Pierre Jean-Marie Bernard Raboisson

In Vitro Activity and Preclinical Profile of TMC435350, a Potent Hepatitis C Virus Protease Inhibitor

ABSTRACT The hepatitis C virus (HCV) NS3/4A serine protease has been explored as a target for the inhibition of viral replication in preclinical models and in HCV-infected patients. TMC435350 is a highly specific and potent inhibitor of NS3/4A protease selected from a series of novel macrocyclic inhibitors. In biochemical assays using NS3/4A proteases of genotypes 1a and 1b, inhibition constants of 0.5 and 0.4 nM, respectively, were determined. TMC435350 inhibited HCV replication in a cellular assay (subgenomic 1b replicon) with a half-maximal effective concentration (EC50) of 8 nM and a selectivity index of 5,875. The compound was synergistic with alpha interferon and an NS5B inhibitor in the replicon model and additive with ribavirin. In rats, TMC435350 was extensively distributed to the liver and intestinal tract (tissue/plasma area under the concentration-time curve ratios of >35), and the absolute bioavailability was 44% after a single oral administration. Compound concentrations detected in both plasma and liver at 8 h postdosing were above the EC99 value measured in the replicon. In conclusion, given the selective and potent in vitro anti-HCV activity, the potential for combination with other anti-HCV agents, and the favorable pharmacokinetic profile, TMC435350 has been selected for clinical development.

Structure–activity relationship study on a novel series of cyclopentane-containing macrocyclic inhibitors of the hepatitis C virus NS3/4A protease leading to the discovery of TMC435350

SAR analysis performed with a limited set of cyclopentane-containing macrocycles led to the identification of N-[17-[2-(4-isopropylthiazole-2-yl)-7-methoxy-8-methylquinolin-4-yloxy]-13-methyl-2,14-dioxo-3,13-diazatricyclo [13.3.0.0(4,6)]octadec-7-ene-4-carbonyl](cyclopropyl)sulfonamide (TMC435350, 32c) as a potent inhibitor of HCV NS3/4A protease (K(i)=0.36nM) and viral replication (replicon EC(50)=7.8nM). TMC435350 also displayed low in vitro clearance and high permeability, which were confirmed by in vivo pharmacokinetic studies. TMC435350 is currently being evaluated in the clinics.

Induced-Fit Binding of the Macrocyclic Noncovalent Inhibitor TMC435 to its HCV NS3/NS4A Protease Target

The NS3 protein of hepatitis C virus (HCV), together with the NS4A peptide co-factor, comprises 685 residues and possesses domain-specific RNA helicase and serine protease activities. NS3/NS4A protease activity is essential to the HCV life cycle. Small-molecule inhibitors of NS3/NS4A protease have been widely explored and are typically grouped into two classes: linear peptidomimetics with a ketoamide functionality that reacts with the catalytic Ser to form a reversible enzyme–inhibitor adduct, and noncovalent peptidomimetics containing a macrocycle (e.g. Figure 1); macrocyclic ketoamide inhibitors have also been reported. Macrocycles, underrepresented in synthetic drugs, are helpful in improving the druglike character of molecules. TMC435 (1; Figure 1), a macrocyclic noncovalent inhibitor of NS3/NS4A protease with subnanomolar Ki values for genotype 1a and 1b NS3/ NS4A proteases, 11] was discovered by optimizing an earlier NS3/NS4A protease inhibitor, BILN-2061 (2 ; Figure 1). Key steps in the progression from 2 to 1 include reduction of macrocycle size, truncation of the P4 (P3 capping) group, conversion of the carboxylate “head group” to an acylsulfonamide, replacement of the P2 proline pyrrolidine with a cyclopentyl ring, and optimization of the substituted quinoline-thiazole ring system (Figure 1). 14–16] Despite exceeding three of four Lipinski criteria, 1 shows excellent pharmacokinetics in humans. We have determined the crystal structure of 1 bound to its NS3/NS4A protease target from the BK strain of genotype 1b HCV at a resolution of 2.4 (Figure 2; see Table S1 and Figure S1 in the Supporting Information). The threedimensional structure of the NS3 protease domain in complex with a truncated version of the NS4A cofactor was first reported in 1996, and that of an engineered single-chain NS3/NS4A protease–helicase construct in 1999. Currently there are multiple covalent NS3/NS4A protease–inhibitor complexes accessible at the PDB. This structure is the first noncovalent NS3/NS4A protease–inhibitor complex to be deposited at the PDB. Additionally, the new structure shows that the large P2 substituent of 1 induces an extended S2 subsite to accommodate this group; none of the previously available complex structures share this feature. We analyze the observed induced-fit binding of 1 to HCV NS3/NS4A protease, discuss key in vitro resistance mutations in the context of the complex, and disclose the new crystal structure for public analysis. The structure of the NS3/NS4A–1 complex shows the expected trypsin-like fold for the enzyme, with the inhibitor bound at the active site, spanning the S3–S1’ subsites (Figure 2; see Figure S1 in the Supporting Information). Unlike many other macrocyclic drugs that can be divided into functional (binding) and modulator (nonbinding) domains, essentially all of 1 is involved in binding to its target site (Figure 2). Two canonical substrate-like intermolecular hydrogen bonds are observed: the P1–P2 backbone amide N contacts Arg155:O, and the carbonyl O of the P2–P3 amide Figure 1. Macrocyclic (1, 2) and ketoamide (3) inhibitors of HCV NS3/ NS4A protease. Substrate positions from NS3/NS4A protease complex structures are indicated for 1 and 3.

