Design, synthesis and biological evaluation of novel benzothiadiazine derivatives as potent PI3Kδ-selective inhibitors for treating B-cell-mediated malignancies.

A series of 24 benzothiadiazine derivatives with structural novelty were designed, synthesized and biologically evaluated as PI3Kδ-selective inhibitors. As a consequence of the structure-activity relationship (SAR) study, compounds 63 and 71 were identified with single-digit nanomolar IC50 values against PI3Kδ and submicromolar GI50 values against human malignant B-cell line SU-DHL-6. Furthermore, chiral resolution of the key amine intermediate of these two compounds was performed to achieve corresponding enantiomers. In subsequent biological evaluation, S-63 (IC50: 4.6 nM) and S-71 (IC50: below 0.32 nM) demonstrated comparable and superior PI3Kδ inhibitory activity, respectively, to that of idelalisib. Additionally, both S-63 (GI50: 33.2 nM) and S-71 (GI50: 15.9 nM) exerted enhanced anti-proliferative activity against the SU-DHL-6 cell line than that of idelalisib. Moreover, both S-63 and S-71 exhibited excellent PI3Kδ selectivity. In the further in vivo pharmacokinetic (PK) study, S-63 displayed a good plasma exposure and an acceptable oral bioavailability of 29.2%. By virtue of its biological performance, S-63 merits further development as a potential therapeutic agent for battling B-cell-mediated malignancies.

Pharmacokinetics and pharmacodynamics of setrobuvir, an orally administered hepatitis C virus non-nucleoside analogue inhibitor.

PURPOSE: New antiviral agents with activity against hepatitis C virus (HCV) are needed to optimize treatment for chronic hepatitis C (CHC). We evaluated the pharmacokinetics of setrobuvir (a non-nucleoside HCV polymerase inhibitor) in healthy volunteers (study 1 & 2) and its antiviral efficacy in patients with genotype 1, noncirrhotic treatment-naive CHC (study 3). METHODS: Three studies investigated the pharmacokinetics and pharmacodynamics of setrobuvir. First, sequential cohorts of volunteers were randomly assigned to receive single oral doses of setrobuvir 400 to 3000 mg or placebo in a double-blind, ascending dose study. In the second study, volunteers were randomly assigned to receive multiple doses of setrobuvir (400 or 800 mg once daily [QD] or 600 mg twice a day [BID]). In the third study, patients with genotype 1 CHC received setrobuvir (200, 400, or 800 mg) or placebo BID for 3 days. FINDINGS: After single doses of setrobuvir (400-3000 mg) to volunteers in a fasted state, peak Cmax and AUC0-∞ increased in a less than dose-proportional manner. The mean apparent t½z ranged from 22.0 to 31.3 hours and was not dose related. Cmax and AUC increased significantly (4.3- and 6.3-fold, respectively) in volunteers who received 2000 mg with a high-fat meal versus fasting. After multiple oral doses, steady state was achieved after 7 days of dosing (400 or 800 mg QD and 600 mg BID) and accumulation was dose-independent. Mean day 14 plasma exposure increased in a less than dose-proportional manner in volunteers who received 400 and 800 mg QD, but it was more than dose-proportional in volunteers receiving 600 mg BID. Dose did not affect the mean t½z (range, 24.1-26.6 hours), apparent oral clearance (0.254-0.516 L/h), or apparent volume of distribution (9.60-18.1 L). In patients with CHC, dose-related reductions in HCV RNA concentration were apparent within 24 hours of the start of treatment. Reductions from baseline to the end of treatment (day 3) in patients treated with setrobuvir 200, 400, and 800 mg BID were -2.1, -2.2, and -2.9 log10 IU/mL, respectively (vs ≤0.1 log10 IU/mL with placebo). Reductions in HCV RNA were greater in patients with genotype 1b (range, -2.7 to -3.1 log10 IU/mL) than in patients with genotype 1a (range, -1.3 to -2.7 log10 IU/mL). Setrobuvir was well tolerated, with no serious adverse events. IMPLICATIONS: The steady state pharmacokinetics of setrobuvir appear to be dose proportional, and setrobuvir produces a mean reduction of 2.9 log10 IU/mL in HCV RNA over 3 days in patients with genotype 1 (a and b) treated with 800 mg BID. ClinicalTrials.gov identifier: NCT00782353.

Human skin in vitro permeation of bentazon and isoproturon formulations with or without protective clothing suit.

