Facile synthesis of carbon-11-labeled sEH/PDE4 dual inhibitors as new potential PET agents for imaging of sEH/PDE4 enzymes in neuroinflammation

To develop PET tracers for imaging of neuroinflammation, new carbon-11-labeled sEH/PDE4 dual inhibitors have been synthesized. The reference standard N-(4-methoxy-2-(trifluoromethyl)benzyl)benzamide (1) and its corresponding desmethylated precursor N-(4-hydroxy-2-(trifluoromethyl)benzyl)benzamide (2) were synthesized from (4-methoxy-2-(trifluoromethyl)phenyl)methanamine and benzoic acid in one and two steps with 84% and 49% overall chemical yield, respectively. The standard N-(4-methoxy-2-(trifluoromethyl)benzyl)-1-propionylpiperidine-4-carboxamide (MPPA, 4) and its precursor N-(4-hydroxy-2-(trifluoromethyl)benzyl)-1-propionylpiperidine-4-carboxamide (5) were synthesized from methyl 4-piperidinecarboxylate, propionyl chloride and (4-methoxy-2-(trifluoromethyl)phenyl)methanamine in two and three steps with 62% and 34% overall chemical yield, respectively. The target tracers N-(4-[¹¹C]methoxy-2-(trifluoromethyl)benzyl)benzamide ([¹¹C]1) and N-(4-[¹¹C]methoxy-2-(trifluoromethyl)benzyl)-1-propionylpiperidine-4-carboxamide ([¹¹C]MPPA, [¹¹C]4) were prepared from their corresponding precursors 2 and 5 with [¹¹C]CH₃OTf through O-[¹¹C]methylation and isolated by HPLC combined with SPE in 25–35% radiochemical yield, based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB). The radiochemical purity was >99%, and the molar activity (A_M) at EOB was 370–740 GBq/μmol with a total synthesis time of 35–40-minutes from EOB.     

Discovery of 3,3-disubstituted piperidine-derived trisubstituted ureas as highly potent soluble epoxide hydrolase inhibitors

3,3-Disubstituted piperidine-derived trisubstituted urea entA-2b was discovered as a highly potent and selective soluble epoxide hydrolase (sEH) inhibitor. Despite the good compound oral exposure, excellent sEH inhibition in whole blood, and remarkable selectivity, compound entA-2b failed to lower blood pressure acutely in spontaneously hypertensive rats (SHRs). This observation further challenges the premise that sEH inhibition can provide a viable approach to the treatment of hypertensive patients.     

Synthesis and biological evaluation of substituted 1,2,3-benzotriazines and pyrido[3,2-d]-1,2,3-triazines as inhibitors of vascular endothelial growth factor receptor-2

A novel series of substituted 1,2,3-benzotriazines and pyrido[3,2-d]-1,2,3-triazines were synthesized. The abilities of these compounds to inhibit the VEGFR-2 kinase activity and the proliferation of human microvascular endothelial cells (MVECs) were determined. 6-Methoxy-4-substituted-1,2,3-benzotriazines and 4-substituted-6-chloro-pyrido[3,2-d]-1,2,3-triazines have the abilities of inhibiting the VEGFR-2 kinase activity, but only the 4-substituted-6-chloro-pyrido[3,2-d]-1,2,3-triazines exhibit good growth inhibitory effects on MVECs. Compound 6-chloro-4-(3-trifluoromethylanilino)-pyrido[3,2-d][1,2,3]triazin (11d) is less half active than PTK787 to inhibit the VEGFR-2 kinase activity, but is more active than PTK787 to inhibit the growth of MVECs. The potential binding modes of 6d, 11d, and CTZ12 in complex with their putative intracellular target, VEGFR-2, were predicted using Surflex-Dock.     

