Synthesis and biological evaluation of 4-morpholino-2-phenylquinazolines and related derivatives as novel PI3 kinase p110alpha inhibitors

A series of 4-morpholino-2-phenylquinazolines and related derivatives were prepared and evaluated as inhibitors of PI3 kinase p110alpha. In this series, the thieno[3,2-d]pyrimidine derivative 15e showed the strongest inhibitory activity against p110alpha, with an IC(50) value of 2.0 nM, and inhibited proliferation of A375 melanoma cells with an IC(50) value of 0.58 microM. Moreover, 15e was found to be selective for p110alpha over other PI3K isoforms and protein kinases, making it the first example of a selective PI3K p110alpha inhibitor.     

A stabilized demethoxyviridin derivative inhibits PI3 kinase

The viridins like demethoxyviridin (Dmv) and wortmannin (Wm) are nanomolar inhibitors of the PI3 kinases, a family of enzymes that play key roles in a host of regulatory processes. Central to the use of these compounds to investigate the role of PI3 kinase in biological systems, or as scaffolds for drug development, are the interrelated issues of stability, chemical reactivity, and bioactivity as inhibitors of PI3 kinase. We found that Dmv was an even more potent inhibitor of PI3 kinase than Wm. However, Dmv was notably less stable than Wm in PBS, with a half-life of 26 min versus Wm’s half-life of 3470 min. Dmv, like Wm, disappeared in culture media with a half-life of less than 1 min. To overcome Dmv’s instability, it was esterified at the C1 position, and then reacted with glycine at the C20 position. The resulting Dmv derivative, termed SA–DmvC20-Gly had a half-life of 218 min in PBS and 64 min in culture media. SA–DmvC20-Gly underwent an exchange reaction at the C20 position with N-acetyl lysine in a manner similar to a WmC20 derivative, WmC20-Proline. SA–DmvC20-Gly inhibited PI3 kinase with an IC₅₀ of 44 nM, compared to Wm’s IC₅₀ of 12 nM. These results indicate that the stability of Dmv can be manipulated by reactions at the C1 and C20 positions, while substantially maintaining its ability to inhibit PI3 kinase. Our results indicate it may be possible to obtain stabilized Dmv derivatives for use as PI3 kinase inhibitors in biological systems.     

Design and synthesis of benzofuro[3,2-b]pyridin-2(1H)-one derivatives as anti-leukemia agents by inhibiting Btk and PI3Kδ

Btk inhibitors and PI3Kδ inhibitors play crucial roles in the treatment of leukemia, and studies confirmed that the synergetic inhibition against Btk and PI3Kδ could gain an optimal response. Herein, a series of novel benzofuro[3,2-b]pyridin-2(1H)-one derivatives were designed and synthesized as dual Btk/PI3Kδ kinases inhibitors for the treatment of leukemia. Studies indicated that most compounds could suppress the proliferation of multiple leukemia or lymphoma cells (Raji, HL60 and K562 cells) at low micromolar concentrations in vitro. Further kinase assays identified several compounds could simultaneously inhibit Btk kinase and PI3Kδ kinase. Thereinto, compound 16b exhibited the best inhibitory activity (Btk: IC₅₀?=?139?nM; PI3Kδ: IC₅₀?=?275?nM) and showed some selectivity against PI3Kδ compared to PI3Kβ/γ. Finally, the SAR of target compounds was preliminarily discussed combined with docking results. In brief, 16b possessed of the potency for the further optimization as anti-leukemia drugs by inhibiting simultaneously Btk kinase and PI3Kδ kinase.     

Novel compounds, '1,3-selenazine derivatives' as specific inhibitors of eukaryotic elongation factor-2 kinase

