Hydroxamic Moiety Research Articles

Design and Synthesis of sEH/HDAC6 Dual-Targeting Inhibitors for the Treatment of Inflammatory Pain.

Soluble epoxide hydrolase (sEH) and HDAC6 mediate the NF-κB pathway in inflammatory responses, and their inhibitors exhibit powerful anti-inflammatory and analgesic activities in treating both inflammation and pain. Therefore, a series of dual-targeting inhibitors containing urea or squaramide and hydroxamic acid moieties were designed and synthesized, and their role as a new sEH/HDAC6 dual-targeting inhibitor in inflammatory pain was evaluated in a formalin-induced mice model and a xylene-induced mouse ear swelling model. Among them, compounds 28g and 28j showed the best inhibitory and selectivity of sEH and HDAC6. Compound 28g had satisfactory pharmacokinetic characteristics in rats. Following administration at 30 mg/kg, compound 28g exhibited more effective analgesic activity than either an sEH inhibitor (GL-B437) or an HDAC6 inhibitor (Rocilinostat) alone and coadministration of both inhibitors. Thus, these novel sEH/HDAC6 dual-targeting inhibitors exhibited powerful analgesic activity in nociceptive behavior and are worthy of further development.

Enhancing anti-cancer capacity: Novel class I/II HDAC inhibitors modulate EMT, cell cycle, and apoptosis pathways

Cancer has been a leading cause of death over the last few decades in western countries as well as in Taiwan. However, traditional therapies are limited by the adverse effects of chemotherapy and radiotherapy, and tumor recurrence may occur. Therefore, it is critical to develop novel therapeutic drugs. In the field of HDAC inhibitor development, apart from the hydroxamic acid moiety, 2-aminobenzamide also functions as a zinc-binding domain, which is shown in well-known HDAC inhibitors such as Entinostat and Chidamide. With recent successful experiences in synthesizing 1-(phenylsulfonyl)indole-based compounds, in this study, we further combined two features of the above chemical compounds and generated indolyl benzamides. Compounds were screened in different cancer cell lines, and enzyme activity was examined to demonstrate their potential for anti-HDAC activity. Various biological functional assays evidenced that two of these compounds could suppress cancer growth and migration capacity, through regulating epithelial–mesenchymal transition (EMT), cell cycle, and apoptosis mechanisms. Data from 3D cancer cells and the in vivo zebrafish model suggested the potential of these compounds in cancer therapy in the future.

Bifunctional octadentate pseudopeptides as Zirconium-89 chelators for immuno-PET applications

BackgroundPositron emission tomography (PET) is a highly sensitive method that provides fine resolution images, useful in the field of clinical diagnostics. In this context, Zirconium-89 (89Zr)-based imaging agents have represented a great challenge in molecular imaging with immuno-PET, which employs antibodies (mAbs) as biological vectors. Indeed, immuno-PET requires radionuclides that can be attached to the mAb to provide stable in vivo conjugates, and for this purpose, the radioactive element should have a decay half-life compatible with the time needed for the biodistribution of the immunoglobulin. In this regard, 89Zr is an ideal radioisotope for immuno-PET because its half-life perfectly matches the in vivo pharmacokinetics of mAbs.ResultsThe main objective of this work was the design and synthesis of a series of bifunctional octadentate pseudopeptides able to generate stable 89Zr complexes. To achieve this, here we investigated hydroxamate, N-methylhydroxamate and catecholate chelating moieties in complexing radioactive zirconium. N-methylhydroxamate proved to be the most effective 89Zr-chelating group. Furthermore, the increased flexibility and hydrophilicity obtained by using polyoxyethylene groups spacing the hydroxamate units led to chelators capable of rapidly forming (15 min) stable and water-soluble complexes with 89Zr under mild reaction conditions (aqueous environment, room temperature, and physiological pH) that are mandatory for complexation reactions involving biomolecules. Additionally, we report challenge experiments with the competitor ligand EDTA and metal ions such as Fe3+, Zn2+ and Cu2+. In all examined conditions, the chelators demonstrated stability against transmetallation. Finally, a maleimide moiety was introduced to apply one of the most promising ligands in bioconjugation reactions through Thiol-Michael chemistry.ConclusionCombining solid phase and solution synthesis techniques, we identified novel 89Zr-chelating molecules with a peptide scaffold. The adopted chemical design allowed modulation of molecular flexibility, hydrophilicity, as well as the decoration with different zirconium chelating groups. Best results in terms of 89Zr-chelating properties were achieved with the N-methyl hydroxamate moiety. The Zirconium complexes obtained with the most effective compounds were water-soluble, stable to transmetallation, and resistant to peptidases for at least 6 days. Further studies are needed to assess the potential of this novel class of molecules as Zirconium-chelating agents for in vivo applications.

