Facile preparation of new 4-phenylamino-3-quinolinecarbonitrile Src kinase inhibitors via 7-fluoro intermediates: identification of potent 7-amino analogs

A more efficient preparation of 4-[(2,4-dichloro-5-methoxyphenyl)amino]-7-fluoro-6-methoxy-3-quinolinecarbonitrile (2), the penultimate intermediate in the synthesis of bosutinib (1a), was developed. New 7-alkoxy-4-phenylamino-3-quinolinecarbonitrile Src inhibitors were prepared from 5 and 9, the 6-ethoxy and 6-hydrogen analogs of 2. In addition, the fluoro group of 2 was readily displaced by primary and secondary amines to give 7-amino analogs. Two of these 7-amino analogs, 15 and 18, were potent Src inhibitors with in vivo activity.     

7-(aryl/heteroaryl-2-ylethynyl)-4-phenylamino-3-quinolinecarbonitriles as new Src kinase inhibitors: addition of water solubilizing groups

New 4-phenylamino-3-quinolinecarbonitriles with a 7-ethynyl group substituted by a pyridine, phenyl or thiophene ring containing basic water solubilizing groups were prepared and evaluated as Src kinase inhibitors. Of these new analogs, potent activity was observed with compounds having a (2,4-dichloro-5-methoxyphenyl)amino group at C-4, a methoxy or ethoxy group at C-6, and a pyridyl group bearing a dimethylamine or N-methylpiperazine on the ethynyl group at C-7.     

Annonaceous acetogenins: Naturally occurring inhibitors of ATP synthesis and photosystem II in spinach chloroplasts

The effects of squamocin ( 1 ), bullatacin ( 2 ) and motrilin ( 3 ), 3 bis-tetrahydrofuran Annonaceous acetogenins, isolated from Annona purpurea (Annonaceae), were investigated on several photosynthetic activities in spinach thylakoids. The results indicated that compounds 1 – 3 significantly inhibited both ATP synthesis and uncoupled electron transport. In addition, they enhanced light-activated Mg2+-ATPase, and basal electron flow. Therefore, acetogenins 1 – 3 behave as uncouplers and Hill reaction inhibitors. Natural products 1 – 3 did not affect photosystem I (PSI) activity but they inhibited photosystem II (PSII) electron flow. The study of the partial PSII reactions from H2O to DCPIPox, H2O to SiMo and diphenylcarbazide to DCPIP established that the site of inhibition was at the oxygen-evolving complex (OEC). Chlorophyll a fluorescence measurements confirmed the behavior of the Annonaceous acetogenins as water-splitting enzyme inhibitors.     

Studies of pesticide effects on Chlorella metabolism I. Effect of herbicides on complex lipid biosynthesis

Various herbicides were tested for possible effects on biosynthesisof complex lipids in Chlorella using ¹⁴C-U-acetate as a precursor.Among the herbicides tested, Hill reaction inhibitors causedinhibition of ¹⁴C-incorporation by 60 to 80 per cent at a concentrationof 5 mg/liter, and noticeable alteration in the labeling patternof glyco- and phospholipids. Proportions of mono- and digalactolipidsmarkedly decreased while those of phosphatidyl ethanolamineand phosphatidyl glycerol increased. In terms of net synthesis,phosphatidyl ethanolamine was affected, if at all, only slightlyby Hill reaction inhibitors, while the other complex lipidsdecreased to various degrees. (Received February 9, 1973; )     

Inhibition of Respiration of Yeast by Photosynthesis Inhibiting Herbicides

In order to elucidate the mode of action of some herbicides, effect of several anilide type herbicides on the respiration of yeast cells was studied. The results obtained were as follows: 1) DCPA (3,4-dichloropropionanilide) and DCMU (3-(3,4-dichlorophenyl)-1,1- dimethylurea), the powerful inhibitors of the Hill reaction in photosynthesis, inhibited the oxygen uptake of yeast cells at low concentrations. 2) DCPA and DCMU inhibited the enzymic reduction of cytochrome-c by the yeast cell-free preparation, but not the reduction of dye. 3) The oxidation of cytochrome-b was inhibited in the yeast cells treated with DCPA or DCMU.     

Non-enzymatic reduction of a 1,2,4-thiadiazolium derivative

It was discovered that 2,3-bis-(2-methoxy-phenyl)-5-phenylamino-[1,2,4]-thiadiazolium bromide (1), a 1,2,4-thiadiazolium derivative, could be reduced to the corresponding imidoylthiourea, 1-[(2-methoxy-phenyl)-(2-methoxy-phenylimino)-methyl]-3-phenyl-thiourea (3), by some biologically interesting reducing reagents including glutathione, cysteine, and ascorbic acid. The reduction also occurred in Sprague-Dawley rat and Yorkshire swine plasma, suggesting that thiol containing biological molecules existing in the plasma are mainly responsible for this reaction. A facile method for preparation of 3 from 1 was established by using 2-thioethanol as reaction reagent as well as solvent. The structure of 3 was fully characterized using nuclear magnetic resonance (NMR) and mass spectrometry with electrospray ionization source (ESI-MS). Those new findings could shed light on the development of 1,2,4-thiadiazolium derivatives for their potential pharmaceutical applications.     

