[Changing Concepts About Optimal Target Blood Pressure and the Therapeutic Advantages of Azilsartan for Achieving it].

The article discusses current issues of the treatment of arterial hypertension. According to presented data, so-called therapeutic nihilism is becoming one of the main barriers to achieving target blood pressure (BP). This nihilism is that despite evidence of the effectiveness of achieving lower BP values, practitioners do not intensify antihypertensive therapy sufficiently to achieve such values. The article specially addresses new criteria for the effectiveness of antihypertensive therapy, which reflect the therapy sustainability. The most commonly used indicator is the duration of the period, during which systolic BP remains in the therapeutic range. The prognostic significance of such indicators is discussed. In these conditions, it is very important to use the most effective antihypertensive drugs for initial antihypertensive therapy, including as a part of combination therapy. This tactic provides more frequent achievement of BP goals without the need for dose adjustment. In this regard, a systematic review was performed, which included sufficiently large randomized studies of the antihypertensive effectiveness of azilsartan medoxomil. This systematic review will provide comprehensive information on a possible role of using the angiotensin II receptor blocker azilsartan as a basic drug for the treatment of a wide range of patients with high BP. Most of the studies included in the systematic review assessed the effectiveness of combination therapy including azilsartan.

Bovine carbonic anhydrase (bCA) inhibitors: Synthesis, molecular docking and theoretical studies of bisoxadiazole-substituted sulfonamide derivatives.

This paper describes the in vitro inhibition potential of bisoxadiazole-substituted sulfonamide derivatives (6a-t) against bovine carbonic anhydrase (bCA) after they were designed through computational analyses and evaluated the predicted interaction via molecular docking. First, in silico ADMET predictions and physicochemical property analysis of the compounds provided insights into solubility and permeability, then density functional theory (DFT) calculations were performed to analyse their ionization energies, nucleophilicity, in vitro electron affinity, dipole moments and molecular interactions under vacuum and dimethyl sulfoxide (DMSO) conditions. After calculating the theoretical inhibition constants, IC50 values determined from enzymatic inhibition were found between 12.93 and 45.77 µM. Molecular docking evaluation revealed favorable hydrogen bonding and π-interactions of the compounds within the bCA active site. The experimentally most active compound, 6p, exhibited the strongest inhibitory activity with a theoretical inhibition constant value of 9.41 nM and H-bonds with Gln91, Thr198, and Trp4 residues and His63 Pi-cation interactions with His63 residues. Overall, the study reveals promising bCA blocking potential for the synthesized derivatives, similar to acetazolamide.

A host-directed oxadiazole compound potentiates antituberculosis treatment via zinc poisoning in human macrophages and in a mouse model of infection.

Antituberculosis drugs, mostly developed over 60 years ago, combined with a poorly effective vaccine, have failed to eradicate tuberculosis. More worryingly, multiresistant strains of Mycobacterium tuberculosis (MTB) are constantly emerging. Innovative strategies are thus urgently needed to improve tuberculosis treatment. Recently, host-directed therapy has emerged as a promising strategy to be used in adjunct with existing or future antibiotics, by improving innate immunity or limiting immunopathology. Here, using high-content imaging, we identified novel 1,2,4-oxadiazole-based compounds, which allow human macrophages to control MTB replication. Genome-wide gene expression analysis revealed that these molecules induced zinc remobilization inside cells, resulting in bacterial zinc intoxication. More importantly, we also demonstrated that, upon treatment with these novel compounds, MTB became even more sensitive to antituberculosis drugs, in vitro and in vivo, in a mouse model of tuberculosis. Manipulation of heavy metal homeostasis holds thus great promise to be exploited to develop host-directed therapeutic interventions.

New oxadiazole and pyrazoline derivatives as anti-proliferative agents targeting EGFR-TK: design, synthesis, biological evaluation and molecular docking study.