Potentiating Activity of GmhA Inhibitors on Gram-Negative Bacteria.

Inhibition of the biosynthesis of bacterial heptoses opens novel perspectives for antimicrobial therapies. The enzyme GmhA responsible for the first committed biosynthetic step catalyzes the conversion of sedoheptulose 7-phosphate into d-glycero-d-manno-heptose 7-phosphate and harbors a Zn2+ ion in the active site. A series of phosphoryl- and phosphonyl-substituted derivatives featuring a hydroxamate moiety were designed and prepared from suitably protected ribose or hexose derivatives. High-resolution crystal structures of GmhA complexed to two N-formyl hydroxamate inhibitors confirmed the binding interactions to a central Zn2+ ion coordination site. Some of these compounds were found to be nanomolar inhibitors of GmhA. While devoid of HepG2 cytotoxicity and antibacterial activity of their own, they demonstrated in vitro lipopolysaccharide heptosylation inhibition in Enterobacteriaceae as well as the potentiation of erythromycin and rifampicin in a wild-type Escherichia coli strain. These inhibitors pave the way for a novel treatment of Gram-negative infections.

In Silico Profiling of Histone Deacetylase 8 Inhibitory Activity: A Computational Analysis of Novel Dipeptide-Based Compounds Cross-Linked with Hydroxamic Acid

This study involved the development of innovative compounds consisting of dipeptide cross-links combined with hydroxamic acid. Our objective was to assess their binding affinities with histone deacetylase 8 (HDAC8) by conducting a docking study, comparing the results with the reference ligand, suberoylanilide hydroxamic acid (SAHA). Docking scores were measured in terms of ΔG (Kcal/mol), and the recorded scores for compounds 2A-D were found to be higher than that of SAHA, with values of 87.36, 80.46, 79.42, and 74.14, respectively. Notably, compound 2A, a dipeptide consisting of L-tryptophyl-L-tyrosine linked to a hydroxamic acid moiety, exhibited the highest docking score of 87.36. This finding suggests that compound 2A may possess the most potent HDAC8 inhibitory activity among the other designed compounds. Furthermore, we utilized the SwissADME server to predict the physicochemical properties and additional ADME parameters for the designed compounds. The analysis revealed that all investigated compounds exhibited a high potential for passive oral absorption and demonstrated no penetration into the blood-brain barrier. Compound 2A, 2B, and 2D exhibited one Lipinski's rule violation each, whereas Compound 2C demonstrated no such violations in all parameters. Additionally, compounds 2A and 2C exhibited potential as P-glycoprotein (P-gp) substrates. SAHA did not exhibit inhibition of any of the cytochrome P450 (CYP) enzymes used in this study, whereas compounds 2B, 2C and 2D displayed possible inhibitory activities. These compelling findings provide encouraging prospects for the future synthesis of the designed compounds and warrant further evaluation through in vitro and in vivo biological studies.

A Mild and Modular Approach to the Total Synthesis of Desferrioxamine B.

A mild and modular approach to the total synthesis of the WHO-listed essential medicine desferrioxamine B is described. Hydroxamic acid fragments were installed under mild conditions, a generalized divergent acylation procedure used to access two monomer precursors, and a transfer hydrogenation reaction used to unmask the hydroxamic acid moieties. Desferrioxamine B was generated over ten linear steps as the formate salt in 17% overall yield using standard amide coupling conditions or in 13% overall yield using microwave-assisted amide coupling conditions.

