Ted Halmos

Peptide-based inhibitors of the hepatitis C virus serine protease

Hexapeptide DDIVPC-OH is a competitive inhibitor of the hepatitis C virus (HCV) NS3 protease complexed with NS4A cofactor peptide. This hexapeptide corresponds to the N-terminal cleavage product of an HCV dodecapeptide substrate derived from the NS5A/5B cleavage site. Structure-activity studies on Ac-DDIVPC-OH revealed that side chains of the P4, P3 and P1 residues contribute the most to binding and that the introduction of a D-amino acid at the P5 position improves potency considerably. Furthermore, there is a strong preference for cysteine at the P1 position and conservative replacements, such as serine, are not well tolerated.

Studies on the C-terminal of hexapeptide inhibitors of the hepatitis C virus serine protease

Replacement of the C-terminal carboxylic acid functionality of peptide inhibitors of hepatitis C virus (HCV) NS3 protease (complexed with NS4A peptide cofactor) by activated carbonyl groups does not produce any substantial increase in potency. These latter inhibitors also inhibit a variety of other serine and cysteine proteases whereas the carboxylic acids are specific. Norvaline was identified as a chemically stable replacement for the P1 residue of Ac-DDIVPC-OH which was also compatible with activated carbonyl functionalities.

Highly potent and selective peptide-based inhibitors of the hepatitis C virus serine protease : Towards smaller inhibitors

Structure-activity studies on a hexapeptide N-terminal cleavage product of a dodecamer substrate led to the identification of very potent and highly specific inhibitors of the HCV NS3 protease/NS4A cofactor peptide complex. The largest increase in potency was accomplished by the introduction of a (4R)-naphthalen-1-yl-4-methoxy substituent to the P2 proline. N-Terminal truncation resulted in tetrapeptides containing a C-terminal carboxylic acid, which exhibited low micromolar activity against the HCV serine protease.

Discovery of a potent and selective noncovalent linear inhibitor of the hepatitis C virus NS3 protease (BI 201335).

C-Terminal carboxylic acid containing inhibitors of the NS3 protease are reported. A novel series of linear tripeptide inhibitors that are very potent and selective against the NS3 protease are described. A substantial contribution to the potency of these linear inhibitors arises from the introduction of a C8 substituent on the B-ring of the quinoline moiety found on the P2 of these inhibitors. The introduction of a C8 methyl group results not only in a modest increase in the cell-based potency of these inhibitors but more importantly in a much better pharmacokinetic profile in rats as well. Exploration of C8-substitutions led to the identification of the bromo derivative as the best group at this position, resulting in a significant increase in the cell-based potency of this class of inhibitors. Structure-activity studies on the C8-bromo derivatives ultimately led to the discovery of clinical candidate 29 (BI 201335), a very potent and selective inhibitor of genotype1 NS3 protease with a promising PK profile in rats.

Discovery of non-peptidic P2-P3 butanediamide renin inhibitors with high oral efficacy.

A new series of non-peptidic renin inhibitors having a 2-substituted butanediamide moiety at the P2 and P3 positions has been identified. The optimized inhibitors have IC50 values of 0.8 to 1.4 nM and 2.5 to 7.6 nM in plasma renin assays at pH 6.0 and 7.4, respectively. When evaluated in the normotensive cynomolgus monkey model, two of the most potent inhibitors were orally active at a dose as low as 3 mg/kg. These potent renin inhibitors are characterized by oral bioavailabilities of 40 and 89% in the cynomolgus monkey. Inhibitor 3z (BILA 2157 BS) was selected as candidate for pre-development.

The use of chemical double-mutant cycles in biomolecular recognition studies: application to HCV NS3 protease inhibitors.

