Biological and computational evaluation of resveratrol inhibitors against Alzheimer's disease.

It has been reported that beta amyloid induces production of radical oxygen species and oxidative stress in neuronal cells, which in turn upregulates ß-secretase (BACE-1) expression and beta amyloid levels, thereby propagating oxidative stress and increasing neuronal injury. A series of resveratrol derivatives, known to be inhibitors of oxidative stress-induced neuronal cell death (oxytosis) were biologically evaluated against BACE-1 using homogeneous time-resolved fluorescence (TRF) assay. Correlation between oxytosis inhibitory and BACE-1 inhibitory activity of resveratrol derivatives was statistically significant, supporting the notion that BACE-1 may act as pivotal mediator of neuronal cell oxytosis. Four of the biologically evaluated resveratrol analogs demonstrated considerably higher activity than resveratrol in either assay. The discovery of some "hits" led us to initiate detailed docking studies associated with Molecular Dynamics in order to provide a plausible explanation for the experimental results and understand their molecular basis of action.

Design and synthesis of potent hydroxyethylamine (HEA) BACE-1 inhibitors carrying prime side 4,5,6,7-tetrahydrobenzazole and 4,5,6,7-tetrahydropyridinoazole templates.

A set of low molecular weight compounds containing a hydroxyethylamine (HEA) core structure with different prime side alkyl substituted 4,5,6,7-tetrahydrobenzazoles and one 4,5,6,7-tetrahydropyridinoazole was synthesized. Striking differences were observed on potencies in the BACE-1 enzymatic and cellular assays depending on the nature of the heteroatoms in the bicyclic ring, from the low active compound 4 to inhibitor 6, displaying BACE-1 IC(50) values of 44 nM (enzyme assay) and 65 nM (cell-based assay).

Design and synthesis of potent macrocyclic renin inhibitors.

Two types of P1-P3-linked macrocyclic renin inhibitors containing the hydroxyethylene isostere (HE) scaffold just outside the macrocyclic ring have been synthesized. An aromatic or aliphatic substituent (P3sp) was introduced in the macrocyclic ring aiming at the S3 subpocket (S3sp) in order to optimize the potency. A 5-6-fold improvement in both the K(i) and the human plasma renin activity (HPRA)IC(50) was observed when moving from the starting linear peptidomimetic compound 1 to the most potent macrocycle 42 (K(i) = 3.3 nM and HPRA IC(50) = 7 nM). Truncation of the prime side of 42 led to 8-10-fold loss of inhibitory activity in macrocycle 43 (K(i) = 34 nM and HPRA IC(50) = 56 nM). All macrocycles were epimeric mixtures in regard to the P3sp substituent and X-ray crystallographic data of the representative renin macrocycle 43 complex showed that only the S-isomer buried the substituent into the S3sp. Inhibitory selectivity over cathepsin D (Cat-D) and BACE-1 was also investigated for all the macrocycles and showed that truncation of the prime side increased selectivity of inhibition in favor of renin.

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.

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.

Novel potent macrocyclic inhibitors of the hepatitis C virus NS3 protease: use of cyclopentane and cyclopentene P2-motifs.

Several highly potent novel HCV NS3 protease inhibitors have been developed from two inhibitor series containing either a P2 trisubstituted macrocyclic cyclopentane- or a P2 cyclopentene dicarboxylic acid moiety as surrogates for the widely used N-acyl-(4R)-hydroxyproline in the P2 position. These inhibitors were optimized for anti HCV activities through examination of different ring sizes in the macrocyclic systems and further by exploring the effect of P4 substituent removal on potency. The target molecules were synthesized from readily available starting materials, furnishing the inhibitor compounds in good overall yields. It was found that the 14-membered ring system was the most potent in these two series and that the corresponding 13-, 15-, and 16-membered macrocyclic rings delivered less potent inhibitors. Moreover, the corresponding P1 acylsulfonamides had superior potencies over the corresponding P1 carboxylic acids. It is noteworthy that it has been possible to develop highly potent HCV protease inhibitors that altogether lack the P4 substituent. Thus the most potent inhibitor described in this work, inhibitor 20, displays a K(i) value of 0.41 nM and an EC(50) value of 9 nM in the subgenomic HCV replicon cell model on genotype 1b. To the best of our knowledge this is the first example described in the literature of a HCV protease inhibitor displaying high potency in the replicon assay and lacking the P4 substituent, a finding which should facilitate the development of orally active small molecule inhibitors against the HCV protease.

Synthesis of novel potent hepatitis C virus NS3 protease inhibitors: discovery of 4-hydroxy-cyclopent-2-ene-1,2-dicarboxylic acid as a N-acyl-L-hydroxyproline bioisostere.

