Development of a novel, CNS-penetrant, metabotropic glutamate receptor 3 (mGlu3) NAM probe (ML289) derived from a closely related mGlu5 PAM

Herein we report the discovery and SAR of a novel metabotropic glutamate receptor 3 (mGlu(3)) NAM probe (ML289) with 15-fold selectivity versus mGlu(2). The mGlu(3) NAM was discovered via a 'molecular switch' from a closely related, potent mGlu(5) positive allosteric modulator (PAM), VU0092273. This NAM (VU0463597, ML289) displays an IC(50) value of 0.66 μM and is inactive against mGlu(5).     

Discovery of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221) as a functional antagonist of the apelin (APJ) receptor

The recently discovered apelin/APJ system has emerged as a critical mediator of cardiovascular homeostasis and is associated with the pathogenesis of cardiovascular disease. A role for apelin/APJ in energy metabolism and gastrointestinal function has also recently emerged. We disclose the discovery and characterization of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221), a potent APJ functional antagonist in cell-based assays that is >37-fold selective over the closely related angiotensin II type 1 (AT1) receptor. ML221 was derived from an HTS of the ～330,600 compound MLSMR collection. This antagonist showed no significant binding activity against 29 other GPCRs, except to the κ-opioid and benzodiazepinone receptors (<50/<70%I at 10 μM). The synthetic methodology, development of structure–activity relationship (SAR), and initial in vitro pharmacologic characterization are also presented.     

Convolutamines I and J, antitrypanosomal alkaloids from the bryozoan Amathia tortusa

Mass-directed isolation of the CH(2)Cl(2)/CH(3)OH extract from the marine bryozoan Amathia tortusa resulted in the purification of two new brominated alkaloids, convolutamines I (1) and J (2). The structures of 1 and 2 were determined following spectroscopic data analysis. Both compounds were isolated during a drug discovery program aimed at identifying new antitrypanosomal leads from a prefractionated natural product library. Compounds 1 and 2 were shown to be active toward the parasite Trypanosoma brucei brucei with IC(50) values of 1.1 and 13.7 μM, respectively. Preliminary toxicity profiling was also performed on both 1 and 2 using the human embryonic kidney cell line, HEK293. Compound 1 was shown to exhibit cytotoxicity against HEK293 with an IC(50) of 22.0 μM whilst 2 was inactive at 41.0 μM.     

The discovery of 4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazolo-[1 ,5-a]-pyrimidine: a corticotropin-releasing factor (hCRF1) antagonist

Structure activity relationship studies led to the discovery of 4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazo lo-[1,5-a]-pyrimidine 11-31 (DMP904), whose pharmacological profile strongly supports the hypothesis that hCRF1 antagonists may be potent anxiolytic drugs. Compound 11-31 (hCRF1 Ki = 1.0+/-0.2 nM (n = 8)) was a potent antagonist of hCRF1-coupled adenylate cyclase activity in HEK293 cells (IC50= 10.0+/-0.01 nM versus 10 nM r/hCRF, n = 8); alpha-helical CRF(9-41) had weaker potency (IC50 = 286+/-63 nM, n = 3). Analogue 11-31 had good oral activity in the rat situational anxiety test; the minimum effective dose for 11-31 was 0.3 mg/kg (po). Maximal efficacy (approximately 57% reduction in latency time in the dark compartment) was observed at this dose. Chlordiazepoxide caused a 72% reduction in latency at 20 mg/kg (po). The literature compound 1 (CP154526-1, 30 mg/kg (po)) was inactive in this test. Compound 11-31 did not inhibit open-field locomotor activity at 10, 30, and 100 mg/kg (po) in rats. In beagle dogs, this compound (5 mg/kg, iv, po) afforded good plasma levels. The key iv pharmacokinetic parameters were t1/2, CL and Vd,ss values equal to 46.4+/-7.6 h. 0.49+/-0.08 L/kg/h and 23.0+/-4.2 L/kg, respectively. After oral dosing, the mean Cmax, Tmax t1/2 and bioavailability values were equal to 1260+/-290 nM, 0.75+/-0.25 h. 45.1+/-10.2 h and 33.1%, respectively. The overall rat behavioral profile of this compound suggests that it may be an anxiolytic drug with a low motor side effect liability.     

