Emanuela Nizi

Discovery of Raltegravir, a Potent, Selective Orally Bioavailable HIV-Integrase Inhibitor for the Treatment of HIV-AIDS Infection

Human immunodeficiency virus type-1 (HIV-1) integrase is one of the three virally encoded enzymes required for replication and therefore a rational target for chemotherapeutic intervention in the treatment of HIV-1 infection. We report here the discovery of Raltegravir, the first HIV-integrase inhibitor approved by FDA for the treatment of HIV infection. It derives from the evolution of 5,6-dihydroxypyrimidine-4-carboxamides and N-methyl-4-hydroxypyrimidinone-carboxamides, which exhibited potent inhibition of the HIV-integrase catalyzed strand transfer process. Structural modifications on these molecules were made in order to maximize potency as HIV-integrase inhibitors against the wild type virus, a selection of mutants, and optimize the selectivity, pharmacokinetic, and metabolic profiles in preclinical species. The good profile of Raltegravir has enabled its progression toward the end of phase III clinical trials for the treatment of HIV-1 infection and culminated with the FDA approval as the first HIV-integrase inhibitor for the treatment of HIV-1 infection.

Discovery of MK-5172, a Macrocyclic Hepatitis C Virus NS3/4a Protease Inhibitor.

A new class of HCV NS3/4a protease inhibitors containing a P2 to P4 macrocyclic constraint was designed using a molecular modeling-derived strategy. Building on the profile of previous clinical compounds and exploring the P2 and linker regions of the series allowed for optimization of broad genotype and mutant enzyme potency, cellular activity, and rat liver exposure following oral dosing. These studies led to the identification of clinical candidate 15 (MK-5172), which is active against genotype 1-3 NS3/4a and clinically relevant mutant enzymes and has good plasma exposure and excellent liver exposure in multiple species.

Novel Peptidomimetics Containing a Vinyl Ester Moiety as Highly Potent and Selective Falcipain-2 Inhibitors

This paper describes the synthesis and biological evaluation of a new class of peptidomimetic falcipain-2 inhibitors based on a 1,4-benzodiazepine scaffold combined with various alpha,beta-unsaturated electrophilic functions such as vinyl-ketone, -amide, -ester, and -nitrile. The profile of reactivity of this class of derivatives has been evaluated and 4c, containing a vinyl ester warhead, proved to be a highly potent and selective falcipain-2 inhibitor.

Design and synthesis of bicyclic pyrimidinones as potent and orally bioavailable HIV-1 integrase inhibitors.

HIV integrase is one of the three enzymes encoded by HIV genome and is essential for viral replication, but integrase inhibitors as marketed drugs have just very recently started to emerge. In this study, we show the evolution from the N-methylpyrimidinone structure to bicyclic pyrimidinones. Introduction of a suitably substituted amino moiety modulated the physical-chemical properties of the molecules and conferred nanomolar activity in the inhibition of spread of HIV-1 infection in cell culture. An extensive SAR study led to sulfamide (R)- 22b, which inhibited the strand transfer with an IC50 of 7 nM and HIV infection in MT4 cells with a CIC95 of 44 nM, and ketoamide (S)- 28c that inhibited strand transfer with an IC50 of 12 nM and the HIV infection in MT4 cells with a CIC95 of 13 nM and exhibited a good pharmacokinetic profile when dosed orally to preclinical species.

