Preparation of 1,5-Dihydropyrazolo[3',4':5,6]pyrano[3,4- b]pyridines via a Microwave-Assisted, Palladium-Catalyzed Regioselective C-H Heteroarylation of Electron-Rich Pyrazoles.

Here we report the first synthesis of a family of novel heterocyclic compounds based on a 5-dihydropyrazolo[3',4':5,6]pyrano[3,4- b]pyridine core. In the course of our drug discovery programs, we had need to access the previously unknown 5-dihydropyrazolo[3',4':5,6]pyrano[3,4- b]pyridine core. Initial attempts required long reaction times, which led to degradation and side products. Reaction optimization identified a Pd-catalyzed, microwave-assisted C-H heteroarylation protocol for the rapid, general, and high yielding synthesis of this tricyclic core (as well as related analogs) suitable to drive optimization efforts.

Total Synthesis of Hinduchelins A-D, Stereochemical Revision of Hinduchelin A, and Biological Evaluation of Natural and Unnatural Analogues.

Here, we report the first total synthesis of hinduchelins A-D, a family of nontoxic catechol derivatives from Streptoalloteichus hindustanus, possessing a druglike chemotype and modest iron-chelating ability. A concise synthesis was developed employing methyl 5-methyloxazole-4-carboxylate as a single starting material to provide hinduchelins A-D (and unnatural analogues) in only four steps and 5-15% overall yields; moreover, the stereochemistry of hinduchelin A was reassigned from ( S) to ( R). Biological evaluation confirmed that natural and unnatural hinduchelins are weak iron chelators (siderophores).

Structure-activity relationship studies of the lipophilic tail region of sphingosine kinase 2 inhibitors.

Sphingosine-1-phosphate (S1P) is a ubiquitous, endogenous small molecule that is synthesized by two isoforms of sphingosine kinase (SphK1 and 2). Intervention of the S1P signaling pathway has attracted significant attention because alteration of S1P levels is linked to several disease states including cancer, fibrosis, and sickle cell disease. While intense investigations have focused on developing SphK1 inhibitors, only a limited number of SphK2-selective agents have been reported. Herein, we report our investigations on the structure-activity relationship studies of the lipophilic tail region of SLR080811, a SphK2-selective inhibitor. Our studies demonstrate that the internal phenyl ring is a key structural feature that is essential in the SLR080811 scaffold. Further, we show the dependence of SphK2 activity and selectivity on alkyl tail length, suggesting a larger lipid binding pocket in SphK2 compared to SphK1.

Ratiometric fluorescence detection of mercury ions in water by conjugated polymer nanoparticles.

We present dye-doped polymer nanoparticles that are able to detect mercury in aqueous solution at parts per billion levels via fluorescence resonance energy transfer (FRET). The nanoparticles are prepared by reprecipitation of highly fluorescent conjugated polymers in water and are stable in aqueous suspension. They are doped with rhodamine spirolactam dyes that are nonfluorescent until they encounter mercury ions, which promote an irreversible reaction that converts the dyes to fluorescent rhodamines. The rhodamine dyes act as FRET acceptors for the fluorescent nanoparticles, and the ratio of nanoparticle-to-rhodamine fluorescence intensities functions as a ratiometric fluorescence chemodosimeter for mercury. The light harvesting capability of the conjugated polymer nanoparticles enhances the fluorescence intensity of the rhodamine dyes by a factor of 10, enabling sensitive detection of mercury ions in water at levels as low as 0.7 parts per billion.

Mitochondrial uncoupler SHC517 reverses obesity in mice without affecting food intake.

AIMS: Mitochondrial uncouplers decrease caloric efficiency and have potential therapeutic benefits for the treatment of obesity and related metabolic disorders. Herein we investigate the metabolic and physiologic effects of a recently identified small molecule mitochondrial uncoupler named SHC517 in a mouse model of diet-induced obesity. METHODS: SHC517 was administered as an admixture in food. The effect of SHC517 on in vivo energy expenditure and respiratory quotient was determined by indirect calorimetry. A dose-finding obesity prevention study was performed by starting SHC517 treatment concomitant with high fat diet for a period of 12 days. An obesity reversal study was performed by feeding mice western diet for 4 weeks prior to SHC517 treatment for 7 weeks. Biochemical assays were used to determine changes in glucose, insulin, triglycerides, and cholesterol. SHC517 concentrations were determined by mass spectrometry. RESULTS: SHC517 increased lipid oxidation without affecting body temperature. SHC517 prevented diet-induced obesity when administered at 0.05% and 0.1% w/w in high fat diet and reversed established obesity when tested at the 0.05% dose. In the obesity reversal model, SHC517 restored adiposity to levels similar to chow-fed control mice without affecting food intake or lean body mass. SHC517 improved glucose tolerance and fasting glucose levels when administered in both the obesity prevention and obesity reversal modes. CONCLUSIONS: SHC517 is a mitochondrial uncoupler with potent anti-obesity and insulin sensitizing effects in mice. SHC517 reversed obesity without altering food intake or compromising lean mass, effects that are highly sought-after in anti-obesity therapeutics.

[1,2,5]Oxadiazolo[3,4-b]pyrazine-5,6-diamine Derivatives as Mitochondrial Uncouplers for the Potential Treatment of Nonalcoholic Steatohepatitis.