VEGF inhibitor (Iressa) arrests histone deacetylase expression: single-cell cotransfection imaging cytometry for multi-target-multi-drug analysis

Multi-target-multi-drug approaches are needed to accelerate the process of drug discovery screening and to design efficient therapeutic strategies against diseases that involve alterations in multiple cellular targets. Herein we report single-cell cotransfection imaging cytometry to quantitatively screen drug-induced off-target effects. Vascular endothelial growth factor (VEGF) and histone deacetylase (HDAC) genes amplified from the genomic DNA were cloned in fluorescently tagged gene constructs (RFP-HDAC/YFP-VEGF). These gene constructs were cotransfected in HEK-293 cells to explore the possibility of off-target effects of 4-phenylbutyrate and Iressa on the expression of VEGF and HDAC through single-cell imaging cytometry. Iressa (10 μM) treatment at the time of cotransfection or 48 h after cotransfection of RFP-HDAC/YFP-VEGF plasmids in HEK-293 cells resulted in off-target effects on HDAC expression. These results suggest possible applications of Iressa in the treatment of diseases in which expression of both HDAC and VEGF should be inhibited. 4-Phenylbutyrate (2.0 mM) did not show any off-target effects on VEGF expression. The developed quantitative multicolor live single-cell cotransfection imaging can be employed to select better drug combinations for faster screening and greater accuracy in multi-target-multi-drug analysis by increasing the on-target/desired off-target effects and eliminating the undesirable off-target effects.     

1-[(4′-Chlorophenyl) carbonyl-4-(aryl) thiosemicarbazide derivatives as potent urease inhibitors: Synthesis,in vitro and in silico studies

A series of 1-[(4′-chlorophenyl)carbonyl-4-(aryl)thiosemicarbazide derivatives 1–25 was synthesized and characterized by spectroscopic techniques such as EI-MS and ¹H NMR. All compounds were screened for urease inhibitory activity in vitro and demonstrated excellent inhibitory activity in the range of IC₅₀ = 0.32 ± 0.01–25.13 ± 0.13 μM as compared to the standard thiourea (IC₅₀ = 21.25 ± 0.13 μM). Amongst the potent analogs, compounds 3 (IC₅₀ = 2.31 ± 0.01 μM), 6 (IC₅₀ = 2.14 ± 0.04 μM), 10 (IC₅₀ = 1.14 ± 0.06 μM), 20 (IC₅₀ = 2.15 ± 0.05 μM), and 25 (IC₅₀ = 0.32 ± 0.01 μM) are many folds more active than the standard. Structure-activity relationship (SAR) was rationalized by looking at the effect of diversely substituted aryl ring on inhibitory potential which predicted that regardless of the nature of substituents, their positions on aryl ring is worth important for the potent activity. Furthermore, to verify these interpretations, in silico study was performed on all compounds and a good correlation was perceived between the biological evaluation and docking study of compounds.     

Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels

Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable nonselective cation channels implicated in diverse physiological functions, including smooth muscle contractility and synaptic transmission. However, lack of potent selective pharmacological inhibitors for TRPC channels has limited delineation of the roles of these channels in physiological systems. Here we report the identification and characterization of ML204 as a novel, potent, and selective TRPC4 channel inhibitor. A high throughput fluorescent screen of 305,000 compounds of the Molecular Libraries Small Molecule Repository was performed for inhibitors that blocked intracellular Ca(2+) rise in response to stimulation of mouse TRPC4β by μ-opioid receptors. ML204 inhibited TRPC4β-mediated intracellular Ca(2+) rise with an IC(50) value of 0.96 μm and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4β currents activated through either μ-opioid receptor stimulation or intracellular dialysis of guanosine 5'-3-O-(thio)triphosphate (GTPγS), suggesting a direct interaction of ML204 with TRPC4 channels rather than any interference with the signal transduction pathways. Selectivity studies showed no appreciable block by 10-20 μm ML204 of TRPV1, TRPV3, TRPA1, and TRPM8, as well as KCNQ2 and native voltage-gated sodium, potassium, and calcium channels in mouse dorsal root ganglion neurons. In isolated guinea pig ileal myocytes, ML204 blocked muscarinic cation currents activated by bath application of carbachol or intracellular infusion of GTPγS, demonstrating its effectiveness on native TRPC4 currents. Therefore, ML204 represents an excellent novel tool for investigation of TRPC4 channel function and may facilitate the development of therapeutics targeted to TRPC4.     

