Discovery of Pyridone‐Based Histone Deacetylase Inhibitors: Approaches for Metabolic Stability

Histone deacetylases (HDACs) are important enzymes in epigenetic regulation and are therapeutic targets for cancer. Most zinc‐dependent HDACs induce proliferation, dedifferentiation, and anti‐apoptotic effects in cancer cells. We designed and synthesized a new series of pyridone‐based HDAC inhibitors that have a pyridone ring in the core structure and a conjugated system with an olefin connecting the hydroxamic acid moiety. Consequently, most of the selected pyridone‐based HDAC inhibitors showed similar or higher inhibition profiles in addition to remarkable metabolic stability against hydrolysis relative to the corresponding lactam‐based HDAC inhibitors. Furthermore, the selectivity of the novel pyridine‐based compounds was evaluated across all of the HDAC isoforms. One of these compounds, (E)‐N‐hydroxy‐3‐{1‐[3‐(naphthalen‐2‐yl)propyl]‐2‐oxo‐1,2‐dihydropyridin‐3‐yl}acrylamide, exhibited the highest level of HDAC inhibition (IC₅₀=0.07 μM ), highly selective inhibition of class I HDAC1 and class II HDAC6 enzymes, metabolic stability in mouse liver microsomal studies, and effective growth inhibition of various cancer cell lines. Docking studies indicated that a long alkyl linker and bulky hydrophobic cap groups affect in vitro activities. Overall, the findings reported herein regarding pyridone‐based HDAC inhibitors can be used to guide future research efforts to develop new and effective anticancer therapeutics.     

Synthesis of N‐Hydroxycinnamides Capped with a Naturally Occurring Moiety as Inhibitors of Histone Deacetylase

Histone deacetylase (HDAC) inhibitors are regarded as promising therapeutics for the treatment of cancer. All reported HDAC inhibitors contain three pharmacophoric features: a zinc‐chelating group, a hydrophobic linker, and a hydrophobic cap for surface recognition. In this study we investigated the effectiveness of osthole, a hydrophobic Chinese herbal compound, as the surface recognition cap in hydroxamate‐based compounds as inhibitors of HDAC. Nine novel osthole‐based N‐hydroxycinnamides were synthesized and screened for enzyme inhibition activity. Compounds 9 d , 9 e , 9 g exhibited inhibitory activities (IC₅₀=24.5, 20.0, 19.6 nM ) against nuclear HDACs in HeLa cells comparable to that of suberoylanilide hydroxamic acid (SAHA; IC₅₀=24.5 nM ), a potent inhibitor clinically used for the treatment of cutaneous T‐cell lymphoma (CTCL). While compounds 9 d and 9 e showed SAHA‐like activity towards HDAC1 and HDAC6, compound 9 g was more selective for HDAC1. Compound 9 d exhibited the best cellular effect, which was comparable to that of SAHA, of enhancing acetylation of either α‐tubulin or histone H3. Molecular docking analysis showed that the osthole moiety of compound 9 d may interact with the same hydrophobic surface pocket exploited by SAHA and it may be modified to provide class‐specific selectivity. These results suggest that osthole is an effective hydrophobic cap when incorporated into N‐hydroxycinnamide‐derived HDAC inhibitors.     

1‐Arylsulfonyl‐5‐(N‐hydroxyacrylamide)indolines Histone Deacetylase Inhibitors Are Potent Cytokine Release Suppressors

A series of 1‐arylsulfonyl‐5‐(N‐hydroxyacrylamide)indolines ( 7 – 15 ) has been developed; the compounds exhibited potent histone deacetylase (HDAC) inhibitory activities. Notably, almost all of this series exhibited better HDAC‐inhibitory and antiproliferative activities than 3‐(1‐benzenesulfonyl‐1H‐indol‐5‐yl)‐N‐hydroxyacrylamide ( 6 ), as reported in a previous study. Among these compounds, 3‐[1‐(4‐methoxybenzenesulfonyl)‐2,3‐dihydro‐1H‐indol‐5‐yl]‐N‐hydroxyacrylamide ( 9 ) showed a two‐ to tenfold increase in activity compared to SAHA ( 1 ) in the suppression of lipopolysaccharide‐induced cytokine production. Compound 9 also caused a marked reduction in carrageenan‐induced acute inflammation in a rat model. Taken together, these data indicated that 1‐arylsulfonyl‐5‐(N‐hydroxyacrylamide)indolines HDAC inhibitors exhibit potent anti‐inflammatory activity.     