Potent tetrapeptidic inhibitors of the HCV NS3 protease have been developed incorporating 4-hydroxy-cyclopent-2-ene-1,2-dicarboxylic acid as a new N-acyl-l-hydroxyproline mimic. The hydroxycyclopentene template was synthesized in eight steps from commercially available (syn)-tetrahydrophthalic anhydride. Three different amino acids were explored in the P1-position and in the P2-position the hydroxyl group of the cyclopentene template was substituted with 7-methoxy-2-phenyl-quinolin-4-ol. The P3/P4-positions were then optimized from a set of six amino acid derivatives. All inhibitors were evaluated in an in vitro assay using the full-length NS3 protease. Several potent inhibitors were identified, the most promising exhibiting a K(i) value of 1.1nM.

Potent inhibitors of the hepatitis C virus NS3 protease: use of a novel P2 cyclopentane-derived template.

The HCV NS3 protease is essential for replication of the hepatitis C virus (HCV) and therefore constitutes a promising new drug target for anti-HCV therapy. Several potent and promising HCV NS3 protease inhibitors, some of which display low nanomolar activities, were identified from a series of novel inhibitors incorporating a trisubstituted cyclopentane dicarboxylic acid moiety as a surrogate for the widely used N-acyl-(4R)-hydroxyproline in the P2 position.

Macrocyclic inhibitors of the malarial aspartic proteases plasmepsin I, II, and IV.

The first macrocyclic inhibitor of the Plasmodium falciparum aspartic proteases plasmepsin I, II, and IV with considerable selectivity over the human aspartic protease cathepsin D has been identified. A series of macrocyclic compounds were designed and synthesized. Cyclizations were accomplished using ring-closing metathesis with the second generation Grubbs catalyst. These compounds contain either a 13-membered or a 16-membered macrocycle and incorporate a 1,2-dihydroxyethylene as transition state mimicking unit. The binding mode of this new class of compounds was predicted with automated docking and molecular dynamics simulations, with an estimation of the binding affinities through the linear interaction energy (LIE) method.

Synthesis of malarial plasmepsin inhibitors and prediction of binding modes by molecular dynamics simulations.

A series of inhibitors of the malarial aspartic proteases Plm I and II have been synthesized with L-mannitol as precursor. These inhibitors are characterized by either a diacylhydrazine or a five-membered oxadiazole ring replacing backbone amide functionalities. Molecular dynamics simulations were applied in the design process. The computationally predicted Plm II Ki values were generally in excellent agreement with the biological results. The diacylhydrazine was found to be superior over the oxadiazole as an amide bond replacement in the Plm I and II inhibitors studied. An extensive flexibility of the S2' pocket was captured by the simulations predicting the binding mode of the unsymmetrical inhibitors. Plm I and II inhibitors with single digit nanomolar Ki values devoid of inhibitory activity toward human Cat D were identified. One compound, lacking amide bonds, was found to be Plm IV selective and very potent, with a Ki value of 35 nM.

Synthesis, biological evaluation, and modeling studies of inhibitors aimed at the malarial proteases plasmepsins I and II.

The increasing resistance of the malarial parasite to antimalarial drugs is a major contributor to the reemergence of the disease and increases the need for new drug targets. The two aspartic proteases, plasmepsins I and II, from Plasmodium falciparum have recently emerged as potential targets. In an effort to inhibit these hemoglobinases, a series of inhibitors encompassing a basic hydroxyethylamine transition state isostere as a central fragment were prepared. The synthesized compounds were varied in the P1' position and exhibited biological activities in the range of 31 to >2000 nM. To try to rationalize the results, molecular docking and 3D-QSAR analysis were used.

Design and synthesis of potent inhibitors of plasmepsin I and II: X-ray crystal structure of inhibitor in complex with plasmepsin II.

New and potent inhibitors of the malarial aspartic proteases plasmepsin (Plm) I and II, from the deadliest malaria parasite Plasmodium falciparum, have been synthesized utilizing Suzuki coupling reactions on previously synthesized bromobenzyloxy-substituted statine-like inhibitors. The enzyme inhibition activity has been improved up to eight times by identifying P1 substituents that effectively bind to the continuous S1-S3 crevice of Plasmepsin I and II. By replacement of the bromo atom in the P1 p-bromobenzyloxy-substituted inhibitors with different aryl substituents, several inhibitors exhibiting K(i) values in the low nanomolar range for both Plm I and II have been identified. Some of these inhibitors are also effective in attenuating parasite growth in red blood cells, with the best inhibitors, compounds 2 and 4, displaying 70% and 83% inhibition, respectively, at a concentration of 5 microM. The design was partially guided by the X-ray crystal structure disclosed herein of the previously synthesized inhibitor 1 in complex with plasmepsin II.

Design and synthesis of potent inhibitors of the malaria aspartyl proteases plasmepsin I and II. Use of solid-phase synthesis to explore novel statine motifs.