The discovery of [1-(4-dimethylamino-benzyl)-piperidin-4-yl]-[4-(3,3-dimethylbutyl)-phen yl]-(3-methyl-but-2-enyl)-amine, an N-type Ca+2 channel blocker with oral activity for analgesia

Our drug discovery efforts for N-type calcium channel blockers in the 4-piperidinylaniline series led to the discovery of an orally active analgesic agent 26.1-[4-Dimethylamino-benzyl)-piperidin-4-yl]-[4-(3,3-dimethyl-but yl)-phenyl]-(3-methyl-but-2-enyl)amine (26) showed high affinity to functionally block N-type calcium channels (IC50=0.7 microM in the IMR32 assay) and exhibited high efficacy in the anti-writhing analgesia test with mice (ED50=12 mg/kg by po and 4 mg/kg by iv). In this report, the rationale for the design, synthesis, biological evaluation, and pharmacokinetics of this series of blockers is described.     

Boric acid increases the expression levels of human anion exchanger genes SLC4A2 and SLC4A3

Boron is an important micronutrient in plants and animals. The role of boron in living systems includes coordinated regulation of gene expression, growth and proliferation of higher plants and animals. There are several well-defined genes associated with boron transportation and tolerance in plants and these genes show close homology with human anion exchanger genes. Mutation of these genes also characterizes some genetic disorders. We investigated the toxic effects of boric acid on HEK293 cells and mRNA expression of anion exchanger (SLC4A1, SLC4A2 and SLC4A3) genes. Cytotoxicity of boric acid at different concentrations was tested by using the methylthiazolyldiphenyl-tetrazolium bromide assay. Gene expression profiles were examined using quantitative real-time PCR. In the HEK293 cells, the nontoxic upper concentration of boric acid was 250 μM; more than 500 μM caused cytotoxicity. The 250 μM boric acid concentration increased gene expression level of SLC4A2 up to 8.6-fold and SLC4A3 up to 2.6-fold, after 36-h incubation. There was no significant effect of boric acid on SLC4A1 mRNA expression levels.     

Synthesis and SAR of a novel metabotropic glutamate receptor 4 (mGlu4) antagonist: unexpected 'molecular switch' from a closely related mGlu4 positive allosteric modulator

Herein we report the discovery and SAR of a novel antagonist of metabotropic glutamate receptor 4 (mGlu(4)). The antagonist was discovered via a molecular switch from a closely related mGlu(4) positive allosteric modulator (PAM). This antagonist (VU0448383) displays an IC(50) value of 8.2±0.4 μM and inhibits an EC(80) glutamate response by 63.1±6.6%.     

Effects of oral clonidine on plasma 3-methoxy-4-hydroxyphenethyleneglycol (MHPG) in man: Preliminary report

Repeated (N=15) administration of clonidine (0,1,5 μg/kg,p.o.) to three normotensive male subjects resulted in significant decreases in plasma free 3-methoxy-4-hydroxyphenethyleneglycol (MHPG) at three hours for both the 1 μg/kg dose (p < .05) and the 5 μg/kg dose (p < .01) when compared to concentrations following placebo. The mean decrement in plasma free MHPG following a 5 μg/kg dose was 36%. Systolic blood pressure fell a mean of 17 mmHg after 1 μg/kg and 37 mmHg after 5 μg/kg of clonidine. The application of a clonidine challenge test to assess noradrenergic receptor sensitivity $\text{in}$$\text{vivo}$ is discussed.     