The inhibitory activities of 5,6-dihydro-4H-1,3-selenazine derivatives on protein kinases were investigated. In a multiple protein kinase assay using a postnuclear fraction of v-src-transformed NIH3T3 cells, 4-ethyl-4-hydroxy-2-p-tolyl-5, 6-dihydro-4H-1,3-selenazine (TS-2) and 4-hydroxy-6-isopropyl-4-methyl-2-p-tolyl-5,6-dihydro-4H-1, 3-selenazine (TS-4) exhibited selective inhibitory activity against eukaryotic elongation factor-2 kinase (eEF-2K) over protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK). In further experiments using purified kinases, TS-2 (IC(50)=0.36 microM) and TS-4 (IC(50)=0.31 microM) inhibited eEF-2K about 25-fold more effectively than calmodulin-dependent protein kinase-I (CaMK-I), and about 6-fold (TS-2) or 33-fold (TS-4) more effectively than calmodulin-dependent protein kinase-II (CaMK-II), respectively. TS-2 and TS-4 showed much weaker inhibitory activity toward PKA and PKC, while TS-4, but not TS-2, moderately inhibited immunoprecipitated v-src kinase. TS-2 (10.7-fold) and TS-4 (12.5-fold) demonstrated more potent and more specific eEF-2K inhibitory activity than rottlerin, a previously identified eEF-2K inhibitor. TS-2 inhibited ATP or eEF-2 binding to eEF-2K in a competitive or non-competitive manner, respectively. In cultured v-src-transformed NIH3T3 cells, TS-2 also decreased phospho-eEF-2 protein level (IC(50)=4.7 microM) without changing the total eEF-2 protein level. Taken together, these results suggest that TS-2 and TS-4 are the first identified selective eEF-2K inhibitors and should be useful tools for studying the function of eEF-2K.     

Synthesis and structure-activity relationships of novel IKK-beta inhibitors. Part 3: Orally active anti-inflammatory agents

A series of 2-amino-3-cyano-4-alkyl-6-(2-hydroxyphenyl)pyridine derivatives was synthesized and evaluated as I kappaB kinase beta (IKK-beta) inhibitors. Modification of a novel IKK-beta inhibitor 1 (IKK-beta IC(50)=1500 nM, Cell IC(50)=8000 nM) at the 4-phenyl ring and 6-phenol group on the pyridine core ring resulted in a marked increased in biological activities. An optimized compound, 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl nicotinonitrile, exhibited excellent in vitro profiles (IKK-beta IC(50)=8.5 nM, Cell IC(50)=60 nM) and a strong oral efficacy in in vivo anti-inflammatory assays (significant effects at 1mg/kg, po in arachidonic acid-induced ear edema model in mice).     

Direct effects of caffeine and theophylline on p110 delta and other phosphoinositide 3-kinases. Differential effects on lipid kinase and protein kinase activities

We investigated the effects of methylxanthines on enzymatic activity of phosphoinositide 3-kinases (PI3Ks). We found that caffeine inhibits the in vitro lipid kinase of class I PI3Ks (IC(50) = 75 microm for p110 delta, 400 microm for p110 alpha and p110 beta, and 1 mm for p110 gamma), and theophylline has similar effects (IC(50) = 75 microm for p110 delta, 300 microm for p110 alpha, and 800 microm for p110 beta and p110 gamma) and also inhibits the alpha isoform of class II PI3K (PI3K-C2 alpha) (IC(50) approximately 400 microm). However, four other xanthine derivatives tested (3-isobutyl-1-methylxanthine, 3-propylxanthine, alloxazine, and PD116948 (8-cyclopentyl-1,3-dipropylxanthine)) were an order of magnitude less effective. Surprisingly the triazoloquinazoline CGS15943 (9-chloro-2-(2-furyl)(1,2,d)triazolo(1,5-c)quinazolin-5-amine) also selectively inhibits p110 delta (IC(50) < 10 microm). Caffeine and theophylline also inhibit the intrinsic protein kinase activity of the class IA PI3Ks and DNA-dependent protein kinase, although with a much lower potency than that for the lipid kinase (IC(50) approximately 10 mm for p110 alpha, 3 mm for p110 beta, and 10 mm for DNA-dependent protein kinase). In CHO-IR cells and rat soleus muscle, theophylline and caffeine block the ability of insulin to stimulate protein kinase B with IC(50) values similar to those for inhibition of PI3K activity, whereas insulin stimulation of ERK1 or ERK2 was not inhibited at concentrations up to 10 mm. Theophylline and caffeine also blocked insulin stimulation of glucose transport in CHO-IR cells. These results demonstrate that these methylxanthines are direct inhibitors of PI3K lipid kinase activity but are distinctly less effective against serine kinase activity and thus could be of potential use in dissecting these two distinct kinase activities. Theophylline, caffeine, and CGS15943 may be of particular use in dissecting the specific role of the p110 delta lipid kinase. Finally, we conclude that inhibition of PI3K (p110 delta in particular) is likely explain some of the physiological and pharmacological properties of caffeine and theophylline.     

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.     

Exploration of the P1 SAR of aldehyde cathepsin K inhibitors

The synthesis and biological activity of a series of aldehyde inhibitors of cathepsin K are reported. Exploration of the properties of the S(1) subsite with a series of alpha-amino aldehyde derivatives substituted at the P(1) position afforded compounds with cathepsin K IC(50)s between 52 microM and 15 nM.     

