Discovery of 7, 4'-dimethoxy-3-hydroxyflavone as a protease-activated receptor 4 antagonist with antithrombotic activity and less bleeding tendency in mice.

There is growing evidence of the importance of protease-activated receptor 4 (PAR4), one of thrombin receptors, as a therapeutic target in thrombotic cardiovascular diseases. In the present study, we utilized ligand-based virtual screening, bioassay, and structure-activity relationship study to discover PAR4 antagonists with new chemical scaffolds from natural origin, and examined their application as antiplatelet agents. By using these approaches, we have identified a flavonoid, 7, 4'-dimethoxy-3-hydroxyflavone, that exhibits anti-PAR4 activity. 7, 4'-Dimethoxy-3-hydroxyflavone inhibited PAR4-mediated human platelet aggregation, GPIIb/IIIa activation, and P-selectin secretion. Also, it inhibited PAR4 downstream signaling pathways, including Ca2+/protein kinase C, Akt, and MAP kinases ERK and p38, in human platelets, and suppressed PAR4-mediated ß-arrestin recruitment in CHO-K1 cells exogenously expressed human PAR4. In a microfluidic system, 7, 4'-dimethoxy-3-hydroxyflavone reduced thrombus formation on collagen-coated chambers at an arterial shear rate in recalcified whole blood. Furthermore, mice treated with 7, 4'-dimethoxy-3-hydroxyflavone were significantly protected from FeCl3-induced carotid arterial occlusions, without significantly affecting tail bleeding time. In conclusion, 7, 4'-dimethoxy-3-hydroxyflavone represents a new class of nature-based PAR4 antagonist, it shows effective in vivo antithrombotic properties with less bleeding tendency, and could be a potential candidate for developing new antiplatelet agents.

Synthesis of Dibenzosuberones Bearing an Isoxazole Group via Palladium-Catalyzed Intramolecular C-H/C-Br Bond Cross-Coupling of Ortho-Aroylated 3,5-Diarylisoxazoles.

A facile and efficient synthetic methodology for preparing dibenzosuberones via a C-H bond activation strategy is presented. The ortho-aroylated 3,5-diarylisoxazole was employed as the starting substrate to undergo palladium-catalyzed intramolecular C-H/C-Br bond cross-coupling to produce a variety of dibenzosuberones bearing an isoxazole group in 24 to >99% 1H NMR yields. The dibenzosuberone structure was further confirmed by X-ray crystallography. The developed methodology exhibits very good functional group tolerance. In addition, a rational mechanism was presented for describing the reaction process. For the prepared dibenzosuberone, the use of Mo(CO)6 as the catalyst can easily transform the isoxazole ring into the ß-aminoenone group. Finally, the structure of the anticipated ring-opening product, dibenzosuberenone, bearing a ß-amino-α-ketone group was secured by X-ray crystallography.

Halogen-Mediated Cascade Cyclization Reaction of Aryldiynes to Indeno[1,2-c]chromene Derivatives.

The halogen-mediated cyclization reaction of aryldiynes to produce halogenated indeno[1,2-c]chromene derivatives is described. Treatment of aryldiynes 1 with one equivalent of iodine gave iodinated indeno[1,2-c]chromenes 3 in good chemical yields. When two equivalents of iodine were employed into the reaction mixture, dimer 9 was obtained as the major products. On the other hand, reaction of two equivalents of CuBr2 with compounds 1 gave the brominated indeno[1,2-c]chromenes 4. The DFT calculation of the iodine-mediated cyclization reactions for molecules containing methoxy, carboxy, amino, and sulfide substituents were carried out in order to understand how the substituent affects the cyclization pathway.

Base-Mediated Cyclization Reaction of 2-(5-Hydroxy-1-pentynyl)benzonitriles to 4-Amino-2,3-dihydronaphtho[2,3-b]furanes and Synthesis of Furanonaphthoquinones.

An efficient transformation of 2-(5-hydroxy-1-pentynyl)benzonitriles 5 to furanonaphthoquinones 11 is presented. Treatment of 5 with 1.5 equiv of NaOMe in DMSO at 140 °C for 0.5 h gave 6 in good yields. Conversion of 6 to 11 was carried out by oxidation of 6 with Fremy's salt and KH2PO4 in acetone and water, followed by dehydrogenation using palladium on charcoal in diphenylether at reflux temperature.