Two new series of oxadiazole and pyrazoline derivatives were designed and synthesized as promising EGFR-TK inhibitors. The in vitro antiproliferative activity was studied against three human cancer cell lines; HCT116, HepG-2 and MCF7 using MTT assay. Compound 10c showed the most potent anticancer activity against all cancer cell lines, with IC50 range of 1.82 to 5.55 µM, while proving safe towards normal cells WI-38 (IC50 = 41.17 µM) compared to the reference drug doxorubicin (IC50 = 6.72 µM). The most active candidates 5a, 9b, 10a, 10b and 10c were further assessed for their EGFR-TK inhibition. The best of which, compounds 5a and 10b showed IC50 of 0.09 and 0.16 µM respectively compared to gefitinib (IC50 = 0.04 µM). Further investigation against other EGFR family members, showed that 5a displayed good activities against HER3 and HER4 with IC50 values 0.18 and 0.37 µM, respectively compared to gefitinib (IC50 = 0.35 and 0.58 µM, respectively). Furthermore, 5a was evaluated for cell cycle distribution and apoptotic induction on HepG-2 cells. It induced mitochondrial apoptotic pathway and increased accumulation of ROS. Molecular docking study came in agreement with the biological results. Compounds 5a and 10b showed promising drug-likeness with good physicochemical properties.

Selective Potentiation of the (α4)3(ß2)2 Nicotinic Acetylcholine Receptor Response by NS9283 Analogues.

NS9283, 3-(3-pyridyl)-5-(3-cyanophenyl)-1,2,4-oxadiazole, is a selective positive allosteric modulator of (α4)3(ß2)2 nicotinic acetylcholine receptors (nAChRs). It has good subtype selective therapeutic potential afforded by its specific binding to the unique α4-α4 subunit interface present in the (α4)3(ß2)2 nAChR. However, there is currently a lack of structure activity relationship (SAR) studies aimed at developing a class of congeners endowed with the same profile of activity that can help consolidate the druggability of the α4-α4 subunit interface. In this study, new NS9283 analogues were designed, synthesized, and characterized for their ability to selectively potentiate the ACh activity at heterologous (α4)3(ß2)2 nAChRs vs nAChR subtypes (α4)2(ß2)3, α5α4ß2, and α7. With few exceptions, all the NS9283 analogues exerted positive modulation of the (α4)3(ß2)2 nAChR ACh-evoked responses. Above all, those modified at the 3-cyanophenyl moiety by replacement with 3-nitrophenyl (4), 4-cyanophenyl (10), and N-formyl-4-piperidinyl (20) showed the same efficacy as NS9283, although with lower potency. Molecular dynamics simulations of NS9283 and some selected analogues highlighted consistency between potentiation activity and pose of the ligand inside the α4-α4 site with the main interaction being with the complementary (-) side and induction of a significant conformational change of the Trp156 residue in the principal (+) side.

Discovery of novel polyheterocyclic neuraminidase inhibitors with 1,3,4-oxadiazole thioetheramide as core backbone.

Inspired by our earlier findings regarding neuraminidase (NA) inhibitors interacting with 150-cavity or 430-cavity of NA, sixteen novel polyheterocyclic NA inhibitors with 1,3,4-oxadiazole thioetheramide as core backbone were designed and synthesized based on the lead compound ZINC13401480. Of the synthesized compounds, compound N5 targeting 150-cavity exerts the best inhibitory activity against the wild-type H5N1 NA, with IC50 value of 0.14 µM, which is superior to oseltamivir carboxylate (OSC) (IC50 = 0.31 µM). Compound N10 targeting 430-cavity exhibits the best activity against the H5N1-H274Y mutant NA. Although the activity of N10 is comparable to that of OSC for wild-type H5N1 inhibition, it is approximately 60-fold more potent than OSC against the H274Y mutant, suggesting that it is not easy for the virus to develop drug resistance and is attractive for drug development. N10 (EC50 = 0.11 µM) also exhibits excellent antiviral activity against H5N1, which is superior to the positive control OSC (EC50 = 1.47 µM). Molecular docking study shows that the occupation of aromatic fused rings and oxadiazole moiety at the active site and the extension of the substituted phenyl to the 150-cavity or 430-cavity make great contributions to the good potency of this series of polyheterocyclic NA inhibitors. Some advancements in the discovery of effective target-specific NA inhibitors in this study may offer some assistance in the development of more potent anti-influenza drugs.

New daphnane diterpenoidal 1,3,4-oxdiazole derivatives as potential anti-hepatoma agents: Synthesis, biological evaluation and molecular modeling studies.