1,5-Benzodiazepine inhibitors of HCV NS5B polymerase

Optimization through parallel synthesis of a novel series of hepatitis C virus (HCV) NS5B polymerase inhibitors led to the identification of (R)-11-(4-benzyloxy-2-fluorophenyl)-6-hydroxy-3,3-dimethyl-10-(6-methylpyridine-2-carbonyl)-2,3,4,5,10,11-hexahydro-dibenzo[b,e][1,4]diazepin-1-one 11zc and (R)-11-(4-benzyloxy-2-fluorophenyl)-6-hydroxy-3,3-dimethyl-10-(2,5-dimethyloxazol-4-carbonyl)-2,3,4,5,10,11-hexahydro-dibenzo[b,e][1,4]diazepin-1-one 11zk as potent (replicon EC(50)=400nM and 270nM, respectively) and selective (CC(50)>20muM) inhibitors of HCV replication. These data warrant further lead-optimization efforts.

Discovery of novel potent and selective dipeptide hepatitis C virus NS3/4A serine protease inhibitors

Starting from the previously reported HCV NS3/4A protease inhibitor BILN 2061, we have used a fast-follower approach to identify a novel series of HCV NS3/4A protease inhibitors in which (i) the P3 amino moiety and its capping group have been truncated, (ii) a sulfonamide is introduced in the P1 cyclopropyl amino acid, (iii) the position 8 of the quinoline is substituted with a methyl or halo group, and (iv) the ring size of the macrocycle has been reduced to 14 atoms. SAR analysis performed with a limited set of compounds led to the identification of N-{17-[8-chloro-2-(4-isopropylthiazol-2-yl)-7-methoxyquinolin-4-yloxy]-2,14-dioxo-3,15-diazatricyclo [13.3.0.0 [Bartenschlager, R.; Lohmann, V. J. Gen. Virol. 2000, 81, 1631; Vincent Soriano, Antonio Madejon, Eugenia Vispo, Pablo Labarga, Javier Garcia-Samaniego, Luz Martin-Carbonero, Julie Sheldon, Marcelle Bottecchia, Paula Tuma, Pablo Barreiro Expert Opin. Emerg. Drugs, 2008, 13, 1-19]]octadec-7-ene-4-carbonyl}(1-methylcyclopropyl)(1-methylcyclopropyl)sulfonamide 19l an extremely potent (K(i)=0.20 nM, EC(50)=3.7 nM), selective, and orally bioavailable dipeptide NS3/4A protease inhibitor, which has features attractive for further preclinical development.

Discovery of novel, potent and bioavailable proline-urea based macrocyclic HCV NS3/4A protease inhibitors

A novel series of P3-truncated macrocyclic HCV NS3/4A protease inhibitors containing a P2 proline-urea or carbamate scaffold was synthesized. Very potent inhibitors were obtained through the optimization of the macrocycle size, urea and proline substitution, and bioisosteric replacement of the P1 carboxylic acid moiety. Variation of the lipophilicity by introduction of small lipophilic substituents resulted in improved PK profiles, ultimately leading to compound 13Bh, an extremely potent (K(i)=0.1 nM, EC(50)=4.5 nM) and selective (CC(50) (Huh-7 cells)>50 microM) inhibitor, displaying an excellent PK profile in rats characterized by an oral bioavailability of 54% and a high liver exposure after oral administration.

Antiviral Activity and Mode of Action of TMC647078, a Novel Nucleoside Inhibitor of the Hepatitis C Virus NS5B Polymerase

ABSTRACT Chronic infection with hepatitis C virus (HCV) is a major global health burden and is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma. Current therapy for HCV infection has limited efficacy, particularly against genotype 1 virus, and is hampered by a range of adverse effects. Therefore, there is a clear unmet medical need for efficacious and safe direct antiviral drugs for use in combination with current treatments to increase cure rates and shorten treatment times. The broad genotypic coverage achievable with nucleosides or nucleotides and the high genetic barrier to resistance of these compounds observed in vitro and in vivo suggest that this class of inhibitors could be a valuable component of future therapeutic regimens. Here, we report the in vitro inhibitory activity and mode of action of 2′-deoxy-2′-spirocyclopropylcytidine (TMC647078), a novel and potent nucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase that causes chain termination of the nascent HCV RNA chain. In vitro combination studies with a protease inhibitor resulted in additive efficacy in the suppression of HCV RNA replication, highlighting the potential for the combination of these two classes in the treatment of chronic HCV infection. No cytotoxic effects were observed in various cell lines. Biochemical studies indicated that TMC647078 is phosphorylated mainly by deoxycytidine kinase (dCK) without inhibiting the phosphorylation of the natural substrate, and high levels of triphosphate were observed in Huh7 cells and in primary hepatocytes in vitro. TMC647078 is a potent novel nucleoside inhibitor of HCV replication with a promising in vitro virology and biology profile.