Synthesis and structure-activity relationships of 5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-based hydroxamic acids as HB-EGF shedding inhibitors

HB-EGF Shedding inhibitors have been expected to become effective medicines for skin diseases caused by the proliferation of keratinocytes. In order to discover novel HB-EGF shedding inhibitors and clarify their structure-activity relationships, 5,6,7,8-tetrahydronaphthylidine-based hydroxamic acid and 5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-based hydroxamic acids have been synthesized. Among the synthesized compounds, the ethoxyethoxy derivative 3o and the methoxypropoxy derivative 3p exhibited much more potent HB-EGF shedding inhibitory activity than CGS 27023A. The structural modification of 5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-based hydroxamic acids enabled us to establish the following structure-activity relationships; the existence of the hydroxamic acid, the sulfonamide, and the phenyl moieties are crucial for a potent HB-EGF shedding inhibitory activity, and the stereochemistry of the alpha carbon of hydroxamic acid is also important. In addition, from the comparison of their HB-EGF shedding inhibitory activities with their MMPs inhibitory activities, we found that the S1' pocket of the responsible enzyme for HB-EGF shedding is deep unlike that of MMP-1.     

Synthesis and biological evaluation of hydroxamate-Based inhibitors of glutamate carboxypeptidase II

A series of hydroxamic acids has been prepared as potential inhibitors of glutamate carboxypeptidase II (GCP II). Compounds based on a P1' residue (primed-side inhibitors) were more potent than those based on a P1 group (unprimed-side inhibitors). Inhibitory potency of the primed-side GCP II inhibitors was found to be dependent on the number of methylene units between the hydroxamate group and pentanedioic acid. Succinyl hydroxamic acid derivative, 2-(hydroxycarbamoylmethyl)pentanedioic acid, is the most potent GCP II inhibitor with an IC(50) value of 220nM. The comparison of the results to those of other classes of GCP II inhibitors as well as hydroxamate-based MMP inhibitors provides further insight into the structure-activity relationships of GCP II inhibition.     

Azaindole N-methyl hydroxamic acids as HIV-1 integrase inhibitors-II. The impact of physicochemical properties on ADME and PK

HIV-1 integrase is one of three enzymes encoded by the HIV genome and is essential for viral replication, and HIV-1 IN inhibitors have emerged as a new promising class of therapeutics. Recently, we reported the discovery of azaindole hydroxamic acids that were potent inhibitors of the HIV-1 IN enzyme. N-Methyl hydroxamic acids were stable against oxidative metabolism, however were cleared rapidly through phase 2 glucuronidation pathways. We were able to introduce polar groups at the β-position of the azaindole core thereby altering physical properties by lowering calculated log D values (c Log D) which resulted in attenuated clearance rates in human hepatocytes. Pharmacokinetic data in dog for representative compounds demonstrated moderate oral bioavailability and reasonable half-lives. These ends were accomplished without a large negative impact on enzymatic and antiviral activity, thus suggesting opportunities to alter clearance parameters in future series.     

Analysis of the binding of hydroxamic acid and carboxylic acid inhibitors to the stromelysin-1 (matrix metalloproteinase-3) catalytic domain by isothermal titration calorimetry.

Matrix metalloproteinases (MMPs) are implicated in diseases such as arthritis and cancer. Among these enzymes, stromelysin-1 can also activate the proenzymes of other MMPs, making it an attractive target for pharmaceutical design. Isothermal titration calorimetry (ITC) was used to analyze the binding of three inhibitors to the stromelysin catalytic domain (SCD). One inhibitor (Galardin) uses a hydroxamic acid group (pK(a) congruent with 8.7) to bind the active site zinc; the others (PD180557 and PD166793) use a carboxylic acid group (pK(a) congruent with 4.7). Binding affinity increased dramatically as the pH was decreased over the range 5.5-7.5. Experiments carried out at pH 6.7 in several different buffers revealed that approximately one and two protons are transferred to the enzyme-inhibitor complexes for the hydroxamic and carboxylic acid inhibitors, respectively. This suggests that both classes of inhibitors bind in the protonated state, and that one amino acid residue of the enzyme also becomes protonated upon binding. Similar experiments carried out with the H224N mutant gave strong evidence that this residue is histidine 224. DeltaG, DeltaH, DeltaS, and DeltaC(p) were determined for the three inhibitors at pH 6.7, and DeltaC(p) was used to obtain estimates of the solvational, translational, and conformational components of the entropy term. The results suggest that: (1) a polar group at the P1 position can contribute a large favorable enthalpy, (2) a hydrophobic group at P2' can contribute a favorable entropy of desolvation, and (3) P1' substituents of certain sizes may trigger an entropically unfavorable conformational change in the enzyme upon binding. These findings illustrate the value of complete thermodynamic profiles generated by ITC in discovering binding interactions that might go undetected when relying on binding affinities alone.     