Hepatocellular carcinoma (HCC) is a major challenge for human healthy. Daphnane-type diterpenes have attracted increasingly attention due to remarkable pharmaceutical potential including anti-HCC activity. To further develop this class of compounds as inhibitors of HCC, the daphnane diterpenoids 12-O-debenzoyl-Yuanhuacine (YHC) and 12-hydroxydaphnetoxin (YHE) were prepared by a standard chemical transformation from dried flower buds of the Daphne genkwa plant. Subsequently, 22 daphnane diterpenoidal 1,3,4-oxdiazole derivatives were rationally designed and synthesized based on YHC and YHE. The assessment of the target compound's anti-hepatocellular carcinoma activity revealed that YHC1 exhibited comparable activity to sorafenib in the Hep3B cell line, while demonstrating higher selectivity. The mechanistic investigation demonstrates that compound YHC1 induces cell cycle arrest at the G0/G1 phase, cellular senescence, apoptosis, and elevates cellular reactive oxygen species levels. Moreover, molecular docking and CETSA results confirm the interaction between YHC1 and YAP1 as well as TEAD1. Co-IP experiments further validated that YHC1 can effectively inhibit the binding of YAP1 and TEAD1. In conclusion, YHC1 selectively targets YAP1 and TEAD1, exhibiting its anti-hepatocellular carcinoma effects through the inhibition of their interaction.

Synthesis and antimicrobial activity of thiophene-based heterocycles derived from thiophene-2-carbohydrazide.

Aim: Thiophene-based heterocycles were synthesized and evaluated for their antimicrobial activity against methicillin-resistant Staphylococcus aureus, Escherichia coli, Clostridium difficile and Candida albicans strains. Methods: Antimicrobial activity was determined using the broth microdilution method. Results: Spiro-indoline-oxadiazole 17 displayed the highest activity against C. difficile while having no effects against other bacterial strains. Compounds 8 and 16 displayed strong effects against TolC, an outer membrane protein, mutant E. coli. The results of computational chemical study and outcomes of experiments were in good agreement. A molecular docking study was conducted using a molecular operating environment to simulate the binding energies of the potent compounds with D-alanine ligase protein. Conclusion: This study suggests that spiro-indoline-oxadiazole 17 could be a good anticlostridial agent.

A series of thiophene-based heterocycles was synthesized and evaluated for their antimicrobial activity against methicillin-resistant Staphylococcus aureus, Escherichia coli, Clostridium difficile and Candida albicans strains. Notablly, a spiro­indoline­oxadiazole derivative displayed the highest activity against C. difficile with minimum inhibitory concentration values of 2 to 4 µg/ml. Interestingly, this compound exhibited no effects against other tested bacterial strains. For C. difficile, drugs that can inhibit it without affecting other Gram-positive or Gram-negative bacteria (not affecting the normal microbiota) are needed. This compound could be a good anticlostridial agent.

Stimuli-responsive biotin-anchored prodrug for the targeted delivery of anti-cancer agent NBDHEX with turn-on NIR fluorescence.

Biothiol-activatable prodrug RK-296 was designed for the delivery of potent anti-cancer agent NBDHEX with concomitant turn-on near infrared (NIR) fluorescence. NBDHEX exhibits anti-cancer activity by selectively inhibiting glutathione-S-transferase pi (GSTP1), which is overexpressed in cancer cells and responsible for the inactivation of chemotherapeutic drugs. The sustained release of NBDHEX from the prodrug would be useful for ameliorating the off-target side-effects of NBDHEX.

Design, Synthesis, and Target Identification of Novel Phenylalanine Derivatives by Drug Affinity Responsive Target Stability (DARTS) in Xanthomonas oryzae pv Oryzae.

The increasing resistance displayed by plant phytopathogenic bacteria to conventional pesticides has heightened the urgency for the exploration of novel antibacterial agents possessing distinct modes of action (MOAs). In this study, a series of novel phenylalanine derivatives with the unique structure of acylhydrazone dithioether have been designed and synthesized. Bioassay results demonstrated that most target compounds exhibited excellent in vitro antibacterial activity against Xanthomonas oryzae pv oryzae (Xoo) and Xanthomonas axonopodis pv citri (Xac). Among them, the EC50 values of L3, L4, L6, L21, and L22 against Xoo were 7.4, 9.3, 6.7, 8.9, and 5.1 µg/mL, respectively, superior to that of bismerthiazol (BT) and thiodiazole copper (TC) (41.5 and >100 µg/mL); the EC50 values of L3, L4, L5, L6, L7, L8, L20, L21, and L22 against Xac were 5.6, 2.5, 6.2, 4.1, 4.2, 6.4, 6.3, 3.6, and 5.2 µg/mL, respectively, superior to that of BT and TC (43.3 and >100 µg/mL). An unmodified drug affinity responsive target stability (DARTS) technology was used to investigate the antibacterial MOAs of active compound L22, and the 50S ribosomal protein L2 (RL2) as an unprecedented target protein in Xoo cells was first discovered. The target protein RL2 was then expressed and purified. Furthermore, the in vitro interactions by microscale thermophoresis (Kd = 0.050 µM) and fluorescence titration (Ka = 1.4 × 105 M-1) experiments also demonstrated a strong binding force between compound L22 and RL2. Overall, these results not only facilitate the development of novel antibacterial agents but also establish a reliable method for exploring the targets of bactericides.