Novel 4-aminoquinazoline derivatives induce growth inhibition,cell cycle arrest and apoptosis via PI3Kα inhibition

Phosphatidylinositol 3-kinase (PI3K) signaling pathway has diverse functions, including the regulation of cellular survival, proliferation, cell cycle, migration, angiogenesis and apoptosis. Among class I PI3Ks (PI3Kα, β, γ, δ), the PIK3CA gene encoding PI3K p110α is frequently mutated and overexpressed in a large portion of human cancers. Therefore, the inhibition of PI3Kα has been considered as a promising target for the development of a therapeutic treatment of cancer. In this study, we designed and synthesized a series of 4-aminoquinazoline derivatives and evaluated their antiproliferative activities against six cancer cell lines, including HCT-116, SK-HEP-1, MDA-MB-231, SNU638, A549 and MCF-7. Compound 6b with the most potent antiproliferative activity and without obvious cytotoxicity to human normal cells was selected for further biological evaluation. PI3K kinase assay showed that 6b has selectivity for PI3Kα distinguished from other isoforms. The western blot assay and PI3K kinase assay indicated that 6b effectively inhibited cell proliferation via suppression of PI3Kα kinase activity with an IC₅₀ of 13.6?nM and subsequently blocked PI3K/Akt pathway activation in HCT116 cells. In addition, 6b caused G1 cell cycle arrest owing to the inhibition of PI3K signaling and induced apoptosis via mitochondrial dependent apoptotic pathway. Our findings suggested that 6b has a therapeutic value as an anticancer agent via PI3Kα inhibition.     

Discovery of pyrazolo[1,5-a]pyridines as p110α-selective PI3 kinase inhibitors

We have made a novel series of pyrazolo[1,5-a]pyridines as PI3 kinase inhibitors, and demonstrated their selectivity for the p110α isoform over the other Class Ia PI3 kinases. We investigated the SAR around the pyrazolo[1,5-a]pyridine ring system, and found compound 5x to be a particularly potent example (p110α IC(50) 0.9nM). This compound inhibits cell proliferation and phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity, and showed in vivo activity in an HCT-116 human xenograft model.     

Identification of 4-quinolone derivatives as inhibitors of reactive oxygen species production from human umbilical vein endothelial cells

Oxidative stress is widely recognized as being associated with a number of disorders, including metabolic dysfunction and atherosclerosis. A series of substituted 4-quinolone derivatives were prepared and evaluated as inhibitors of reactive oxygen species (ROS) production from human umbilical vein endothelial cells (HUVECs). One compound in particular, 2-({[4-(3-hydroxy-3-methylbutoxy)pyridin-2-yl]oxy}methyl)-3-methylquinolin-4(1H)-one (25b), inhibited ROS production from HUVECs with an IC(50) of 140 nM. This compound also exhibited low CYP2D6 inhibitory activity, high aqueous solubility, and good in vitro metabolic stability. An in vivo pharmacokinetic study of this compound in SD rats revealed high oral bioavailability and a long plasma half-life.     

Discovery of a novel CCR3 selective antagonist

In searching for a novel CCR3 receptor antagonist, we designed a library that included a variety of carboxamide derivatives based on the structure of our potent antagonists for human CCR1 and CCR3 receptors, and screened the new compounds for inhibitory activity against 125I-Eotaxin binding to human CCR3 receptors expressed in CHO cells. Among them, two 2-(benzothiazolethio)acetamide derivatives (1a and 2a) showed binding affinities with IC50 values of 750 and 1000 nM, respectively, for human CCR3 receptors. These compounds (1a and 2a) also possessed weak binding affinities for human CCR1 receptors. We selected la as a lead compound for derivatization to improve in vitro potency and selectivity for CCR3 over CCRI receptors. Derivatization of la by incorporating substituents into each benzene ring of the benzothiazole and piperidine side chain resulted in the discovery of a compound (1b) exhibiting 820-fold selectivity for CCR3 receptors (IC50 = 2.3 nM) over CCR1 receptors (IC50 = 1900 nM). This compound (1b) also showed potent functional antagonist activity for inhibiting Eotaxin (IC50 = 27 nM)- or RANTES (IC50 = 13 nM)-induced Ca2+ increases in eosinophils.     

Synthesis and inhibitory activity of acyl-peptidyl-pyrrolidine derivatives toward post-proline cleaving enzyme; a study of subsite specificity.

