SAR studies of 3-cyclopropanecarbonyloxy-2-cyclohexen-1-one as inhibitors of 4-hydroxyphenylpyruvate dioxygenase

Various 3-cyclopropanecarbonyloxy-2-cyclohexen-1-one 1 derivatives have been synthesized and tested as inhibitors of 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) from pig liver. The inhibition results indicated that well-positioned dicarbonyl groups as well as the cyclopropyl group of 1 were essential for potent inhibition. Substitution at the 2-position of the ring system has a significant effect on inhibitor potency, while the 5-position can undergo substantial variations and retain inhibitor potency. In the compounds examined, 2-chloro substituted 12 is the best inhibitor of all with IC(50) of 15 nM, the rest of the synthesized analogues were less potent inhibitors than the parent compound.     

Design,synthesis, and evaluation of postulated transient intermediate and substrate analogues as inhibitors of 4-hydroxyphenylpyruvate dioxygenase

An epoxybenzoquinone, 4-hydroxyphenoxypropionic acid, and 2-hydroxy-3-phenyl-3-butenoic acid derivatives have been designed, synthesized, and evaluated for in vitro inhibition activity against 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) from pig liver by the spectrophotometric enol-borate method. The biological data demonstrated that neither epoxybenzoquinone ester nor 2-hydroxy-3-phenyl-3-butenoic acid is an inhibitor of 4-HPPD. The most potent 4-HPPD inhibitor tested was 3-hydroxy-4-phenyl-2(5H)-furanone with an IC(50) value of 0.5 microM, which may serve as a lead compound for further design of more potent 4-HPPD inhibitors.     

Alternate substrates and inhibitors of bacterial 4-hydroxyphenylpyruvate dioxygenase

A variety of analogues of (4-hydroxyphenyl)pyruvic acid were synthesized, and the reactions of these compounds with the 4-hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. P.J. 874 were examined. Several of the ring-substituted substrate analogues are reversible inhibitors of the enzyme, the most potent being the competitive inhibitor (2,6-difluoro-4-hydroxyphenyl) pyruvate (Ki = 1.3 microM). Two substrate analogues (2-fluoro-4-hydroxyphenyl)pyruvate and [(4-hydroxyphenyl)thio]pyruvate proved to be alternate substrates for the enzyme. The former compound is converted to (3-fluoro-2,5-dihydroxyphenyl)acetate in an essentially normal catalytic sequence including oxidative decarboxylation, ring hydroxylation, and side-chain migration. The latter compound, however, undergoes oxidative decarboxylation and sulfoxidation to give [(4-hydroxyphenyl)sulfinyl]acetate; ring oxidation is not observed. The implications of these results with regard to the catalytic mechanism of 4-hydroxyphenylpyruvate dioxygenase are discussed.     

Acylcyclohexanedione derivatives as potential in vivo sequential inhibitors of 4-hydroxyphenylpyruvate dioxygenase and GA(20) 3beta-hydroxylase

Acylcyclohexanedione derivatives have been designed, synthesized, and evaluated for in vitro inhibition activity against the enzyme 4-hydroxyphenylpyruvate dioxygenase (4-HPPD). The biological data demonstrated that 7 is a potent inhibitor of 4-HPPD with an IC(50) value of 40 nM. After metabolism, compound 7 has the potential to become a potent inhibitor of a second enzyme, GA(20) 3beta-hydroxylase.     

SAR studies of 2-o-substituted-benzoyl- and 2-alkanoyl-cyclohexane-1,3-diones as inhibitors of 4-hydroxyphenylpyruvate dioxygenase

Inhibition studies of 4-hydroxyphenylpyruvate dioxygenase (HPPD) with various synthesized 2-o-substituted-benzoyl- and 2-alkanoyl-cyclohexane-1,3-diones suggest that the presence of a strongly electronegative group at the ortho position and the conformation of the benzene ring moiety on the benzoylcyclohexane-1,3-dione inhibitors are crucial for potent HPPD inhibition.     