Alkoxy-Functionalized Hydroxamate/Zinc Metal-Organic Frameworks and the Effects of Substituents and Acid Addition on Their Structures.

Single crystals of alkoxy-functionalized hydroxamate/zinc metal-organic frameworks (MOFs) were obtained by fixating the hydroxamate moiety via intramolecular hydrogen bonding. The resulting MOF structures depend on the steric demand of the alkoxy groups, whereby the incorporation of bulky isopropyl groups affords porous hydroxamate/zinc MOFs. The topological structures of the isopropyl-substituted MOFs could be controlled by adding acid.

Identification of novel phenylalanine derivatives bearing a hydroxamic acid moiety as potent quorum sensing inhibitors.

Phenylalanine derivatives are a well-known small moiety responsible for controlling the virulence factors of several bacteria. Herein, for the first time, we report novel structures of phenylalanine derivatives bearing a hydroxamic acid moiety which were designed, synthesized, and evaluated for use as quorum sensing inhibitors. Biological results reveal that six compounds showed good quorum sensing inhibitors properties with an IC50 ranging from 7.12 ± 2.11 μM-92.34 ± 2.09 μM (4NPO, a reference compound, IC50 = 29.13 ± 0.88 μM). In addition, three out of the six compounds (4a, 4c, 4h) showed strong anti-biofilm formation and CviR inhibitory activity when compared to that of 4NPO. These biological data were also confirmed by computational studies. In this series of compounds, 4h is the most promising compound for future drug development targeting quorum sensing. Our results concluded that the fragment-based drug design is a good approach for the discovery of novel quorum-sensing inhibitors in the future.

Inhibition of DXR in the MEP pathway with lipophilic N-alkoxyaryl FR900098 analogs.

In Mycobacterium tuberculosis (Mtb) and Plasmodium falciparum (Pf), the methylerythritol phosphate (MEP) pathway is responsible for isoprene synthesis. This pathway and its products are vital to bacterial/parasitic metabolism and survival, and represent an attractive set of drug targets due to their essentiality in these pathogens but absence in humans. The second step in the MEP pathway is the conversion of 1-deoxy-d-xylulose-5-phosphate (DXP) to MEP and is catalyzed by 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR). Natural products fosmidomycin and FR900098 inhibit DXR, but are too polar to reach the desired target inside some cells, such as Mtb. Synthesized FR900098 analogs with lipophilic substitution in the position α to the phosphorous atom showed promise, resulting in increased activity against Mtb and Pf. Here, an α substitution, consisting of a 3,4-dichlorophenyl substituent, in combination with various O-linked alkylaryl substituents on the hydroxamate moiety is utilized in the synthesis of a novel series of FR900098 analogs. The purpose of the O-linked alkylaryl substituents is to further enhance DXR inhibition by extending the structure into the adjacent NADPH binding pocket, blocking the binding of both DXP and NADPH. Of the initial O-linked alkylaryl substituted analogs, compound 6e showed most potent activity against Pf parasites at 3.60 μM. Additional compounds varying the phenyl ring of 6e were synthesized. The most potent phosphonic acids, 6l and 6n, display nM activity against PfDXR and low μM activity against Pf parasites. Prodrugs of these compounds were less effective against Pf parasites but showed modest activity against Mtb cells. Data from this series of compounds suggests that this combination of substituents can be advantageous in designing a new generation of antimicrobials.