Skin exposures to chemicals may lead, through percutaneous permeation, to a significant increase in systemic circulation. Skin is the primary route of entry during some occupational activities, especially in agriculture. To reduce skin exposures, the use of personal protective equipment (PPE) is recommended. PPE efficiency is characterized as the time until products permeate through material (lag time, Tlag). Both skin and PPE permeations are assessed using similar in vitro methods; the diffusion cell system. Flow-through diffusion cells were used in this study to assess the permeation of two herbicides, bentazon and isoproturon, as well as four related commercial formulations (Basagran(®), Basamais(®), Arelon(®) and Matara(®)). Permeation was measured through fresh excised human skin, protective clothing suits (suits) (Microchem(®) 3000, AgriSafe Pro(®), Proshield(®) and Microgard(®) 2000 Plus Green), and a combination of skin and suits. Both herbicides, tested by itself or as an active ingredient in formulations, permeated readily through human skin and tested suits (Tlag < 2 h). High permeation coefficients were obtained regardless of formulations or tested membranes, except for Microchem(®) 3000. Short Tlag, were observed even when skin was covered with suits, except for Microchem(®) 3000. Kp values tended to decrease when suits covered the skin (except when Arelon(®) was applied to skin covered with AgriSafe Pro and Microgard(®) 2000), suggesting that Tlag alone is insufficient in characterizing suits. To better estimate human skin permeations, in vitro experiments should not only use human skin but also consider the intended use of the suit, i.e., the active ingredient concentrations and type of formulations, which significantly affect skin permeation.

Synthesis and biological characterization of B-ring amino analogues of potent benzothiadiazine hepatitis C virus polymerase inhibitors.

Benzothiadiazine inhibitors of the HCV NS5B RNA-dependent RNA polymerase are an important class of non-nucleoside inhibitors that have received considerable attention in the search for novel HCV therapeutics. Research in our laboratories has identified a novel series of tetracyclic benzothiadiazine inhibitors of HCV polymerase bearing a benzylamino substituent on the B-ring. Compounds in this series exhibit low-nanomolar activities in both genotypes 1a and 1b polymerase inhibition assays and subgenomic replicon assays. Optimization of pharmacokinetic properties in rat led to compound 30, which has good oral bioavailability (F = 56%) and a favorable tissue distribution drug profile, with high liver to plasma ratios. Compound 30 is a potent inhibitor in replicon assays, with EC(50) values of 10 and 6 nM against genotypes 1a and 1b, respectively.

Inhibitors of hepatitis C virus polymerase: synthesis and biological characterization of unsymmetrical dialkyl-hydroxynaphthalenoyl-benzothiadiazines.

The hepatitis C virus (HCV) NS5B polymerase is essential for viral replication and has been a prime target for drug discovery research. Our efforts directed toward the discovery of HCV polymerase inhibitors resulted in the identification of unsymmetrical dialkyl-hydroxynaphthalenoyl-benzothiadiazines 2 and 3. The most active compound displayed activity in genotypes 1a and 1b polymerase and replicon cell culture inhibition assays at subnanomolar and low nanomolar concentrations, respectively. It also displayed an excellent pharmacokinetic profile in rats, with a plasma elimination half-life after intravenous dosing of 4.5 h, oral bioavailability of 77%, and a peak liver concentration of 21.8 microg/mL.

Characterizing pesticide sorption and degradation in microscale biopurification systems using column displacement experiments.

Biopurification systems treating pesticide contaminated water are very efficient, however they operate as a black box. Processes inside the system are not yet characterized. To optimize the performance, knowledge of degradation and retention processes needs to be generated. Therefore, displacement experiments were carried out for four pesticides (isoproturon, bentazone, metalaxyl, linuron) in columns containing different organic mixtures. Bromide, isoproturon and bentazone breakthrough curves (BTCs) were well described using the convection-dispersion equation (CDE) and a first-order degradation kinetic approach. Metalaxyl and linuron BTCs were well described using the CDE model expanded with Monod-type kinetics. Freundlich sorption, first-order degradation and Monod kinetics coefficients were fitted to the BTCs. Fitted values of the distribution coefficient K(f,column) were much lower than those determined from batch experiments. Based on mobility, pesticides were ranked as: bentazone>metalaxyl-isoproturon>linuron. Based on degradability, pesticides were ranked as: linuron>metalaxyl-isoproturon>bentazone.

Second generation of hydroxyethylamine BACE-1 inhibitors: optimizing potency and oral bioavailability.

BACE-1 inhibition has the potential to provide a disease-modifying therapy for the treatment of Alzheimer's disease. Optimization of a first generation of BACE-1 inhibitors led to the discovery of novel hydroxyethylamines (HEAs) bearing a tricyclic nonprime side. These derivatives have nanomolar cell potency and are orally bioavailable.

