly increases the likelihood of identifying compounds that bind to the enzyme active site with high specificity for its target. For hepatitis C the decapeptide recognition sequences would need to be truncated to more drug-like dimensions, on the order of P1’ to P4 as a practical maximum for a deliverable drug (Figure 5.7). The SAR delineated in TubZes 5.3-5.6 is indicative of the intrinsic difficulty of inhibiting this enzyme. The right hand terminus of peptidic inhibitors may contribute to the poor potencies observed, since the carboxylic acid represents a non-covalent

Figure 5.7. Generalized inhibitor design.

232

HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY Table 5.7. OPTIMIZED PRODUCTS

Compound

P6

P5

P4

P3

P2

PI

(20) (21) (22) (23) (24) (25)

AcAsp

Glu AcGlu

AcGlu

Asp AcGlu

Dif Dif AcDif Dif Dif AcDif

Glu Glu Glu Ile Ile Ile

Cha Cha Cha Cha Cha Cha

CysOH CysOH CysOH CysOH CysOH CysOH

G o (IN)

0.05 1.4 30 0.06 2.4 100

warhead. The electrostatic characteristic of the terminal carboxylate does not compensate for the covalency achieved for warheads such as aldehydes or other active carbonyl compounds, for example. WARHEADS

Studies of functional groups that can react covalently with the catalytic serine of the NS3 have focussed almost entirely on reversible systems. A survey of warheads clearly demonstrates the inferior binding ability of the non-covalent carboxylate (33) and is summarized in Table 5.8 [122]. Simple aldehydes provide significant improvement relative to the carboxylic acid. Also evident from Table 5.8 the ketoamide warhead is the best among the limited group. Dicarbonyl compounds, particularly ketoamides are ubiquitous protease inhibitor warheads. Series of a-keto derivatives, ketoamides, esters and acids and diketones have been reported and found to be potent warheads (vide infra) [123, 1241. Other warheads commonly studied as protease inhibitors have been found to be surprisingly ineffective. For example the trifluoroketone (35) is more than 20 times less active than the corresponding aldehyde. Aldehydes Aldehydes are the simplest reversible warheads. Though they are highly reactive toward attack by the proteolytic serine they are problematical because of the inherent instability of aliphatic aldehydes. A series of peptidic aldehydes have been studied and the SAR remains consistent with previous truncation studies of petides with carboxylate termini (Table 5.9). An aldehyde derived from an E D W scaffold (50) shows less than a 14-fold loss of potency relative to the bis-carboxylate containing analogue (5 1) [ 1 171.

R.B. PERNI AND A.D. KWONG

233

Table 5.8. ACTIVATED CARBONYL WARHEADS Compound

Peptide aldehyde sequence'

ICsn (PM)

17

(33)

1.1

(34)

22

(35)

12

0.64

(37)2

'Lower case letters denote

D

amino acids. 2cc-Carbon is racemic

This is a significantly smaller decrease in potency than the 150-fold decrease observed for non-covalent inhibitors (1) and (6) when the P6 and P5 acids are removed (Table 5.4).

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234

Table 5.9. PROTEASE INHIBITORY ACTIVITY OF PEPTIDE ALDEHYDES

Compound

Peptide aldehyde sequence

IC50

Ac-Val-Val-Abu-Cys-H Ac-Val-Val- Abu-Abu-H Ac-Asp-Val-Val-Nva-H Ac- Asp-Val-Val- Abu-Cys-H Ac-Asp-Val-Val- Abu-Abu-H Ac-Asp-Val-Val- Abu-Nva-H Ac-Glu-Asp-Val-Val-Abu-Cys-H Ac-Glu-Asp-Val-Val-Abu-Abu-H Ac-Glu-Asp-Val-Val- Abu-Nva-H Ac-Glu-Asp-Val-Val- Abu-Abu-H Ac-Glu- Asp-Val-Val- Abu-Nva-H Ac-Glu-Asp-Val-Val-Abu-DAbuH

>i00 >i00 >100 >100 >100

fM.