Structure‐Based Design of Microsomal Prostaglandin E2 Synthase‐1 (mPGES‐1) Inhibitors using a Virtual Fragment Growing Optimization Scheme

A small library of 2,3‐dihydroxybenzamide‐ and N‐(2,3‐dihydroxyphenyl)‐4‐sulfonamide‐based microsomal prostaglandin E₂ synthase‐1 (mPGES‐1) inhibitors was identified following a step‐by‐step optimization of small aromatic fragments selected to interact in focused regions in the active site of mPGES‐1. During the virtual optimization process, the 2,3‐dihydroxybenzamide moiety was first selected as a backbone of the proposed new chemical entities; the identified compounds were then synthesized and biologically evaluated, identifying derivatives with very promising inhibitory activities in the micromolar range. Subsequent structure‐guided replacement of the 2,3‐dihydroxybenzamide by the N‐(2,3‐dihydroxyphenyl)sulfonamide moiety led to the identification of N‐(2,3‐dihydroxyphenyl)‐4‐biphenylsulfonamide ( 6 ), the most potent small molecule of the series (IC₅₀=0.53±0.04 μm ). The simple synthetic procedure and the possibility of enhancing the potency of this class of inhibitors through additional structural modifications pave the way for further development of new molecules with mPGES‐1‐inhibitory activity, with potential application as anti‐inflammatory and anticancer agents.     

Designing Dual Transglutaminase 2/Histone Deacetylase Inhibitors Effective at Halting Neuronal Death

In recent years there has been a clear consensus that neurodegenerative conditions can be better treated through concurrent modulation of different targets. Herein we report that combined inhibition of transglutaminase 2 (TG2) and histone deacetylases (HDACs) synergistically protects against toxic stimuli mediated by glutamate. Based on these findings, we designed and synthesized a series of novel dual TG2–HDAC binding agents. Compound 3 [(E)‐N‐hydroxy‐5‐(3‐(4‐(3‐oxo‐3‐(pyridin‐3‐yl)prop‐1‐en‐1‐yl)phenyl)thioureido)pentanamide] emerged as the most interesting of the series, being able to inhibit TG2 and HDACs both in vitro (TG2 IC₅₀=13.3±1.5 μm , HDAC1 IC₅₀=3.38±0.14 μm , HDAC6 IC₅₀=4.10±0.13 μm ) and in cell‐based assays. Furthermore, compound 3 does not exert any toxic effects in cortical neurons up to 50 μm and protects neurons against toxic insults induced by glutamate (5 mm ) with an EC₅₀ value of 3.7±0.5 μm .     

Stereoselective HDAC Inhibition from Cysteine‐Derived Zinc‐Binding Groups

A series of small‐molecule histone deacetylase (HDAC) inhibitors, which feature zinc binding groups derived from cysteine, were synthesized. These inhibitors were tested against multiple HDAC isoforms, and the most potent, compound 10 , was determined to have IC₅₀ values below 1 μM . The compounds were also tested in a cellular assay of oxidative stress‐induced neurodegeneration. Many of the inhibitors gave near‐complete protection against cell death at 10 μM without the neurotoxicity seen with hydroxamic acid‐based inhibitors, and were far more neuroprotective than HDAC inhibitors currently in clinical trials. Both enantiomers of cysteine were used in the synthesis of a variety of novel zinc‐binding groups (ZBGs). Derivatives of L ‐cysteine were active in the HDAC inhibition assays, while the derivatives of D ‐cysteine were inactive. Notably, the finding that both the D ‐ and L ‐cysteine derivatives were active in the neuroprotection assays suggests that multiple mechanisms are working to protect the neurons from cell death. Molecular modeling was employed to investigate the differences in inhibitory activity between the HDAC inhibitors generated from the two enantiomeric forms of cysteine.     