Exploring aminomethylcoumarins: versatile synthesis, structural diversity, and ADME prediction

This research presents a highly efficient method for synthesizing diverse aminomethylcoumarin libraries through the interaction of Mannich bases of coumarins and primary amines. The developed amination process demonstrated versatility and compatibility with various substituents. Reactions were completed within short timeframes, yielding high-purity products with substantial yields, as well as facilitating the scale-up of the process. The synthesized derivatives exhibited structural diversity, incorporating carboxylic and amino groups, as well as amide, hydrazide, and hydroxamic acid moieties. In silico ADME predictions highlighted the potential of these aminomethylcoumarins as promising candidates for further optimization in the development of oral chemotherapeutic agents

Discovery of benzamide-based PI3K/HDAC dual inhibitors with marked pro-apoptosis activity in lymphoma cells

Inhibition of PI3K and histone deacetylase (HDAC) activity simultaneously using a single molecule appears to be a promising approach for cancer treatment. Current PI3K/HDAC dual inhibitors commonly use hydroxamate moiety as zinc binding group, which lack HDAC isoform selectivity and have potential genotoxicity. In this study, a novel series of benzamide-based PI3K/HDAC dual inhibitors were rationally designed and synthesized. Representative compound PH14 showed potent inhibitory activity toward PI3Kα and HDAC3, with IC50 values of 20.3 nM and 24.5 nM, respectively. This was further supported by the blockage of AKT phosphorylation and an increase in acetylated histone H3 levels in Western blot study. The advantage of simultaneously targeting PI3Kα and HDAC is not only reflected in the significant antiproliferative activity, but also in its ability to promote the apoptosis in Jeko-1 cells. Moreover, PH14 had weak inhibitory effects on CYP450 enzymes and hERG. In the pharmacokinetic study, the administration of 1 mg/kg of PH14 the administration of 1 mg/kg of PH14 resulted in a t1/2 of 10 h and an AUC (0-∞) of 2772 h ng/mL. Our study may provide ideas for the further development of novel HDAC/PI3K dual inhibitors.

Probing a distinct druggable tubulin binding site with gatorbulins 1–7, their metabolic and physicochemical properties, and pharmacological consequences

Microtubules, consisting of α/β-tubulin heterodimers, are prime targets for anticancer drug discovery. Gatorbulin-1 (GB1, 1a) is a recently described marine natural product that targets tubulin at a new, seventh pharmacological site at the tubulin intradimer interface. Using our previously developed robust route towards GB1 (1a), we synthesized simplified, first-generation gatorbulins, GB2–7 (1b–1g) of this highly modified cyclodepsipeptide (GB1) that does not contain any proteinogenic amino acid. We systematically investigated the structure-activity relationship at the biochemical and cellular level using GB1-susceptible ovarian and cervical cancer cells. We validated that the hydroxamate moiety in the N-methyl-alanine residue is critical for activity. All other structural modifications present in GB1, including C-hydroxylation of asparagine, methylation at C-4 of proline, and sp2 hybridization in dehydro-alanine, were proven to be functionally relevant. Replacement of the primary amide with a methyl ester also resulted in reduced activity, indicating the intricate scaffold optimization by the GB1-producing cyanobacterium. Inhibition of tubulin polymerization in vitro and binding affinities correlated very well, translating into differentials in cellular efficacy. We used docking and molecular dynamics to evaluate the effects of the chemical simplification at the structural level, indicating that each modification resulted in loss of target interactions, although energetically modest. Similar to cevipabulin that targets two different sites on the tubulin dimer, GB1 promotes proteasome-mediated tubulin degradation but by an unknown mechanism, presumably distinct from that of cevipabulin. Comparison with cevipabulin indicated that this compound binds to the same tubulin region as GB1 (1a), although the binding mode is distinct. Cevipabulin almost exclusively interacts with α-tubulin, including nonexchangeable GTP. In contrast, GB1 (1a) makes extensive contact and hydrogen bonds with both α- and β-chains of tubulin. GB1-7 showed excellent solubility and much higher than that of paclitaxel. Hepatic microsome stability was excellent, human cytochrome P450s were not inhibited and plasma binding was minimal with high free fractions. Passive permeability was predicted to be high based on PAMPA. Parent compound GB1 (1a) was further evaluated using a cellular model with MDCK cells stably transduced with the human efflux transporter MDR1/P-gp, showing similar permeability with and against transporter gradient, indicating that GB1 (1a) is a poor P-gp substrate.

Exploring new histone deacetylase 6 inhibitors and their effects on reversing the α-tubulin deacetylation and cell morphology changes caused by methamphetamine.