100 2&30 30-40 1&12 5.5

12.4 >loo

Boronates Boronic acid analogues of peptidomimetic inhibitors have also been investigated. Boronates have been previously utilized as warheads for the inhibition of serine proteases [ 1251 and consequently anti-HCV petide boronic acids have been described [ 126-1281. Though boronates are reversible inhibitors the formation of an 0-B bond between protein and inhibitor results in very tight binding. Boronic acid (52) displays in vitro potency of 38 nM versus the NS3e4A protease [129].

R.B. PERNI AND A.D. KWONG

235

a-Ketoacids, amides and diketones The a-ketoacid warhead has been identified as particularly efficient in binding to the NS3*4A complex [ 1301. Detailed mechanistic studies have demonstrated that peptidomimetic inhibitors with ketoacid warheads are slow binding inhibitors which occupy the NS3 active site in the same mode as ketoacids interact with thrombin or trypsin [130, 1311.

COOH I

F-F

(53)

(54)

f

The X-ray crystal structures of tripeptides (53) and (54) show that the tetrahedral intermediate of the keto moiety does not bind in the oxy anion hole, as would be expected based on other serine proteases such as thrombin. Instead, both carboxylate oxygens are hydrogen bound to the oxyanion hole residues Ser-139 and Gly-137 (Figure 5.8). The ketone derived hydroxyl forms a hydrogen bond to His-57 [ 1311. This particularly tight binding motif has allowed the truncation to a tripeptide scaffold to retain significant binding potency though a terminal charge group is present. Possibly more significant than a-keto-acids, from the perspective of a potential drug, are cc-ketoamides. This warhead incorporates all positive attributes of the keto-acids in a neutral moiety that will more likely be able to penetrate

236

HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY

138

/

Gly 137

.vvn

Figure 5.8. u-Ketoacid binding motif:

cells. A number of derivatives have been prepared [123, 1321. The P1’ region has been shown to accommodate a variety of spatially configured and sized groups. Excellent potency is achieved for non-substituted amide (55) in Table 5.10. Similarly, potency is observed for an inhibitor with an amide substituent extending to the prime side of the enzyme. The stereochemistry of

Table 5.1 0. a-KETOAMIDES: PRIME-SIDE SAR I

(55)

(56)

(57)

I

/

4

1100

231

R.B. PERNI AND A.D. KWONG

the prime side group plays a crucial role as demonstrated by the dramatic 250fold decrease in binding affinity for (57) relative to (56) simply by inverting the methyl group (Table 5.10) [ 1321. Similar results are obtained for a-diketones though, in general, poorer potency is observed [ 1231. Compound (58) exhibits ICso = 4.8 pM, about 1000-fold less potent than the corresponding ketoamide (55). Poorer binding and increased synthetic complexity relative to ketoamides and ketoacids make the diketone series of inhibitors less attractive.

THE P1 SPECIFICITY POCKET

When the catalytic effects of substitution of the PI site on the 5A/5B substrate were examined it became apparent that this site serves as a specificity pocket for substrates as it does for other serine proteases [60]. These data are summarized in Table 5.11. It is clear that significant bulk and/or basicity is detrimental to the efficiency of proteolytic catalysis as measure by the K,,,/K, ratio.

Table 5.1 1. CATALYTIC EFFECTS OF P1 SUBSTlTUTIONS ON 5A/5B SUBSTRATE HYDROLYSIS

CYS Aha SMeCysb Thr Ala Val Leu TYr ASP hPheC 2-WaphthAld

0.61 f 0 . 2 0.06 f 0.003 0.049 f 0.003 0.024 f 0.002 0.022 f 0.005 0.005 f 0.0004

32f2 11Of18 130*20 145 f 25 815i205 550 f 67

20000 580 385 165 25 9 5

aAminoburyric acid, bS-Methylcysteine, ‘homophenylalanine, d2-naphthyl alanine

-

>700 >700 >700 >700

23 8

HEPATITIS C VIRUS A N OVERDUE LINE OF THERAPY

Table 5.12. PI SUBSTITUTION AcDDlVPO JH

Ri

Compound

(@)!