Clara cell damage and inhibition of pulmonary mixed-function oxidase activity by naphthalene

The photosystem II electron acceptor 3,6-dichloro-2,5-dimethoxy-p-benzoquinone [DCDMQ] is suggested to replace the second quinone-type two electron acceptor B (or R); the DCDMQ Hill reaction is sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, but is insensitive to dry heptane extraction of thylakoids and other photosystem II inhibitors. Addition of HCO₃^? to CO₂-depleted thylakoids in silicomolybdate, DCDMQ, diaminodurene and ferricyanide Hill reactions brought about 1,3,10 and 10 fold increase in the electron transport rates; these data confirm that HCO₃^? affects both Q^? to B and B^2? to PQ reactions.     

Discovery of TAK-733, a potent and selective MEK allosteric site inhibitor for the treatment of cancer

A novel 5-phenylamino-8-methylpyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione series of MEK inhibitors has been developed using structure-based drug design. Lead optimization of this series led to the discovery of TAK-733. This was advanced to Phase I clinical studies for cancer treatment.     

alpha-1-Anti-trypsin, an inhibitor of renin.

A naturally occurring competitive inhibitor of pig kidney renin has been identified in human plasma. The inhibitor was shown to be alpha-1 anti-trypsin and the effect in vitro on the renin activity was examined. The slope in the Hill plot is compatible with the assumption of one-site competitive inhibition. Other proteinase inhibitors, such as alpha-2-macroglobulin and C1 inactivator, however, have no inhibitory effect on the renin-angiotensinogen reaction.     

Inhibition of human serum arylesterase by metal chlorides

The inhibition of arylesterase (paraoxonase, EC 3.1.8.1) by metal chlorides was studied with both pooled human serum (A phenotype) and purified enzyme, using phenyl acetate as substrate. Inhibition data were analysed with the Hill equation. Results obtained with whole serum and purified enzyme were very similar. On the basis of the Hill coefficient, n(H), three groups of inhibitors were distinguished: (1) Cu(2+) and Hg(2+) for which n(H)=1, suggesting a single binding site (probably the free cysteine at position 283); these metals were mixed inhibitors, with more affinity for the free enzyme than for the enzyme-substrate complex; (2) Mn(2+), Co(2+), Ni(2+), Zn(2+), and Cd(2+) for which n(H)>1, suggesting several cooperative binding sites; (3) La(3+), for which n(H)<1. Within groups (1) and (2) the inhibiting potency followed the order of the periodic table. For the 3d elements the inhibiting order followed the Irving-Williams series, with the classical exception of Cu(2+). Only Zn(2+) was inhibitory at its physiological concentration.     

β(1–3)Glucan synthase activity ofNeurospora crassa: Kinetic analysis of negative effectors

β(1–3)Glucan synthase activity ofNeurospora crassa was inhibited by a number of compounds. Uridine nucleotides were linear competitive inhibitors of enzyme activity. Hill plots for the competitive inhibitors and for the substrate (UDP-glucose) resulted in straight lines with Hill numbers near unity suggesting a single substrate binding site. Tunicamycin, dolichol, or dolichol phosphate did not affect enzyme activity and a lipid-linked intermediate was not detected. Sorbose, gluconolactone, echinocandin B, and papulacandin B were uncompetitive inhibitors. Mixed inhibitor studies revealed that the binding of one uncompetitive inhibitor blocked completely the binding of each of the other uncompetitive inhibitors.     

Requirement for plastoquinone a in the hill reaction of isolated chloroplasts

In order to avoid the problem of an uncoupling effect in stimulation of potential Hill reaction activity during extraction with heptane. Hill reaction activity was measured with uncoupled spinach chloroplasts. The reactions were also run under aerobic conditions. Under these conditions a correction for a 3-(p-chlorophenyl)-1, 1-dimethylurea insensitive, photostimulated O₂ uptake reaction is necessary, especially with extracted chloroplasts. The reaction displayed proper Hill reaction stoichiometry in untreated and restored systems if these corrections were made. Plastoquinone A always restored some activity to the extracted chloroplasts but never restored activity to the original level. Additional activity could be restored by the crude extract. It appears that there is a clear requirement for plastoquinone A in the Hill reaction but the restoration of maximum activity depends upon additional factors. The variability of response to plastoquinone A which has been reported in various laboratories may be determined by the degree of extraction of the other materials.     

Photosynthetic electron transport and phosphorylation reactions in thylakoid membranes from the blue-green alga Anacystis nidulans.