Indazole-based HDAC6 inhibitors with novel zinc-binding modifications were synthesized and evaluated to determine their potential to inhibit HDAC6. The analogs were subjected to a histone deacetylase (HDAC) enzyme assay, which led to identification of compounds 3a and 3b. Both compounds demonstrated higher potency and selectivity as HDAC6 inhibitors with IC50 values of 9.1nM and 9.0nM, respectively, and highlighted the importance of the hydroxamic acid moiety for binding to Zn2+ inside the catalytic pocket of HDAC enzymes. In the neuroblastoma SH-SY5Y cell line, both compounds efficiently acetylated α-tubulin but not histone H3 at a low concentration of 0.5µM. Moreover, compounds 3a and 3b effectively reversed the deacetylation of α-tubulin caused by methamphetamine in the SH-SY5Y cell line, suggesting the potential usefulness of HDAC6 selective inhibition in restoring blood brain barrier integrity by reversing methamphetamine-induced deacetylation.

A chemical catalyst enabling histone acylation with endogenous acyl-CoA

Life emerges from a network of biomolecules and chemical reactions catalyzed by enzymes. As enzyme abnormalities are often connected to various diseases, a chemical catalyst promoting physiologically important intracellular reactions in place of malfunctional endogenous enzymes would have great utility in understanding and treating diseases. However, research into such small-molecule chemical enzyme surrogates remains limited, due to difficulties in developing a reactive catalyst capable of activating inert cellular metabolites present at low concentrations. Herein, we report a small-molecule catalyst, mBnA, as a surrogate for a histone acetyltransferase. A hydroxamic acid moiety of suitable electronic characteristics at the catalytic site, paired with a thiol-thioester exchange process, enables mBnA to activate endogenous acyl-CoAs present in low concentrations and promote histone lysine acylations in living cells without the addition of exogenous acyl donors. An enzyme surrogate utilizing cellular metabolites will be a unique tool for elucidation of and synthetic intervention in the chemistry of life and disease.

Heterologous Production and Biosynthesis of Threonine-16:0dioic acids with a Hydroxamate Moiety.

Dereplication and genome mining in Streptomyces aureus LU18118 combined with heterologous expression of selected biosynthetic gene clusters (BGCs) led to the discovery of various threonine-16:0dioic acids named lipothrenins. Lipothrenins consist of the core elements l-Thr, d-allo-Thr, or Dhb, which are linked to hexadecanedioic acid by an amide bond. The main compound lipothrenin A (1) carries the N-hydroxylated d-allo form of threonine and expresses a siderophore activity. The lipothrenin BGC was analyzed by a series of deletion experiments. As a result, a variety of interesting genes involved in the recruitment and selective activation of linear 16:0dioic acids, amide bond formation, and the epimerization of l-Thr were revealed. Furthermore, a diiron N-oxygenase was identified that may be directly involved in the monooxygenation of the amide bond. This is divergent from the usual hydroxamate formation mechanism in siderophores, which involves hydroxylation of the free amine prior to amide bond formation. Siderophore activity was observed for all N-hydroxylated lipothrenins by application of the CAS assay method.

Talarolides Revisited: Cyclic Heptapeptides from an Australian Marine Tunicate-Associated Fungus, Talaromyces sp. CMB-TU011.

Application of a miniaturized 24-well plate system for cultivation profiling (MATRIX) permitted optimization of the cultivation conditions for the marine-derived fungus Talaromyces sp. CMB-TU011, facilitating access to the rare cycloheptapeptide talarolide A (1) along with three new analogues, B-D (2-4). Detailed spectroscopic analysis supported by Marfey's analysis methodology was refined to resolve N-Me-l-Ala from N-Me-d-Ala, l-allo-Ile from l-Ile and l-Leu, and partial and total syntheses of 2, and permitted unambiguous assignment of structures for 1 (revised) and 2-4. Consideration of diagnostic ROESY correlations for the hydroxamates 1 and 3-4, and a calculated solution structure for 1, revealed how cross-ring H-bonding to the hydroxamate moiety influences (defines/stabilizes) the cyclic peptide conformation. Such knowledge draws attention to the prospect that hydroxamates may be used as molecular bridges to access new cyclic peptide conformations, offering the prospect of new biological properties, including enhanced oral bioavailability.

