Piperidines: a new class of urease inhibitors

A variety of piperidines (2-12, 14-26) with variable substituents at N-atoms have been synthesized and evaluated as urease inhibitors. The synthesized compounds showed varying degree of urease inhibitory activity ranging from 31.97 to 254 microM. The size and electron-donating or -withdrawing effects of substituents influence the activity, which lead to the formation of urease inhibitors.     

Synthesis of a new class of uridine phosphorylase inhibitors

A new series of potent uridine phosphorylase inhibitors have been prepared from barbituric acid. Among them, 1-[(2-hydroxyethoxy)methyl]-5-)(m--benzyloxy)benzylbarbituric acid ( 37 , BBBA) is the most promising having a K_i value of 1.1 ± 0.2 nM with uridine phosphorylase from human liver. The new inhibitors are easily synthesized and are better inhibitors of human uridine phosphorylase than their uracil counterparts.     

Beta-N-cyanoethyl acyl hydrazide derivatives: a new class of beta-glucuronidase inhibitors

Eight new beta-N-substituted acyl hydrazides along with their corresponding acyl derivatives were synthesized and screened for in vitro beta-glucuronidase inhibition and found to be active against the enzyme. All of these compounds were found to be noncompetitive inhibitors except for N'-(2-cyanoethyl)-4-hydroxy benzohydrazide (10), which was found to be an uncompetitive inhibitor. Structure-activity relationship studies indicated that the benzyloxy group present in compounds 12 and 13 is responsible for the beta-glucuronidase inhibition activity.     

Trihydroxy-2-thiaquinolizidine derivatives as a new class of bicyclic glycosidase inhibitors

Trihydroxy-2-thiaquinolizidines, a new class of bicyclic dideoxy-iminohexitol glycosidase inhibitor derivatives with nominally the d-gluco, l-ido, d-manno and l-gulo configurations were synthesized. X-ray analyses indicated that the preferred conformation for d-gluco and d-manno derivatives was a flat trans-fused system. Unlike deoxynojirimycin, the compound with d-gluco configuration was selective for α-glucosidases (yeast and rice) and showed no inhibitory activity towards β-glucosidase (almond), α-galactosidase (green coffee beans), α-galactosidase (E. coli) and α-mannosidase (jack bean), while the l-ido derivative was specific for β-glucosidase (almond).     

Substrate activity screening: a fragment-based method for the rapid identification of nonpeptidic protease inhibitors

A new fragment-based method for the rapid development of novel and distinct classes of nonpeptidic protease inhibitors, Substrate Activity Screening (SAS), is described. This method consists of three steps: (1) a library of N-acyl aminocoumarins with diverse, low molecular weight N-acyl groups is screened to identify protease substrates using a simple fluorescence-based assay, (2) the identified N-acyl aminocoumarin substrates are optimized by rapid analogue synthesis and evaluation, and (3) the optimized substrates are converted to inhibitors by direct replacement of the aminocoumarin with known mechanism-based pharmacophores. The SAS method was successfully applied to the cysteine protease cathepsin S, which is implicated in autoimmune diseases. Multiple distinct classes of nonpeptidic substrates were identified upon screening an N-acyl aminocoumarin library. Two of the nonpeptidic substrate classes were optimized to substrates with >8000-fold improvements in cleavage efficiency for each class. Select nonpeptidic substrates were then directly converted to low molecular weight, novel aldehyde inhibitors with nanomolar affinity to cathepsin S. This study demonstrates the unique characteristics and merits of this first substrate-based method for the rapid identification and optimization of weak fragments and provides the framework for the development of completely nonpeptidic inhibitors to many different proteases.     