R'

Ic50

CH2SH CH2SCH3 CH2CH2SCH3 CH2OH CH2NHz CHJ CH2CH-j CH2CH2CH3 CH2CH(CH3)2 CH2CH2CH2CH3

28 160 500 >loo0

> 1000 750 250

150

780 190 630 800 500

The HCV NS3 P1 specificity pocket differs from that of most serine proteases. Unlike thrombin for example, basic groups are deleterious to binding to NS304A allowing for good selectivity versus the clotting enzymes. The small shallow pocket is defined by the Leu-135, Phe-154, and Ala-157 side-chains [52, 531. Phe-154 is primarily responsible for the observed specificity by interacting with the cysteine thiol found in trans cleavage sites [133]. Table 5.12 presents a brief survey of small replacement groups for the P1 cysteine found in the natural substrate. Interestingly non-polar groups function at least as well as polar substituents. Ethyl (65), and N-propyl (66) derivatives demonstrate the best potencies in this, albeit, limited series. Surprisingly, despite the poor activity of (69), a compound possessing a gemdimethyl substitution, it was found that a cyclopropyl P1 moiety is advantageous [ 1191. This result has been exploited [ 118, 134-1361 with a non-covalent carboxylate warhead. The cyclopropyl PI (73) provides a 3-fold improvement in binding relative to a norvaline P1 group (72). An optimized hexapeptide inhibitor (74) with larger hydrophobic groups at P2 and P4, displays nanomolar potency.

R.B. PERNI AND A.D. KWONG

239

COOH

(72) Ki=150pM COOH

(73) Ki = 54 p FOOH

M

8

s

(74) Ki = 0.013 pM

The cyclopropyl P1 group has also been combined with a boronate warhead [128]. The boromate was only slighly more potent than the corresponding carboxylate. P2 VARIANTS AND SOLVENT SHIELDING

Recent NMR calculations on an a-ketoacid containing peptidomimetic inhibitor (53) show that a leucine side-chain at P2 has a particular stabilizing effect on the catalytic His-57 imidazole to Asp-81 carboxylate hydrogen bond [ 1 121. This stabilization appears to be effected by the shielding of that site from solvent exposure. This effect can be exploited by the use of large hydrophobic substituents at the P2 position and may be part of the reason for the good potency observed for the naphthylmethylether proline derivative (74). Cyclohexylalanine at P2 has been shown to be superior to smaller side-chains such as leucine [ 1211. As peptidomimetic inhibitors have evolved in recent years the cyclohexylalanine group (cha) has given way to 0-substituted 4-hydroxylproline as the P2 residue of choice. The 0-benzyl group is among the most studied substituent ( e g (50)) but compounds incorporating larger groups are displaying excellent inhibitory potencies. Compound (75) with a tricyclic proline

240

HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY

substituent possesses an ICs0 of less than 500 nM against the NS3-4A [118]. Truncation to tripetide derivatives was effective in this series maintaining significant potencies [ 1341.

P3, P4 SUBSITUENTS

Both P3 and P4 subsites benefit from the presence of hydrophobic substitution in regard to enzyme inhibition. The P4 SAR clearly shows the effects of hydrophobic substitution (Table 5.13) [ 12 11. The Dif (diphenylalanine) containing inhibitor (81) is the most potent but from a practical perspective, is very expensive. Cheaper alternatives are available for substitution at P4 but the trade-off is a reduction in potency. Although a Glu residue at P3 is close to optimal, non-charged side-chains are obviously preferred since charged groups hinder cellular penetration. Small hydrophobic amino acids such as valine or iso-leucine have been shown to be comparable to the Glu at P3. D-amino acids are unacceptable at both P3 and P4 [121].

Table 5.13. P4 OPTIMIZATION

Compound

Ac-Asp-Glu-X-Glu-Cha-Cys

~CSO(FW

X Val Nleu Cha Ileu Leu Dif

0.330 0.224 0.140 0.122 0.118 0.055

R.B. PERNI AND A.D. KWONG

24 1

P3 Indolinyl derivatives A particularly interesting class of inhibitors contains a novel P3 group, a 2-substituted indolyl moiety. These compounds incorporating an a-ketoacid warhead display surprising potency despite the presence, in some cases, of isomeric mixtures [72, 1371. The SAR is summarized in Table 5.14.