Several pyrrolidine derivatives have been synthesized and examined for their inhibitory activity on post-proline cleaving enzymes from Flavobacterium meningosepticum and bovine brain. Almost all the compounds tested in this study inhibited the activity of both enzymes at low IC50 values (from nM to microM) but a specificity difference was observed with alkylacyl-peptidyl-pyrrolidine derivatives which strongly inhibited only the bacterial enzyme. The most effective inhibitors have a proline residue on their P2 sites and a substituted or unsubstituted phenoxybutyryl moiety on their P3 sites. Thus phenoxybutyryl-prolyl-pyrrolidine is the most effective partial structure of the inhibitors. The best inhibitors found were: 4-(4-benzylphenoxy)butyryl-prolyl-pyrrolidine for bacterial enzyme (IC50 1.4 nM) and 4-phenylbutyryl-thioprolyl-pyrrolidine for bovine brain enzyme (IC50 67 nM). In the passive avoidance test, using amnesic rats experimentally induced with scopolamine, the pyrrolidine derivatives which had potent inhibitory activity toward post-proline cleaving enzymes also showed strong anti-amnesic activities at doses of 1-5 mg/kg, i.p.     

Synthesis and biological evaluation of 4-(piperid-3-yl)amino substituted 6-pyridylquinazolines as potent PI3Kδ inhibitors

PI3Kδ is an intriguing target for developing anti-cancer agent. In this study, a new series of 4-(piperid-3-yl)amino substituted 6-pyridylquinazoline derivatives were synthesized. After biological evaluation, compounds A5 and A8 were identified as potent PI3Kδ inhibitors, with IC₅₀ values of 1.3 and 0.7 nM, respectively, which are equivalent to or better than idelalisib (IC₅₀ = 1.2 nM). Further PI3K isoforms selectivity evaluation showed that compound A5 afforded excellent PI3Kδ selectivity over PI3Kα, PI3Kβ and PI3Kγ. A8 exhibited superior PI3Kδ/γ selectivity over PI3Kα and PI3Kβ. Moreover, compounds A5 and A8 selectively exhibited anti-proliferation against SU-DHL-6 in vitro with IC₅₀ values of 0.16 and 0.12 μM. Western blot analysis indicated that A8 could attenuate the AKT^S473 phosphorylation. Molecular docking study suggested that A8 formed three key H-bonds action with PI3Kδ, which may account for its potent inhibition of PI3Kδ. These findings indicate that 4-(piperid-3-yl)amino substituted 6-pyridylquinazoline derivatives were potent PI3Kδ inhibitors with distinctive PI3K-isoforms and anti-proliferation profiles.     

Synthesis and structure-activity relationships of novel IKK-beta inhibitors. Part 2: Improvement of in vitro activity

A series of 2-amino-3-cyano-4-alkyl-6-(2-hydroxyphenyl)pyridine derivatives was synthesized and evaluated as IkappaB kinase beta (IKK-beta) inhibitors. Substitution of an aminoalkyl group for the aromatic group at the 4-position on the core pyridine ring resulted in a marked increase in both kinase enzyme and cellular potencies, and provided potent IKK-beta inhibitors with IC(50) values of below 100 nM.     

Synthesis and structure-activity relationships of dual PI3K/mTOR inhibitors based on a 4-amino-6-methyl-1,3,5-triazine sulfonamide scaffold

Phosphoinositide 3-kinase (PI3K) is an important target in oncology due to the deregulation of the PI3K/Akt signaling pathway in a wide variety of tumors. A series of 4-amino-6-methyl-1,3,5-triazine sulfonamides were synthesized and evaluated as inhibitors of PI3K. The synthesis, in vitro biological activities, pharmacokinetic and in vivo pharmacodynamic profiling of these compounds are described. The most promising compound from this investigation (compound 3j) was found to be a pan class I PI3K inhibitor with a moderate (>10-fold) selectivity over the mammalian target of rapamycin (mTOR) in the enzyme assay. In a U87 MG cellular assay measuring phosphorylation of Akt, compound 3j displayed low double digit nanomolar IC(50) and exhibited good oral bioavailability in rats (F(oral)=63%). Compound 3j also showed a dose dependent reduction in the phosphorylation of Akt in a U87 tumor pharmacodynamic model with a plasma EC(50)=193nM (91ng/mL).     