Design of gem‐Difluoro‐bis‐Tetrahydrofuran as P2 Ligand for HIV‐1 Protease Inhibitors to Improve Brain Penetration: Synthesis,X‐ray Studies,and Biological Evaluation

The structure‐based design, synthesis, biological evaluation, and X‐ray structural studies of fluorine‐containing HIV‐1 protease inhibitors are described. The synthesis of both enantiomers of the gem‐difluoro‐bis‐THF ligands was carried out in a stereoselective manner using a Reformatskii–Claisen reaction as the key step. Optically active ligands were converted into protease inhibitors. Two of these inhibitors, (3R,3aS,6aS)‐4,4‐difluorohexahydrofuro[2,3‐b]furan‐3‐yl(2S,3R)‐3‐hydroxy‐4‐((N‐isobutyl‐4‐methoxyphenyl)sulfonamido)‐1‐phenylbutan‐2‐yl) carbamate ( 3 ) and (3R,3aS,6aS)‐4,4‐difluorohexahydrofuro[2,3‐b]furan‐3‐yl(2S,3R)‐3‐hydroxy‐4‐((N‐isobutyl‐4‐aminophenyl)sulfonamido)phenylbutan‐2‐yl) carbamate ( 4 ), exhibited HIV‐1 protease inhibitory K_i values in the picomolar range. Both 3 and 4 showed very potent antiviral activity, with respective EC₅₀ values of 0.8 and 3.1 nM against the laboratory strain HIV‐1_LAI. The two inhibitors exhibited better lipophilicity profiles than darunavir, and also showed much improved blood–brain barrier permeability in an in vitro model. A high‐resolution X‐ray structure of inhibitor 4 in complex with HIV‐1 protease was determined, revealing that the fluorinated ligand makes extensive interactions with the S2 subsite of HIV‐1 protease, including hydrogen bonding interactions with the protease backbone atoms. Moreover, both fluorine atoms on the bis‐THF ligand formed strong interactions with the flap Gly 48 carbonyl oxygen atom.     

Design,Synthesis, in vitro MAO‐B Inhibitory Evaluation,and Computational Studies of Some 6‐Nitrobenzothiazole‐Derived Semicarbazones

Monoamine oxidase B (MAO‐B) is an important drug target for the treatment of neurological disorders. A series of 6‐nitrobenzothiazole‐derived semicarbazones were designed, synthesized, and evaluated as inhibitors of the rat brain MAO‐B isoenzyme. Most of the compounds were found to be potent inhibitors of MAO‐B, with IC₅₀ values in the nanomolar to micromolar range. Molecular docking studies were performed with AutoDock 4.2 to deduce the affinity and binding mode of these inhibitors toward the MAO‐B active site. The free energies of binding (ΔG) and inhibition constants (K_i) of the docked compounds were calculated by the Lamarckian genetic algorithm (LGA) of AutoDock 4.2. Good correlations between the calculated and experimental results were obtained. 1‐[(4‐Chlorophenyl)(phenyl)methylene]‐4‐(6‐nitrobenzothiazol‐2‐yl)semicarbazide emerged as the lead MAO‐B inhibitor, with top ranking in both the experimental MAO‐B assay (IC₅₀: 0.004±0.001 μM ) and in computational docking studies (K_i: 1.08 μM ). Binding mode analysis of potent inhibitors suggests that these compounds are well accommodated by the MAO‐B active site through stable hydrophobic and hydrogen bonding interactions. Interestingly, the 6‐nitrobenzothiazole moiety is stabilized in the substrate cavity with the aryl or diaryl residues extending up into the entrance cavity of the active site. According to our results, docking experiments could be an interesting approach for predicting the activity and binding interactions of this class of semicarbazones against MAO‐B. Thus, a binding site model consisting of three essential pharmacophoric features is proposed, and this can be used for the design of future MAO‐B inhibitors.     