Nonpeptidic Vinyl and Allyl Phosphonates as Falcipain-2 Inhibitors

Malaria remains one of the most deadly parasitic diseases, affecting 500 million people all over the world and causing more than one million deaths each year. The limitations of antimalarial chemotherapy underscore the urgent need to discover new drugs that are able to interact with new targets. Research efforts are currently focused on the design of inhibitors of malarial proteases, among which falcipain-2 (FP-2) plays a key role. FP-2 is a papain-family cysteine protease of the most virulent species of the malaria-causing parasite Plasmodium falciparum, and is required by mature schizonts for the cleavage of erythrocytic cytoskeletal proteins and by intraerythrocytic trophozoites for hemoglobin degradation, which provides free amino acids for parasite protein synthesis. Thus, selective and irreversible inhibition of FP-2 would be advantageous for the control and elimination of the parasite. Peptides and peptidomimetics containing an activated double bond, such as vinyl sulfones and vinyl esters, have been shown to be highly potent irreversible cysteine protease inhibitors. Whereas the former are active on papain-like enzymes, the latter are known in particular as inhibitors of viral proteases. Both inhibitor types interact with the target enzyme by forming a covalent bond with the thiol group of the active site cysteine. Peptidyl vinyl sulfones are stable and unreactive toward nucleophiles, and require the “catalytic machinery” of cysteine proteases for their activation. Peptidyl vinyl sulfones containing a homoPhe residue at the P1 site have been proven to be highly specific FP-2 inhibitors, with the aromatic side chain being a key structural requirement for greater selectivity toward the target enzyme. In this context we recently reported a new class of peptidomimetic FP-2 inhibitors based on a rigid benzodiazepine scaffold as a conformationally constrained form of the d-Ser-Gly fragment and on a terminal electrophilic vinyl sulfone moiety on the P1 site that reacts as classical Michael acceptor (such as compounds 1a–d, Figure 1). All the synthesized compounds showed a high level of inhibitory potency and are quite selective toward FP-2, as they weakly inhibit human cysteine proteases cathepsin B and L. In particular, compound 1b displayed potent enzymatic inhibition (k2=307000m min ) coupled with a good activity against cultured P. falciparum (IC50= 9.1 mm). Among other irreversible cysteine protease inhibitors containing a vinyl moiety conjugated to electron withdrawing groups (EWGs), vinyl phosphonates showed good inhibitory activity against cultured P. falciparum. On this basis, we designed and synthesized nonpeptidic unsaturated phosphonate structures 2 and 3 (Scheme 1) to evaluate their ability to inhibit FP-2 and to make a head-to-head

Capped dipeptide α-ketoacid inhibitors of the HCV NS3 protease

Abstract The N-terminal aminoacid of α-ketotripeptide inhibitors of the hepatitis C virus NS3 protease can be replaced with an α-hydroxy acid, leading to capped dipeptide inhibitors such as 20 with an IC50 value of 3.0 μM. The importance of the lipophilic side chain interactions at S3 of the protease and the requirement of the capping residue with R configuration have been explained by molecular modeling studies.

Solid phase synthesis of 2,6-disubstituted-4(3H)-pyrimidinones targeting HIV-1 reverse transcriptase

The solution and solid phase synthesis of 6-substituted 2-[(4-hydroxybutyl)amino]-4(3H)-pyrimidinones 1 as HIV-1 RT inhibitors is described.

Dihydroxy-pyrimidine and N-methylpyrimidone HIV-integrase inhibitors: Improving cell based activity by the quaternarization of a chiral center

In the context of HIV-integrase, dihydroxypyrimidine and N-methyl pyrimidone inhibitors the cellular activity of this class of compounds has been optimized by the introduction of a simple methyl substituent in the alpha-position of the C-2 side chains. Enhanced passive membrane permeability has been identified as the key factor driving the observed cell-based activity improvement. The rat PK profile of the alpha-methyl derivative 26a was also improved over its des-methyl exact analog.

Discovery of a Selective Series of Inhibitors of Plasmodium falciparum HDACs

The identification of a new series of P. falciparum growth inhibitors is described. Starting from a series of known human class I HDAC inhibitors a SAR exploration based on growth inhibitory activity in parasite and human cells-based assays led to the identification of compounds with submicromolar inhibition of P. falciparum growth (EC50 < 500 nM) and good selectivity over the activity of human HDAC in cells (up to >50-fold). Inhibition of parasital HDACs as the mechanism of action of this new class of selective growth inhibitors is supported by hyperacetylation studies.

Studies of metabolism and disposition of potent human immunodeficiency virus (HIV) integrase inhibitors using 19F-NMR spectroscopy

19F-nuclear magnetic resonance (NMR) has been extensively used in a drug-discovery programme to support the selection of candidates for further development. Data on an early lead compound, N-(4-fluorobenzyl)-5-hydroxy-1-methyl-2-(4-methylmorpholin-3-yl)-6-oxo-1,6-dihydropyrimidine-4-carboxamide (compound A (+)), and MK-0518 (N-(4-fluorobenzyl)-5-hydroxy-1-methyl-2-(1-methyl-1-{[(5-methyl-1,3,4-oxadiazol-2-yl)carbonyl]amino}ethyl)-6-oxo-1,6-dihydropyrimidine-4-carboxamide), a potent inhibitor of this series currently in phase III clinical trials, are described. The metabolic fate and excretion balance of compound A (+) and MK-0518 were investigated in rats and dogs following intravenous and oral dosing using a combination of 19F-NMR-monitored enzyme hydrolysis and solid-phase extraction chromatography and NMR spectroscopy (SPEC-NMR). Dosing with the 3H-labelled compound A (+) enabled the comparison of standard radiochemical analysis with 19F-NMR spectroscopy to obtain quantitative metabolism and excretion data. Both compounds were eliminated mainly by metabolism. The major metabolite identified in rat urine and bile and in dog urine was the 5-O-glucuronide.