Åsa Rosenquist

Discovery and Development of Simeprevir (TMC435), a HCV NS3/4A Protease Inhibitor

Hepatitis C virus is a blood-borne infection and the leading cause of chronic liver disease (including cirrhosis and cancer) and liver transplantation. Since the identification of HCV in 1989, there has been an extensive effort to identify and improve treatment options. An important milestone was reached in 2011 with the approval of the first-generation HCV NS3/4A protease inhibitors. However, new therapies are needed to improve cure rates, shorten treatment duration, and improve tolerability. Here we summarize the extensive medicinal chemistry effort to develop novel P2 cyclopentane macrocyclic inhibitors guided by HCV NS3 protease assays, the cellular replicon system, structure-based design, and a panel of DMPK assays. The selection of compound 29 (simeprevir, TMC435) as clinical candidate was based on its excellent biological, PK, and safety pharmacology profile. Compound 29 has recently been approved for treatment of chronic HCV infection in combination with pegylated interferon-α and ribavirin in Japan, Canada, and USA.

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.

Design, synthesis and SAR of potent statine-based BACE-1 inhibitors: exploration of P1 phenoxy and benzyloxy residues.

Several BACE-1 inhibitors with low nanomolar level activities, encompassing a statine-based core structure with phenyloxymethyl- and benzyloxymethyl residues in the P1 position, are presented. The novel P1 modification introduced to allow the facile exploration of the S1 binding pocket of BACE-1, delivered highly promising inhibitors.

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.

Synthesis, X-Ray Analysis, and Biological Evaluation of a New Class of Stereopure Lactam-Based HIV-1 Protease Inhibitors.

In an effort to identify a new class of druglike HIV-1 protease inhibitors, four different stereopure β-hydroxy γ-lactam-containing inhibitors have been synthesized, biologically evaluated, and cocrystallized. The impact of the tether length of the central spacer (two or three carbons) was also investigated. A compound with a shorter tether and (3R,4S) absolute configuration exhibited high activity with a Ki of 2.1 nM and an EC50 of 0.64 μM. Further optimization by decoration of the P1′ side chain furnished an even more potent HIV-1 protease inhibitor (Ki = 0.8 nM, EC50 = 0.04 μM). According to X-ray analysis, the new class of inhibitors did not fully succeed in forming two symmetric hydrogen bonds to the catalytic aspartates. The crystal structures of the complexes further explain the difference in potency between the shorter inhibitors (two-carbon spacer) and the longer inhibitors (three-carbon spacer).

Synthesis and SAR of potent inhibitors of the Hepatitis C virus NS3/4A protease: Exploration of P2 quinazoline substituents

Novel NS3/4A protease inhibitors comprising quinazoline derivatives as P2 substituent were synthesized. High potency inhibitors displaying advantageous PK properties have been obtained through the optimization of quinazoline P2 substituents in three series exhibiting macrocyclic P2 cyclopentane dicarboxylic acid and P2 proline urea motifs. For the quinazoline moiety it was found that 8-methyl substitution in the P2 cyclopentane dicarboxylic acid series improved on the metabolic stability in human liver microsomes. By comparison, the proline urea series displayed advantageous Caco-2 permeability over the cyclopentane series. Pharmacokinetic properties in vivo were assessed in rat on selected compounds, where excellent exposure and liver-to-plasma ratios were demonstrated for a member of the 14-membered quinazoline substituted P2 proline urea series.

New inhibitors of the malaria aspartyl proteases plasmepsin I and II.

New inhibitors of plasmepsin I and II, the aspartic proteases of the malaria parasite Plasmodium falciparum, are described. From paralell solution phase chemistry, several reversed-statine type isostere inhibitors, many of which are aza-peptides, have been prepared. The synthetic strategy delivers the target compounds in good to high overall yields and with excellent stereochemical control throughout the developed route. The final products were tested for their plasmepsin I and II inhibiting properties and were found to exhibit modest but promising activity. The best inhibitor exhibits K(i) values of 250 nM and 1.4 microM for Plm I and II, respectively.

Design and synthesis of BACE-1 inhibitors utilizing a tertiary hydroxyl motif as the transition state mimic

Two series of drug-like BACE-1 inhibitors with a shielded tertiary hydroxyl as transition state isostere have been synthesized. The most potent inhibitor exhibited a BACE-1 IC(50) value of 0.23 microM.