Biphenyl derivatives incorporating urea unit as novel VEGFR-2 inhibitors: Design,synthesis and biological evaluation

A series of novel biphenyl urea derivates were synthesized and investigated for their potential to inhibit vascular endothelial growth factor receptor-2 (VEGFR-2). In particular, A7, B3 and B4 displayed significant enzymatic inhibitory activities, with IC₅₀ values of 4.06, 4.55 and 5.26 nM. Compound A7 exhibited potent antiproliferative activity on several cell lines. SAR study suggested that the introduction of methyl at ortho-position of the biphenyl urea and tertiary amine moiety could improve VEGFR-2 inhibitory activity and antitumor effects. Molecular docking indicated that the urea moiety formed four hydrogen bonds with DFG residue. These biphenyl ureas could serve as promising lead compounds for further optimization.     

Discovery of arylamide-5-anilinoquinazoline-8-nitro derivatives as VEGFR-2 kinase inhibitors: Synthesis,in vitro biological evaluation and molecular docking

Herein, we embarked on a structural optimization campaign aiming at the discovery of novel anticancer agents with our previously reported XL-6f as a lead compound. A library of 23 compounds has been synthesized based on the highly conserved active site of VEGFR-2. Several title compounds exhibited selective inhibitory activities against VEGFR-2, which also displayed selective anti-proliferation potency against HepG2 cell. All synthesized compounds were evaluated for anti-angiogenesis capability. Compound 7o showed the most potent anti-angiogenesis ability, the efficient cytotoxic activities (in vitro against HUVEC and HepG2 cell lines with IC₅₀ values of 0.58 and 0.23 µM, respectively). The molecular docking analysis revealed 7o is a Type-II inhibitor of VEGFR-2 kinase. In general, these results indicated these arylamide-5-anilinoquinazoline-8-nitro derivatives are promising inhibitors of VEGFR-2 for the potential treatment of anti-angiogenesis.     

Discovery of 1-(1,3,5-triazin-2-yl)piperidine-4-carboxamides as inhibitors of soluble epoxide hydrolase

1-(1,3,5-Triazin-yl)piperidine-4-carboxamide inhibitors of soluble epoxide hydrolase were identified from high through-put screening using encoded library technology. The triazine heterocycle proved to be a critical functional group, essential for high potency and P450 selectivity. Phenyl group substitution was important for reducing clearance, and establishing good oral exposure. Based on this lead optimization work, 1-[4-methyl-6-(methylamino)-1,3,5-triazin-2-yl]-N-{[[4-bromo-2-(trifluoromethoxy)]-phenyl]methyl}-4-piperidinecarboxamide (27) was identified as a useful tool compound for in vivo investigation. Robust effects on a serum biomarker, 9, 10-epoxyoctadec-12(Z)-enoic acid (the epoxide derived from linoleic acid) were observed, which provided evidence of robust in vivo target engagement and the suitability of 27 as a tool compound for study in various disease models.     

Exploring the size of the lipophilic unit of the soluble epoxide hydrolase inhibitors

Soluble epoxide hydrolase (sEH) inhibitors are potential drugs for several diseases. Adamantyl ureas are excellent sEH inhibitors but have limited metabolic stability. Herein, we report the effect of replacing the adamantane group by alternative polycyclic hydrocarbons on sEH inhibition, solubility, permeability and metabolic stability. Compounds bearing smaller or larger polycyclic hydrocarbons than adamantane yielded all good inhibition potency of the human sEH (0.4 ≤ IC₅₀ ≤ 21.7 nM), indicating that sEH is able to accommodate inhibitors of very different size. Human liver microsomal stability of diamantane containing inhibitors is lower than that of their corresponding adamantane counterparts.     

Synthesis and biological evaluation of 2-anilino-4-substituted-7H-pyrrolopyrimidines as PDK1 inhibitors

PDK1, a biological target that has attracted a large amount of attention recently, is responsible for the positive regulation of the PI3K/Akt pathway that is often activated in a large number of human cancers. A series of second-generation 2-anilino-4-substituted-7H-pyrrolopyrimidines were synthesised by installation of various functions at the 4-position of the 7H-pyrrolopyrimidine scaffold. All compounds were screened against the isolated PDK1 enzyme and dose response analysis was obtained on the best compounds of the series.     