New Alpha9 nAChR Ligands Based on a 5-(Quinuclidin-3-ylmethyl)-1,2,4-oxadiazole Scaffold.

Several lines of evidence have indicated that nicotinic acetylcholine receptors (nAChR) that contain α9 subunits, probably in combination with α10 subunits, may be valuable targets for the management of pain associated with inflammatory diseases through a cholinergic anti-inflammatory system (CAS), which has also been associated with α7 nAChR. Both α7- and α9-containing neuronal nAChR can be pharmacologically distinguished from the high-affinity nicotinic receptors of the brain by their sensitivity to α-bungarotoxin, but in other ways, they have quite distinct pharmacological profiles. The early association of α7 with CAS led to the development of numerous new ligands, variously characterized as α7 agonists, partial agonists, or silent agonists that desensitized α7 receptors without activation. Subsequent reinvestigation of one such family of α7 ligands based on an N,N-diethyl-N'-phenylpiperazine scaffold led to the identification of potent agonists and antagonists for α9. In this paper, we characterize the α9/α10 activity of a series of compounds based on a 5-(quinuclidin-3-ylmethyl)-1,2,4-oxadiazole (QMO) scaffold and identify two new potent ligands of α9, QMO-28, an agonist, and QMO-17, an antagonist. We separated the stereoisomers of these compounds to identify the most potent agonist and discovered that only the 3R isomer of QMO-17 was an α9 antagonist, permitting an in silico model of α9 antagonism to be developed. The α9 activity of these compounds was confirmed to be potentially useful for CAS management of inflammatory pain in cell-based assays of cytokine release.

Discovery of dual-active ethionamide boosters inhibiting the Mycobacterium tuberculosis ESX-1 secretion system.

Drug-resistant Mycobacterium tuberculosis (Mtb) remains a major public health concern requiring complementary approaches to standard anti-tuberculous regimens. Anti-virulence molecules or compounds that enhance the activity of antimicrobial prodrugs are promising alternatives to conventional antibiotics. Exploiting host cell-based drug discovery, we identified an oxadiazole compound (S3) that blocks the ESX-1 secretion system, a major virulence factor of Mtb. S3-treated mycobacteria showed impaired intracellular growth and a reduced ability to lyse macrophages. RNA sequencing experiments of drug-exposed bacteria revealed strong upregulation of a distinct set of genes including ethA, encoding a monooxygenase activating the anti-tuberculous prodrug ethionamide. Accordingly, we found a strong ethionamide boosting effect in S3-treated Mtb. Extensive structure-activity relationship experiments revealed that anti-virulence and ethionamide-boosting activity can be uncoupled by chemical modification of the primary hit molecule. To conclude, this series of dual-active oxadiazole compounds targets Mtb via two distinct mechanisms of action.

"Antibacterial effect and possible mechanism of action of 1,3,4-oxadiazole in Staphylococcus aureus".

Staphylococcus aureus is one of the main etiological agents causing foodborne diseases, and the development of new antibacterial agents is urgent. This study evaluated the antibacterial activity and the possible mechanism of action of the 1,3,4-oxadiazole LMM6 against S. aureus. The minimum inhibitory concentration (MIC) of LMM6 ranged from 1.95 to 7.81 µg ml-1. The time-kill assay showed that 48-h treatment at 1× to 8× MIC reduced S. aureus by 4 log colony forming unit (CFU), indicating a bacteriostatic effect. Regarding the possible mechanism of action of LMM6, there was accumulation of reactive oxygen species (ROS) and an increase in the absorption of crystal violet (∼50%) by the cells treated with LMM6 at 1× and 2× MIC for 6-12 h. In addition, there was increased propidium iodide uptake (∼84%) after exposure to LMM6 for 12 h at 2× MIC. After 48 h of treatment, 100% of bacteria had been injured. Scanning electron microscopy observations demonstrated that LMM6-treated cells were smaller compared with the untreated group. LMM6 exhibited bacteriostatic activity and its mechanism of action involves increase of intracellular ROS and disturbance of the cell membrane, which can be considered a key target for controlling the growth of S. aureus.