Thylakoid membranes were prepared from the blue-green alga, Anacystis nidulans with lysozyme treatment and a short period of sonic oscillation. The thylakoid membrane preparation was highly active in the electron transport reactions such as the Hill reactions with ferricyanide and with 2,6-dichlorophenolindophenol, the Mehler reaction mediated by methyl viologen and the system 1 reaction with methyl viologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system. The Hill reaction with ferricyanide and the system 1 reaction was stimulated by the phosphorylating conditions. The cyclic and non-cyclic phosphorylation was also active. These findings suggest that the preparation of thylakoid membranes retained the electron transport system from H2O to reaction center 1, and that the phosphorylation reaction was coupled to the Hill reaction and the system 1 reaction.     

Hg ++ - a DCMU independent electron acceptor of photosystem II

Mercuric chloride functions as a direct electron acceptor from the quencher of fluorescence in Photosystem II. The photoreduction of ferricyanide, dichlorophenol-indophenol or methyl viologen is inhibited by mercuric ion while oxygen evolution is uneffected. Mercuric chloride supported oxygen evolution (mercury Hill reaction) is not prevented by DCMU or other similar electron transport inhibitors.     

Photosynthetic electron transport and phosphorylation reactions in thylakoid membranes from the blue-green alga Anacystis nidulans

Thylakoid membranes were prepared from the blue-green alga, Anacystis nidulans with lysozyme treatment and a short period of sonic oscillation. The thylakoid membrane preparation was highly active in the electron transport reactions such as the Hill reactions with ferricyanide and with 2,6-dichlorophenolindophenol, the Mehler reaction mediated by methyl viologen and the system 1 reaction with methyl viologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system. The Hill reaction with ferricyanide and the system 1 reaction was stimulated by the phosphorylating conditions. The cyclic and non-cyclic phosphorylation was also active.These findings suggest that the preparation of thylakoid membranes retained the electron transport system from H₂O to reaction center 1, and that the phosphorylation reaction was coupled to the Hill reaction and the system 1 reaction.     

Mechanisms of the acido- and thermo-phily of Cyanidium caldarium Geitler I. Effects of temperature, pH and light intensity on the photosynthetic oxygen evolution of intact and treated cells

Photosynthetic oxygen evolution in an acido- and thermo-philicunicellular alga, Cyanidium caldarium, was measured under variousconditions, using a Clark-type oxygen electrode. 1). Maximum Hill reaction activity with p-benzoquinone as theHill oxidant was obtained at 45°C in a wide pH range from1.0 to 7.0. 2) The pH activity curve showed two peaks at pH3.0 and 7.0. The Hill activity had an optimum at pH 3.0 in cellspreilluminated under strong light (300,000 lux, 30 min, 40°C).Sonication of algal cells abolished the pH 3.0 component ofthe Hill reaction producing an activity maximum at pH 7.0. 3)Endogenous O₂ evolution in the absence of the Hill oxidant,which lasted for several minutes after illumination, had a maximumat pH 7.0. 4) This endogenous O₂ evolution was abolished bysonication. 5) KCN inhibited endogenous O₂ evolution, but notthe Hill reaction in the presence of p-benzoquinone. (Received August 19, 1974; )     

Complex Inhibition of [3H]Imipramine Binding by Serotonin and Nontricyclic Serotonin Uptake Blockers

Tricyclic antidepressant drugs inhibit [3H]imipramine binding to the rat brain cortex in a competitive manner, giving linear Hofstee plots and Hill coefficients of approximately 1.0. Serotonin, the only neurotransmitter to inhibit [3H]imipramine binding, does so in a complex manner, exhibiting a Hill coefficient of 0.40-0.50. Nontricyclic inhibitors of serotonin uptake such as fluoxetine, paroxetine, norzimelidine, and citalopram inhibit [3H]imipramine binding in the same complex manner as serotonin. These results are interpreted as suggesting that [3H]imipramine binds to a site associated with the serotonin uptake system but different from either the substrate recognition site for serotonin or the site of action of the nontricyclic inhibitors of neuronal uptake of serotonin.     

Effect of imidazole on the activity of photosystem II in spinach chloroplasts

The activity of the Hill reaction was accelerated about 2–3times in the presence of 1–5 mM imidazole. However, inchloroplasts uncoupled by carbonyl cyanide m-chlorophenylhydrazone,the acceleration of the Hill activity was not observed in thesame imidazole concentration range, but a moderate inhibitionof the activity was observed. These results suggest that inthe coupled chloroplasts, the enhanced Hill activity by imidazoleis due to uncoupling of phosphorylation. Imidazole-washing of chloroplasts at neutral pH caused a moderateinhibition of the Hill reaction to about 60% of the control,while a complete loss of the Hill activity was observed afterwashing at pH 10.5. In chloroplasts washed with imidazole atpH 10.5, the variable fluorescence yield was also diminished.These activities were restored by adding an artificial electrondonor to photosystem II. (Received October 14, 1978; )