Activity of a potent hepatitis C virus polymerase inhibitor in the chimpanzee model.

A-837093 is a potent and specific nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase. It possesses nanomolar potencies in both enzymatic and replicon-based cell culture assays. In rats and dogs this compound demonstrated an oral plasma half-life of greater than 7 h, and its bioavailability was >60%. In monkeys it had a half-life of 1.9 h and 15% bioavailability. Its antiviral efficacy was evaluated in two chimpanzees infected with HCV in a proof-of-concept study. The design included oral dosing of 30 mg per kg of body weight twice a day for 14 days, followed by a 14-day posttreatment observation. Maximum viral load reductions of 1.4 and 2.5 log(10) copies RNA/ml for genotype 1a- and 1b-infected chimpanzees, respectively, were observed within 2 days after the initiation of treatment. After this initial drop in the viral load, a rebound of plasma HCV RNA was observed in the genotype 1b-infected chimpanzee, while the genotype 1a-infected chimpanzee experienced a partial rebound that lasted throughout the treatment period. Clonal analysis of NS5B gene sequences derived from the plasma of A-837093-treated chimpanzees revealed the presence of several mutations associated with resistance to A-837093, including Y448H, G554D, and D559G in the genotype 1a-infected chimpanzee and C316Y and G554D in the genotype 1b-infected chimpanzee. The identification of resistance-associated mutations in both chimpanzees is consistent with the findings of in vitro selection studies, in which many of the same mutations were selected. These findings validate the antiviral efficacy and resistance development of benzothiadiazine HCV polymerase inhibitors in vivo.

Effects of cyclothiazide on GluR1/AMPA receptors.

Cyclothiazide (CTZ), a positive allosteric modulator of ionotropic alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors, is used frequently to block the desensitization of both native and heterologously expressed AMPA receptors. Specifically, CTZ is known to produce a fast inhibition of AMPA receptor desensitization and a much slower potentiation of the AMPA current. By using patch-clamp techniques, the effects of CTZ were studied in HEK 293 cells stably transfected with the rat flip GluR1 subunit. Upon CTZ treatment, we found an increased apparent affinity for the agonist, a slow whole-cell current potentiation, a fast inhibition of desensitization, and a lengthening of single-channel openings. Furthermore, we show that CTZ alters the channel gating events modifying the relative contribution of different single-channel classes of conductance (gamma), increasing and decreasing, respectively, the contributions of gammaM (medium) and gammaL (low) without altering that of the gammaH (high) conductance channels. We also present a kinetic model that predicts well all of the experimental findings of CTZ action. Finally, we suggest a protocol for standard cell treatment with CTZ to attain maximal efficacy of CTZ on GluR1 receptors.

Neuropharmacokinetics of a new alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) modulator, S18986 [(S)-2,3-dihydro-[3,4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide], in the rat.

The aim of our study was to determine the neuropharmacokinetics of S18986 [(S)-2,3-dihydro-[3,4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide], a new positive allosteric modulator of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type receptors, in the rat. We focused on its blood-brain barrier (BBB) uptake and on its brain intra- and extracellular fluid (bICF-bECF) partitioning. BBB transport of S18986 was measured using the in situ brain perfusion technique. bECF concentrations were determined by microdialysis in the two effector areas, i.e., frontal cortex (FC) and dorsal hippocampus (DH), and blood samples were collected simultaneously through a femoral catheter. Cerebrospinal fluid and brain tissue concentrations were determined using a conventional pharmacokinetic approach. Using all the experimental data, pharmacokinetic modeling was applied to describe the S18986 blood-brain disposition. The brain uptake clearance of S18986 was found to be high, about 20 mul s(-1) g(-1). Terminal half-lives were similar in plasma and brain, at around 1 h. Experimental and predicted blood and brain concentrations were a good fit with the pharmacokinetic model, which assumed first-order rate constants at each interface. Ratios of bECF to the unbound plasma area under the curve (AUC) were 0.24 in FC and 0.25 in DH, whereas ratios of bICF/plasma AUC were 1 in FC and 1.5 in DH. We conclude that despite the ratio of bECF/plasma AUC below 1, there is nevertheless an elevated BBB uptake of S18986. This can be explained by the S18986 nonhomogenous bECF/bICF partitioning, since S18986 mainly distributes into hippocampal bICF. This illustrates the importance of taking bECF/bICF partitioning into account when interpreting the neuropharmacokinetics of a drug.