Substituted indoles as selective protease activated receptor 4 (PAR-4) antagonists: Discovery and SAR of ML354

Herein we report the discovery and SAR of an indole-based protease activated receptor-4 (PAR-4) antagonist scaffold derived from a similarity search of the Vanderbilt HTS collection, leading to MLPCN probe ML354 (VU0099704). Using a novel PAC-1 fluorescent α_IIbβ3 activation assay this probe molecule antagonist was found to have an IC₅₀ of 140 nM for PAR-4 with 71-fold selectivity versus PAR-1 (PAR-1IC₅₀ = 10 μM).     

Synthesis and bioassay evaluation of 1-(4-substitutedideneaminooxymethyl)-phenyl-3-(2,6-difluorobenzoyl)ureas

A variety of novel 1-(4-substitutedideneaminooxymethyl)-phenyl-3-(2,6-difluorobenzoyl)ureas were designed and synthesized by the reaction of 4-substitutedideneaminooxymethyl aniline with 2,6-difluorobenzoyl isocyanates in good yields. The title compounds were soluble in most organic solvents, which should make them easier to use. The preliminary bioassay showed that some of the title compounds show excellent insecticidal activity against Mythimna separata at the dosage of 25 mg kg(-1) and moderate insecticidal activity against Nephotettix cincticeps at the dosage of 500 mg kg(-1). Toxicity assays indicated that these title compounds cause in M. separata and N. cincticeps such symptoms of toxicity as discolouration, and weight loss, and cessation of feeding and lethal. One title compound exhibited acaricidal activity against Tetranychus urticae.     

The pulmonary pharmacology of [4-methoxy-N1-(4-trans-nitrooxycyclohexyl)-N3-(3-pyridinylmethyl)-1,3-benzenedicarboxamide] (2NTX-99), an anti-atherotrombotic compound with therapeutic potential in pathological conditions that target lung vasculature

The pharmacological activity of 2NTX-99 ([4-methoxy-N1-(4-trans-nitrooxycyclohexyl)-N3-(3-pyridinylmethyl)-1,3-benzenedicarboxamide]) was investigated in vitro in the intact, rat pulmonary vasculature and in guinea pig airways. Rat lungs were perfused at constant flow and changes in vascular tone recorded. Challenge with the TXA? analogue 9,11-dideoxy-9α11α-methanoepoxy ProstaglandinF? (U46619, 0.5 μM) increased vessel tone (32.48±1.5 vs 13.13±0.56 mmHg; n=12). 2NTX-99 (0.1-100 μM; n=5), caused a concentration-dependent relaxation, prevented by 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 10 μM, n=4), an inhibitor of soluble guanylate cyclase. Acetylcholine (0.1-10 μM; n=3) and a reference NO-donor, isosorbide-5-mononitrate (5-100 μM; n=4), were ineffective. Intraluminal perfusion of washed human platelets (2 × 10? cells/ml) increased intravascular pressure after challenge with arachidonic acid (AA, 2 μM; n=5), an increase abolished by acetylsalicylic acid and significantly reduced by 2NTX-99 (40 μM; n=5). TXB? in the lung perfusate was detected after platelet activation, 2NTX-99 inhibited TXA? synthesis (6.45±0.6 and 1.10±0.2 ng/ml, respectively). 2NTX-99 did not alter central or peripheral airway responsiveness to Histamine (0.001-300 μM; n=6), U46619 (0.001-3 μM, n=3) or LTD? (1 pM-1 μM; n=6). 2NTX-99 vasodilates the pulmonary vasculature via the release of nitric oxide (NO) and reduces intraluminal, AA-induced, TXA? formation. The combined activity of 2NTX-99 as an NO-donor and a TXA?-synthesis inhibitor provides strong support for its potential therapeutic use in pathologies of the pulmonary vascular bed (e.g. pulmonary hypertension).     