Fluorophore‐Labeled Cyclooxygenase‐2 Inhibitors for the Imaging of Cyclooxygenase‐2 Overexpression in Cancer: Synthesis and Biological Studies

A group of cyclooxygenase‐2 (COX‐2)‐specific fluorescent cancer biomarkers were synthesized by linking the anti‐inflammatory drugs ibuprofen, (S)‐naproxen, and celecoxib to the 7‐nitrobenzofurazan (NBD) fluorophore. In vitro COX‐1/COX‐2 inhibition studies indicated that all of these fluorescent conjugates are COX‐2 inhibitors (IC₅₀ range: 0.19–23.0 μM ) with an appreciable COX‐2 selectivity index (SI≥4.3–444). In this study the celecoxib–NBD conjugate N‐(2‐((7‐nitrobenzo[c][1,2,5]oxadiazol‐4‐yl)amino)ethyl)‐4‐(5‐(p‐tolyl)‐3‐(trifluoromethyl)‐1H‐pyrazol‐1‐yl)benzenesulfonamide ( 14 ), which displayed the highest COX‐2 inhibitory potency and selectivity (COX‐2 IC₅₀=0.19 μM ; SI=443.6), was identified as an impending COX‐2‐specific biomarker for the fluorescence imaging of cancer using a COX‐2‐expressing human colon cancer cell line (HCA‐7).     

From Type I to Type II: Design,Synthesis, and Characterization of Potent Pyrazin‐2‐ones as DFG‐Out Inhibitors of PDGFRβ

Reversible protein kinase inhibitors that bind in the ATP cleft can be classified as type I or type II binders. Of these, type I inhibitors address the active form, whereas type II inhibitors typically lock the kinase in an inactive form. At the molecular level, the conformation of the flexible activation loop holding the key DFG motif controls access to the ATP site, thereby determining an active or inactive kinase state. Accordingly, type I and type II kinase inhibitors bind to so‐called DFG‐in or DFG‐out conformations, respectively. Based on our former study on highly selective platelet‐derived growth factor receptor β (PDGFRβ) pyrazin‐2‐one type I inhibitors, we expanded this scaffold toward the deep pocket, yielding the highly potent and effective type II inhibitor 5 (4‐[(4‐methylpiperazin‐1‐yl)methyl]‐N‐[3‐[[6‐oxo‐5‐(3,4,5‐trimethoxyphenyl)‐1H‐pyrazin‐3‐yl]methyl]phenyl]benzamide). In vitro characterization, including selectivity panel data from activity‐based assays (300 kinases) and affinity‐based assays (97 kinases) of these PDGFRβ type I ( 1 ; 5‐(4‐hydroxy‐3‐methoxy‐phenyl)‐3‐(3,4,5‐trimethoxyphenyl)‐1H‐pyrazin‐2‐one) and II ( 5 ) inhibitors showing the same pyrazin‐2‐one chemotype are compared. Implications are discussed regarding the data for selectivity and efficacy of type I and type II ligands.     

ω‐Imidazolyl‐ and ω‐Tetrazolylalkylcarbamates as Inhibitors of Fatty Acid Amide Hydrolase: Biological Activity and in vitro Metabolic Stability

Fatty acid amide hydrolase (FAAH) is a serine hydrolase that terminates the analgesic and anti‐inflammatory effects of endocannabinoids such as anandamide. Herein, structure–activity relationship studies on a new series of aryl N‐(ω‐imidazolyl‐ and ω‐tetrazolylalkyl)carbamate inhibitors of FAAH were investigated. As one result, a pronounced increase in inhibitory potency was observed if a phenyl residue attached to the carbamate oxygen atom was replaced by a pyridin‐3‐yl moiety. The most active compounds exhibited IC₅₀ values in the low nanomolar range. In addition, investigations on the metabolic properties of these inhibitors were performed. In rat liver homogenate and in porcine plasma, the extent of their degradation was shown to be strongly dependent on the kind of aryl residue bound to the carbamate as well as on the length and type of the alkyl spacer connecting the carbamate group with the heterocyclic system. With the aid of esterase inhibitors it was shown that in porcine plasma, carboxylesterase‐like enzymes and paraoxonase are involved in carbamate cleavage. Moreover, it was found that highly active pyridin‐3‐yl carbamates reacted with albumin, which led to covalent albumin adducts.     