Picomolar to low nanomolar inhibitors of the two aspartic proteases plasmepsin (Plm) I and II, from the malaria parasite Plasmodium falciparum, have been identified from sets of libraries containing novel statine-like templates modified at the amino and carboxy terminus. The syntheses of the novel statine templates were carried out in solution phase using efficient synthetic routes and resulting in excellent stereochemical control. The most promising statine template was attached to solid support and diversified by use of parallel synthesis. The products were evaluated for their Plm I and II inhibitory activity as well as their selectivity over cathepsin D. Selected inhibitors were, in addition, evaluated for their inhibition of parasite growth in cultured infected human red blood cells. The most potent inhibitor in this report, compound 16, displays Ki values of 0.5 and 2.2 nM for Plm I and II, respectively. Inhibitor 16 is also effective in attenuating parasite growth in red blood cells showing 51% inhibition at a concentration of 5 microM. Several inhibitors have been identified that exhibit Ki values between 0.5 and 74 nM for both Plm I and II. Some of these inhibitors also show excellent selectivity vs cathepsin D.

Potent inhibitors of the Plasmodium falciparum enzymes plasmepsin I and II devoid of cathepsin D inhibitory activity.

The hemoglobin-degrading aspartic proteases plasmepsin I (Plm I) and plasmepsin II (Plm II) of the malaria parasite Plasmodium falciparum have lately emerged as putative drug targets. A series of C(2)-symmetric compounds encompassing the 1,2-dihydroxyethylene scaffold and a variety of elongated P1/P1' side chains were synthesized via microwave-assisted palladium-catalyzed coupling reactions. Binding affinity calculations with the linear interaction energy method and molecular dynamics simulations reproduced the experimental binding data obtained in a Plm II assay with very good accuracy. Bioactive conformations of the elongated P1/P1' chains were predicted and agreed essentially with a recent X-ray structure. The compounds exhibited picomolar to nanomolar inhibition constants for the plasmepsins and no measurable affinity to the human enzyme cathepsin D. Some of the compounds also demonstrated significant inhibition of parasite growth in cell culture. To the best of our knowledge, these plasmepsin inhibitors represent the most selective reported to date and constitute promising lead compounds for further optimization.

High-speed optimization of inhibitors of the malarial proteases plasmepsin I and II.

Four focused libraries targeted for inhibition of the malarial proteases plasmepsin I and II were designed, synthesized, purified, and screened. Selected carboxylic acids and organometallic reactants with diverse physical properties were attached to the hydroxylethylamine scaffold in the P3 and P1' positions to furnish inhibitors with highly improved activity. The concept of controlled and sequential microwave heating was employed for rapid library generation. This combinatorial optimization protocol afforded plasmepsin inhibitors not only with K(i) values in the low nanomolar range, but also with high selectivity versus the human protease cathepsin D. With this class of inhibitory agents, modifications of the P1' substituents resulted in the largest impact on the plasmepsin/cathepsin D selectivity.

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.

Solid-phase library synthesis of reversed-statine type inhibitors of the malarial aspartyl proteases plasmepsin I and II.

With the aim to develop inhibitors of the plasmepsin I and II aspartic proteases of the malaria parasite Plasmodium falciparum, we have synthesized sets of libraries from novel reversed-statine isosteres, using a combination of solution phase and solid phase chemistry. The synthetic strategy furnishes the library compounds in good to high overall yields and with excellent stereochemical control throughout the developed route. The products were evaluated for their plasmepsin I and II inhibiting properties and were found to exhibit modest but promising activity. The best inhibitor exhibits an in vitro activity of 28% inhibition of plasmepsin II at an inhibitor concentration of 0.5 microM (K(i) for Plm II=5.4 microM).

New potent C2-symmetric malaria plasmepsin I and II inhibitors.

A series of malaria plasmepsin (Plm) I and II inhibitors containing a C(2)-symmetric core structure have been synthesised and tested for protease inhibition activity. These compounds can be prepared using a straightforward synthesis involving a phenol nucleophilic ring opening of a diepoxide. Exemplar compounds synthesised exhibited remarkable inhibitory activity against both Plm I and II, notably 15c with K(i) values of 2.7nM and 0.25nM respectively, as well as showing >100-fold selectivity against Cathepsin D.

Design and synthesis of plasmepsin I and plasmepsin II inhibitors with activity in Plasmodium falciparum-infected cultured human erythrocytes.

A series of protease inhibitors targeted at the malarial enzymes plasmepsin I and II, and encompassing a basic hydroxyethylamine transition state isostere scaffold, was prepared. The substituents in the P1' position were varied and the biological activities expressed in K(i)-values ranged from 60 to >2000 nM. A more than 4-fold selectivity for either of the plasmepsins could be achieved. All of the active compounds exhibited high preference for the plasmepsins over cathepsin D, the most closely related human protease. A few active compounds were shown to inhibit parasite growth in cultured infected human erythrocytes. An ED(50) value as low as 1.6 microM was observed for one of the inhibitors despite K(i) values of 115 nM (Plm I) and 121 nM (Plm II).