Dipeptidyl aspartyl fluoromethylketones as potent caspase inhibitors: peptidomimetic replacement of the P2 alpha-amino acid by a alpha-hydroxy acid

As a continuation of our SAR studies of dipeptidyl aspartyl-fmk as caspase inhibitors, we explored the replacement of the P2 alpha-amino acid by a peptidomimetic alpha-hydroxy acid. These alpha-carbamoyl-alkylcarbonyl-aspartyl fluoromethylketones were found to be potent caspase inhibitors, and the SAR of these compounds is similar to the corresponding dipeptidyl aspartyl-fmk. MX1153, (S)-3-methyl-2-(phenylcarbamoyl)butanoyl-Asp-fmk, is identified as a potent broad-spectrum caspase inhibitor, and is selective for caspases versus other proteases. MX1153 also has good activity in the cell apoptosis protection assays and is active in the mouse liver apoptosis model.     

Dipeptidyl aspartyl fluoromethylketones as potent caspase-3 inhibitors: SAR of the P2 amino acid

This article describes the synthesis and biological evaluation of a series of dipeptidyl aspartyl fluoromethylketones as caspase-3 inhibitors. Structure-activity relationship (SAR) studies showed that for caspase-3 inhibition, Val is the best P(2) amino acid. The SAR studies also showed that the Asp free carboxylic acid in P(1) is important for caspase inhibiting activities, as well as for selectivity over other proteases.     

Dipeptidyl aspartyl fluoromethylketones as potent caspase inhibitors: SAR of the N-protecting group

This article describes the synthesis and biological evaluation of a group of N-protected Val-Asp-fmk as caspase inhibitors. The protecting group was found to contribute to caspase-3 inhibiting activity, and compounds with a large group such as Cbz are more active than compounds with a small group such as Ac. Compounds with more hydrophobic protecting groups were found to be more active in cell apoptosis protection assays, probably due to increased cell permeability. MX1122, 2,4-di-Cl-Cbz-Val-Asp-fmk, is identified as a potent broad-spectrum caspase inhibitor and is selective for caspases versus other proteases, with good activity in the cell apoptosis protection assays as well as good efficacy in the mouse liver apoptosis model.     

Introduction of non-natural amino acid residues into the substrate-specific P1 position of trypsin inhibitor SFTI-1 yields potent chymotrypsin and cathepsin G inhibitors

A series of trypsin inhibitor SFTI-1compounds modified in substrate-specific P₁ position was synthesized by the solid-phase method. Lys5 present in the wild inhibitor was replaced by Phe derivatives substituted in para position of the phenyl ring, l-pyridylalanine and N-4-nitrobenzylgycine. Their inhibitory activities with bovine α-chymotrypsin and cathepsin G were estimated by determination of association equilibrium constants (K_a). All analogues inhibited bovine α-chymotrypsin. The highest inihbitory activity displayed peptides with the fluorine, nitro and methyl substituents. They were 13–15-fold more active than [Phe⁵]SFTI-1 used as a reference. They are the most potent chymotrypsin inhibitors of this size. Substitution of Lys5 by Phe did not change the cathepsin G inhibitory activity. Introduction of Phe(p-F), Phe(p-NH₂) and Phe(p-CH₃) in this position retained the affinity towards this proteinase, whereas Phe(p-guanidine) gave an inhibitor more than twice as active, which appeared to be stable in human serum. On the other hand, a peptomeric analogue with N-4-nitrobenzylglycine failed to inhibit cathepsin G. Despite the fact the introduced amino acids were non-coded, the peptide bonds formed by them were hydrolyzed by chymotrypsin. We postulate that additional interaction of para-substitutents with the enzyme are responsible for the enhanced inhibitory activity of the analogues.     

Lipophilic versus hydrogen-bonding effect in P3 on potency and selectivity of valine aspartyl ketones as caspase 3 inhibitors

Caspase 3 is a cysteinyl protease that mediates apoptotic cell death. Its inhibition may have an important impact in the treatment of several degenerative diseases. The P1 aspartic acid residue is a required element of recognition for this enzyme that was maintained constant along with the adjacent natural valine as the P2 group. The thiobenzylmethylketone warhead on the aspartate was conveniently handled through solid-phase synthesis allowing modification in the P3 region that eventually led to simpler derivatives with increased potency against caspase 3. The key to such an effect is the introduction of hydroxyl group alpha to the P3 carbonyl.     