Cyclic derivatives

A series of novel macrocyclic peptidomimetics have been studied [ 1381. Compound (89) incorporates a 15-membered ring structure tying PI to P3. Cellular activity is claimed for this series of inhibitors in Huh-7 cells. These cells express Table 5.14. P3 INDOLINYL DERIVATIVES

Y-F

F

Compound

R

ICsu (PM)

Isomer Ratio

50

single

92

1.5:l:l:l

16

1:l:l

45

1:l:I:l

5

single

0.8

>10:1

H

gc?z H

H

I

242

HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY

a portion of the HCV polyprotein from NS3 through NSSA ending with the first six amino acids of NSSB and utilizes SEAP as the reporter construct [93].

Table 5.15. PI’-REGIONBASED INHIBITORS Compound

Peptide Sequence

Ac-Asp-Glu-Dif-Ile-Cha-Cys-Pro-Nle-Ser-Leu-NH2 Ac-Asp-Glu-Dif-Ile-Cha-Cys-Pro-Nle-Gln-Leu-NH2 Ac-Asp-Glu-Dif-Ile-Cha-Cys-Pro-Nle-Hyp-Leu-NH2 Ac-Asp-Glu-Dif-Ile-Cha-Cys-Pro-Nle-Asp-Leu-NH2 Ac-Asp-Glu-Dif-Ile-Cha-Cys-Pro-Cha-Ser-Leu-NH2 Ac-Asp-Glu-Dif-Ile-C ha-Cys-Pro-Cha-(D)-Trp-Leu-NH2 Ac-Asp-Glu-Dif-lle-C ha-Cys-Pro-Cha-Gln-Leu-NH2 Ac-Asp-GIu-Dif-Ile-Cha-Cys-Pro-Cha-Hyp-Leu-NH2 Ac-Asp-Glu-Dif-lle-C ha-Cys-Pro-ChaAsp-Leu-NH2 Ac-Asp-Glu-Dif-Ile-Cha-Cys-Pro-H~f-Gln-Leu-NH~ Ac-Asp-Glu-Di f-Ile-Cha-C y s-Pro-Hof-Hyp-Leu-NH2 Ac-Asp-Glu-Di f-Ile-Cha-Cy s-Pro-Hof-Asp-Leu-NH2 Ac-Asp-Glu-Dif-lle-Cha-Cys-Pro-Phg-Asp-Leu-NH2

Ic50

(nM)

64 32 26 1.8

23 820 14 11

1.3 18 15 1.8 7

Table 5.16. OPTIMIZED PI’-BASEDINHIBITORS Compound

Peptide Sequence Ac-Glu-A~p-Val-Val- Abu-Cys-Pro-Nle-Ser-NH~

I G n (nM)

8500 876 3100 64 Ac-Asp-Glu-Dif-Ile-Cha-Cys-Pro-Cha-Ser-Leu-NH2 23 Ac-Asp-Glu-Dif-Ile-Cha-Cys-Pro-Cha-Asp-Leu-NH2 1.3 Ac-Asp-D-Glu-Leu-Ile-Cha-Cys-Pro-Cha-Asp-Leu-NH2 <0.2 Ac-Asp-D-Glu-Leu-Ile-Cha-Cys-Pro-Cha-Asp-Leu-Pro-Tyr-Ly~-NH~ <0.2

Ac-Asp-Glu-Dif-Ile-Cha-Cys-Pro-Nle-Ser-Tyr-NH2 Ac-Asp-Glu-Dif-Ile-C ha-Cys-NH2 Ac-Asp-Glu-Dif-Ile-Cha-Cys-Pro-Nle-Ser-Leu-NH2

243

R.B. PERNI AND A.D. KWONG PI’ REGION BASED INHIBITORS

While the S’ region of the NS3 polypeptide is essential for catalytic function it is less important relative to the S-region in regard to substrate binding. This is clear from the truncation studies previously discussed. Specifically, that study showed that removal of P2’ and P3’ from the substrate had little effect on Ki. A recent study has determined optimal peptidic substitution to provide optimal prime side binding in subsites little used by the natural substrate [139]. This study, while not actually evolving drug-like compounds, nevertheless demonstrates the possibility that peptidomimetic compounds that bind on the prime-side represent potential therapeutics. NON-PEPTIDIC INHIBITORS SCREENING LEADS