C3(OH)2mim][BF4]-Au/Pt biosensor for glutamate sensing in vivo integrated with on-line microdialysis system

A new type of hydroxyl functionalized room temperature ionic liquid (RTIL), [C(3)(OH)(2)mim][BF(4)], was synthesized herein and a novel H(2)O(2) biosensor is fabricated with [C(3)(OH)(2)mim][BF(4)] as the substrate and electrodepositing bimetallic Au/Pt nanoparticles (NPs) onto the [C(3)(OH)(2)mim][BF(4)] film. The functionalization of RTIL with hydroxyl groups provided an appropriate environment for the preparation of more uniform and smaller Au/Pt NPs with the diameter of 2.5 nm±0.2 nm. Immobilized with glutamate oxidase (GlutaOx), the resulting GlutaOx-[C(3)(OH)(2)mim][BF(4)]-Au/Pt-Nafion biosensor displayed excellent electrocatalytic response to glutamate at a potential of -200 mV. An effective on-line microdialysis system, which was powered by a microdialysis pump, was set up and used for the detection of glutamate successively in the striatum of rats. The glutamate biosensor in on-line microdialysis system showed good linear range from 0.5 μM to 20.0 μM with the detection limit of 0.17 μM (S/N=3). The basal level of glutamate in the striatum of anaesthetic rats was calculated to be 3.01±0.67 μM (n=3). The application of the GlutaOx-[C(3)(OH)(2)mim][BF(4)]-Au/Pt-Nafion electrode is further demonstrated for in vivo sensing of the variation of glutamate level in the striatum when rats received intraperitoneal (i.p.) injection of 100 mM KCl and brain electrical stimulation of the subthalamic nucleus area (STN). Both of the two kinds of stimulation resulted in an increase in the extracellular concentration of glutamate. This method has proved to be sensitive and reproducible, which enables its promising application in physiology and pathology.     

Novel tandem biotransformation process for the biosynthesis of a novel compound, 4-(2,3,5,6-tetramethylpyrazine-1)-4'-demethylepipodophyllotoxin

According to the structure of podophyllotoxin and its structure-function relationship, a novel tandem biotransformation process was developed for the directional modification of the podophyllotoxin structure to directionally synthesize a novel compound, 4-(2,3,5,6-tetramethylpyrazine-1)-4'-demethylepipodophyllotoxin (4-TMP-DMEP). In this novel tandem biotransformation process, the starting substrate of podophyllotoxin was biotransformed into 4'-demethylepipodophyllotoxin (product 1) with the demethylation of the methoxyl group at the 4' position by Gibberella fujikuroi SH-f13, which was screened out from Shennongjia prime forest humus soil (Hubei, China). 4'-Demethylepipodophyllotoxin (product 1) was then biotransformed into 4'-demethylpodophyllotoxone (product 2) with the oxidation of the hydroxyl group at the 4 position by Alternaria alternata S-f6, which was screened out from the gathered Dysosma versipellis plants in the Wuhan Botanical Garden, Chinese Academy of Sciences. Finally, 4'-demethylpodophyllotoxone (product 2) and ligustrazine were linked with a transamination reaction to synthesize the target product 4-TMP-DMEP (product 3) by Alternaria alternata S-f6. Compared with podophyllotoxin (i.e., a 50% effective concentration [EC(50)] of 529 μM), the EC(50) of 4-TMP-DMEP against the tumor cell line BGC-823 (i.e., 0.11 μM) was significantly reduced by 5,199 times. Simultaneously, the EC(50) of 4-TMP-DMEP against the normal human proximal tubular epithelial cell line HK-2 (i.e., 0.40 μM) was 66 times higher than that of podophyllotoxin (i.e., 0.006 μM). Furthermore, compared with podophyllotoxin (i.e., log P = 0.34), the water solubility of 4-TMP-DMEP (i.e., log P = 0.66) was significantly enhanced by 94%. For the first time, the novel compound 4-TMP-DMEP with superior antitumor activity was directionally synthesized from podophyllotoxin by the novel tandem biotransformation process developed in this work.     

p-Aminophenol-induced cytotoxicity in Jurkat T cells: protective effect of 2(RS)-n-propylthiazolidine-4(R)-carboxylic acid