Design and synthesis of novel histone deacetylase inhibitor derived from nuclear localization signal peptide

We describe herein the synthesis and characterization of a new class of histone deacetylase (HDAC) inhibitors derived from conjugation of a suberoylanilide hydroxamic acid-like aliphatic-hydroxamate pharmacophore to a nuclear localization signal peptide. We found that these conjugates inhibited the histone deacetylase activities of HDACs 1, 2, 6, and 8 in a manner similar to suberoylanilide hydroxamic acid (SAHA). Notably, compound 7b showed a threefold improvement in HDAC 1/2 inhibition, a threefold increase in HDAC 6 selectivity and a twofold increase in HDAC 8 selectivity when compared to SAHA.     

Histone deacetylase inhibitors suppress natural killer cell cytolytic activity

Treatment of transformed cells from leukemia or solid tumors with histone deacetylase inhibitors (HDACi) was shown to increase their sensitivity to NK cell lysis. In this study, treatment of IL-2-activated NK cells with HDACi including suberoylanilide hydroxamic acid and valproic acid was studied. Both drugs at therapeutic concentrations inhibited NK cell cytotoxicity on human leukemic cells. This inhibition was associated with decreased expression and function of NK cell activating receptors NKp46 and NKp30 as well as impaired granule exocytosis. NFkappaB activation in IL-2-activated NK cells was inhibited by both HDACi. Pharmacologic inhibition of NFkappaB activity resulted in similar effects on NK cell activity like those observed for HDACi. These results demonstrate for the first time that HDACi prevent NK cytotoxicity by downregulation of NK cell activating receptors probably through the inhibition of NFkappaB activation.     

Synthesis, enzymatic inhibition, and cancer cell growth inhibition of novel delta-lactam-based histone deacetylase (HDAC) inhibitors

delta-Lactam-based hydroxamic acids, inhibitors of histone deacetylase (HDAC), have been synthesized via ring closure metathesis of key diene intermediates followed by conversion to hydroxamic acid analogues. The hydroxamic acids 12a, 12b, and 17c showed potent inhibitory activity in HDAC enzyme assay. The hydroxamic acid 12b exhibited growth inhibitory activity on five human tumor cell lines, showing good sensitivity on the MDA-MB-231 breast tumor cell.     

Pimelic diphenylamide 106 is a slow, tight-binding inhibitor of class I histone deacetylases

Histone deacetylase (HDAC) inhibitors, including various benzamides and hydroxamates, are currently in clinical development for a broad range of human diseases, including cancer and neurodegenerative diseases. We recently reported the identification of a family of benzamide-type HDAC inhibitors that are relatively non-toxic compared with the hydroxamates. Members of this class of compounds have shown efficacy in cell-based and mouse models for the neurodegenerative diseases Friedreich ataxia and Huntington disease. Considerable differences in IC(50) values for the various HDAC enzymes have been reported for many of the HDAC inhibitors, leading to confusion as to the HDAC isotype specificities of these compounds. Here we show that a benzamide HDAC inhibitor, a pimelic diphenylamide (106), is a class I HDAC inhibitor, demonstrating no activity against class II HDACs. 106 is a slow, tight-binding inhibitor of HDACs 1, 2, and 3, although inhibition for these enzymes occurs through different mechanisms. Inhibitor 106 also has preference toward HDAC3 with K(i) of approximately 14 nm, 15 times lower than the K(i) for HDAC1. In comparison, the hydroxamate suberoylanilide hydroxamic acid does not discriminate between these enzymes and exhibits a fast-on/fast-off inhibitory mechanism. These observations may explain a paradox involving the relative activities of pimelic diphenylamides versus hydroxamates as gene activators.     