Synthesis and calpain inhibitory activity of peptidomimetic compounds with constrained amino acids at the P2 position

The effect of incorporating α,α′-diethylglycine and α-aminocyclopentane carboxylic acid at the P₂ position of inhibitors on μ-calpain inhibition was studied. Compound 3 with α,α′-diethylglycine was over 20-fold more potent than 2 with α-aminocyclopentane carboxylic acid. Additionally, 3 was over 35-fold selective for μ-calpain compared to cathepsin B, while 2 was 3-fold selective for cathepsin B compared to μ-calpain. Thus, the conformation induced by the P₂ residue influenced the activities of the compounds versus the closely related cysteine proteases, and suggests an approach to the discovery of selective μ-calpain inhibitors.     

Succinimide hydroxamic acids as potent inhibitors of histone deacetylase (HDAC)

A series of succinimide hydroxamic acids was prepared and evaluated in vitro for HDAC inhibition and tumor cell antiproliferation. While the original macrocyclic analogue 6 was quite potent in both assays, several appropriately substituted non-macrocyclic succinimides, such as 23, were equipotent.     

Discovery of novel aspartyl ketone dipeptides as potent and selective caspase-3 inhibitors

The discovery of a series of potent, selective and reversible dipeptidyl caspase-3 inhibitors are reported. The iterative discovery process of using combinatorial chemistry, parallel synthesis, moleculare modelling and structural biology will be discussed.     

N-Benzoyl amino acids as LFA-1/ICAM inhibitors 1: amino acid structure-activity relationship

The association of ICAM-1 with LFA-1 plays a critical role in several autoimmune diseases. N-2-Bromobenzoyl L-tryptophan, compound 1, was identified as an inhibitor to the formation of the LFA-1/ICAM complex. The SAR of the amino acid indicates that the carboxylic acid is required for inhibition and that L-histidine is the most favored amino acid.     

The discovery of fused pyrrole carboxylic acids as novel, potent D-amino acid oxidase (DAO) inhibitors

The 'NMDA hypofunction hypothesis of schizophrenia' can be tested in a number of ways. DAO is the enzyme primarily responsible for the metabolism of d-serine, a co-agonist for the NMDA receptor. We identified novel DAO inhibitors, in particular, acid 1, which demonstrated moderate potency for DAO in vitro and ex vivo, and raised plasma d-serine levels after dosing ip to rats. In parallel, analogues were prepared to survey the SARs of 1.     

Serine protease inhibitors N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK) and N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK) are potent inhibitors of activated caspase proteases

Serine protease inhibitors N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK) and N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK) exhibit multiple effects on cell death pathways in mammalian cells. Thus, they are able to induce apoptosis by itself or promote cell death induced by other cytotoxic stimuli [King et al., 2004; Murn et al., 2004]. On the other hand, TLCK and TPCK were reported to prevent apoptosis by inhibiting the processing of caspases in response to some cell death inducing stimuli [Stefanis et al., 1997; Jones et al., 1998]. We observed that the pretreatment of HL-60 cells with TLCK or TPCK diminished caspases 3 and -7 (DEVDase) and caspase-6 (VEIDase) activity in response to various cell death inducing stimuli such as staurosporine (STS), etoposide (ETP), or N6-(2-isopentenyl)adenosine. In addition, TLCK but not TPCK inhibited collapse of mitochondrial transmembrane potential Delta Psi m (delta psi) in dying HL-60 cells. Such effects used to be considered as protective, however, the protection was only presumable since neither TLCK nor TPCK actually prevented cells from death. Our results further indicated that serine protease inhibitors TLCK and particularly TPCK acted as efficient direct inhibitors of mature caspases. Indeed, experiments with human recombinant caspases provided unequivocal evidence that TLCK and TPCK are very potent but non-specific inhibitors of activated caspases, namely caspases 3, -6, and -7. Interestingly, TPCK exhibited similar efficiency towards human recombinant caspases to that found for panspecific caspase inhibitor Boc-D-CMK. Such properties of TLCK and TPCK, previously considered as specific inhibitors of serine proteases, might offer novel consistent explanation for several protective or protective-like effects on apoptotic cells.     

Convenient introduction of 2-(trimethylsilyl)ethylsulfonyl (SES) amino protection on different amino acids and its use in peptidomimetic chemistry

A direct synthesis of a series of N-SES amino acids is described. N-SES Ala has been further utilized in the synthesis of a perhydro-1,4-diazepin-2-one.     