Triple-helical transition state analogues: a new class of selective matrix metalloproteinase inhibitors

Alterations in activities of one family of proteases, the matrix metalloproteinases (MMPs), have been implicated in primary and metastatic tumor growth, angiogenesis, and pathological degradation of extracellular matrix (ECM) components, such as collagen and laminin. Since hydrolysis of the collagen triple-helix is one of the committed steps in ECM turnover, we envisioned modulation of collagenolytic activity as a strategy for creating selective MMP inhibitors. In the present study, a phosphinate transition state analogue has been incorporated within a triple-helical peptide template. The template sequence was based on the alpha1(V)436-450 collagen region, which is hydrolyzed at the Gly(439)-Val(440) bond selectively by MMP-2 and MMP-9. The phosphinate acts as a tetrahedral transition state analogue, which mimics the water-bound peptide bond of a protein substrate during hydrolysis. The phosphinate replaced the amide bond between Gly-Val in the P1-P1' subsites of the triple-helical peptide. Inhibition studies revealed Ki values in the low nanomolar range for MMP-2 and MMP-9 and low to middle micromolar range for MMP-8 and MMP-13. MMP-1, MMP-3, and MT1-MMP/MMP-14 were not inhibited effectively. Melting of the triple-helix resulted in a decrease in inhibitor affinity for MMP-2. The phosphinate triple-helical transition state analogue has high affinity and selectivity for the gelatinases (MMP-2 and MMP-9) and represents a new class of protease inhibitors that maximizes potential selectivity via interactions with both prime and nonprime active site subsites as well as with secondary binding sites (exosites).     

Synthesis and evaluation of a new class of tertiary alcohol based BACE-1 inhibitors

BACE-1 has emerged as one of the best characterized targets for future Alzheimer therapy. In accordance with the successful identification of masked inhibitors of HIV-1 protease, we envisioned that tert-alcohol containing transition-state mimicking structures would also be worthwhile evaluating as BACE-1 inhibitors. Twelve novel inhibitors were prepared via synthetic routes using epoxyalcohol derivates as key intermediates. The best synthesized tert-hydroxy inhibitor exhibited a BACE-1 IC₅₀ value of 0.38 μM.     

Dithiocarbamates: a new class of carbonic anhydrase inhibitors. Crystallographic and kinetic investigations

The zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) is inhibited by several classes of zinc-binders (sulfonamides, sulfamates, and sulfamides) as well as by compounds which do not interact with the metal ion (phenols, polyamines and coumarins). Here we report a new class of potent CA inhibitors which bind the zinc ion: the dithiocarbamates (DTCs). They coordinate to the zinc ion from the enzyme active site in monodentate manner and establish many favorable interactions with amino acid residues nearby. Several low nanomolar CA I, II and IX inhibitors were detected.     

Dinucleosides with non-natural backbones: a new class of ribonuclease A and angiogenin inhibitors

Ribonuclease?A (RNase A) serves as a convenient model enzyme in the identification and development of inhibitors of proteins that are members of the ribonuclease superfamily. This is principally because the biological activity of these proteins, such as angiogenin, is linked to their catalytic ribonucleolytic activity. In an attempt to inhibit the biological activity of angiogenin, which involves new blood vessel formation, we employed different dinucleosides with varied non-natural backbones. These compounds were synthesized by coupling aminonucleosides with dicarboxylic acids and amino- and carboxynucleosides with an amino acid. These molecules show competitive inhibition with inhibition constant (K(i)) values of (59±3) and (155±5) μM for RNase A. The compounds were also found to inhibit angiogenin in a competitive fashion with corresponding K(i) values in the micromolar range. The presence of an additional polar group attached to the backbone of dinucleosides was found to be responsible for the tight binding with both proteins. The specificity of different ribonucleolytic subsites were found to be altered because of the incorporation of a non-natural backbone in between the two nucleosidic moieties. In spite of the replacement of the phosphate group by non-natural linkers, these molecules were found to selectively interact with the ribonucleolytic site residues of angiogenin, whereas the cell binding site and nuclear translocation site residues remain unperturbed. Docked conformations of the synthesized compounds with RNase A and angiogenin suggest a binding preference for the thymine-adenine pair over the thymine-thymine pair.     

