Phthalazinones 2: Optimisation and synthesis of novel potent inhibitors of poly(ADP-ribose)polymerase

We have previously described the discovery of poly(ADP-ribose)polymerase-1 (PARP-1) inhibitors based on a phthalazinone scaffold. Subsequent optimisation of inhibitory activity, metabolic stability and pharmacokinetic parameters has led to a novel series of meta-substituted 4-benzyl-2H-phthalazin-1-one PARP-1 inhibitors which retain low nM cellular activity and show good stability in vivo and efficacy in cell based models.     

Phthalazinones. Part 1: The design and synthesis of a novel series of potent inhibitors of poly(ADP-ribose)polymerase

Screening of the Maybridge compound collection identified 4-arylphthalazinones as micromolar inhibitors of PARP-1 catalytic activity. Subsequent optimisation of both inhibitory activity and metabolic stability led to a novel series of meta-substituted 4-benzyl-2H-phthalazin-1-ones with low nanomolar, cellular activity as PARP-1 inhibitors and promising metabolic stability in vitro.     

Discovery and optimization of 2-(4-substituted-pyrrolo[2,3-b]pyridin-3-yl)methylene-4-hydroxybenzofuran-3(2H)-ones as potent and selective ATP-competitive inhibitors of the mammalian target of rapamycin (mTOR)

We discovered 2-(4-substituted-pyrrolo[2,3-b]pyridin-3-yl)methylene-4-hydroxybenzofuran-3(2H)-ones as potent and selective ATP-competitive inhibitors of the mammalian target of rapamycin (mTOR). Since phenolic OH groups pose metabolic liability, one of the two hydroxyl groups was selectively removed. The SAR data showed the structural features necessary for subnanomolar inhibitory activity against mTOR kinase as well as selectivity over PI3Kα. An X-ray co-crystal structure of one inhibitor with the mTOR-related PI3Kγ revealed the key hydrogen bonding interactions.     

ORL1 and opioid receptor preferences of nociceptin and dynorphin A analogues with Dmp substituted for N-terminal aromatic residues

Nociceptin (NOC) and dynorphin A (DYN) analogues containing 2',6'-dimethylphenylalanine (Dmp) in place of Phe or Tyr in position 1 and/or 4 were synthesized and their metabolic stability and receptor-binding properties were investigated. [Dmp1]NOC(1-13)-NH2 (1) possessed high ORL1 receptor affinity comparable to that of the parent peptide with substantially improved affinities for kappa-, mu-, and delta-opioid receptors. However, Dmp4 substitution of NOC peptide (2) reduced ORL1 receptor affinity. [Dmp1]DYN(1-13)-NH2 (4) and its Dmp4 analogue (5) possessed a 3-fold greater kappa-opioid receptor affinity and improved kappa-receptor selectivity compared to the parent peptide. Analogue 4 however exhibited an unexpectedly low in vitro bioactivity (GPI assay), suggesting, the phenolic hydroxyl group at the N-terminal residue in DYN peptide is extremely important for activation of the kappa-opioid receptor. Analogue 5 possessed an improved kappa-opioid receptor selectivity with an IC50 ratio of 1(kappa)/509(mu)/211598(delta); thus, this peptide may serve as a highly selective kappa-receptor agonist for pharmacological study. Dmp1 substitution in both the NOC and DYN peptides improved metabolic stability toward these peptides, while Dmp4 substitution provided no additional metabolic stability.     

Novel and orally active 5-(1,3,4-oxadiazol-2-yl)pyrimidine derivatives as selective FLT3 inhibitors

5-(1,3,4-Oxadiazol-2-yl)pyrimidine derivative 1 was identified as a new class of FLT3 inhibitor from our compound library. With the aim of enhancement of antitumor activity of 2 prepared by minor modification of 1, structure optimization of side chains at the 2-, 4-, and 5-positions of the pyrimidine ring of 2 was performed to improve the metabolic stability. Introduction of polar substituents on the 1,3,4-oxadiazolyl group contributed to a significant increase in the metabolic stability. As a result, a series of compounds showed increased efficacy against MOLM-13 xenograft model in mice by oral administration.     