Purification and properties of hog liver 4-hydroxyphenylpyruvate dioxygenase

The purification of hog liver 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27), and the determination of some of its characteristics, are reported. The enzyme was purified 330-fold in 22% yield from an acetone powder extract by ammonium sulfate fractionation, chromatography twice using sulfopropyl Sephadex under carefully controlled pH conditions (once at pH 5.36 and a second time with a pH gradient from 5.25 to 5.80), and a final chromatography on DEAE-cellulose. The purified enzyme was found to be homogeneous by several standard criteria, but activity measurements indicated that a small amount (less than 5%) of a carboxylesterase (EC 3.1.1.1) isoenzyme is present as a minor impurity. On long-term storage at ?20 °C the enzyme forms polymers but this can be reversed with thiols. The molecular weight of the freshly prepared or depolymerized enzyme was estimated to be 89,000 ± 2000 by equilibrium ultracentrifugation, and 50,000 to 54,000 by gel filtration. Sodium dodecyl sulfate-gel electrophoresis experiments, performed in the presence and absence of mercaptoethanol, indicate that the enzyme is composed of two nonidentical subunits with similar molecular weights (44,000 ± 2000). The enzyme gives a typical protein ultraviolet absorption spectrum with no noticeable peaks above 300 nm, it has no detectable carbohydrate content, and it contains 0.9 atom iron and 0.4 atom copper/89,000 daltons. Added iron and copper salts activate the enzyme to some extent but by less than a factor of 2. The enzymatic reaction has a large temperature coefficient (the rate increases ca. fivefold for each 10 °C rise) and is markedly stimulated (up to sixfold) by the presence of some organic solvents in concentrations up to 10% of the medium. These results suggest that a protein conformation change, possibly aided by binding of the organic solvent, is involved in the rate-determining step of the reaction. The similarities and differences of this 4-hydroxyphenylpyruvate dioxygenase to those from other sources, and to prolyl hydroxylase, are discussed.     

Design and synthesis of 2-oxo-imidazolidine-4-carboxylic acid hydroxyamides as potent matrix metalloproteinase-13 inhibitors

A novel series of imidazolidinone-based matrix metalloproteinase (MMP) inhibitors was discovered by structural modification of pyrrolidinone la. Potent inhibition of MMP-13 was exhibited by the analogues having 4-(4-fluorophenoxy)phenyl (4a, IC50 = 3 nM) and 4-(naphth-2-yloxy)phenyl (4h, IC50 = 4 nM) as P1' groups.     

2-Cyano-4-fluoro-1-thiovalylpyrrolidine analogues as potent inhibitors of DPP-IV

We report the synthesis and biological activity of a series of 2-cyano-4-fluoro-1-thiovalylpyrrolidine inhibitors of DPP-IV. Within this series, compound 19 provided a potent, selective, and orally active DPP-IV inhibitor which demonstrated a very long duration of action in both rat and dog.     

Design,synthesis, and evaluation of novel 2-phenylpropionic acid derivatives as dual COX inhibitory-antibacterial agents

A series of 2-(4-substitutedmethylphenyl)propionic acid derivatives (6a–6m) were synthesized, characterized and evaluated for cyclooxygenase (COX) enzyme inhibitory and antimicrobial activity. Test compounds that exhibited good COX inhibition and antibacterial activity were further screened for their cytotoxicity and genotoxicity. Compounds 6h and 6l showed better COX-1 and COX-2 inhibition when compared to ibuprofen. Inhibition potency of these compounds against COX-2 was very close to that of nimesulide. The compounds 6d, 6h, 6l and 6m displayed promising antibacterial property when compared to chloramphenicol. However, the compound 6l was emerged as the best dual COX inhibitory-antibacterial agent in this study. The ADME prediction of the compounds revealed that they may have a good pharmacokinetic profile. Docking results of the compounds 6h and 6l with COX-1 (PDB ID: 1EQG) also exhibited a strong binding profile.

     

Design,synthesis and evaluation of 1,2-benzisothiazol-3-one derivatives as potent caspase-3 inhibitors

A number of 1,2-benzisothiazol-3-one derivatives were prepared through structural modification of the original compound from high-throughput screening. Some analogues (e.g., 6b, 6r, 6s and 6w) were identified as novel and potent caspase inhibitors with IC₅₀ of nanomolar. Structure–activity relationship (SAR) studies for caspase-3 inhibition were evaluated in vitro. Molecular modeling studies provided further insight into the interaction of this class of compounds with activated caspase-3. The present small molecule caspase-3 inhibitor with novel structures different from structures of known caspase inhibitors revealed a new direction for therapeutic strategies directed against diseases involving abnormally up-regulated apoptosis.     