AMPA receptor potentiators for the treatment of CNS disorders.

Glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors mediate most of the excitatory neurotransmission in the mammalian central nervous system and also participate in forms of synaptic plasticity thought to underlie memory and learning, and the formation of neural networks during development. Molecular cloning techniques have shown that the AMPA receptor family is composed of four different subunits named GluR1-4 or GluRA-D (newly termed as Glu(A1)-Glu(A4)) and native AMPA receptors are most likely tetramers generated by the assembly of one or more of these subunits, yielding homomeric or heteromeric receptors. Additional complexity among AMPA receptors is conferred by alternative splicing of RNA for each subunit giving rise to flip and flop variants. Clinical and experimental data have suggested that positive modulation of AMPA receptors may be therapeutically effective in the treatment of cognitive deficits. Several classes of AMPA receptor potentiators have been reported, including pyrroliddones (piracetam, aniracetam), benzothiazides (cyclothiazide), benzylpiperidines (CX-516, CX-546) and more recently biarylpropylsulfonamides (LY392098, LY404187 and LY503430). These molecules enhance cognitive function in rodents, which appears to correlate with increased hippocampal activity. In addition, clinical studies have suggested that AMPA receptor modulators enhance cognitive function in elderly subjects, as well as patients suffering from neurological and psychiatric disorders. Several independent studies have suggested that AMPA receptors can increase BDNF expression by both calcium-dependent and independent pathways. For example, recent studies have shown that AMPA receptors interact with the protein tyrosine kinase, Lyn. Activation of Lyn can recruit the mitogen-activated protein kinase (MAPK) signalling pathway and increase the expression of BDNF. Therefore, in addition to directly enhancing glutamatergic synaptic transmission, AMPA receptor activation can increase the expression of BDNF in vitro and in vivo. This may account for activity of AMPA receptor potentiators in rodent models predictive of antidepressant activity (forced swim and tail suspension tests). The increase in neurotrophin expression also may contribute to the functional, neuroprotective and neurotrophic actions of LY404187 and LY503430 after infusion of 6-OHDA into the substantia nigra. In conclusion, several potent, selective and systemically active AMPA receptor potentiators have been reported. Data indicate that these molecules modulate glutamatergic transmission, enhance synaptic transmission, long-term potentiation (LTP) and increase neurotrophin expression. Therefore, these AMPA receptor potentiators offer an exciting new class of drugs with potential for treating (1) cognitive impairment associated with Alzheimer's disease and schizophrenia, (2) depression, (3) slowing the progression and potentially enhancing recovery from Parkinson's disease.

Bioaccumulation and toxicity of sediment associated herbicides (ioxynil, pendimethalin, and bentazone) in Lumbriculus variegatus (Oligochaeta) and Chironomus riparius (Insecta).

The benthic macroinvertebrates Lumbriculus variegatus and Chironomus riparius were used in toxicity and bioaccumulation tests to determine the toxic concentrations and accumulation potential of sediment associated herbicides. The tested chemicals were ioxynil, bentazone, and pendimethalin. The bioaccumulation tests with L. variegatus were performed in four different sediments, each having different characteristics. Water-only LC(50) tests were performed with both L. variegatus and C. riparius. A sublethal effect of model compounds in sediments was assessed by a C. riparius larvae growth-inhibition test. Of the model compounds, ioxynil appeared to be the most toxic, with LC(50) values 1.79 and 2.79 mgL(-1) for L. variegatus and C. riparius, respectively. The LC(50) water concentrations for bentazone were 79.11 and 62.31 mgL(-1) for L. variegatus and C. riparius, respectively. Similarly, ioxynil revealed the highest bioaccumulation potential in bioaccumulation tests. The most important characters affecting chemical fate in the sediment seemed to be the organic matter content and the particle size fraction. The sediments with low organic material and coarse particle size consistently showed high bioaccumulation potential and vice versa. In C. riparius growth tests bentazone had a statistically significant effect on larval growth at sediment concentrations of 1160 and 4650 mgkg(-1) (P<0.05). It is noteworthy that standard deviations tend to be greater at high chemical concentrations, which addresses the fact that part of the individuals started to suffer. Ioxynil had an effect on the larval growth in other test sediment at the highest concentration (15.46 mgkg(-1)dw), in which head capsule length correlated with larval weight, decreasing toward higher exposure concentrations. The current results show the importance of sediment organic matter as a binding site of xenobiotics.

Effects of a centrally active benzoylpyrrolidine drug on AMPA receptor kinetics.