Involvement of components of the phospholipid-signaling pathway in wound-induced phenylpropanoid metabolism in lettuce (Lactuca sativa) leaf tissue

In plant tissue, a wound signal is produced at the site of injury and propagates or migrates into adjacent tissue where it induces increased phenylalanine ammonia lyase (PAL, EC 4.3.1.5) activity and phenylpropanoid metabolism. We used excised mid-rib leaf tissue from Romaine lettuce (Lactuca sativa L., Longifolia) as a model system to examine the involvement of components of the phospholipid-signaling pathway in wound-induced phenolic metabolism. Exposure to 1-butanol vapors or solutions inhibited wound-induced increase in PAL activity and phenolic metabolism. Phospholipases D (EC 3.1.4.4), an enzyme involved in the phospholipid-signaling pathway is specifically inhibited by 1-butanol. Re-wounding tissue, in which an effective 1-butanol concentration had declined below active levels by evaporation, did not elicit the normal wound response. It appears the 1-butanol-treated tissue developed resistance to wound-induced increases in phenylpropanoid metabolism that persisted even when active levels of 1-butanol were no longer present. However, a metabolic product of 1-butanol, rather than 1-butanol itself, may be the active compound eliciting persistence resistance. Inhibiting a subsequent enzyme in the phospholipid-signaling pathway, lipoxygenase (LOX; EC 1.13.11.12) with 1-phenyl-3-pyrazolidinone (1P3P) or reducing the product of LOX activity with diethyldithio-carbamic acid (DIECA) also inhibited wound-induced PAL activity and phenolic accumulation. The effectiveness of 1-butanol, DIECA, and 1P3P declined as the beginning of the 1-h immersion period was delayed from 0 to 4 h after excision. This decline in effectiveness is consistent with involvement of the inhibitors in the production or propagation of a wound signal. The wound signal in lettuce moves into adjacent tissue at 0.5 cm h⁻¹, so delaying application would allow the signal to move into and induce the wound response in adjacent tissue before the delayed application inhibited synthesis of the signal. Salicylic acid (SA) inhibits allene oxide synthase (AOS, EC 4.2.1.92), another enzyme in the phospholipid-signaling pathway. Exposure to 1 or 10 mM SA for 60 min reduced wound-induced phenolic accumulation by 26 or 56%, respectively. However, 1 mM SA lost its effectiveness if applied 3 h after excision, while 10 mM SA remained effective even when applied 4 h after excision. At 1 mM, SA may be perturbing the wound signal through inhibition of AOS, while at 10 mM it appears to have some generally inhibitory effect on subsequent phenolic metabolism. These data further implicate the phospholipid-signaling pathway in the generation of a wound signal that induces phenolic metabolism in wounded leaf tissue.     

Inhibition of the mutagenicity of benzo[a]pyrene in the V79/HGPRT system by bioantioxidants

In the presence of metabolic activation (S9 microsomal fraction of mouse-liver homogenate) the mutagenicity of benzo[a]pyrene (BP) in Chinese hamster V79 cells was inhibited by the phenolic bioantioxidants (BA) Dibunol (2,6-di-tert-butyl-4-methylphenol-D) and 5-methylresorcine(5-MR). The mixture BP + D and BP + 5-MR at molar ratios of 1:1 and 1:85 respectively showed no mutagenic activity compared to the control. One can assume that D and 5-MR inhibited BP-induced mutagenesis by binding the free radicals of BP metabolites with the formation of less active phenolic derivatives and also by linkage with cytochrome P-450, which prevents further metabolic activation of BP.     

Evaluation of a new biosensor-based mushroom (Agaricus bisporus) tissue homogenate: investigation of certain phenolic compounds and some inhibitor effects

A biosensor based on mushroom tissue homogenate for detecting some phenolic compounds (PCs) and usage of the biosensor for quantifying certain substances that inhibit the polyphenol oxidase activity in mushroom (Agaricus bisporus) tissue homogenate is described. The mushroom tissue homogenate was immobilized to the top of a Clark-type oxygen electrode with gelatin and glutaraldehyde. Optimization of the experimental parameters was done by buffer system, pH, buffer concentration, and temperature. Besides, the detection range of eight phenolic compounds were obtained with the help of the calibration graphs. Thermal stability, storage stability, and repeatability of the biosensor were also investigated. A linear response was observed from 20 x 10(-3) to 200 x 10(-3) mM phenol. The biosensor retained approximately 74% of its original activity after 25 days of storage at 4 degrees C. In repeatability studies, variation coefficient (C.V.) and standard deviation (S.D.) were calculated as 2.44% and +/-0.002, respectively. Inhibition studies revealed that the proposed biosensor was applicable for monitoring benzoic acid and thiourea in soft drinks and fruit juices.     