Substituent electronic effects govern direct intramolecular C-N cyclization of N-(Biphenyl)pyridin-2-amines induced by hypervalent iodine(III) reagents.

The hypervalent iodine(III) reagent-induced the direct intramolecular C-N cyclization of N-(biphenyl)pyridin-2-amines to 6-arylbenzimidazoles and N-pyridinyl-9H-carbazoles is presented. The substituent electronic effects governing the formation of benzimidazoles and carbazoles from the reaction of N-(biphenyl)pyridin-2-amines with hypervalent iodine(III) reagents is investigated. Radical trapping and UV-vis spectroscopic experiments on the detection of the cation radical are carried out. Rational mechanisms for these reactions are presented. The selective intramolecular C-N and C-O cyclization of N-(biphenyl)acetamides based on the substituent electronic effects is also presented.

Transition metal-catalyzed cascade cyclization of aryldiynes to halogenated benzo[b]naphtho[2,1-d]thiophene derivatives.

Treatment of 2-(2-(2-(2-substituted ethynyl)phenyl)ethynyl)thioanisoles (1) with 5 mol % of Ph3PAuCl/AgSbF6 and 2 equiv of NIS at refluxing CH2Cl2 gave iodo-substituted benzo[b]naphtho[2,1-d]thiophene (6) in good yields. Chloro- and bromo-substituted benzo[b]naphtho[2,1-d]thiophene derivatives (8 and 9) were also generated by treating compound 1 with 5 mol % of PdX2 and 3 equiv of CuX2 at refluxing THF.

Design, synthesis, biological evaluation and molecular modeling studies of 1-aryl-6-(3,4,5-trimethoxyphenyl)-3(Z)-hexen-1,5-diynes as a new class of potent antitumor agents.

A series of novel enediyne-containing molecules, 1-aryl-6-(3,4,5-trimethoxyphenyl)-3(Z)-hexen-1,5-diynes, were synthesized and displayed significant IC50 values of 10(-7) to 10(-6) M against various cancer cell lines. Of these compounds, 1-(2-pyridinyl)-6-(3,4,5-trimethoxyphenyl)-3(Z)-hexen-1,5-diyne (8) demonstrated the greatest growth inhibition activity. Compound 8 also arrested cancer cells in the G2/M phase and induced apoptosis via activation of Caspase-3. In addition to the G2/M block, compound 8 caused microtubule depolymerization at low concentrations and markedly decreased tumor size in xenographic studies.

Au(I)-catalyzed and iodine-mediated cyclization of enynylpyrazoles to provide pyrazolo[1,5-a]pyridines.

Pyrazolo[1,5-a]pyridines and 6-iodopyrazolo[1,5-a]pyridines were synthesized by gold-catalyzed and iodine-mediated cyclization of enynylpyrazoles in good to excellent yields, respectively. The iodinated adducts were further converted to 6-arylpyrazolo[1,5-a]pyridines via Suzuki-Miyaura coupling reaction and 6-cyanopyrazolo[1,5-a]pyridine by Ullmann condensation reaction. One of the cyclization adducts, 2-(4-fluorophenyl)pyrazolo[1,5-a]pyridine, was converted to a p38 kinase inhibitor, 2-(4-fluorophenyl)-3-(4-pyridinyl)pyrazolo[1,5-a]pyridine, in two steps.

Iodine-mediated cascade cyclization of enediynes to iodinated benzo[a]carbazoles.

Treatment of N,N-dimethyl 2-[2-(2-ethynylphenyl)ethynyl]anilines (1) with 1.2 equiv of iodine in CH(2)Cl(2) gave benzo[a]carbazoles (2) in good yields. Mechanistic studies showed this reaction must go through the haloindole (3) followed by iodonium ion catalyzed atom-transfer cyclization reaction to give the benzo[a]carbazoles.

Palladium(II)-catalyzed one-pot syntheses of 9-(pyridin-2-yl)-9H-carbazoles through a tandem C-H activation/C-X (X=C or N) formation process.