Identification of a Small‐Molecule Inhibitor of HIV‐1 Assembly that Targets the Phosphatidylinositol (4,5)‐bisphosphate Binding Site of the HIV‐1 Matrix Protein

The development of drug resistance remains a critical problem for current HIV‐1 antiviral therapies, creating a need for new inhibitors of HIV‐1 replication. We previously reported on a novel anti‐HIV‐1 compound, N²‐(phenoxyacetyl)‐N‐[4‐(1‐piperidinylcarbonyl)benzyl]glycinamide ( 14 ), that binds to the highly conserved phosphatidylinositol (4,5)‐bisphosphate (PI(4,5)P₂) binding pocket of the HIV‐1 matrix (MA) protein. In this study, we re‐evaluate the hits from the virtual screen used to identify compound 14 and test them directly in an HIV‐1 replication assay using primary human peripheral blood mononuclear cells. This study resulted in the identification of three new compounds with antiviral activity; 2‐(4‐{[3‐(4‐fluorophenyl)‐1,2,4‐oxadiazol‐5‐yl]methyl})‐1‐piperazinyl)‐N‐(4‐methylphenyl)acetamide ( 7 ), 3‐(2‐ethoxyphenyl)‐5‐[[4‐(4‐nitrophenyl)piperazin‐1‐yl]methyl]‐1,2,4‐oxadiazole ( 17 ), and N‐[4‐ethoxy‐3‐(1‐piperidinylsulfonyl)phenyl]‐2‐(imidazo[2,1‐b][1,3]thiazol‐6‐yl)acetamide ( 18 ), with compound 7 being the most potent of these hits. Mechanistic studies on 7 demonstrated that it directly interacts with and functions through HIV‐1 MA. In accordance with our drug target, compound 7 competes with PI(4,5)P₂ for MA binding and, as a result, diminishes the production of new virus. Mutation of residues within the PI(4,5)P₂ binding site of MA decreased the antiviral effect of compound 7 . Additionally, compound 7 displays a broadly neutralizing anti‐HIV activity, with IC₅₀ values of 7.5–15.6 μM for the group M isolates tested. Taken together, these results point towards a novel chemical probe that can be used to more closely study the biological role of MA and could, through further optimization, lead to a new class of anti‐HIV‐1 therapeutics.     

Design,Synthesis, and Evaluation of an 18F‐Labeled Radiotracer Based on Celecoxib–NBD for Positron Emission Tomography (PET) Imaging of Cyclooxygenase‐2 (COX‐2)

A series of novel fluorine‐containing cyclooxygenase‐2 (COX‐2) inhibitors was designed and synthesized based on the previously reported fluorescent COX‐2 imaging agent celecoxib–NBD ( 3 ; NBD=7‐nitrobenzofurazan). In vitro COX‐1/COX‐2 inhibitory data show that N‐(4‐fluorobenzyl)‐4‐(5‐p‐tolyl‐3‐trifluoromethylpyrazol‐1‐yl)benzenesulfonamide ( 5 ; IC₅₀=0.36 μM , SI>277) and N‐fluoromethyl‐4‐(5‐p‐tolyl‐3‐trifluoromethylpyrazol‐1‐yl)benzenesulfonamide ( 6 ; IC₅₀=0.24 μM , SI>416) are potent and selective COX‐2 inhibitors. Compound 5 was selected for radiolabeling with the short‐lived positron emitter fluorine‐18 (¹⁸F) and evaluated as a positron emission tomography (PET) imaging agent. Radiotracer [¹⁸F] 5 was analyzed in vitro and in vivo using human colorectal cancer model HCA‐7. Although radiotracer uptake into COX‐2‐expressing HCA‐7 cells was high, no evidence for COX‐2‐specific binding was found. Radiotracer uptake into HCA‐7 tumors in vivo was low and similar to that of muscle, used as reference tissue.     