Phenolic acids and peroxidase activity in alfalfa (Medicago sativa) embryogenic cultures after ethephon treatment

Treatment with ethephon increased the concentration of exogenous ethylene in Medicago sativa L. embryogenic cell suspension cultures (consisting of single cells, small cellular clumps and globular somatic embryos) and induced changes in the metabolism of phenolic substances, activities of peroxidase (EC 1.11.1.7) and caused significant suppression of suspension culture growth. Treatment with the ethylene-releasing substance, ethephon, resulted in a several-fold increase in phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) activity above the basal level and was accompanied by an elevated accumulation of phenolic acids (significant increase of methoxy-substituted acids). The majority of newly synthesised phenolic acids was incorporated into the fractions of glycosides and esters bound to the cell wall. Phenolic glycosides seemed to serve as a metabolic pool from which the phenolics were utilised during further culture. The increased activity of wall-bound ionic peroxidase after ethephon application correlated with the pronounced incorporation of ferulic acid in the cell walls. In contrast, the increased level of exogenous ethylene did not influence the growth of culture of more advanced embryos nor did it significantly alter phenylpropanoid metabolism.     

N-Alkylidenearylcarboxamides as new potent and selective CB(2) cannabinoid receptor agonists with good oral bioavailability

A novel series of N-alkylidenearylcarboxamides 4, a CB(2) receptor agonist, were synthesized and evaluated for activity against the human CB(2) receptor. In a previous paper, we reported that sulfonamide derivative 1 acted as a potent CB(2) receptor agonist (IC(50)=65 nM, EC(50)=19 nM, E(max)=90%). However, compound 1 also exhibited poor metabolic stability in human liver microsomes. During the structural modification of 1, we found that a novel series of N-alkylidenearylcarboxamide, 4-1, had a moderate affinity for the CB(2) receptor (IC(50)=260 nM, EC(50)=86 nM, E(max)=100%) and good metabolic stability in human liver microsomes. We explored its analogues to discover compounds with a high affinity for the CB(2) receptor and with good oral bioavailability. Among them, compounds 4-9 and 4-27 had high affinities for the human CB(2) receptor (CB(2) IC(50)=13 nM and 1.2 nM) and a high selectivity for CB(2) (CB(1) IC(50)/CB(2) IC(50)=270 and 1600); furthermore, significant plasma levels were observed following oral administration in rats (C(max)=233 ng/mL and 148 ng/mL, respectively, after a dose of 10 mg/kg). Furthermore, compound 4-9 had good oral bioavailability (F=52%, 3mg/kg).     

Clonal response to cold tolerance in creeping bentgrass and role of proline-associated pentose phosphate pathway

Single seed origin creeping bentgrass (‘Penncross’) clonal lines were screened to find genetic heterogeneity, which reflected diversity of phenolic production linked to cold stress within a cross-pollinated cultivar. In this study, total soluble phenolic and antioxidant activity varied among 20 creeping bentgrass clonal lines, confirming wide heterogeneity in this cross-pollinated species. Correlations between phenolic content and proline-associated pentose phosphate pathway were also found among the clonal lines. The active metabolic role of proline in cellular metabolic adjustment to cold stress and its support for likely energy synthesis via mitochondrial oxidative phosphorylation was inferred in creeping bentgrass clonal lines based on the activity of proline dehydrogenase. Results of photochemical efficiency of these clonal lines after cold temperature treatment (4 °C) also indicated a close association between stress tolerance and proline-associated pentose phosphate pathway regulation for phenolic biosynthesis and antioxidant response. This study provides a sound metabolic based rationale to screen bentgrass clonal lines for enhanced cold stress tolerance.     