Significant efforts have also been expended in identifying non-peptidic small molecule inhibitors of the HCV NS304A protease. These compounds have almost always been identified from natural product library screening. Several structural classes have been described but there is a distinct lack of structural diversity even among the classes. Binding has usually been shown to be nonspecific. In some cases multiple inhibitor molecules appear to associate with the enzyme simultaneously. Several diarylamide derivatives, for example (1 11) and (1 12), that incorporate a naphthyl ether moiety have been identified [ 1401. The compounds display reasonable selectivity for HCV NS304A over a number of other serine proteases.

(111)

(112)

CI

Thiazolidine derivatives such as (1 13) and (1 14) are related to the diarylamide series of compounds exemplified by (1 1 1) and (1 12) and have also been shown to inhibit NS304A protease. The haloaryl groups common to both series of compounds are intriguing but the significance of this observation is thus far unknown.

244

HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY

(113) JCm = 2.3 pglmL

(114) IC5,= 3.2 pM

Modest potency has been observed for a series of 15 such inhibitors. Although potency varies for various double bond substitutions from low single digit pM to >50 pg/mL no clear structural relationships are observed [140, 1411. A series of arylalkylidene rhodanine based inhibitors of NS3*4A, for example (1 15), have been reported [ 1421. These rhodanine derivatives resemble a combination of the diary1 amides and thiazolidines described previously. Interestingly these compounds appear to be at least as effective as chymotrypsin and plasmin inhibitors.

H3C0

K 0

,OCHJ

T0

Identified from natural product screening is the fungal metabolite (1 16) [ 1431. Not many HCV protease inhibitors have been reported from the

screening of natural product libraries but (1 16) exhibits potency against the NS3 enzyme comparable to the diarylamides and the thiazolidines. Acetylation of the hemiketal reduced potency approximately twofold. OH

(116) ICs0= 3.8 pg/mL

R.B. PERNI AND A.D. KWONG

245

SYNTHESIS OF NS304A PROTEASE INHIBITORS GENERAL COMMENTS

The vast majority of the synthetic work has been carried out on peptidomimetic inhibitors. Some of the non-peptidic inhibtors are natural products (e.g., hemiketal 116) [ 1431 and some are commercially available (e.g., diary1 amides) [140]. Clever syntheses of warhead hnctionalities, which allow coupling to polymer supports, have allowed for the partially automated synthesis of the peptidomimetics analogues. As may be expected, the syntheses of peptidomoimetic inhibitors are generally long with low overall yields. This situation may result in lengthy process development programmes and commercialization/cost issues similar to those that originally beset HIV programmes. BORONATES

A solid phase procedure was used to prepare the boronate series of peptidomimetic inhibitors. The route shown in Figure 5.9 provides the PI-warhead synthon in moderate yields [126]. The resin-based chemistry allows for the preparation of directed libraries of boronate inhibitors. The solid supported amino acid is elaborated using a standard deprotection-coupling sequence. A final acid-mediated hydrolysis frees the completed molecule. This route was also utilized for a solution phase synthesis of boronic acid inhibitors with only minor modifications. ALDEHYDE SYNTHESIS

A number of groups have prepared aldehydic inhibitors and almost all routes are based on the Weinreb amide-reduction strategy. A typical solution phase methodology is shown in Figure 5.10 wherein a protected amino acid is converted to a Weinreb amide followed by lithium aluminium hydride reduction [129]. The P1 amino aldehyde is protected as a dimethylacetal and standard coupling protocols are carried out. A final deprotection step affords the polypeptide aldehyde. Aldehydic inhibitors are also readily prepared via efficient solid-supported chemistry. A SynPhase-MD-1 polymeric support was used (Figure 5.11). An fmoc protected aminoaldehyde is converted to an aminal via a tether to a polymeric support [ 1441. Peptide elaboration followed by simple TFA deprotection gives the aldehydic inhibitor.

HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY

246

Figure 5.9. Synthesis of boronic acid inhibitors.