Synthesis,biological evaluation,and molecular docking of new series of antitumor and apoptosis inducers designed as VEGFR-2 inhibitors

Based on quinazoline, quinoxaline, and nitrobenzene scaffolds and on pharmacophoric features of VEGFR-2 inhibitors, 17 novel compounds were designed and synthesised. VEGFR-2 IC₅₀ values ranged from 60.00 to 123.85 nM for the new derivatives compared to 54.00 nM for sorafenib. Compounds 15_a, 15_b, and 15_d showed IC₅₀ from 17.39 to 47.10 µM against human cancer cell lines; hepatocellular carcinoma (HepG2), prostate cancer (PC3), and breast cancer (MCF-7). Meanwhile, the first in terms of VEGFR-2 inhibition was compound 15_d which came second with regard to antitumor assay with IC₅₀ = 24.10, 40.90, and 33.40 µM against aforementioned cell lines, respectively. Furthermore, Compound 15_d increased apoptosis rate of HepG2 from 1.20 to 12.46% as it significantly increased levels of Caspase-3, BAX, and P53 from 49.6274, 40.62, and 42.84 to 561.427, 395.04, and 415.027 pg/mL, respectively. Moreover, 15_d showed IC₅₀ of 253 and 381 nM against HER2 and FGFR, respectively.     

Synthesis and evaluation of potent and selective MGL inhibitors as a glaucoma treatment

Monoacylglycerol lipase (MGL) inhibition provides a potential treatment approach to glaucoma through the regulation of ocular 2-arachidonoylglycerol (2-AG) levels and the activation of CB1 receptors. Herein, we report the discovery of new series of carbamates as highly potent and selective MGL inhibitors. The new inhibitors showed potent nanomolar inhibitory activity against recombinant human and purified rat MGL, were selective (>1000-fold) against serine hydrolases FAAH and ABHD6 and lacked any affinity for the cannabinoid receptors CB1 and CB2. Protein-based ¹H NMR experiments indicated that inhibitor 2 rapidly formed a covalent adduct with MGL with a residence time of about 6?h. This interconversion process “intrinsic reversibility” was exploited by modifications of the ligand’s size (length and bulkiness) to generate analogs with “tunable’ adduct residence time (τ). Inhibitor 2 was evaluated in a normotensive murine model for assessing intraocular pressure (IOP), which could lead to glaucoma, a major cause of blindness. Inhibitor 2 was found to decrease ocular pressure by ～4.5?mmHg in a sustained manner for at least 12?h after a single ocular application, underscoring the potential for topically-administered MGL inhibitors as a novel therapeutic target for the treatment of glaucoma.     

Association of the sEH gene promoter polymorphisms and haplotypes with preeclampsia

BackgroundThe epoxyeicosatrienoic acids (EETs) have antihypertensive, anti-inflammatory, and organ protective properties and their circulation levels are related to hypertension, diabetes mellitus, cardiovascular diseases, and preeclampsia. Soluble epoxide hydrolase (sEH) catalyses the degradation of EETs to less biologically active dihydroxyeicosatrienoic acids. Here, we sequenced the promoter region of EPHX2 to investigate the association between promoter sequence alterations that we thought to affect the expression levels of the enzyme and preeclampsia (PE).MethodsNucleotide sequencing of the promoter region of the EPHX2, spanning from position -671 to +30, was performed on 100 pregnant women with PE and, 20 or more weeks pregnant normotensive, healthy women (n=100).ResultsPregnant women who carry rs4149235, rs4149232, rs73227309, and rs62504268 polymorphisms have 4.4, 2.4, 2.3, and 2.8 times significantly increased risk of PE, respectively. CCGG (OR: 3.11; 95% CI: 1.12-8.62) and CCCA (OR: 0.45; 95% CI: 0.36-0.55) haplotypes were associated with an increased and decreased risk of PE, respectively.ConclusionsFour SNPs (rs4149232, rs4149235, rs73227309, and rs62504268) in the promoter region of the EPHX2, and CCGG and CCCA haplotypes of these 4 SNPs were significantly associated with PE. These SNPs in the promoter region may affect sEH expression and thus enzyme activity and may play a role in PE pathogenesis by causing individual differences in EET levels. However, future studies are needed to confirm our findings and examine the effect of these SNPs on the sEH expression and/or enzyme activity.     