Design, synthesis, and biological activity of novel factor Xa inhibitors: improving metabolic stability by S1 and S4 ligand modification

Serine protease factor xa (fXa) inhibitor 1 showed good ex vivo anti-fXa activity upon oral administration in rats. However, it has been revealed that 1 had low metabolic stability against human liver microsomes. To improve the metabolic stability, we attempted to modify the S1 and S4 ligands of 1. These modifications resulted in compound 34b, which exhibited selective anti-fXa activity and excellent anti-coagulation activity.     

Chemosystematic value of chemical constituents from Scabiosa hymettia (Dipsacaceae)

The first phytochemical investigation of Scabiosa hymettia led to the isolation and characterization of nine known compounds, 2-10, and of the new kaempferol derivative 1. In total, two flavonoids, three coumarins, three iridoids, and two phenolic constituents were obtained. The chemosystematic value of these compounds, as well as the metabolic relationship between swertiamarin (6) and the other isolated coumarins, are discussed. Both the extracts as well as all isolated constituents of S. hymettia were evaluated for their antimicrobial activities against six Gram-positive or Gram-negative bacteria, and against three human pathogenic fungi. The new compound 1 was found to exhibit the highest activity against all organisms tested.     

2,6-Diaryl-4-acylaminopyrimidines as potent and selective adenosine A(2A) antagonists with improved solubility and metabolic stability

In this report, the strategy and outcome of expanding SAR exploration to improve solubility and metabolic stability are discussed. Compound 35 exhibited excellent potency, selectivity over A(1) and improved solubility of >4 mg/mL at pH 8.0. In addition, compound 35 had good metabolic stability with a scaled intrinsic clearance of 3 mL/min/kg (HLM) and demonstrated efficacy in the haloperidol induced catalepsy model.     

A novel 3,4-dihydropyrimidin-2(1H)-one: HIV-1 replication inhibitors with improved metabolic stability

Following the previous SAR of a novel dihydropyrimidinone scaffold as HIV-1 replication inhibitors a detailed study directed towards optimizing the metabolic stability of the ester functional group in the dihydropyrimidinone (DHPM) scaffold is described. Replacement of the ester moiety by thiazole ring significantly improved the metabolic stability while retaining antiviral activity against HIV-1 replication. These novel and potent DHPMs with bioisosteres could serve as advanced leads for further optimization.     

Effects of pentachlorophenol (Na PCP) on survival,activity and metabolism in Rhinomugil corsula (Hamilton), Cyprinus carpio (Linnaeus) and Tilapia mossambica (Peters)

The median lethal time (LT 50) at any one initial nominal concentration of PCP tested (1.0–4.0 mg · l^-1), for example 3.0 mg · l^-1, is found to be longest for Tilapia mossambica (175 minutes), followed by Cyprinus carpio (50 minutes) and Rhinomugil corsula (31 minutes) suggesting that the difference in resistance (survival) to PCP may be species-specific. When R. corsula was exposed to 0.1 and 0.133 mg · l^-1 PCP, the standard metabolic rate was elevated by 37 and 45% respectively at 30 °C, when compared to control fish (112 mg kg^-1 hr^-1). In carp, the metabolic rate showed an increase to begin with, but later decreased during the test period (20 days), but the random (spontaneous) activity was seen to slope down as the number of exposure days increased. During exercise (35 cm s^-1) the rate of oxygen consumption in the control and PCP treated Tilapia decreased with the duration of exercise (5 h), but the metabolic rate in the treated fish increased considerably.     