R

HCI"H(OCHs)CH3 HOBT, NEM. CHzClz EtN=C=N(CH&N(CH&

-

R

PCH3 1 ) LiAIH, / THF

Fmocx!%N'CH30

c

2) HCI / MeOH

Figure 5.10. Solution-phase aldehyde synthesis.

u-KETO ACIDS AND DERIVATIVES

There are numerous methodologies available for preparing a-keto-acids, esters and amides. As might be expected, all are not equally amenable to the preparation of peptidomimetic derivatives. Two recent syntheses appear useful.

R.B. PERNI AND A.D. KWONG

241

Figure 5.11. Solid-phase aldehyde synthesis

A method starting with aldehydes derived from aminoacids has been found useful and reasonably efficient [123]. The route allows the coupling of a previously assembled tetramer to an a-hydroxy synthon. The aldehyde is condensed with acetone cyanohydrin and the resulting adduct is subsequently hydrolyzed. Following P 1 amide formation the hydroxyamide is coupled with the tetrapeptide. A final Dess-Martin oxidation provides the target compound. The DessMartin periodate has been found to be one of the few oxidants which effectively oxidizes the hydroxyl group without destroying the rest of the molecule.

TEA, CHzCIz

BOP / DlEA / DMF

2) HCI/ HzO/ dioxane 3) (BOC)~O / NazCO3/H20

1) HCI /dioxane

Boc-Asp(tBu)-Glu(tBu)-Val-Val-Pro’ 2) tetrapeptide BOP / D I E N DMF

1) Dess-Martin Oxidation

H-Asp-Glu-Val-Val-Pro’ 2) TFA / CHZCI2

Figure 5.12. a-Ketoamide synthesis

L

HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY

248

DHQPAL

T

O

C

H

3

AOCH, 1) Hz Pd IC

KzIOSOZ(OH)~I CbrNH

MeOH

Cbz-NCI Na H,O-iPrOH

1 Des-Martin Oxidation

2) TFA / CHzCIz

.Ir

2) Tetrapeptide BOP / DIEA / DMF

t

-

i

0

~

Figure 5.13. Alternative a-kefoamide synthesis

An alternative method begins with an olefin and a Sharpless aminohydroxylation protocol for the initial formation of the hydroxy ester intermediate [123]. For the target compound shown in Figure 5.13, a high enantiomeric excess of 95% was obtained after recrystallization of the Sharpless product. Ester hydrolysis and coupling affords the hydroxyamide. The convergent synthesis again culminates in the usual Dess-Martin oxidation/deprotection step. Novel PI carboxylic acids The novel ally1 cyclopropyl P 1 derivatives are prepared according to the route shown in Figure 5.14 [ 1 18, 1341. The condensation of 1,4-dibromo-2-butene with a protected glycine aldehyde gives the cyclopropane carboxylic acid. While the procedure is relatively economical a mixture of isomers is invariably obtained. After separation of diastereomers, the racemic product is resolved to its 1R-2S isomer via an enzymatic reaction with Alcalase.

HCI'H2NCH2C02Et NazSO, I TBME I Et3N

-

srN-co2Et B

r

A

B

r

LiOtBu I toluene I RT

Figure 5.14. Cycloprop,yl PI .synthesis.

then H30* then NaOH

*

R . B . PERNI AND A.D. KWONG

249

P3 INDOLINYL DERIVATIVES

Inhibitors incorporating the novel P3 capping substituent, the 2-indolinyl group, are prepared in a straightforward manner starting with commercially available indoline-2-carboxylic acid [72]. Protection followed by a-alkylation affords the capping synthon which is subsequently coupled to the peptide in standard fashion (Figure 5.15).