Synthesis and biological evaluation of pyrrolopyridazine derivatives as novel HER-2 tyrosine kinase inhibitors

A series of novel pyrrolopyridazine derivatives have been discovered to be HER-2 inhibitors. These compounds selectively inhibited HER-2 kinase activity at low nanomolar concentrations. Compound 7d was identified as a potent HER-2 inhibitor with an IC₅₀ of 4 nM.     

Design,synthesis and biological evaluation of pyrimidine-based derivatives as VEGFR-2 tyrosine kinase inhibitors

Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a crucial role in tumor angiogenesis, and inhibition of the VEGFR-2 signaling pathway has already become an attractive approach for cancer therapy. In this study, a novel pyrimidine-based derivative 7j was designed as lead compound, and three series of potent VEGFR-2 inhibitors were synthesized and biologically evaluated against A549 and HepG2 cell lines. Compounds 7d, 9s and 13n exhibited superior inhibitory activities against A549 cell with IC₅₀ ranged from 9.19 to 13.17 μM and HepG2 cell with IC₅₀ ranged from 11.94 to 18.21 μM compared to those of Pazopanib (IC₅₀ = 21.18 and 36.66 μM). In addition, molecular docking study was performed to investigate the binding capacity and binding mode between target compounds and VEGFR-2.     

Preparation and evaluation of soluble epoxide hydrolase inhibitors with improved physical properties and potencies for treating diabetic neuropathic pain

Soluble epoxide hydrolase (sEH), a novel therapeutic target for neuropathic pain, is a largely cytosolic enzyme that degrades epoxy-fatty acids (EpFAs), an important class of lipid signaling molecules. Many inhibitors of sEH have been reported, and to date, the 1,3-disubstituted urea has the highest affinity reported for the sEH among the central pharmacophores evaluated. An earlier somewhat water soluble sEH inhibitor taken to the clinic for blood pressure control had mediocre potency (both affinity and kinetics) and a short in vivo half-life. We undertook a study to overcome these difficulties, but the sEH inhibitors carrying a 1,3-disubstituted urea often suffer poor physical properties that hinder their formulation. In this report, we described new strategies to improve the physical properties of sEH inhibitors with a 1,3-disubstituted urea while maintaining their potency and drug-target residence time (a complementary in vitro parameter) against sEH. To our surprise, we identified two structural modifications that substantially improve the potency and physical properties of sEH inhibitors carrying a 1,3-disubstituted urea pharmacophore. Such improvements will greatly facilitate the movement of sEH inhibitors to the clinic.     

Synthesis and biological evaluation of 4-quinazolinones as Rho kinase inhibitors

Rho kinase (ROCK) is an attractive therapeutic target for various diseases including glaucoma, hypertension, and spinal cord injury. Herein, we report the development of a series of ROCK-II inhibitors based on 4-quinazolinone and quinazoline scaffolds. SAR studies at three positions of the quinazoline core led to the identification of analogs with high potency against ROCK-II and good selectivity over protein kinase A (PKA).     

Bioisosteric substitution of adamantane with bicyclic lipophilic groups improves water solubility of human soluble epoxide hydrolase inhibitors

A series of inhibitors of the soluble epoxide hydrolase (sEH) containing lipophilic groups of natural origin (camphanyl, norcamphanyl, furan-2-yl) were developed. Inhibitory potency ranging from 0.4 nM to 2.16 μM were obtained. While having the same level of inhibitory activity bicyclic ureas are up to 10-fold more soluble than the corresponding ureas containing adamantyl or 4-trifluoromethoxyphenyl substituents. This makes them easier to formulate, more bioavailable and thus more promising as therapeutic sEH inhibitors. Endo/exo-form of compound 2b derived from l-camphor is 14-fold more potent than the corresponding analogue derived from d-camphor (IC₅₀ = 3.7 nM vs. 50.6 nM) indicating enantiomeric preference.     