Synthesis, biological evaluation and molecular docking studies of novel 2-(1,3,4-oxadiazol-2-ylthio)-1-phenylethanone derivatives

In present study, a series of new 2-(1,3,4-oxadiazol-2-ylthio)-1-phenylethanone derivatives (6a-6x) as potential focal adhesion kinase (FAK) inhibitors were synthesized. The bioassay assays demonstrated that compound 6i showed the most potent activity, which inhibited the growth of MCF-7 and A431 cell lines with IC(50) values of 140 ± 10 nM and 10 ± 1 nM, respectively. Compound 6i also exhibited significant FAK inhibitory activity (IC(50)=20 ± 1 nM). Docking simulation was performed to position compound 6i into the active site of FAK to determine the probable binding model.     

Insulin-like growth factor I prevents the development of sensitivity to kainate neurotoxicity in cerebellar granule cells

This study reports that insulin-like growth factor I (IGF-I) prevents cerebellar granule cells from developing sensitivity to kainate neurotoxicity. Sensitivity to kainate neurotoxicity normally develops 5-6 days after switching cultures to a serum-free medium containing 25 mM K(+). Addition of either IGF-I or insulin to the serum-free medium at the time of the switch prevented the development of sensitivity to kainate, whereas brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, and nerve growth factor did not. The dose-response curves indicated IGF-I was more potent than insulin, favoring the assignment of the former as the physiological protective agent. The phosphatidylinositol 3-kinase (PI 3-K) inhibitors wortmannin (10-100 nM) and LY 294002 (0.3-1 microM) abolished the protection afforded by IGF-I. The p70 S6 kinase (p70(S6k)) inhibitor rapamycin (5-50 nM:) also abolished the protection afforded by IGF-I. The activities of both enzymes decreased in cultures switched to serum-free medium but increased when IGF-I was included; wortmannin (100 nM) lowered the activity of PI 3-K from 2 to 5 days after medium switch, whereas rapamycin (50 nM) prevented the increase observed for p70(S6k) activity over the same interval. The mitogen-activated protein kinase kinase inhibitor U 0126 and the mitogen-activated protein kinase inhibitor SB 203580 did not abolish IGF-I protection. Kainate neurotoxicity was not prevented by Joro spider toxin; therefore, the development of kainate neurotoxicity could not be explained by the formation of calcium-permeable alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors. These results indicate that IGF-I functions through a signal transduction pathway involving PI 3-K and p70(S6k) to prevent the development of sensitivity to kainate neurotoxicity in cerebellar granule cells.     

Synthesis and biological evaluation of glycogen synthase kinase 3 (GSK-3) inhibitors: an fast and atom efficient access to 1-aryl-3-benzylureas

The glycogen synthase kinase 3 (GSK-3) is implicated in multiple cellular processes and has been linked to the pathogenesis of Alzheimer's disease (AD). In the course of our research topic we synthesized a library of potent GSK-3 inhibitors. We utilized the urea scaffold present in the potent and highly selective GSK-3 inhibitor AR-A014418 (AstraZeneca). This moiety suits both (a) a convergent approach utilizing readily accessible building blocks and (b) a divergent approach based on a microwave heating assisted Suzuki coupling. We established a chromatography-free purification method to generate products with sufficient purity for the biological assays. The structure-activity relationship of the library provided the rationale for the synthesis of the benzothiazolylurea 66 (IC(50)=140 nM) and the pyridylurea 62 (IC(50)=98 nM), which displayed two to threefold enhanced activity versus the reference compound 18 (AR-A014418: IC(50)=330 nM) in our assays.     

Identification of Novel Piperazinylquinoxaline Derivatives as Potent Phosphoinositide 3-Kinase (PI3K) Inhibitors

Background

Development of small-molecule inhibitors targeting phosphoinositide 3-kinase (PI3K) has been an appealing strategy for the treatment of various types of cancers.

Methodology/Principal Finding

Our approach was to perform structural modification and optimization based on previously identified morpholinoquinoxaline derivative WR1 and piperidinylquinoxaline derivative WR23 with a total of forty-five novel piperazinylquinoxaline derivatives synthesized. Most target compounds showed low micromolar to nanomolar antiproliferative potency against five human cancer cell lines using MTT method. Selected compounds showed potent PI3Kα inhibitory activity in a competitive fluorescent polarization assay, such as compound 22 (IC₅₀ 40 nM) and 41 (IC₅₀: 24 nM), which induced apoptosis in PC3 cells. Molecular docking analysis was performed to explore possible binding modes between target compounds and PI3K.

Conclusions/Significance

The identified novel piperazinylquinoxaline derivatives that showed potent PI3Kα inhibitory activity and cellular antiproliferative potency may be promising agents for potential applications in cancer treatment.