THE FUTURE: SUMMARY AND OUTLOOK One cannot separate the medicinal chemistry research into HCV NS3*4A protease inhibitors from the virological research devoted to the development of assays and models. The lack of cellular and animal model systems has hampered progress in the field. This is beginning to change. The construction of an HCV replicon [98], for example, should lead to assay systems that include most of the HCV genome in cell culture. Unfortunately, the development of animal models is progressing more slowly. While the chimpanzee model is generally accepted as the most reliable, it is still not validated. The cost associated with such primate models as well as ethical considerations preclude its use for screening large numbers of molecules. While clear progress in inhibitor design is being made, much work remains to be done. Inhibitory potencies against the NS3*4A protease are improving rapidly particularly with peptide-based inhibitors but the most potent compounds are still not drug-like: they are large, charged, and unlikely to penetrate cells. Removing the charge will provide compounds that are likely to improve cell penetration but will be overall less potent, very hydrophobic and

Figure 5.15. fndofinvl P3 .synthesis

250

HEPATITIS C VIRUS AN OVERDUE LINE OF THERAPY

consequently will be difficult to formulate into a suitable dosage form. While cellular model systems are becoming available no clear demonstration of protease inhibition in cellular or animal systems has been published. Non-peptic inhibitors are even further from the finishing line. Library screening has yielded a number of classes of inhibitory compounds but the potencies are less than those of peptidomimetic compounds. In addition, for the majority of non-peptidic inhibitors, simple inhibition kinetics are not observed. In some cases more than one inhibitor molecule is involved in binding to a single protease moiety. Moreover, the SAR has not been easily exploitable as evidenced by the lack of improved activity in analogues. Given this backdrop, structure-based drug design is clearly the best bet. On the brighter side, the recent advances in cellular replication systems, X-ray, NMR and SAR analyses are allowing researchers to begin to crack HCV’s secrets. The understanding of the subtleties of the enzyme function and structure will, in the not too distant future, yield useful, marketable protease inhibitors which will revolutionize the treatment of hepatitis C in the same manner that HIV protease inhibitors revolutionized therapy for AIDS patients. REFERENCES 1 Alberti, A,, Chemello, L. and Benvegnu, L. (1999) J. Hepatol. 31 (Suppl I), 17-24. 2 Hagedorn, C.H. and Rice, C.M. (2000) Curr. Top. Microbiol. Immunol. Vol. 242. Springer, Berlin, New York. 3 Lavanchy, D. (1999) J. Viral Hep. 6, 35-47. 4 Steedman, A., Sarbah, M.D. and Younossi, Z.M. (2000) J. Clin. Gastroenterol. 30, 125-143. 5 Feinstone, S.M., Kapikian, A.Z., Purcell, R.H., Alter, M.J. and Holland, P.V. (1975) New Engl. J. Med. 292, 767-770. 6 Schreiber, G.B., Busch, M.P., Kleinman, S.H. and Korelitz, J.T. (1996) New Engl. J. Med. 334, 1685-1690. 7 Alter. M.J., Kruszon-Moran, D. and Nainan, O.V. (1999) N. Engl. J. Med. 341, 556562. 8 Lam, N.P. (1999) Am. J. Health-Syst. Pharm. 56, 961-973. 9 MacDonald, M., Crofts, N. and Kaldor, J. (1996) Epidemiologic Rev. 18, 137-148. 10 Niederau, C., Lange, S . , Heintges, T., Erhardt, A., Buschkamp, M., Hurter, D., Nawrocki, M., Kruska, L., Hensel, F., Petry, W. and Haussinger, D. (1998) Hepatology 28, 1687-1695. 11 Seef, L.B. (1997) Hepatology 26(Suppl. l ) , 21S28S. 12 Huang, L. and Koziel, M. (2000) Curr. Opin. Immun. 16, 55%564. 13 Kew, M.C. (1994) FEMS Microbiol. Rev. 14,211-220. 14 National Institutes of Health Consensus Development Statement. Management of Hepatitis C. (1997) Hepatology 26(Supplement I), 2’SIOS. 15 KoK R.S. (2000) Am. J. Gastroenter. 95, 1392-1393. 16 Malone, D.C. (2000) Formulatory 35, 681. 17 Wong, J.B., Poynard. T., Ling, M.H., Albrecht, J.K. and Pauker, S.G. (2000) Am. J. Gastroenter. 95, 1524-1530. 18 Radkowski, M.. Kubicka, J., Kisiel, E., Cianciara, J., Nowicki, M., Rakela, J. and Laskus, T. (2000) Blood 95, 39863989.

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