Carboxyl residues in the active site of human phenol sulfotransferase (SULT1A1)

The carboxyl-specific amino acid modification reagent, Woodward's reagent K (WK), was utilized to characterize carboxyl residues (Asp and Glu) in the active site of human phenol sulfotransferase (SULT1A1). SULT1A1 was purified using the pMAL-c2 expression system in E. coli. WK inactivated SULT1A1 activity in a time- and concentration-dependent manner. The inactivation followed first-order kinetics relative to both SULT1A1 and WK. Both phenolic substrates and adenosine 3'-phosphate 5'-phosphosulfate (PAPS) protected against the inactivation, which suggests the carboxyl residue modification causing the inactivation took place within the active site of the enzyme. With partially inactivated SULT1A1, both V(max) and K(m) changed for PAPS, while for phenolic substrates, V(max) decreased and K(m) did not change significantly. A computer model of the three-dimensional structure of SULT1A1 was constructed based on the mouse estrogen sulfotransferase (mSULT1E1) X-ray crystal structure. According to the model, Glu83, Asp134, Glu246, and Asp263 are the residues likely responsible for the inactivation of SULT1A1 by WK. According to these results, five SULT1A1 mutants, E83A, D134A, E246A, D263A, and E151A, were generated (E151A as control mutant). Specific activity determination of the mutants demonstrated that E83A and D134A lost almost 100% of the catalytic activity. E246A and D263A also decreased SULT1A1 activity, while E151A did not change SULT1A1 catalytic activity significantly. This work demonstrates that carboxyl residues are present in the active site and are important for SULT1A1 catalytic activity. Glu83 and E134 are essential amino acids for SULT1A1 catalytic activity.     

Antimicrobial Activity of Kumazasa (Sasa albo-marginata)

The organic solvent extract of Kumazasa leaves (Sasa albo-marginata) showed antimicrobial activity against bacteria, fungi and yeast. Kumazasa at a concentration of 0.2-1.0% showed stronger antimicrobial activity than potassium sorbate or sodium benzoate at the same concentration. Both acidic and phenolic fractions of the extract showed strong antimicrobial activity. Thirty acidic and phenolic compounds were identified by GC and GC-MS analysis. Acetic, propionic, benzoic, phenylacetic, salicylic, 3-hydroxybenzoic and o-anisic acids, and guaiacol, phenol, 4-ethylphenol, xylenol and 4-vinylphenol were the main components. It was estimated that these components play an important role in the formation of the antimicrobial activity of Kumazasa extract.     

Interactions of the anticancer antibiotic altromycin B with copper(II), palladium(II) and platinum(II) ions and in vitro activity of the formed complexes

Interaction of the anticancer antibiotic altromycin B with Cu(II), Pd(II) and Pt(II) ions was studied using 1H-NMR, EPR, electronic absorption and circular dichroism spectroscopy. The results derived from NMR studies where that the Pt(II) and Pd(II) ions interact with the nitrogen atom of the dimethylamino group of the C(10)-disaccharide, while the C(2)-epoxide group does not participate and remains intact. Cu(II) ions interact in a different way with altromycin B as was concluded by EPR and circular dichroism spectra. Altromycin B coordinates to the Cu(II) ions via the oxygen atoms of the C(11) phenolic and the C(12) carbonyl group while the nitrogen atom does not participate in the complexation. The presence of these metal ions improves the stability of altromycin B in solution. These complexes were studied in vitro against K562 leukemia sensitive and doxorubicin-resistant cells and GLC4 lung tumor cells, sensitive and doxorubicin-resistant. The activity of the complexes compared to the free drug is improved against resistant cells and is affected moderately against sensitive cells. Finally, 20% of platinum added as altromycin B metal complex entered GLC4 cells.     

Quantitative relationship between protonophoric and uncoupling activities of analogs of SF6847 (2,6-di-t-butyl-4-(2',2'-dicyanovinyl)phenol)

Uncoupling activity with rat liver mitochondria and protonophoric activity across the lecithin liposomal membranes were measured for a series of non-classical uncouplers related to the most potent uncoupler known until now, SF6847 (2,6-di-t-butyl-4-(2',2'-dicyanovinyl)phenol). The correlation between uncoupling and protonophoric activities for a number of uncouplers, both non-classical and classical (simply substituted phenols), was examined quantitatively. Correlation was excellent when such factors as the stability of anionic species in the membrane phase and the difference in the pH conditions of the extramembranous aqueous phase were taken into account. Carbonylcyanide m-chlorophenylhydrazone (CCCP) and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), which are structurally different, were correlated in a way that resembled the correlation of phenolic compounds, so we think that the mode of action of weakly acidic uncouplers was the same regardless of the structural type. Our findings were evidence for the shuttle-type mechanism of uncoupling action.