Redox-Optimized ROS Balance and the relationship between mitochondrial respiration and ROS

The Redox-Optimized ROS Balance [R-ORB] hypothesis postulates that the redox environment [RE] is the main intermediary between mitochondrial respiration and reactive oxygen species [ROS]. According to R-ORB, ROS emission levels will attain a minimum vs. RE when respiratory rate (VO₂) reaches a maximum following ADP stimulation, a tenet that we test herein in isolated heart mitochondria under forward electron transport [FET]. ROS emission increased two-fold as a function of changes in the RE (~ 400 to ~ 900 mV·mM) in state 4 respiration elicited by increasing glutamate/malate (G/M). In G/M energized mitochondria, ROS emission decreases two-fold for RE ~ 500 to ~ 300 mV·mM in state 3 respiration at increasing ADP. Stressed mitochondria released higher ROS, that was only weakly dependent on RE under state 3. As a function of VO₂, the ROS dependence on RE was strong between ~ 550 and ~ 350 mV·mM, when VO₂ is maximal, primarily due to changes in glutathione redox potential. A similar dependence was observed with stressed mitochondria, but over a significantly more oxidized RE and ~ 3-fold higher ROS emission overall, as compared with non-stressed controls. We conclude that under non-stressful conditions mitochondrial ROS efflux decreases when the RE becomes less reduced within a range in which VO₂ is maximal. These results agree with the R-ORB postulate that mitochondria minimize ROS emission as they maximize VO₂ and ATP synthesis. This relationship is altered quantitatively, but not qualitatively, by oxidative stress although stressed mitochondria exhibit diminished energetic performance and increased ROS release.     

Design,synthesis, antiviral and cytostatic activity of ω-(1H-1,2,3-triazol-1-yl)(polyhydroxy)alkylphosphonates as acyclic nucleotide analogues

The efficient synthesis of a new series of polyhydroxylated dibenzyl ω-(1H-1,2,3-triazol-1-yl)alkylphosphonates as acyclic nucleotide analogues is described starting from dibenzyl ω-azido(polyhydroxy)alkylphosphonates and selected alkynes under microwave irradiation. Selected O,O-dibenzylphosphonate acyclonucleotides were transformed into the respective phosphonic acids. All compounds were evaluated in vitro for activity against a broad variety of DNA and RNA viruses and for cytostatic activity against murine leukemia L1210, human T-lymphocyte CEM and human cervix carcinoma HeLa cells. Compound (1S,2S)-16b exhibited antiviral activity against Influenza A H3N2 subtype (EC₅₀ = 20 μM—visual CPE score; EC₅₀ = 18 μM—MTS method; MCC >100 μM, CC₅₀ >100 μM) in Madin Darby canine kidney cell cultures (MDCK), and (1S,2S)-16k was active against vesicular stomatitis virus and respiratory syncytial virus in HeLa cells (EC₅₀ = 9 and 12 μM, respectively). Moreover, compound (1R,2S)-16l showed activity against both herpes simplex viruses (HSV-1, HSV-2) in HEL cell cultures (EC₅₀ = 2.9 and 4 μM, respectively) and feline herpes virus in CRFK cells (EC₅₀ = 4 μM) but at the same time it exhibited cytotoxicity toward uninfected cell (MCC ⩾ 4 μM). Several other compounds have been found to inhibit proliferation of L1210, CEM as well as HeLa cells with IC₅₀ in the 4–50 μM range. Among them compounds (1S,2S)- and (1R,2S)-16l were the most active (IC₅₀ in the 4–7 μM range).     

Synthesis and biological evaluation of novel 2-(substituted phenylaminocarbonylmethylthio)-6-(2,6-dichlorobenzyl)-pyrimidin-4(3H)-ones as potent HIV-1 NNRTIs

A series of novel 2-(phenylaminocarbonylmethylthio)-6-(2,6-dichlorobenzyl)-pyrimidin-4(3H)-ones have been designed and synthesized. All of the new compounds were evaluated for their anti-HIV activities in MT-4 cells. Most of these new compounds showed moderate to potent activities against wild-type HIV-1 with an EC₅₀ ranging from 4.48 μM to 0.18 μM. Among them, 2-[(4-bromophenylamino)carbonylmethylthio]-6-(2,6-dichlorobenzyl)-5-methylpyrimidin-4(3H)-one 4b3 was identified as the most promising compound (EC₅₀ = 0.18 ± 0.06 μM, CC₅₀ >243.56 μM, SI >1326). The structure–activity relationship (SAR) of these new congeners is discussed.     