Convenient introduction of 2-(trimethylsilyl)ethylsulfonyl (SES) amino protection on different amino acids and its use in peptidomimetic chemistry

Summary A direct synthesis of a series ofN-SES amino acids is described.N-SES Ala has been further utilized in the synthesis of a perhydro-1,4-diazepin-2-one.     

Synthesis of enantiopure non-natural alpha-amino acids using tert-butyl (2S)-2-[bis-(tert-butoxycarbonyl)amino]-5-oxopentanoate as key-intermediate:the first synthesis of(S)-2-amino-oleic acid.

A general method for the synthesis of enantiopure non-natural alpha-amino acids is described. The key intermediate tert-butyl (2S)-2-[bis(tert-butoxycarbonyl)amino]-5-oxopentanoate was obtained from l-glutamic acid after suitable protection and selective reduction of the gamma-methyl ester group by DIBALH. Wittig reaction of this chiral aldehyde with various ylides led to a variety of delta,epsilon-unsaturated alpha-amino acids. This methodology was applied to the synthesis of (S)-2-amino-oleic acid.     

Structure-activity relationships of natural and non-natural amino acid-based amide and 2-oxoamide inhibitors of human phospholipase A(2) enzymes

A variety of 2-oxoamides and related amides based on natural and non-natural amino acids were synthesized. Their activity on two human intracellular phospholipases (GIVA cPLA(2) and GVIA iPLA(2)) and one human secretory phospholipase (GV sPLA(2)) was evaluated. We show that an amide based on (R)-gamma-norleucine is a highly selective inhibitor of GV sPLA(2).     

Discovery of pyrazole carboxylic acids as potent inhibitors of rat long chain L-2-hydroxy acid oxidase

Long chain L-2-hydroxy acid oxidase 2 (Hao2) is a peroxisomal enzyme expressed in the kidney and the liver. Hao2 was identified as a candidate gene for blood pressure (BP) quantitative trait locus (QTL) but the identity of its physiological substrate and its role in vivo remains largely unknown. To define a pharmacological role of this gene product, we report the development of selective inhibitors of Hao2. We identified pyrazole carboxylic acid hits 1 and 2 from screening of a compound library. Lead optimization of these hits led to the discovery of 15-XV and 15-XXXII as potent and selective inhibitors of rat Hao2. This report details the structure activity relationship of the pyrazole carboxylic acids as specific inhibitors of Hao2.     

N-i-Propoxy-N-biphenylsulfonylaminobutylhydroxamic acids as potent and selective inhibitors of MMP-2 and MT1-MMP

Structural manipulation of the pharmacophoric model of type A selective MMP inhibitors (MMPi), obtained by the insertion of some alkyl substituents R2 possessing an appropriate geometry, steric bulkiness and lipophilicity, is able to improve potency, in the subnanomolar range on MMP-2, and to give a good MMP inhibition on MMP-14 (MT1-MMP) in the designed MMPi of type C, while maintaining a good MMP-1/MMP-2 selectivity profile. The simultaneous inhibition of these two enzymes yields type C compounds, which are potent antiangiogenic agents, able to block a chemoinvasion model on HUVEC cells in the micromolar range.     

Rapid assessment of correlated amino acids from pair-to-pair (P2P) substitution matrices

Identification of correlated amino acids in proteins has been a topic of broad interest in view of its functional implications and importance in protein design. A new set of pair-to-pair (P2P) substitution matrices for amino acids was recently introduced as a useful tool for inferring information on such correlated sites. We present a website developed for automated application of these matrices for analysis of query sequences. The site offers options for graphical analysis of correlations, as well as visualization of correlated amino acids on representative, structurally characterized, members of the examined family of sequences. Availability: http://www.ccbb.pitt.edu/p2p.     

Diaminopimelic acid uptake by Bacillus megaterium: influence of growth conditions and other amino acids

Diaminopimelic acid (DAP) uptake was studied in Bacillus megaterium. The K _m and V _max were determined for bacteria grown with or without DAP. The uptake of DAP was shown to be constitutive and unaffected by the presence of other amino acids (including cystine). The concentration of DAP and lysine in the amino acid pool was examined and a procedure for pulse-labelling cell walls developed.     

Glycosylated dihydrochalcones as potent and selective sodium glucose co-transporter 2 (SGLT2) inhibitors

A series of glucose conjugates was synthesized and tested for inhibition of SGLT1 and SGLT2. The core structure was derived from compound 1a. Modification of the benzofuran moiety and 4'-substituent of the phenyl ring in compound 1a improved selectivity at SGLT2. Select compounds were compared to 1a in metabolic stability and in vivo efficacy studies.