Design,synthesis and bioevaluation of 3-oxo-6-aryl-2,3-dihydropyridazine-4-carbohydrazide derivatives as novel xanthine oxidase inhibitors

In view of expanding the structure activity relationship of xanthine oxidase inhibitors, a series of 3-oxo-6-aryl-2,3-dihydropyridazine-4-carbohydrazide/carboxylic acid derivatives were designed by molecular docking and synthesized. All the target compounds were evaluated for their in vitro XO inhibition by using febuxostat and allopurinol as the standard controls. Most of the hydrazide derivatives exhibited potency levels in the micromolar range. From the view of docking study, hydrazide derivatives bind to the active site of XO through a novel interaction mode, which is different from that of febuxostat bearing a carboxyl group. The most promising compound 8b was further subjected to kinetic analysis to deduce their modes of inhibition.     

Design, synthesis and evaluation of flavonoid derivatives as potent AChE inhibitors

A new series of flavonoid derivatives have been designed, synthesized and evaluated as potent AChE inhibitors. Most of them showed more potent inhibitory activities to AChE than rivastigmine. The most potent inhibitor isoflavone derivative 10d inhibit AChE with a IC₅₀ of 4 nM and showed high BChE/AChE inhibition ratio (4575-fold), superior to donepezil (IC₅₀ = 12 nM, 389-fold). Molecular docking studies were also performed to explore the detailed interaction with AChE.     

Design and synthesis of eugenol derivatives, as potent 15-lipoxygenase inhibitors

A group of 4-allyl-2-methoxyphenol (eugenol) esters were designed, synthesized, and evaluated as potential inhibitors of soybean 15-lipoxygenase (SLO). Compounds 4c, 4d 4f, 4p, and 4q showed the best IC(50) in SLO inhibition (IC(50)=1.7, 2.3, 2.1, 2.2, and 0.017microM, respectively). All compounds were docked into SLO active site and showed that allyl group of compounds is oriented toward the iron atom in the active site of SLO. It is assumed that lipophilic interaction of ligand-enzyme would be in charge of inhibiting the enzyme activity. The selectivity of eugenol derivatives in inhibiting 15-HLOb was also compared with 15-HLOa by molecular modeling and multiple alignment techniques.     

A 2-step synthesis of Combretastatin A-4 and derivatives as potent tubulin assembly inhibitors

A series of combretastatin derivatives were designed and synthesised by a two-step stereoselective synthesis by use of Wittig olefination followed by Suzuki cross-coupling. Interestingly, all new compounds (2a-2i) showed potent cell-based antiproliferative activities in nanomolar concentrations. Among the compounds, 2a, 2b and 2e were the most active across three cancer cell lines. In addition, these compounds inhibited the polymerisation of tubulin in vitro more efficiently than CA-4. They caused cell cycle arrest in G₂/M phase further confirming their ability to inhibit tubulin polymerisation.     

Design and synthesis of disubstituted (4-piperidinyl)-piperazine derivatives as potent acetyl-CoA carboxylase inhibitors

Acetyl-CoA carboxylases (ACCs), the rate limiting enzymes in de novo lipid synthesis, play important roles in modulating energy metabolism. The inhibition of ACC has demonstrated promising therapeutic potential for treating obesity and type 2 diabetes mellitus in transgenic mice and preclinical animal models. We describe herein the structure-based design and synthesis of a novel series of disubstituted (4-piperidinyl)-piperazine derivatives as ACC inhibitors. Our structure-based approach led to the discovery of the indole derivatives 13i and 13j, which exhibited potent in vitro ACC inhibitory activity.     

Design,synthesis and preliminary biological evaluation of 4-aminopyrazole derivatives as novel and potent JAKs inhibitors

JAKs inhibitors were widely applied in the treatment of immunodeficiency diseases, inflammation and cancers. We designed and synthesized a novel series of 4-aminopyrazole derivatives, which showed inhibitory potency against various JAKs. The in vitro protein kinase inhibition experiment indicated that compounds 17k, 17l, 17m and 17n could inhibit JAKs effectively. Among them, compound 17m possessed the highest protein kinase inhibitory rates (%) at 10 μM, which were 97, 96 and 100 to JAK1, JAK2 and JAK3, respectively. Further evaluation revealed that the IC₅₀ values of 17m against JAK1, JAK2 and JAK3 were 0.67 μM, 0.098 μM and 0.039 μM, respectively. Moreover, western blotting results showed compound 17m could inhibit the phosphorylation of JAK2 in Hela cells effectively. The data supports the further investigation of these compounds as novel JAKs inhibitors.     