Design,synthesis and biological evaluation of type-II VEGFR-2 inhibitors based on quinoxaline scaffold

In an effort to develop ATP-competitive VEGFR-2 selective inhibitors, a series of new quinoxaline-based derivatives was designed and synthesized. The target compounds were biologically evaluated for their inhibitory activity against VEGFR-2. The design of the target compounds was accomplished after a profound study of the structure activity relationship (SAR) of type-II VEGFR-2 inhibitors. Among the synthesized compounds, 1-(2-((4-methoxyphenyl)amino)-3-oxo-3,4 dihydroquinoxalin-6-yl)-3-phenylurea (VIIa) displayed the highest inhibitory activity against VEGFR-2. Molecular modeling study involving molecular docking and field alignment was implemented to interpret the variable inhibitory activity of the newly synthesized compounds.     

环氧化物水解酶2在肝细胞癌中的表达水平与功能作用

本研究通过公共数据和实验数据,全面分析环氧化物水解酶2(epoxide hydrolase 2, EPHX2)在肝细胞癌中的表达情况、功能作用以及预后意义。利用GEO和MitoCarta数据集,筛选肝细胞癌中呈差异表达的线粒体相关基因;利用TCGA数据库分析EPHX2及其相关基因在肝细胞癌中的表达水平;运行R包绘制Kaplan-Meier生存曲线和功能富集分析;基于STRING和GSEA构建蛋白质互作网络和基因集富集分析;荧光定量PCR和GEO数据集验证EPHX2在肝细胞癌中的表达水平。本研究共筛选得到15个在肝细胞癌中呈差异表达的线粒体相关基因。EPHX2在肝细胞癌组织中的表达水平显著降低(P<0.01)。EPHX2表达水平与肝癌患者性别、分期和级别有关,而与年龄、T分期等因素无关。与EPHX2低表达组肝癌患者相比,EPHX2高表达组肝癌患者预后较好。功能富集结果显示,EPHX2与补体途径、脂肪酸降解等信号通路有关。蛋白质互作网络结果显示,EPHX2与HAO1、AGXT、ACOX1、GSTκ1、SCP-2、CAT、CYP2C8,CYP2C9,CYP2B6,和CYP2J2等密切相关。GSEA结果显示,EPHX2低表达组与肝癌细胞增殖、肝癌复发等基因集正相关。荧光定量PCR和GEO数据集验证结果显示,EPHX2在肝细胞癌组织和肝癌细胞株中呈显著低表达。EPHX2在肝细胞癌中呈显著低表达,提示其可能在肝细胞癌发生发展过程中发挥抑癌基因作用,但具体作用机制还需进一步验证。     

Synthesis and biological evaluation of substituted 3-anilino-quinolin-2(1H)-ones as PDK1 inhibitors

PDK1 is an important regulator of the PI3K/Akt pathway, which has been found frequently activated in a large number of human cancers. Herein we described the preparation of novel substituted 3-anilino-quinolin-2(1H)-ones as PDK1 inhibitors. The synthesis is based around a Buchwald–Hartwig cross-coupling of various 3-bromo-6-substituted-quinolin-2(1H)-ones with three different functionalised anilines. The modular nature of the designed synthesis allowed access to a series of novel inhibitors through derivatisation of a late-stage intermediate. All compounds were screened against isolated PDK1 enzyme, with modest inhibition observed.     

Synthesis and biological evaluation of benzyl styrylsulfonyl derivatives as potent anticancer mitotic inhibitors

We herein report the synthesis, biological activity and structure activity relationship of derivatives of benzylstyrylsulfone, benzylstyrylsulfine and benzylsulfonyl-N-phenylacetamide. A lead compound 7 represents a new class of mitotic inhibitors that demonstrates potent anti-proliferative activity and selectively induces cancer cell apoptosis while sparing non-transformed lung fibroblast.