Discovery of substituted N′-(2-oxoindolin-3-ylidene)benzohydrazides as new apoptosis inducers using a cell- and caspase-based HTS assay

We report the discovery of a series of substituted N′-(2-oxoindolin-3-ylidene)benzohydrazides as inducers of apoptosis using our proprietary cell- and caspase-based ASAP HTS assay. Through SAR studies, N′-(4-bromo-5-methyl-2-oxoindolin-3-ylidene)-3,4,5-trimethoxybenzohydrazide (3g) was identified as a potent apoptosis inducer with an EC₅₀ value of 0.24 μM in human colorectal carcinoma HCT116 cells, more than a 40-fold increase in potency from the initial screening hit N′-(5-bromo-2-oxoindolin-3-ylidene)-3,4,5-trimethoxybenzohydrazide (2a). Compound 3g also was found to be highly active in a growth inhibition assay with a GI₅₀ value of 0.056 μM in HCT116 cells. A group of potentially more aqueous soluble analogs were prepared and found to be highly active. Among them, compound 4e incorporating a methyl piperazine moiety was found to have EC₅₀ values of 0.17, 0.088 and 0.14 μM in human colorectal carcinoma cells HCT116, hepatocellular carcinoma cancer SNU398 cells and human colon cancer RKO cells, respectively. Compounds 3g and 4e were found to function as inhibitors of tubulin polymerization.     

Studies on the anti-hepatitis C virus activity of newly synthesized tropolone derivatives: Identification of NS3 helicase inhibitors that specifically inhibit subgenomic HCV replication

We synthesized new tropolone derivatives substituted with cyclic amines: piperidine, piperazine or pyrrolidine. The most active anti-helicase compound (IC₅₀ = 3.4 μM), 3,5,7-tri[(4′-methylpiperazin-1′-yl)methyl]tropolone (2), inhibited RNA replication by 50% at 46.9 μM (EC₅₀) and exhibited the lowest cytotoxicity (CC₅₀) >1 mM resulting in a selectivity index (SI = CC₅₀/EC₅₀) >21. The most efficient replication inhibitor, 3,5,7-tri[(4′-methylpiperidin-1′-yl)methyl]tropolone (6), inhibited RNA replication with an EC₅₀ of 32.0 μM and a SI value of 17.4, whereas 3,5,7-tri[(3′-methylpiperidin-1′-yl)methyl]tropolone (7) exhibited a slightly lower activity with an EC₅₀ of 35.6 μM and a SI of 9.8.     

Synthesis and biological evaluation of novel thiadiazole amides as potent Cdc25B and PTP1B inhibitors

A series of novel thiadiazole amide derivatives have been synthesized and evaluated for inhibitory activities against Cdc25B and PTP1B. Most of them showed inhibitory activities against Cdc25B (IC₅₀ = 1.18–8.01 μg/mL) and PTP1B (IC₅₀ = 0.85–8.75 μg/mL), respectively. Moreover, compounds 5b and 4l were most potent with IC₅₀ values of 1.18 and 0.85 μg/mL for Cdc25B and PTP1B, respectively, compared with reference drugs Na₃VO₄ (IC₅₀ = 0.93 μg/mL) and oleanolic acid (IC₅₀ = 0.85 μg/mL). The results of selectivity experiments showed that the target compounds were selective inhibitors against PTP1B and Cdc25B. Enzyme kinetic experiments demonstrated that compound 5k was a specific inhibitor with the typical characteristics of a mixed inhibitor.     

Discovery of N-(4′-(indol-2-yl)phenyl)sulfonamides as novel inhibitors of HCV replication

A series of novel 2-phenylindole analogs were synthesized and evaluated for activity in subgenomic HCV replicon inhibition assays. Several compounds containing small alkyl sulfonamides on the phenyl ring exhibiting submicromolar EC₅₀ values against the genotype 1b replicon were identified. Among these, compound 25d potently inhibited the 1b replicon (EC₅₀ = 0.17 μM) with 147-fold selectivity with respect to cytotoxicity. Compound 25d was stable in the presence of human liver microsomes and had a good pharmacokinetic profile in rats with an IV half-life of 4.3 h and oral bioavailability (F) of 58%.     

Structural modifications of CH(OH)-DAPYs as new HIV-1 non-nucleoside reverse transcriptase inhibitors

A series of CR₂(OH)-diarylpyrimidine derivatives (CR₂(OH)-DAPYs) featuring a hydrophobic group at CH(OH) linker between wing I and the central pyrimidine were synthesized and evaluated for their anti-HIV activity in MT-4 cell cultures. All the target compounds except for compound 3k displayed inhibitory activity against HIV-1 wild-type with EC₅₀ values ranging from 7.21 ± 1.99 to 0.067 ± 0.006 μM. Among them, compound 3d showed the most potent anti-HIV-1 activity (EC₅₀ = 0.067 ± 0.006 μM, SI > 592), which was approximately 2-fold more potent than the reference drugs nevirapine (NVP) and delaviridine (DLV) in the same assay. In addition, the binding modes with HIV-1 RT and the preliminary SAR studies of these new derivatives were also investigated.     