Substituted 2-pyridinemethanol derivatives as potent and selective phosphodiesterase-4 inhibitors

The synthesis and the phosphodiesterase-4 (PDE4) inhibitory activity of 2-pyridinemethanol derivatives is described. The evaluation of the structure-activity relationship (SAR) in this series of novel PDE4 inhibitors led to the identification of compound 9 which exhibits excellent in vitro activity, desirable pharmacokinetic parameters and good efficacy in animal models of bronchoconstriction.     

Design,synthesis and biological evaluation of anthranilamide derivatives as potent SMO inhibitors

A series of anthranilamide derivatives were designed and synthesized as novel smoothened (SMO) inhibitors based on the SMO inhibitor taladegib (LY2940680), which can also inhibit the SMO-D473H mutant, via a ring-opening strategy. The phthalazine core in LY2940680 was replaced with anthranilamide, which retained the inhibitory activity towards the hedgehog (Hh) signaling pathway as evidenced by a dual luciferase reporter gene assay. Compound 12a displayed the best inhibitory activity against the Hh signaling pathway with IC₅₀ value of 34.09 nM, and exhibited better proliferation inhibitory activity towards the Daoy cell line (IC₅₀ = 0.48 μM) than LY2940680 (IC₅₀ = 0.79 μM).     

Discovery of indole-3-butyric acid derivatives as potent histone deacetylase inhibitors

In discovery of HDAC inhibitors (HDACIs) with improved anticancer potency, structural modification was performed on the previous derived indole-3-butyric acid derivative. Among all the synthesised compounds, molecule I13 exhibited high HDAC inhibitory and antiproliferative potencies in the in vitro investigations. The IC₅₀ values of I13 against HDAC1, HDAC3, and HDAC6 were 13.9, 12.1, and 7.71 nM, respectively. In the cancer cell based screening, molecule I13 showed increased antiproliferative activities in the inhibition of U937, U266, HepG2, A2780, and PNAC-1 cells compared with SAHA. In the HepG2 xenograft model, 50 mg/kg/d of I13 could inhibit tumour growth in athymic mice compared with 100 mg/kg/d of SAHA. Induction of apoptosis was revealed to play an important role in the anticancer potency of molecule I13. Collectively, a HDACI (I13) with high anticancer activity was discovered which can be utilised as a lead compound for further HDACI design.     

Design and synthesis of cyanamides as potent and selective N-acylethanolamine acid amidase inhibitors

N-acylethanolamine acid amidase (NAAA) inhibition represents an exciting novel approach to treat inflammation and pain. NAAA is a cysteine amidase which preferentially hydrolyzes the endogenous biolipids palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). PEA is an endogenous agonist of the nuclear peroxisome proliferator-activated receptor-α (PPAR-α), which is a key regulator of inflammation and pain. Thus, blocking the degradation of PEA with NAAA inhibitors results in augmentation of the PEA/PPAR-α signaling pathway and regulation of inflammatory and pain processes. We have prepared a new series of NAAA inhibitors exploring the azetidine-nitrile (cyanamide) pharmacophore that led to the discovery of highly potent and selective compounds. Key analogs demonstrated single-digit nanomolar potency for hNAAA and showed >100-fold selectivity against serine hydrolases FAAH, MGL and ABHD6, and cysteine protease cathepsin K. Additionally, we have identified potent and selective dual NAAA-FAAH inhibitors to investigate a potential synergism between two distinct anti-inflammatory molecular pathways, the PEA/PPAR-α anti-inflammatory signaling pathway,^1–4 and the cannabinoid receptors CB1 and CB2 pathways which are known for their antiinflammatory and antinociceptive properties.^5–8 Our ligand design strategy followed a traditional structure–activity relationship (SAR) approach and was supported by molecular modeling studies of reported X-ray structures of hNAAA. Several inhibitors were evaluated in stability assays and demonstrated very good plasma stability (t_1/2 > 2 h; human and rodents). The disclosed cyanamides represent promising new pharmacological tools to investigate the potential role of NAAA inhibitors and dual NAAA-FAAH inhibitors as therapeutic agents for the treatment of inflammation and pain.