A new one-pot synthesis of 9-(pyridin-2-yl)-9H-carbazoles through the simultaneous C-H activation and palladium(II)-catalyzed cross-coupling of N-phenylpyridin-2-amines with potassium aryltrifluoroborates is presented. Silver acetate and 1,4-dioxane proved to be the best oxidant and solvent, respectively. The product yields fluctuated from modest to excellent and the reaction showed sufficient functional group tolerance. p-Benzoquinone served as an important ligand for the transmetalation and reductive elimination steps in the catalytic process. The kinetic isotope effects (k(H)/k(D)) for the first and second C-H activation/C-C or C-N formation steps were measured as 2.14 and 1.18, respectively. Finally, a rational catalytic mechanism is presented based on all experimental evidence.

A copper-mediated cyclization reaction of hydrazine with enediynones providing pyrazolo[1,5-a]pyridines.

2,7-Disubstituted pyrazolo[1,5-a]pyridines were synthesized in good chemical yields by the reaction of enediynones with hydrazine, followed by addition of copper chloride. This reaction can tolerate many functional groups.

First total synthesis of antrocamphin A and its analogs as anti-inflammatory and anti-platelet aggregation agents.

Naturally occurring antrocamphin A (1) is a potent anti-inflammatory compound from the edible fungus Antrodia camphorata (Taiwanofungus camphoratus), whose wild fruiting body is used as a valuable folk medicine in Taiwan. This study is the first total synthesis of antrocamphin A (1) and its analogs. Their inhibition ability on NO release, superoxide anion generation, elastase release and platelet aggregation are reported herein.

Synthetic development and mechanistic study on Pd(II)-catalyzed cyclization of enediynes to benzo[a]carbazoles.

Treatment of N,N-dimethyl 2-[2-(2-ethynylphenyl)ethynyl]anilines (1) with 10 mol % of palladium chloride and 2 equiv of cupric chloride in refluxing THF gave benzo[a]carbazoles (6) in good yields. A mechanistic study showed that this reaction must proceed through formation of haloindole (7) followed by a palladium(II)-catalyzed atom transfer cyclization reaction to give the benzo[a]carbazoles.

Synthesis and antitumor activity of cis-dichloridoplatinum(II) complexes of 1,1'-biisoquinolines.

A simple approach to 1,1',2,2',3,3',4,4'-octahydro-1,1'-biisoquinolines is described. Reaction of phenethylamines with oxalyl chloride led to N,N'-bis(phenethyl) oxamides (1). Cyclization of oxamides by using Bischler-Napieralski conditions gave 3,3',4,4'-tetrahydro-1,1'-biisoquinoline (3) and unusual products 2, 4, 5. Reduction of 3,3',4,4'-tetrahydro-1,1'-biisoquinolines with sodium boron hydride gave both rac-1,1',2,2',3,3',4,4'-octahydro-1,1'-biisoquinolines (6) and meso-1,1',2,2',3,3',4,4' -octahydro-1,1'-biisoquinolines (7). Compound 6 was resolved to (1S, 1S') (8) and (1R, 1R') (9) furtherly. By treating all the biisoquinolines with K2PtCl4 afforded their cis-dichloridoplatinum (II) complexes (12-18). The antitumor activity of these complexes was evaluated.

1-(2-((Z)-6-(2-(Trifluoromethyl)phenyl)hexa-3-en-1,5-diynyl)phenyl)piperidin-2-one as a new potent apoptosis agent.

Compounds 4a-f, 5a-f and 6-9, showed significant growth inhibition activity against human tumor cell lines. Of these compounds, 1-(2-((Z)-6-(2-(trifluoromethyl)phenyl)hexa-3-en-1,5-diynyl)phenyl)piperidin-2-one (8) displayed the most potent growth inhibition activity. Compound 8 also arrested cancer cells in G2/M phase and induced apoptosis via activation of caspase-3 and -9. According to western-blotting analysis, compound 8 can up-regulate Bax, down-regulate Bcl-2 and XIAP, as well as promote cytochrome c release.

Palladium-catalyzed annulation of 2-(1-alkynyl)biphenyls with aryl iodides to disubstituted methylidenefluorenes.

The palladium-catalyzed annulation reaction of 2-(1-alkynyl)biphenyls (1) with aryl iodides has been developed to prepare a variety of diarylmethylidenefluorenes (2). This method tolerates various functional groups in aryl iodide, such as OCH(3), CH(3), Cl, Br, Cf(3), and NO(2). All proceed in good yield with the exception of 4-iodonitrobenzene.

Membrane-bound conformation and phospholipid components modulate membrane-damaging activity of Taiwan cobra cardiotoxins.