Acetaminophen (APAP) is hepatotoxic and can cause toxicity in Jurkat T cells. p-Aminophenol (PAP), an industrial chemical and APAP metabolite, is nephrotoxic and hepatotoxic. Its potential toxicity in Jurkat T cells was investigated. PAP (10-250 μM) caused toxicity (decreased survival and increased LDH activity in incubation medium) and GSH depletion. At a concentration of 100 μM but not 250 μM, PAP increased DNA fragmentation. It decreased p-Akt levels (Elisa) and at higher concentrations decreased p-Akt expression (Western blotting). It had no effect on FasL expression. The cysteine precursor 2(RS)-n-propylthiazolidine-4(R)-carboxylic acid (250 μM) attenuated the PAP (100 μM)-induced decrease in viability and prevented GSH depletion and increased DNA fragmentation. It attenuated the PAP-induced decrease in p-Akt levels and protected against the decrease in p-Akt expression. The results demonstrate PAP-induced toxicity and suggest that it is due at least in part to apoptosis and involves GSH depletion and p-Akt inactivation.     

Discovery of a substituted 8-arylquinoline series of PDE4 inhibitors: structure-activity relationship, optimization, and identification of a highly potent, well tolerated, PDE4 inhibitor

The discovery and SAR of a new series of substituted 8-arylquinoline PDE4 inhibitors are herein described. This work has led to the identification of several compounds with excellent in vitro and in vivo profiles, including a good therapeutic window of emesis to efficacy in several animal models. Typical optimized compounds from this series are potent inhibitors of PDE4 (IC(50)<1nM) and also of LPS-induced TNF-alpha release in human whole blood (IC(50)<0.5microM). The same compounds are potent inhibitors of ovalbumin-induced bronchoconstriction in conscious guinea pigs (EC(50)<0.1mg/kg ip) but require a dose of about 10mg/kg po in the squirrel monkey to produce an emetic response. From this series of compounds, 23a (L-454,560) was identified as an optimized compound.     

Some 3-(4-Aminophenyl)pyrrolidine-2,5-diones as All- trans -retinoic Acid Metabolising Enzyme Inhibitors (RAMBAs)

A series of (±) -3-(4-aminophenyl) pyrrolidin-2,5-diones substituted in the 1-, 3- or 1,3- position with an aryl or long chain alkyl function are weak inhibitors of the metabolism of all-trans retinoic acid (RA) by rat liver microsomes (68-75% inhibition) compared with ketoconazole (85%). Further studies with the 1-cyclohexyl analogue (1) (IC 50 = 98.8 μM, ketoconazole, 22.15 μM) showed that it was not stereoselective in its inhibition. (±) - (1) was not an inhibitor of pig brain microsomal enzyme (ketoconazole, IC 50 = 20.9 μM), had little effect on human liver microsomal enzyme (19.3%, ketoconazole, 81.6%) or human placental microsomal enzyme (9.8%, ketoconazole 73.9%) but was a weak inhibitor of human and rat skin homogenates (52.6% and IC 50 = 211.6 μM respectively; ketoconazole, 38.8% and 85.95 μM). In RA-induced cell cultures of human male genital fibroblasts and HaCat cells, (±) - (1) was a weak inhibitor (c. 53% at 200 μM) whereas ketoconazole showed high potency (c. 65% at 0.625 μM and 0.25 μM respectively). The nature of the induced target enzyme is discussed.     

Novel inhibitors of Mycobacterium tuberculosis dTDP-6-deoxy-L-lyxo-4-hexulose reductase (RmlD) identified by virtual screening

The complex and highly impermeable cell wall of Mycobacterium tuberculosis (Mtb) is largely responsible for the ability of the mycobacterium to resist the action of chemical therapeutics. An L-rhamnosyl residue, which occupies an important anchoring position in the Mtb cell wall, is an attractive target for novel anti-tuberculosis drugs. In this work, we report a virtual screening (VS) study targeting Mtb dTDP-deoxy-L-lyxo-4-hexulose reductase (RmlD), the last enzyme in the L-rhamnosyl synthesis pathway. Through two rounds of VS, we have identified four RmlD inhibitors with half inhibitory concentrations of 0.9-25 μM, and whole-cell minimum inhibitory concentrations of 20-200 μg/ml. Compared with our previous high throughput screening targeting another enzyme involved in L-rhamnosyl synthesis, virtual screening produced higher hit rates, supporting the use of computational methods in future anti-tuberculosis drug discovery efforts.