A newly developed benzoylpyrrolidine drug (BDP-20) that increases the size of fast, excitatory synaptic responses was examined for its effects on the kinetic properties of alpha-amino-3-hydroxy-5-methyl-4-isoxalepropionic acid (AMPA)-type glutamate receptors. When long pulses of glutamate were applied to excised hippocampal patches of the rat, the compound BDP-20 caused an approximately 15-fold reduction in the rate at which responses desensitized and a similar size increase in steady-state currents. In experiments using 1-ms glutamate pulses, BDP-20 prolonged response deactivation by a factor of about four and greatly reduced the depression in the second response when two consecutive glutamate pulses were given. Two types of equilibrium binding assays indicated that BDP-20 causes a measurable increase in the affinity of AMPA receptors; the EC50 values for this effect were similar to those obtained in excised patch studies. The actions of BDP-20 on physiology and ligand binding could be adequately reproduced in a receptor model by slowing the rate of desensitization and increasing the affinity of the sensitized states. The biochemical and physiological effects of this benzoylpyrrolidine compound were qualitatively different from those obtained with cyclothiazide, although both types of drug increased AMPA receptor-mediated synaptic responses. Moreover, interactions between the drugs were at most only partially competitive; AMPA receptors may thus have multiple modulatory sites with distinct drug preferences and different effects on receptor kinetics.

Environmental behavior of bentazon herbicide.

Bentazon is a postemergence herbicide used in early spring to early summer in many crops, usually at application rates of 1.0 kg a.i./ha. Its selectivity is based on the ability of the crop plants to metabolize bentazon quickly to 6-OH- and 8-OH-bentazon and conjugate these with sugars, while weeds do not, so that photosynthesis is disrupted and the weeds die. Also, there is a further degradation to small fragments, which subsequently are incorporated into natural plant products. Residues in the raw agricultural commodities range from 0.1 to approximately 1.0 mg/kg (straw and other "leftover" plant parts). In the upper soil layer, bentazon is quickly degraded, microbially and aerobically, via the intermediary and instable products 6-OH-, 8-OH-bentazon, and AIBA. These are immediately bound biotically and abiotically to the mostly nonbioavailable soil organic matter fraction. Additionally, a considerable part (24-50%) is mineralized to CO2. Half-lives in field soils ranged from 3 to 21 d with an average of 12 d. Abiotic degradation processes predominantly involve photolyses on plant and soil surfaces and in surface water. The physical-chemical properties and soil column laboratory studies with bentazon would seem, at first glance, to predict a leaching potential. However, several field lysimeter studies unambiguously proved that it does not leach under field situations. Annual averages in the leachates were always < 0.1 microgram/L, also after the second year. Reasons for the favorable field behavior, in contrast to the laboratory studies, are discussed. Reports on bentazon findings in groundwater and drinking water were classified as resulting predominantly from former filtrate along Rhine banks. Since 1988, the bentazon levels in the Rhine River ranged below or near 0.1 microgram/L. Very few isolated point-source contaminations, arising from accidents and other reasons, are marked by sporadic findings of concentrations > 1 microgram/L. Nonvalidated findings at various locations are reported that lie below or near the determination limit. In such situations, it is highly recommended to identify bentazon with a "full" MS-spectrum. The ecotoxicological effects of bentazon lead to "no classification necessary." The small octanol/water partition coefficient precludes bioaccumulation. Bentazon is rapidly excreted by warm-blooded animals without any uptake of residues in edible tissues. Based on its toxicological properties, bentazon was classified as noncarcinogenic ("Group E") by the EPA. The ADI is set at 0.1 mg/kg body weight/d. The WHO drinking water guideline value, based on the toxicological profile of bentazon, was recently raised to 30 micrograms/L.(ABSTRACT TRUNCATED AT 400 WORDS)

Quinolinecarboxylic acids. 3. Synthesis and antibacterial evaluation of 2-substituted-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de] [1,4]benzothiazine-6-carboxylic acids related to rufloxacin.

A series of 2-substituted-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4]benzothiazine-6 -carboxylic acids has been prepared and evaluated for in vitro antibacterial activity. These derivatives were less active than corresponding desmethylated analogues. Among these derivatives, the most active compound 22a was selected for preliminary pharmacokinetics in rats. The pharmacokinetic data indicated that 22a was rapidly absorbed and induced lasting plasma and urinary levels. In comparison with rufloxacin, it was excreted in low quantity in urine; a significant amount of desmethylated piperazinyl urinary metabolite was observed.