Chemical double-mutant cycles (DMCs) have been elegantly used to quantify the energies of noncovalent interactions between specific pairs of functional groups in the molecular recognition of abiotic systems. The method has also been employed to estimate the free energy of a novel noncovalent interaction in an intramolecular context. The analysis assumes that there are no significant structural differences between the four components of the cycle and that the free energies of the individual interactions are additive. It can then be used to determine the energetic contribution of a specific interaction in the context of a more complex, global binding event in such a manner that secondary interactions are effectively cancelled out. Here, we describe the novel use of a particular type of chemical DMC, which serves as a useful formalism to characterize the mutual effect of different substructures within a molecule on a biomolecular recognition process, namely, binding to an enzyme active site. This treatment varies from those described above in that two “deletion mutations” occupy different positions on the same chemical species while the binding partner is kept constant. Contrary to the DMC whose validity rests on the conditions of unchanging structure and the additive contributions of subsites to the total binding energy, the present analysis is useful in providing a clear indication of whether two portions of a ligand contribute to the total binding energy in an additive or cooperative (synergistic or antagonistic) manner, thus affording insight into the binding phenomenon and/or aiding in the understanding of structure–activity relationships (SAR) when structural data is lacking. The treatment is exemplified by thermodynamic cycles constructed from peptidic inhibitors of the hepatitis C virus NS3 protease (HCV protease) where the binding event is competitive inhibition of the enzyme. Examples of all three cases (additive, synergistic and antagonistic) are described. HCV protease represents an important target in the quest for a specific anti ACHTUNGTRENNUNGviral agent against hepatitis C infection, a serious global health problem for which the therapeutic need has not been met. Of central importance in our early efforts was the discovery that this novel serine protease suffers product inhibition by oligopeptide N-terminal cleavage products. Optimization of product-based hexapepACHTUNGTRENNUNGtides entailed N-truncation, elaboration of a novel P1 residue and the incorporation of an aryloxy substituent onto the P2 residue, which led to potent tripeptides bearing a free Cterminal carboxylate. This series ultimately provided the first HCV protease inhibitor demonstrated to reduce viral load in ACHTUNGTRENNUNGinfected patients. A molecule central to this optimization effort was inhibitor 4, which has become a key standard and biological tool in the study of this class of ligands and the enzyme target. As is generally observed for peptidyl inhibitors of serine proteases, compound 4 is bound to HCV protease in an extended conformation (Figure 1), canonical H-bonds involving the P1-NH, P3-

Minimizing the Contribution of Enterohepatic Recirculation to Clearance in Rat for the NCINI Class of Inhibitors of HIV

A scaffold replacement approach was used to identifying the pyridine series of noncatalytic site integrase inhibitors. These molecules bind with higher affinity to a tetrameric form compared to a dimeric form of integrase. Optimization of the C6 and C4 positions revealed that viruses harboring T124 or A124 amino acid substitutions are highly susceptible to these inhibitors, but viruses having the N124 amino acid substitution are about 100-fold less susceptible. Compound 20 had EC50 values <10 nM against viruses having T124 or A124 substitutions in IN and >800 nM in viruses having N124 substitions. Compound 20 had an excellent in vitro ADME profile and demonstrated reduced contribution of biliary excretion to in vivo clearance compared to BI 224436, the lead compound from the quinoline series of NCINIs.

Chemo-enzymatic synthesis of chiral 2-substituted succinic acid derivatives

Abstract Prochiral discrimination by the biocatalyst Alcalase ® , an enzyme preparation of subtilisin Carlsberg, was used to effect enantio- and regioselective monohydrolysis of a variety of ( RS )-2-substituted succinate diesters to afford the corresponding half esters in modest to excellent enantiomeric excesses (>99%). Exploitation of malonate chemistry, as well as recycling of the unhydrolyzed isomer from the enantioselective hydrolysis, has resulted in a process which is both practical and economical.

Identification of potent and orally bioavailable nucleotide competing reverse transcriptase inhibitors: in vitro and in vivo optimization of a series of benzofurano[3,2-d]pyrimidin-2-one derived inhibitors.

Recently, a new class of HIV reverse transcriptase (HIV-RT) inhibitors has been reported. The novel mechanism of inhibition by this class involves competitive binding to the active site of the RT enzyme and has been termed Nucleotide-Competing Reverse Transcriptase Inhibitors (NcRTIs). In this publication we describe the optimization of a novel benzofurano[3,2-d]pyrimidin-2-one series of NcRTIs. The starting point for the current study was inhibitor 2, which had high biochemical and antiviral potency but only moderate permeability in a Caco-2 assay and high B-to-A efflux, resulting in moderate rat bioavailability and low Cmax. We present herein the results and strategies we employed to optimize both the potency as well as the permeability, metabolic stability and pharmacokinetic profile of this series. One of the key observations of the present study was the importance of shielding polar functionality, at least in the context of the current chemotype, to enhance permeability. These studies led to the identification of inhibitors 39 and 45, which display sub-nanomolar antiviral potency in a p24 ELISA assay with significantly reduced efflux ratios (ratios <1.5). These inhibitors also display excellent rat pharmacokinetic profiles with high bioavailabilities and low clearance.

Synthesis and optimization of a novel series of HCV NS3 protease inhibitors: 4-Arylproline analogs

In this report we describe the synthesis and evaluation of diverse 4-arylproline analogs as HCV NS3 protease inhibitors. Introduction of this novel P2 moiety opened up new SAR and, in combination with a synthetic approach providing a versatile handle, allowed for efficient exploitation of this novel series of NS3 protease inhibitors. Multiple structural modifications of the aryl group at the 4-proline, guided by structural analysis, led to the identification of analogs which were very potent in both enzymatic and cell based assays. The impact of this systematic SAR on different drug properties is reported.