Synthesis and pharmacological exploitation of clioquinol-derived copper-binding apoptosis inducers triggering reactive oxygen species generation and MAPK pathway activation

In the present study, we carried out Mannich-type reaction to synthesize clioquinol-derived 7-methyl-arylsulfonylpiperazine analogs with improved growth-inhibitory effects. 11 bearing 5-nitro group on the quinoline ring exhibited 26-fold more potent than that of clioquinol against HeLa cells with a GI₅₀ value of 0.71 μM. In addition, 11 revealed synergistic effects on the growth inhibition of HeLa cells with GI₅₀ values of 0.65, 0.25, and 0.06 μM in the presence of 1, 10, and 50 μM copper, respectively. Consistent to the clioquinol-mediated apoptosis, mechanistic study indicates that 9- and 11-induced growth inhibition is attributed to caspase-dependent pathway. Detection of reactive oxygen species in response to clioquinol, 9 and 11 confirmed that ROS was dramatically stimulated in the presence of copper and partially abolished upon treatment of 1 mM tempol. Further study indicated that 9- and 11-mediated induction of oxidative stress by ROS generation resulted in the activation MAPK pathway.     

Identification of small molecule inhibitors of proline-rich tyrosine kinase 2 (Pyk2) with osteogenic activity in osteoblast cells

A screening campaign of a diverse collection of ～250,000 small molecule compounds was performed to identify inhibitors of proline-rich tyrosine kinase 2 (Pyk2) with potential osteogenic activity in osteoblast cells. Compounds were prioritized based on selectivity following a counter-screen against focal adhesion kinase (FAK), a closely related kinase. 4-Amino and 5-aryl substituted pyridinone series were identified that showed strong biochemical potency against Pyk2 and up to 3700-fold selectivity over FAK. Modeling analysis suggested that structural differences in the substrate binding cleft could explain the high selectivity of these chemical series against FAK. Representative compounds from each series showed inhibition of Pyk2 autophosphorylation in 293T cells (IC₅₀ ～0.11 μM), complete inhibition of endogenous Pyk2 in A7r5 cells and increased levels of osteogenic markers in MC3T3 osteoblast cells (EC₅₀’s ～0.01 μM). These results revealed a new class of compounds with osteogenic-inducing activity in osteoblast cells and a starting point for the development of more potent and selective Pyk2 inhibitors.     

Synthesis and biological evaluation of 3′,4′,5′-trimethoxychalcone analogues as inhibitors of nitric oxide production and tumor cell proliferation

A series of 23 3′,4′,5′-trimethoxychalcone analogues was synthesized and their inhibitory effects on nitric oxide (NO) production in LPS/IFN-γ-treated macrophages, and tumor cell proliferation has been investigated. 4-Hydroxy-3,3′,4′,5′-tetramethoxychalcone (7), 3,4-dihydroxy-3′,4′,5′-trimethoxychalcone (11), 3-hydroxy-3′,4,4′,5′-tetramethoxychalcone (14), and 3,3′,4′,5′-tetramethoxychalcone (15) were the most potent growth inhibitory agents on NO production, with an IC₅₀ value of 0.3, 1.5, 1.3 and 0.3 μM, respectively. The tumor cells proliferation assay results revealed that several compounds exhibited potent inhibition activity against different cancer cell lines. The chalcone 15 was the most potent anti-proliferative compound in the series with IC₅₀ values of 1.8 and 2.2 μM toward liver cancer Hep G2 and colon cancer Colon 205 cell lines, respectively. 2,3,3′,4′,5′-Pentamethoxychalcone (1), 3,3′,4,4′,5,5′-hexamethoxychalcone (3), 2,3′,4,4′,5,5′-hexamethoxychalcone (5), 2-hydroxy-3,3′,4′,5′-tetramethoxychalcone (10), 11 and 14 showed significant anti-proliferation actions in Hep G2 and Colon 205 cells with an IC₅₀ values ranging between 10 and 20 μM. Among the tested agents, compound 7 showed selective NO production inhibition (IC₅₀ = 0.3 μM), while has no effect on tumor cell proliferation (IC₅₀ >100 μM). 3,3′,4,4′,5′-Pentamethoxychalcone (2) showed selective anti-proliferation effect in Hep G2 cells, in addition to its potent NO inhibition, however has no such response in Colon 205 cells. In contrast, 3-formyl-3′,4′,5′-trimethoxychalcone (22) showed moderate growth inhibition in Colon 205 cells, while has no such effect on NO production and Hep G2 cells proliferation. These results provide insight into the correlation between some structural properties of 3′,4′,5′-trimethoxychalcones and their in vitro anti-inflammatory and anti-cancer differentiation activity.     