Unveiling the chemistry of 1,3,4-oxadiazoles and thiadiazols: A comprehensive review.

Oxadiazoles and thiadiazoles are malleable heterocycles that have recently generated major interest in the field of medicinal chemistry. Compounds based on these moieties have versatile biological applications such as anticonvulsant, anticancer, antidiabetic, and antioxidant agents. Due to the versatile nature and stability of the oxadiazole and thiadiazole nucleus, medicinal chemists have changed the structural elements of the ring in numerous ways. These compounds have shown significant anticonvulsant effects, demonstrating their potential in the management of epileptic disorders. In this review, we have covered numerous biological pathways and in silico targeted proteins of oxadiazole and thiadiazole derivatives for treating various biological disorders. The data compiled in this article will be helpful for researchers, research scientists, and research chemists who work in the field of drug discovery and drug development.

1,3,4-Oxadiazoles as Anticancer Agents: A Review.

Among the deadliest diseases, cancer is characterized by tumors or an increased number of a specific type of cell because of uncontrolled divisions during mitosis. Researchers in the current era concentrated on the development of highly selective anticancer medications due to the substantial toxicities of conventional cytotoxic drugs. Several marketed drug molecules have provided resistance against cancer through interaction with certain targets/growth factors/enzymes, such as Telomerase, Histone Deacetylase (HDAC), Methionine Aminopeptidase (MetAP II), Thymidylate Synthase (TS), Glycogen Synthase Kinase-3 (GSK), Epidermal Growth Factor (EGF), Vascular Endothelial Growth Factor (VEGF), Focal Adhesion Kinase (FAK), STAT3, Thymidine phosphorylase, and Alkaline phosphatase. The molecular structure of these drug molecules contains various heterocyclic moieties that act as pharmacophores. Recently, 1,3,4- oxadiazole (five-membered heterocyclic moiety) and its derivatives attracted researchers as these have been reported with a wide range of pharmacological activities, including anti-cancer. 1,3,4- oxadiazoles have exhibited anti-cancer potential via acting on any of the above targets. The presented study highlights the synthesis of anti-cancer 1,3,4-oxadiazoles, their mechanism of interactions with targets, along with structure-activity relationship concerning anti-cancer potential.

Azilsartan prevents muscle loss and fast- to slow-twitch muscle fiber shift in natural ageing sarcopenic rats.

Sarcopenia is a musculoskeletal disease that reduces muscle mass and strength in older individuals. The study investigates the effects of azilsartan (AZL) on skeletal muscle loss in natural sarcopenic rats. Male Sprague-Dawley rats aged 4-6 months and 18-21 months were selected as young-matched control and natural-aged (sarcopenic) rats, respectively. Rats were allocated into young and old control (YC and OC) and young and old AZL treatment (YT and OT) groups, which received vehicles and AZL (8 mg/kg, orally) for 6 weeks. Rats were then sacrificed after muscle function analysis. Serum and gastrocnemius (GN) muscles were isolated for further endpoints. AZL significantly improved muscle grip strength and antioxidant levels in sarcopenic rats. AZL also restored the levels of insulin, testosterone, and muscle biomarkers such as myostatin and creatinine kinase in sarcopenic rats. Furthermore, AZL treatment improved the cellular and ultrastructure of GN muscle and prevented the shift of type II (glycolytic) myofibers to type I (oxidative) myofibers. The results showed that AZL intervention restored protein synthesis in natural sarcopenic rats by increasing p-Akt-1 and decreasing muscle RING-finger protein-1 and tumor necrosis factor alpha immunoexpressions. In conclusion, the present findings showed that AZL could be an effective intervention in treating age-related muscle impairments.

Both Nitro Groups Are Essential for High Antitubercular Activity of 3,5-Dinitrobenzylsulfanyl Tetrazoles and 1,3,4-Oxadiazoles through the Deazaflavin-Dependent Nitroreductase Activation Pathway.