Hybrids from Farnesylthiosalicylic Acid and Hydroxamic Acid as Dual Ras‐Related Signaling and Histone Deacetylase (HDAC) Inhibitors: Design,Synthesis and Biological Evaluation

A novel series of hybrids was designed and synthesized by combining key elements from farnesylthiosalicylic acid (FTS) and hydroxamic acid. Several 3,7,11‐trimethyldodeca‐2,6,10‐trien‐1‐yl) thio)benzamide derivatives, particularly those with branched and linear aliphatic linkers between the hydroxamic zinc binding group (ZBG) and the benzamide core, not only displayed significant antitumor activities against six human cancer cells but also exhibited histone deacetylase (HDAC) inhibitory effects in vitro. Among them, N‐(4‐(hydroxyamino)‐4‐oxobutyl)‐2‐(((2E,6E)‐3,7,11‐trimethyldodeca‐2,6, 10‐trien‐1‐yl)thio)benzamide ( 8 d ) was the most potent, with IC₅₀ values of 4.9–7.6 μM ; these activities are eight‐ to sixteen‐fold more potent than FTS and comparable to that of suberoylanilide hydroxamic acid (SAHA). Derivative 8 d induced cell cycle arrest in the G0/G1 phase, inhibited the acetylation of histone H3 and α‐tubulin, and blocked Ras‐related signaling pathways in a dose‐dependent manner. The improved tumor growth inhibition and cell‐cycle arrest in vitro might result from the dual inhibition. These findings suggest dual inhibitors of Ras‐related signaling pathway and HDAC hold promise as therapeutic agents for the treatment of cancer.     

Iron‐Catalyzed Oxidative Mono‐ and Bis‐Phosphonation of N,N‐Dialkylanilines

The dehydrogenative α‐phosphonation of substituted N,N‐dialkylanilines by dialkyl H‐phosphonates was achieved under mild conditions by using environmentally benign iron(II) chloride as catalyst and tert‐butyl hydroperoxide as oxidant. The reaction proceeded in the presence of electron‐donating (methoxy, methyl, benzyl) and electron‐withdrawing ring‐substitutents (bromo, carbonyl, carboxyl, m‐nitro) in moderate to good yields. The X‐ray crystal structure of N‐(5,5‐dimethyl‐2‐oxo‐2λ⁵‐[1,3,2]dioxaphosphinan‐2‐yl‐methyl)‐N‐methyl‐p‐toluidine was determined. Bis‐(4‐(dimethylamino)phenyl)methane and bis‐4,4′‐(dimethylamino)benzophenone underwent bisphosphonation selectively by respective monophosphonation at the remote dimethylamino groups. Furthermore, the use of excess dialkyl H‐phosphonate and oxidant allowed us to functionalize both methyl groups of N(CH₃)₂ in N,N‐dimethyl‐p‐toluidine and N,N‐dimethylaminomesidine, respectively, to obtain α,α′‐bisphosphonatoamines in high yield.     

Pharmacophore‐Based Design of Novel Oxadiazoles as Selective Sphingosine‐1‐phosphate (S1P) Receptor Agonists with in vivo Efficacy