Membrane-damaging activity of Naja naja atra cardiotoxin 3 (CTX3) on 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC)/1,2-dimyristoyl-phosphatidic acid (DMPA) vesicles was approximately 3-fold that of N. naja atra cardiotoxin 4 (CTX4), while CTX3 and CTX4 displayed insignificantly permeabilizing activity in 1,2-dipalmitoyl-phosphatidylcholine (DPPC)/DMPA vesicles. Phospholipid-binding capability and oligomeric assembly upon binding with lipid vesicles did not closely correlate with membrane-damaging potency of CTX3 and CTX4. Geometrical arrangement of CTX3 in contact with POPC/DMPA vesicles was different from that noted with CTX4, and binding forces between CTX3 and POPC/DMPA were stronger than those between CTX4 and POPC/DMPA. Unlike POPC/DMPA, the interaction between CTXs and DPPC/DMPA was drastically reduced by increasing salt concentration. Color transformation of phospholipid/polydiacetylene membrane assay and FTIR spectra analyses revealed that CTX3 and CTX4 adopted different conformationsand modes upon absorption on POPC/DMPA and DPPC/DMPA vesicles. Taken together, our data show that, in addition to membrane packing density and phospholipid-binding capability, membrane-bound conformation of CTXs plays a vital role in displaying membrane-damaging activity.

Synthesis and antitumor activity of novel enediyne-linked pyrrolo[2,1-c][1,4]benzodiazepine hybrids.

A series of novel pyrrolo[2,1-c][1,4]benzodiazepine (PBD) hybrids linked with enediyne is described. These compounds were prepared by linking C-8 of DC-81 (1) with an enediyne (10-16) through carbon chain linkers to afford PBD hybrid agents 17-23 in good yields. Most of the hybrids on human cancer cell lines exhibited higher cytotoxicity, and an increase in the sub-G1 population than 1. In a previous article, we have demonstrated that DC-81-indole conjugate agents (3-6) are potent inducers of cell apoptosis in melanoma. In the present article, we investigated whether DC-81-enediyne agents possess more cytotoxicity than 6 on human 293T cells. Our data revealed that treatment of 293T cells with DC-81-enediyne resulted in a significant increase of annexin V binding, caspase-3 degradation, and p53 arrest to identify apoptotic cells than 6. These results suggest that the DC-81-enediyne agents are more efficient in inducing apoptosis than DC-81-indole in 293T cells.

Catalytic activity-independent pathway is involved in phospholipase A(2)-induced apoptotic death of human leukemia U937 cells via Ca(2+)-mediated p38 MAPK activation and mitochondrial depolarization.

In view of the controversial role of catalytic activity on the cytotoxicity of phospholipase A(2) (PLA(2)), the present study is conducted to explore whether PLA(2) induces apoptotic process of human leukemia U937 cells through catalytic activity-independent pathway. Modification of His-48 (according to the sequence alignment with porcine pancreatic PLA(2)) with p-bromophenacyl bromide (BPB) caused over 99.9% drop in enzymatic activity Naja naja atra PLA(2). It was found that BPB-PLA(2)-induced apoptotic death of U937 cells was associated with mitochondrial depolarization, modulation of Bcl-2 family members, cytochrome c release and activation of caspases 9 and 3. Upon exposure to BPB-PLA(2), elevation of intracellular Ca(2+) levels and p38 MAPK activation were observed in U937 cells. Pretreatment with BAPTA-AM (Ca(2+) chelator) and nifedipine (L-type Ca(2+) channel blocker) abrogated Ca(2+) increase and p38 MAPK activation, and rescued viability of BPB-PLA(2)-treated U937 cells. BPB-PLA(2)-induced dissipation of mitochondrial membrane potential and down-regulation of Bcl-2 were suppressed by SB202190 (p38MAPK inhibitor). Although PLA(2) mutants in which His-48 and Asp-49 were substituted by Ala and Lys, respectively, did not display detectable PLA(2) activity, they induced death of U937 cells. The signaling pathway of PLA(2) mutants in inducing cell death was indistinguishable from that of BPB-PLA(2). Taken together, our data indicate that catalytic activity-independent pathway is involved in PLA(2)-induced apoptotic death of human leukemia U937 cells via mitochondria-mediated death pathway triggering by Ca(2+)-mediated p38 MAPK activation.