Synthesis and biological evaluation of imidazole thioacetanilides as novel non-nucleoside HIV-1 reverse transcriptase inhibitors

A series of 2-(1-aryl-1H-imidazol-2-ylthio)acetamide [imidazole thioacetanilide (ITA)] derivatives were synthesized and evaluated as potent inhibitors of human immunodeficiency virus type-1 (HIV-1). Among them, the most potent HIV-1 inhibitors were 4a5 (EC₅₀ = 0.18 μM), and 4a2 (EC₅₀ = 0.20 μM), which were more effective than the lead compound L1 (EC₅₀ = 2.053 μM) and the reference drugs nevirapine and delavirdine. The preliminary structure–activity relationship (SAR) of the newly synthesized congeners is discussed.     

Design,synthesis and biological evaluation of 3-benzyloxy-linked pyrimidinylphenylamine derivatives as potent HIV-1 NNRTIs

A novel series of 3-benzyloxy-linked pyrimidinylphenylamine derivatives (8a–8s) was designed, synthesized and evaluated for their in vitro anti-HIV activity in MT-4 cell cultures. Most of the compounds inhibited wild-type (wt) HIV-1 replication in the lower micromolar concentration range (EC₅₀ = 0.05–35 μM) with high selectivity index (SI) values (ranged from 10 to >4870). In particular, 8h and 8g displayed excellent antiretroviral activity against wt HIV-1 with low cytotoxicity (EC₅₀ = 0.07 μM, CC₅₀ >347 μM, SI >4870; EC₅₀ = 0.05 μM, CC₅₀ = 42 μM, SI = 777, respectively), comparable to that of the marked drug nevirapine (EC₅₀ = 0.113 μM, CC₅₀ >15 μM, SI >133). In order to confirm the binding target, 8h was selected to perform the anti-HIV-1 RT assay. Additionally, preliminary structure activity relationship (SAR) analysis and molecular docking studies of newly synthesized compounds were also discussed, as well as the predicted physicochemical properties.     

4′-Substituted pyrimidine nucleosides lacking 5′-hydroxyl function as potential anti-HCV agents

Hepatitis C virus (HCV) infection is one of the major health problems worldwide. If left untreated, it leads to liver cirrhosis, liver cancer and death. Herein, we report synthesis and anti-HCV activity of a new class of pyrimidine nucleosides possessing a 4′-carboxymethyl (9–16, 21 and 23) or 4′-carboxamide function (17–19 and 24). Among these, 10–12 (EC₅₀ = 33.1–42.4 μM), 14 and 21 (EC₅₀ = 43.4–59.5 μM) exhibited potent activity in HCV-1a replicon cells without any toxicity to parent Huh-7 cells (CC₅₀ = >829–1055 μM). The anti-HCV activities demonstrated by this unusual class of compounds were superior to that of ribavirin (EC₅₀ = 81.9 μM). Further, the most active analog, 12, was found to interact synergistically with ribavirin to inhibit HCV RNA replication.     

Synthesis and biological activity of aminophthalazines and aminopyridazines as novel inhibitors of PGE2 production in cells

This Letter reports the synthesis and biological evaluation of a collection of aminophthalazines as a novel class of compounds capable of reducing production of PGE₂ in HCA-7 human adenocarcinoma cells. A total of 28 analogs were synthesized, assayed for PGE₂ reduction, and selected active compounds were evaluated for inhibitory activity against COX-2 in a cell free assay. Compound 2xxiv (R¹ = H, R² = p-CH₃O) exhibited the most potent activity in cells (EC₅₀ = 0.02 μM) and minimal inhibition of COX-2 activity (3% at 5 μM). Furthermore, the anti-tumor activity of analog 2vii was analyzed in xenograft mouse models exhibiting good anti-cancer activity.     

Discovery of ML326: The first sub-micromolar,selective M5 PAM

This Letter describes the further chemical optimization of the M₅ PAM MLPCN probes ML129 and ML172. A multi-dimensional iterative parallel synthesis effort quickly explored isatin replacements and a number of southern heterobiaryl variations with no improvement over ML129 and ML172. An HTS campaign identified several weak M₅ PAMs (M₅ EC₅₀ >10 μM) with a structurally related isatin core that possessed a southern phenethyl ether linkage. While SAR within the HTS series was very shallow and unable to be optimized, grafting the phenethyl ether linkage onto the ML129/ML172 cores led to the first sub-micromolar M₅ PAM, ML326 (VU0467903), (human and rat M₅ EC₅₀s of 409 nM and 500 nM, respectively) with excellent mAChR selectivity (M₁–M₄ EC₅₀s >30 μM) and a robust 20-fold leftward shift of the ACh CRC.