Design, synthesis and molecular modeling study of iminodiacetyl monohydroxamic acid derivatives as MMP inhibitors

As the matrix metalloproteinases (MMPs) can be massively up-regulated in degenerative tissues and degrade the extracellular matrix, these key enzymes are promising targets for the therapy of cancer and other degenerative diseases. Here, we are presenting a series of new non-peptidic hydroxamate-based matrix metalloproteinase inhibitors, MMPIs, incorporating the iminodiacetic (IDA) hydroxamic acid scaffold, as mimics of truncated peptidic MMPIs. A series of alkylaryl and sulfonylaryl groups, on the IDA basic scaffold, was investigated with the aim of improving potency and selectivity against MMPs involved in degenerative diseases. The sulfonamide based IDA derivatives studied (compounds B1-B3) showed to be potent (nM range) against deep S1' pocket MMPs enzymes (i.e., MMP-2).     

Proteoglycan-degrading enzymes. A radiochemical assay method and the detection of a new enzyme cathepsin F.

1. Polyacrylamide beads containing entrapped 35S-labelled proteoglycan molecules have been prepared. 2. The measurement of release of radioactivity provides an extremely sensitive assay for proteoglycan-degrading enzymes, including proteinases and hyaluronidase. 3. The amount of label released is a logarithmic function of enzyme concentration or time of incubation. Experiments were made in an attempt to explain this. 4. Assays were made by the new method at several pH values, and with the inclusion of inhibitors to identify the proteoglycan-degrading enzymes of rabbit ear cartilage. 5. A previously undescribed proteinase active against proteoglycan at pH4.5 but unaffected by pepstatin, was discovered. The enzyme was named cathepsin F, and was partially purified and characterized; it was detected in human articular cartilage.     

Modifications around the hydroxamic acid chelating group of fosmidomycin,an inhibitor of the metalloenzyme 1-deoxyxylulose 5-phosphate reductoisomerase (DXR)

Fosmidomycin derivatives in which the hydroxamic acid group has been replaced by several bidentate chelators as potential hydroxamic alternatives were prepared and tested against the DXR from Escherichia coli. These results illustrate the predominant role of the hydroxamate functional group as the most effective metal binding group in DXR inhibitors.     

A quantitative structure-activity relationship study on matrix metalloproteinase inhibitors: piperidine sulfonamide aryl hydroxamic acid analogs

A quantitative structure-activity relationship (QSAR) study has been made on a series of piperidine sulfonamide aryl hydroxamic acid analogs acting as matrix metalloproteinase (MMP) inhibitors. The inhibitory potencies of the compounds against two MMPs, MMP-2 and MMP-13, are found to be significantly correlated with the hydrophobic properties of the molecules, suggesting that in both enzymes the hydrophobic interaction is playing a dominant role.     

Autophagy induced by suberoylanilide hydroxamic acid in Hela S3 cells involves inhibition of protein kinase B and up-regulation of Beclin 1

Histone deacetylase inhibitors are promising chemotherapeutic agents and some are in clinical trials. Several molecular mechanisms have been invoked to describe their effects on cancer cells in vivo and in vitro. Autophagy has been observed in response to several anticancer reagents and has been demonstrated to be responsible for cell death. However, the exact mechanism of this phenomenon is still not clear. Here we demonstrated that suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, induces nonapoptotic cell death with several specific features characteristic of autophagy in Hela S3 cells. Suberoylanilide hydroxamic acid inhibits the activity of the mammalian target of rapamycin, a negative regulator of macroautophagy which induces the formation of autophagosomes in a Beclin 1- and autophagy-related 7-dependent manner. This process is mediated by Akt and tuberous sclerosis 2 as is demonstrated by inhibition by continuous active Akt plasmid transfection and RNA interference of tuberous sclerosis 2. Our data provide the first evidence that suberoylanilide hydroxamic acid induces autophagy in Hela S3 cells through interference with the mammalian target of rapamycin signaling pathway. These findings suggest that suberoylanilide hydroxamic acid may induce autophagic cancer cell death via its specific pathway, and invite further investigation into the detailed mechanism of this pathway to explore this compound's full potential as a chemotherapeutic agent.     