Sphingosine‐1‐phosphate (S1P) receptor agonists have shown promise as therapeutic agents for multiple sclerosis (MS) due to their regulatory roles within the immune, central nervous system, and cardiovascular system. Here, the design and optimization of novel [1,2,4]oxadiazole derivatives as selective S1P receptor agonists are described. The structure–activity relationship exploration was carried out on the three dominant segments of the series: modification of the polar head group (P), replacement of the oxadiazole linker (L) with different five‐membered heterocycles, and the use of diverse 2,2′‐disubstituted biphenyl moieties as the hydrophobic tail (H). All three segments have a significant impact on potency, S1P receptor subtype selectivity, physicochemical properties, and in vitro absorption, distribution, metabolism, excretion and toxicity (ADMET) profile of the compounds. From these optimization studies, a selective S1P₁ agonist, N‐methyl‐N‐(4‐{5‐[2‐methyl‐2′‐(trifluoromethyl)biphenyl‐4‐yl]‐1,2,4‐oxadiazol‐3‐yl}benzyl)glycine ( 45 ), and a dual S1P_1,5 agonist, N‐methyl‐N‐(3‐{5‐[2′‐methyl‐2‐(trifluoromethyl)biphenyl‐4‐yl]‐1,2,4‐oxadiazol‐3‐yl}benzyl)glycine ( 49 ), emerged as frontrunners. These compounds distribute predominantly in lymph nodes and brain over plasma and induce long lasting decreases in lymphocyte count after oral administration. When evaluated head‐to‐head in an experimental autoimmune encephalomyelitis mouse model, together with the marketed drug fingolimod, a pan‐S1P receptor agonist, S1P_1,5 agonist 49 demonstrated comparable efficacy while S1P₁‐selective agonist 45 was less potent. Compound 49 is not a prodrug, and its improved property profile should translate into a safer treatment of relapsing forms of MS.     

Highly potent and selective 4,4'‐biphenyl‐4‐acylate‐4'‐N‐n‐butylcarbamate inhibitors of Pseudomonas species lipase

4,4'‐Biphenyl‐4‐acylate‐4'‐N‐n‐butylcarbamates ( 1–8 ) are synthesized and characterized as highly potent and selective pseudo‐substrate inhibitors of Pseudomonas species lipase. Thus, the n‐butylcarbamate moieties of the inhibitors bind to the first acyl chain binding site (ACS) of the enzyme. Therefore, the ester moieties of the inhibitors may bind to the second ACS of the enzyme, due to the linear 4,4'‐biphenyl moiety of the inhibitors. –logK_i, logk₂, and logk_i values of carbamates 1–8 are multiply linearly correlated with the Taft steric constant (E_S) and the Hansch hydrophobicity constant (π), but not with the Taft substituent constant (σ*). For –logK_i, logk₂, and logk_i correlations, values of δ are 0.8, 0.34, and 1.0, respectively, and values of ψ are 1.0, 0.4, and 1.3, respectively. Positive δ and ψ values for these correlations indicate that the second ACS of the enzyme prefers to bind to small and hydrophobic ester groups of the inhibitors. Among carbamates 1–8 , carbamate 3 , with a K_i value of 2.5 nM, is the most potent inhibitor.     

Discovery of Non‐ATP‐Competitive Inhibitors of Polo‐like Kinase 1

Polo‐like kinase 1 (Plk1) is an evolutionarily conserved serine/threonine kinase, and its N‐terminal kinase domain (KD) controls cell signaling through phosphorylation. Inhibitors of Plk1 are potential anticancer drugs. Most known Plk1 KD inhibitors are ATP‐competitive compounds, which may suffer from low selectivity. In this study we discovered novel non‐ATP‐competitive Plk1 KD inhibitors by virtual screening and experimental studies. Potential binding sites in Plk1 KD were identified by using the protein binding site detection program Cavity. The identified site was subjected to molecular‐docking‐based virtual screening. The activities of top‐ranking compounds were evaluated by in vitro enzyme assay with full‐length Plk1 and direct binding assay with Plk1 KD. Several compounds showed inhibitory activity, and the most potent was found to be 3‐((2‐oxo‐2‐(thiophen‐2‐yl)ethyl)thio)‐6‐(pyridin‐3‐ylmethyl)‐1,2,4‐triazin‐5(4H)‐one (compound 4 ) with an IC₅₀ value of 13.1±1.7 μm . Our work provides new insight into the design of kinase inhibitors that target non‐ATP binding sites.