3,5-Dinitrobenzylsulfanyl tetrazoles and 1,3,4-oxadiazoles, previously identified as having high in vitro activities against both replicating and nonreplicating mycobacteria and favorable cytotoxicity and genotoxicity profiles were investigated. First we demonstrated that these compounds act in a deazaflavin-dependent nitroreduction pathway and thus require a nitro group for their activity. Second, we confirmed the necessity of both nitro groups for antimycobacterial activity through extensive structure-activity relationship studies using 32 structural types of analogues, each in a five-membered series. Only the analogues with shifted nitro groups, namely, 2,5-dinitrobenzylsulfanyl oxadiazoles and tetrazoles, maintained high antimycobacterial activity but in this case mainly as a result of DprE1 inhibition. However, these analogues also showed increased toxicity to the mammalian cell line. Thus, both nitro groups in 3,5-dinitrobenzylsulfanyl-containing antimycobacterial agents remain essential for their high efficacy, and further efforts should be directed at finding ways to address the possible toxicity and solubility issues, for example, by targeted delivery.

Design, Synthesis, Nematicidal Activity, and Mechanism of Novel Amide Derivatives Containing an 1,2,4-Oxadiazole Moiety.

To discover new nematicides, a series of novel amide derivatives containing 1,2,4-oxadiazole were designed and synthesized. Several compounds showed excellent nematicidal activity. The LC50 values of compounds A7, A18, and A20-A22 against pine wood nematode (Bursaphelenchus xylophilus), rice stem nematode (Aphelenchoides besseyi), and sweet potato stem nematode (Ditylenchus destructor) were 1.39-3.09 mg/L, which were significantly better than the control nematicide tioxazafen (106, 49.0, and 75.0 mg/L, respectively). Compound A7 had an outstanding inhibitory effect on nematode feeding, reproductive ability, and egg hatching. Compound A7 effectively promoted the oxidative stress of nematodes and caused intestinal damage to nematodes. Compound A7 significantly inhibited the activity of succinate dehydrogenase (SDH) in nematodes, leading to blockage of electron transfer in the respiratory chain and thereby hindering the synthesis of adenosine triphosphate (ATP), which consequently affects the entire oxidative phosphorylation process to finally cause nematode death. Therefore, compound A7 can be used as a potential SDH inhibitor in nematicide applications.

Synthesis of 1,3,4-oxadiazoles with 4-methoxynaphthalene ring: discovering new compounds with antimicrobial activity.

Background: Paracoccidioidomycosis (PCM) is a systemic infection caused by Paracoccidioides spp. (Pb). PCM can be associated or clinically confused with tuberculosis (TB), another pulmonary infection, caused by Mycobacterium tuberculosis (Mtb). Futhermore, the long treatment time of TB and PCM and the cases of TB drug resistance impose difficulties for the cure of these diseases. Results: New 1,3,4-oxadiazoles containing the 4-methoxynaphthalene ring were synthesized and their antimicrobial activity was evaluated against Pb and Mtb. The derivative 6n (with 2-hydroxy-5-nitrophenyl subunit) is the most promising of the series. Conclusion: The 1,3,4-oxadiazole 6n can be used as a prototype drug candidate, with anti-Pb and anti-MTb activities, showing a broad-spectrum profile for the treatment of both pulmonary infections.

Difluoromethyl-1,3,4-oxadiazoles Are Selective, Mechanism-Based, and Essentially Irreversible Inhibitors of Histone Deacetylase 6.

Histone deacetylase 6 (HDAC6) is an important drug target in oncological and non-oncological diseases. Most available HDAC6 inhibitors (HDAC6i) utilize hydroxamic acids as a zinc-binding group, which limits therapeutic opportunities due to its genotoxic potential. Recently, difluoromethyl-1,3,4-oxadiazoles (DFMOs) were reported as potent and selective HDAC6i but their mode of inhibition remained enigmatic. Herein, we report that DFMOs act as mechanism-based and essentially irreversible HDAC6i. Biochemical data confirm that DFMO 6 is a tight-binding HDAC6i capable of inhibiting HDAC6 via a two-step slow-binding mechanism. Crystallographic and mechanistic experiments suggest that the attack of 6 by the zinc-bound water at the sp2 carbon closest to the difluoromethyl moiety followed by a subsequent ring opening of the oxadiazole yields deprotonated difluoroacetylhydrazide 13 as active species. The strong anionic zinc coordination of 13 and the binding of the difluoromethyl moiety in the P571 pocket finally result in an essentially irreversible inhibition of HDAC6.