Using steric hindrance to design new inhibitors of class C beta-lactamases

beta-lactamases confer resistance to beta-lactam antibiotics such as penicillins and cephalosporins. However, beta-lactams that form an acyl-intermediate with the enzyme but subsequently are hindered from forming a catalytically competent conformation seem to be inhibitors of beta-lactamases. This inhibition may be imparted by specific groups on the ubiquitous R(1) side chain of beta-lactams, such as the 2-amino-4-thiazolyl methoxyimino (ATMO) group common among third-generation cephalosporins. Using steric hindrance of deacylation as a design guide, penicillin and carbacephem substrates were converted into effective beta-lactamase inhibitors and antiresistance antibiotics. To investigate the structural bases of inhibition, the crystal structures of the acyl-adducts of the penicillin substrate amoxicillin and the new analogous inhibitor ATMO-penicillin were determined. ATMO-penicillin binds in a catalytically incompetent conformation resembling that adopted by third-generation cephalosporins, demonstrating the transferability of such sterically hindered groups in inhibitor design.     

Development and characterization of nonpeptidic small molecule inhibitors of the XIAP/caspase-3 interaction

Elevated expression of inhibitor of apoptosis protein (IAP) family members in various types of cancers is thought to provide a survival advantage to these cells. Thus, antiapoptotic functions of IAPs, and their potential as novel anticancer targets have attracted considerable interest. Among the IAPs, the X chromosome-linked inhibitor of apoptosis protein (XIAP) is regarded as the most potent suppressor of mammalian apoptosis through direct binding and inhibition of caspases. A high-throughput biochemical screen of a combinatorial chemical library led to the discovery of a novel nonpeptidic small molecule that has the ability to disrupt the XIAP/caspase-3 interaction. The activity of this nonpeptidic small molecule inhibitor of the XIAP/caspase-3 interaction has been characterized both in vitro and in cells. Molecules of this type can be used to conditionally inhibit the cellular function of XIAP and may provide insights into the development of therapeutic agents that act by modulating apoptotic pathways.     

Farnesyl transferase inhibitors: the successes and surprises of a new class of potential cancer chemotherapeutics

Proteins that transmit abnormal growth signals offer enticing points of intervention for the treatment of cancer. The discovery that isoprenoid attachment is required for the aberrant biological activity of oncogenic Ras proteins has provided just such a target.     

The first synthesis of substituted azepanes mimicking monosaccharides: a new class of potent glycosidase inhibitors

The synthesis of the first examples of seven-membered ring iminoalditols, molecules displaying an extra hydroxymethyl substituent on their seven-membered ring compared to the previously reported polyhydroxylated azepanes, has been achieved from d-arabinose in 10 steps using RCM of a protected N-allyl-aminohexenitol as a key step. While the (2R,3R,4R)-2-hydroxymethyl-3,4-dihydroxy-azepane 10, a seven-membered ring analogue of fagomine, is a weak inhibitor of glycosidases, the (2R,3R,4R,5S,6S)-2-hydroxymethyl-3,4,5,6-tetrahydroxy-azepane 9 selectively inhibits green coffee bean alpha-galactosidase in the low micromolar range (Ki = 2.2 muM) despite a D-gluco relative configuration.     

Development of a new nonpeptidic self-immolative spacer. Application to the design of protease sensing fluorogenic probes

The design and synthesis of novel self-immolative spacer systems aiming at the release of phenol-containing compounds are described. The newly designed traceless linkers proved to be conveniently stable under physiological conditions and operate through spontaneous decomposition of an hemithioaminal intermediate under neutral aqueous conditions. Their utility was then illustrated by the preparation of original fluorogenic substrates of penicillin amidase whose strong fluorescence is unveiled through enzyme-initiated domino reactions.     

Serine and threonine beta-lactones: a new class of hepatitis A virus 3C cysteine proteinase inhibitors