Synthesis and Biological Evaluation of Carbonic Anhydrase III and IX Inhibitors using Gas Chromatography with Modified pH Sensitive Pellets

Fifteen compounds were synthesized and tested as potential carbonic anhydrase III (CAIII) and carbonic anhydrase IX (CAIX) inhibitors, six of which are novel. Amides (a1-4), hydroxamic acids (b1-2), and imines (c1-9) derivatives were evaluated for their inhibitory activity against CAII and CAIX using gas chromatography with modified pH-sensitive pellets. The derivatives showed inhibition percentages between 12-56% for CAIII and 44-59% for CAIX, compared to 49% and 63% for captopril (the positive control), respectively. Imines showed the best inhibition of CAIII, while all derivatives showed comparable activity against CAIX. It is hypothesized that the nitrogen atom in imine, amide, or hydroxamic acid moieties in the vicinity of an ionizable group is in coordination with the zinc ion in the active site. Furthermore, the candidates were tested for their antimicrobial and antifungal activity. Generally, they showed low to zero activity against some gram-positive and negative bacteria. This supports the theory of their ability to bind to human carbonic anhydrase but not to bacterial one. These compounds could serve as useful scaffolds to develop more potent and selective carbonic anhydrase inhibitors as anti-obesity and anticancer candidates.

Melatonin-vorinostat hybrid ligands show higher histone deacetylase and cancer cell growth inhibition than vorinostat.

Anticancer drug conjugates are an emerging approach for future cancer treatment. Here, we report a series of hybrid ligands merging the neurohormone melatonin with the approved histone deacetylase (HDAC) inhibitor vorinostat, using melatonin's amide side chain (3a-e), its indolic nitrogen (5a-d), and its ether oxygen (7a-d) as attachment points. Several hybrid ligands showed higher potency thanvorinostat in both HDAC inhibition and cellular assays on different cultured cancer cell lines. In the most potent HDAC1 and HDAC6 inhibitors, 3e, 5c, and 7c, the hydroxamic acid moiety of vorinostat is linked to melatonin through a hexamethylene spacer. Hybrid ligands 5c and 7c were also found to be potent growth inhibitors of MCF-7, PC-3M-Luc, and HL-60 cancer cell lines. As these compounds showed only weak agonist activity at melatonin MT1 receptors, the findings indicate that their anticancer actions are driven by HDAC inhibition.

Front Cover: Development of LpxH Inhibitors Chelating the Active Site Dimanganese Metal Cluster of LpxH (ChemMedChem 11/2023)

The Front Cover shows the design of a potent LpxH inhibitor by attaching an acyl hydroxamate moiety that chelates the active site dimanganese cluster. The overall shape of the inhibitor resembles a dragon reaching out to claw the pearl. The dragon art was created/hand-drawn by Julia Zhou, Durham Academy Middle School. More information can be found in the Research Article by Seung-Hwa Kwak, C. Skyler Cochrane, Pei Zhou, Jiyong Hong et al.

Discovery of indole-piperazine derivatives as selective histone deacetylase 6 inhibitors with neurite outgrowth-promoting activities and neuroprotective activities

Novel indole-piperazine derivatives with a hydroxamic acid moiety were designed and synthesized as selective histone deacetylase 6 (HDAC6) inhibitors. In enzymatic assays, all compounds exhibited nanomolar IC50 values. N-hydroxy-4-((4-(7-methyl-1H-indole-3-carbonyl)piperazin-1-yl)methyl)benzamide, 9c, was the most potent HDAC6 inhibitor (IC50, 13.6 nM). In vitro, 9c induced neurite outgrowth of PC12 cells without producing toxic effects, better than Tubastatin A (Tub A). Additionally, 9c demonstrated blatant neuroprotective activity in PC12 cells against H2O2-induced oxidative damage. In western blot assay, 9c could increase the acetylation of α-tubulin in a dose-dependent manner.