Design, synthesis and biological evaluation of tyrosine-based hydroxamic acid analogs as novel histone deacetylases (HDACs) inhibitors

Histone deacetylases (HDACs) are a promising target for treating cancer and some other disorders. Herein, based on the structure of our previously reported tetrahydroisoquinoline-based hydroxamic acids, a novel series of tyrosine-based hydroxamic acid derivatives was designed and synthesized as HDACs inhibitors. Compared with tetrahydroisoquinoline-based hydroxamic acids, tyrosine-based hydroxamic acid derivatives exhibited more potent HDAC8 inhibitory activity. However, the antiproliferative activities and HeLa cell nuclear extract inhibition of several selected tyrosine-based hydroxamic acids were moderate.     

A quantitative structure-activity relationship study on matrix metalloproteinase inhibitors: Piperidine sulfonamide aryl hydroxamic acid analogs

A quantitative structure-activity relationship (QSAR) study has been made on a series of piperidine sulfonamide aryl hydroxamic acid analogs acting as matrix metalloproteinase (MMP) inhibitors. The inhibitory potencies of the compounds against two MMPs, MMP-2 and MMP-13, are found to be significantly correlated with the hydrophobic properties of the molecules, suggesting that in both enzymes the hydrophobic interaction is playing a dominant role.     

Structural requirements of HDAC inhibitors: SAHA analogs functionalized adjacent to the hydroxamic acid

Inhibitors of histone deacetylase (HDAC) proteins such as suberoylanilide hydroxamic acid (SAHA) have emerged as effective therapeutic anti-cancer agents. To better understand the structural requirements of HDAC inhibitors, a small molecule library with a variety of substituents attached adjacent to the metal binding hydroxamic acid of SAHA was synthesized. The presence of a substituent adjacent to the hydroxamic acid led to an 800- to 5000-fold decrease in inhibition compared to SAHA. The observed results have implications for drug design, suggesting that HDAC inhibitors with substituents near the metal binding moiety will have inhibitory activities in the micromolar rather than nanomolar range.     

Inhibition and structure-activity studies of methionine hydroxamic acid derivatives with bacterial peptide deformylase

The posttranslational deformylation of N-formyl-Met-polypeptides by the metalloenzyme, peptide deformylase, is essential for bacterial growth. Methionine hydroxamic acid derivatives were found to inhibit recombinant Escherichia coli peptide deformylase activity containing either zinc or cobalt. The binding of methionine hydroxamate and hydrazide inhibitors to cobalt-substituted deformylase caused spectral changes consistent with the formation of a pentacoordinate metal complex similar to that of actinonin, a psuedopeptide hydroxamate inhibitor. The spectral and kinetic data support the binding of these N-substituted L-methionine derivatives in a reverse orientation with respect to N-formyl-Met-peptide substrates within the active site. Based on this hypothesis a second generation of N-substituted methionyl hydroxamic acids were evaluated and found to possess greater inhibitory potency. These results may provide the basis for the design of more potent and selective deformylase inhibitors as potential antibacterial agents.     

Simple inhibitors of histone deacetylase activity that combine features of short-chain fatty acid and hydroxamic acid inhibitors

Butyric acid and trichostatin A (TSA) are anti-cancer compounds that cause the upregulation of genes involved in differentiation and cell cycle regulation by inhibiting histone deacetylase (HDAC) activity. In this study we have synthesized and evaluated compounds that combine the bioavailability of short-chain fatty acids, like butyric acid, with the bidentate binding ability of TSA. A series of analogs were made to examine the effects of chain length, simple aromatic cap groups, and substituted hydroxamates on the compounds' ability to inhibit rat-liver HDAC using a fluorometric assay. In keeping with previous structure-activity relationships, the most effective inhibitors consisted of longer chains and hydroxamic acid groups. It was found that 5-phenylvaleric hydroxamic acid and 4-benzoylbutyric hydroxamic acid were the most potent inhibitors with IC50's of 5 microM and 133 microM respectively.