Bertil Samuelsson

Stimulation of renin release from rabbit renal cortex by arachidonic acid and prostaglandin endoperoxides.

The mechanism by which renal prostaglandins stimulate renin secretion in vivo is unknown. In this in vitro study we measured the effects of activation of the prostaglandin (PG) system on renin release from slices of rabbit renal cortex. The PG precursor arachidonic add (C20:4), a natural PG endoperoxide (PGG2), two stable synthetic PG endoperoxide analogues (EPA I and H), PGE,, PGF2α, and two different PG synthesis Inhibitors [indomethadn and 5,8,11,14-eicosatetraynoic add (ETA)] were used to evaluate the possibility of a direct action of the cortical PG system on renin secretion. Renin release increased significantly with time after addition of C20:4, PGG2, EPA I, and EPA II to the incubation medium. Stimulation of renin release was se-related for C20:4 in concentrations of 0.6 to 4.5 × 10-6 m, for EPA I in concentrations of 0.7 to 2.8 × 10-6, and for EPA II in concentrations of 1.4 to 14.0 × 10-6 m. Indometbadn (10-4 m) and ETA (10-4 m) significantly decreased basal renin release as well as the renin release stimulated by C20:4 and EPA I. PGE, (10-12 to 10-6 m) had no effect on renin release, whereas PGF2α (10-12 to 10-6 m) decreased renin release in a dose-dependent manner. These data raise the possibility of a direct action of the renal cortical PG system on renin secretion. The results further indicate that stimulation of renin release by C20:4 may depend more specifically on the action of PG endoperoxides than on the primary prostaglandins.

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.

Acyl sulfonamides as potent protease inhibitors of the hepatitis C virus full-length NS3 (protease-helicase/NTPase): A comparative study of different C-terminals

Synthesis and inhibitory potencies of three types of protease inhibitors of the hepatitis C virus (HCV) full-length NS3 (protease-helicase/NTPase) are reported: (i) inhibitors comprising electrophilic serine traps (pentafluoroethyl ketones, alpha-keto acids, and alpha-ketotetrazoles), (ii) product-based inhibitors comprising a C-terminal carboxylate group, and (iii) previously unexplored inhibitors comprising C-terminal carboxylic acid bioisosteres (tetrazoles and acyl sulfonamides). Bioisosteric replacement with the tetrazole group provided inhibitors equally potent to the corresponding carboxylates, and substitution with the phenyl acyl sulfonamide group yielded more potent inhibitors. The hexapeptide inhibitors Suc-Asp-D-Glu-Leu-Ile-Cha-Nva-NHSO(2)Ph and Suc-Asp-D-Glu-Leu-Ile-Cha-ACPC-NHSO(2)Ph with K(i) values of 13.6 and 3.8 nM, respectively, were approximately 20 times more potent than the corresponding inhibitors with a C-terminal carboxylate and were comparable to the carboxylate-based inhibitor containing the native cysteine, Suc-Asp-D-Glu-Leu-Ile-Cha-Cys-OH (K(i)=28 nM). The acyl sulfonamide group constitutes a very promising C-terminal functionality that allows for prime site optimization.

Novel reagent system for converting a hydroxy-group into an iodo-group in carbohydrates with inversion of configuration. Part 2

Isolated primary and secondary hydroxy-groups in carbohydrate derivatives are transformed into iodo-groups with inversion of configuration on treatment with either triphenylphosphine, iodine, and imidazole or triphenyl-phosphine and 2,4,5-tri-iodoimidazole at elevated temperatures. At lower temperatures, primary hydroxy-groups may be selectively replaced by iodo-groups.

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.

Tetrapeptides as Potent Protease Inhibitors of Hepatitis C Virus Full-Length NS3 (Protease-Helicase/NTPase)

A library of tetrapeptides was evaluated for Hepatitis C Virus NS3 protease inhibitor activity in an in vitro assay system comprising the native bifunctional full-length NS3 (protease-helicase/NTPase) protein. Tetrapeptides with K(i) values in the high nanomolar range were identified, for example Suc-Chg-Glu-2-Nal-Cys (K(i)=0.27+/-0.03 microM) and Suc-Dif-Glu-Glu-Cys (K(i)=0.40+/-0.10 microM). Furthermore, it was shown that the inhibitory potencies are not affected significantly by assay ionic strength. As suggested by molecular modelling, potential binding interactions of the tetrapeptide inhibitors with the helicase domain might explain the data and structure-activity relationships thus obtained. Hence, we postulate that the full-length NS3 assay is a relevant system for inhibitor identification, offering new opportunities for inhibitor design.

Characterization of a set of HIV-1 protease inhibitors using binding kinetics data from a biosensor-based screen

The interaction between 290 structurally diverse human immunodeficiency virus type 1 (HIV-1) protease inhibitors and the immobilized enzyme was analyzed with an optical biosensor. Although only a single concentration of inhibitor was used, information about the kinetics of the interaction could be obtained by extracting binding signals at discrete time points. The statistical correlation between the biosensor binding data, inhibition of enzyme activity (K;), and viral replication (EC50) revealed that the association and dissociation rates for the interaction could be resolved and that they were characteristic for the compounds. The most potent inhibitors, with respect to K; and EC50 values, including the clinically used drugs, all exhibited fast association and slow dissociation rates. Selective or partially selective binders for HIV-1 protease could be distinguished from compounds that showed a general protein-binding tendency by using three reference target proteins. This biosensor-based direct binding assay revealed a capacity to efficiently provide high-resolution information on the interaction kinetics and specificity of the interaction of a set of compounds with several targets simultaneously.

Regioselective reductive ring-opening of 4-methoxybenzylidene acetals of hexopyranosides. Access to a novel protecting-group strategy. Part 1

Reduction of fully protected 4,6-O-(4-methoxybenzylidene) hexopyranosides with sodium cyanoboro-hydride–trifluoroacetic acid in NN′-dimethylformamide, or trimethylsilyl chloride in acetonitrile, gives the 6- and 4-O-(4-methoxybenzyl) ethers, respectively, in good yield and good regioselectivity. The 4-methoxybenzyl ether linkage in products containing benzyl ethers or other protective groups is selectively cleaved upon treatment with cerium(IV) ammonium nitrate in aqueous acetonitrile.

Synthesis of 2′,3′-dideoxycyclo-2′-pentenyl-3′-C-hydroxymethyl carbocyclic nucleoside analogues as potential anti-viral agents

Abstract The synthesis of optically pure unsaturated carbocyclic nucleoside analogues is described. (3,4 S )-Bis( t -butyldiphenylsilyloxymethyl)-2-cyclopenten-1 R and 1 S -ol were coupled with 6-chloropurine and 2-amino-6-chloropurine respectively, using a modified Mitsunobu reaction. The products were reacted further using standard procedures to give compounds 12, 14, 16 and 18 which were tested for anti-HIV activity.