Hepatitis A virus (HAV) 3C enzyme is a cysteine proteinase essential for viral replication and infectivity and represents a target for the development of antiviral drugs. A number of serine and threonine beta-lactones were synthesized and tested against HAV 3C proteinase. The D-N-Cbz-serine beta-lactone 5a displays competitive reversible inhibition with a K(i) value of 1.50 x 10(-6) M. Its enantiomer, L-N-Cbz-serine beta-lactone 5b is an irreversible inactivator with k(inact) = 0.70 min(-1), K(Iota) = 1.84 x 10(-4) M and k(inact)/K(Iota) = 3800 M(-1) min(-1). Mass spectrometry and HMQC NMR studies using (13)C-labeled 5b show that inactivation of the enzyme occurs by nucleophilic attack of the cysteine thiol (Cys-172) at the beta-position of the oxetanone ring. Although the N-Cbz-serine beta-lactones 5a and 5b display potent inhibition, other related analogues with an N-Cbz side chain, such as the five-membered ring homoserine gamma-lactones 14a and 14b, the four-membered ring beta-lactam 33, 2-methylene oxetane 34, cyclobutanone 36, and 3-azetidinone 39, fail to give significant inhibition of HAV 3C proteinase, thus demonstrating the importance of the beta-lactone ring for binding.     

2, 3-Diaryl-5-ethylsulfanylmethyltetrahydrofurans as a new class of COX-2 inhibitors and cytotoxic agents

2,3-Diaryl-5-ethylsulfanylmethyltetrahydrofuran-3-ols were designed and synthesized by the allylations of benzoins followed by iodocyclization and nucleophilic replacement reactions with ethanthiol. These molecules exhibit IC(50) for COX-2 at <10 nM concentration and exhibit average GI(50) over all the 59 human tumor cell lines at microM concentration.     

Identification and specificity profiling of protein prenyltransferase inhibitors using new fluorescent phosphoisoprenoids

Posttranslational modification of proteins with farnesyl and geranylgeranyl isoprenoids is a widespread phenomenon in eukaryotic organisms. Isoprenylation is conferred by three protein prenyltransferases: farnesyl transferase (FTase), geranylgeranyl transferase type-I (GGTase-I), and Rab geranylgeranyltransferase (RabGGTase). Inhibitors of these enzymes have emerged as promising therapeutic compounds for treatment of cancer, viral and parasite originated diseases, as well as osteoporosis. However, no generic nonradioactive protein prenyltransferase assay has been reported to date, complicating identification of enzyme-specific inhibitors. We have addressed this issue by developing two fluorescent analogues of farnesyl and geranylgeranyl pyrophosphates {3,7-dimethyl-8-(7-nitro-benzo[1,2,5]oxadiazol-4-ylamino)-octa-2,6-diene-1}pyrophosphate (NBD-GPP) and {3,7,11-trimethyl-12-(7-nitro-benzo[1,2,5]oxadiazo-4-ylamino)-dodeca-2,6,10-trien-1} pyrophosphate (NBD-FPP), respectively. We demonstrate that these compounds can serve as efficient lipid donors for prenyltransferases. Using these fluorescent lipids, we have developed two simple (SDS-PAGE and bead-based) in vitro prenylation assays applicable to all prenyltransferases. Using the SDS-PAGE assay, we found that, in contrast to previous reports, the tyrosine phosphatase PRL-3 may possibly be a dual substrate for both FTase and GGTase-I. The on-bead prenylation assay was used to identify prenyltransferase inhibitors that displayed nanomolar affinity for RabGGTase and FTase. Detailed analysis of the two inhibitors revealed a complex inhibition mechanism in which their association with the peptide binding site of the enzyme reduces the enzyme's affinity for lipid and peptide substrates without competing directly with their binding. Finally, we demonstrate that the developed fluorescent isoprenoids can directly and efficiently penetrate into mammalian cells and be incorporated in vivo into small GTPases.     

Facile, efficient, and enantiospecific syntheses of 1,1'-N-linked pseudodisaccharides as a new class of glycosidase inhibitors

This article describes an efficient synthesis of a potent trehalase inhibitor, 1,1'-N-linked pseudodisaccharide 1 (consisting of two valienamines), in 14 steps with an overall yield of 12% and a first synthesis of 2 (consisting of two 2-epi-valienamines) in 15 steps with an overall yield of 24% from (-)-quinic acid. The synthesis involves a stereospecific palladium-catalyzed coupling reaction between an allylic amine and an allylic chloride as the crucial step. The acetonide blocking groups were shown to be the best hydroxyl protecting groups, compatible with the palladium-catalyzed allylic amination reaction that afforded high yields of the 1,1'-N-linked pseudodisaccharides with a minimum amount of an elimination diene side product.