Small molecule mitochondrial uncouplers are emerging as a new class of molecules for the treatment of nonalcoholic steatohepatitis. We utilized BAM15, a potent protonophore that uncouples the mitochondria without depolarizing the plasma membrane, as a lead compound for structure-activity profiling. Using oxygen consumption rate as an assay for determining uncoupling activity, changes on the 5- and 6-position of the oxadiazolopyrazine core were introduced. Our studies suggest that unsymmetrical aniline derivatives bearing electron withdrawing groups are preferred compared to the symmetrical counterparts. In addition, alkyl substituents are not tolerated, and the N-H proton of the aniline ring is responsible for the protonophore activity. In particular, compound 10b had an EC50 value of 190 nM in L6 myoblast cells. In an in vivo model of NASH, 10b decreased liver triglyceride levels and showed improvement in fibrosis, inflammation, and plasma ALT. Taken together, our studies indicate that mitochondrial uncouplers have potential for the treatment of NASH.

Phenotypic screen for oxygen consumption rate identifies an anti-cancer naphthoquinone that induces mitochondrial oxidative stress.

A hallmark of cancer cells is their ability to reprogram nutrient metabolism. Thus, disruption to this phenotype is a potential avenue for anti-cancer therapy. Herein we used a phenotypic chemical library screening approach to identify molecules that disrupted nutrient metabolism (by increasing cellular oxygen consumption rate) and were toxic to cancer cells. From this screen we discovered a 1,4-Naphthoquinone (referred to as BH10) that is toxic to a broad range of cancer cell types. BH10 has improved cancer-selective toxicity compared to doxorubicin, 17-AAG, vitamin K3, and other known anti-cancer quinones. BH10 increases glucose oxidation via both mitochondrial and pentose phosphate pathways, decreases glycolysis, lowers GSH:GSSG and NAPDH/NAPD+ ratios exclusively in cancer cells, and induces necrosis. BH10 targets mitochondrial redox defence as evidenced by increased mitochondrial peroxiredoxin 3 oxidation and decreased mitochondrial aconitase activity, without changes in markers of cytosolic or nuclear damage. Over-expression of mitochondria-targeted catalase protects cells from BH10-mediated toxicity, while the thioredoxin reductase inhibitor auranofin synergistically enhances BH10-induced peroxiredoxin 3 oxidation and cytotoxicity. Overall, BH10 represents a 1,4-Naphthoquinone with an improved cancer-selective cytotoxicity profile via its mitochondrial specificity.

Evaluation of Synthetic Cytochrome P450-Mimetic Metalloporphyrins To Facilitate "Biomimetic" Biotransformation of a Series of mGlu5 Allosteric Ligands.

Allosteric ligands within a given chemotype can have the propensity to display a wide range of pharmacology, as well as unexpected changes in GPCR subtype selectivity, typically mediated by single-atom modifications to the ligand. Due to the unexpected nature of these "molecular switches", chemotypes with this property are typically abandoned in lead optimization. Recently, we have found that in vivo oxidative metabolism by CYP450s can also engender molecular switches within allosteric ligands, changing the mode of pharmacology and leading to unwanted toxicity. We required a higher-throughput approach to assess in vivo metabolic molecular switches, and we turned to a "synthetic liver", a 96 well kit of biomimetic catalysts (e.g., metalloporphyrins) to rapidly survey a broad panel of synthetic CYP450s' ability to oxidize/"metabolize" an mGlu5 PAM (VU0403602) known to undergo an in vivo CYP450-mediated molecular switch. While the synthetic CYP450s did generate a number of oxidative "metabolites" at known "hot spots", several of which proved to be pure mGlu5 PAMs comparable in potency to the parent, the known CYP450-mediated in vivo ago-PAM metabolite, namely, VU0453103, was not formed. Thus, this technology platform has potential to identify hot spots for oxidative metabolism and produce active metabolites of small-molecule ligands in a high-throughput, scalable manner.

Discovery of Novel Central Nervous System Penetrant Metabotropic Glutamate Receptor Subtype 2 (mGlu2) Negative Allosteric Modulators (NAMs) Based on Functionalized Pyrazolo[1,5- a]pyrimidine-5-carboxamide and Thieno[3,2- b]pyridine-5-carboxamide Cores.

A scaffold hopping exercise from a monocyclic mGlu2 NAM with poor rodent PK led to two novel heterobicyclic series of mGlu2 NAMs based on either a functionalized pyrazolo[1,5- a]pyrimidine-5-carboxamide core or a thieno[3,2- b]pyridine-5-carboxamide core. These novel analogues possess enhanced rodent PK, while also maintaining good mGlu2 NAM potency, selectivity (versus mGlu3 and the remaining six mGlu receptors), and high CNS penetration. Interestingly, SAR was divergent between the new 5,6-heterobicyclic systems.

Small Molecule Mitochondrial Uncouplers and Their Therapeutic Potential.

Small molecule mitochondrial uncouplers transport protons from the mitochondrial inner membrane space into the mitochondrial matrix independent of ATP synthase, uncoupling nutrient metabolism from ATP generation. The therapeutic potential of mitochondrial uncouplers has been investigated for the treatment of metabolic diseases such as obesity and type 2 diabetes (T2D), ischemia-reperfusion injury, and neurodegenerative diseases. This communication will review the small molecule mitochondrial uncouplers reported to date and explore their potential as therapeutics.

VU6007477, a Novel M1 PAM Based on a Pyrrolo[2,3-b]pyridine Carboxamide Core Devoid of Cholinergic Adverse Events.

Herein, we report the chemical optimization of a new series of M1 positive allosteric modulators (PAMs) based on a novel pyrrolo[2,3-b]pyridine core, developed via scaffold hopping and iterative parallel synthesis. The vast majority of analogs in this series proved to display robust cholinergic seizure activity. However, by removal of the secondary hydroxyl group, VU6007477 resulted with good rat M1 PAM potency (EC50 = 230 nM, 93% ACh max), minimal M1 agonist activity (agonist EC50 > 10 µM), good CNS penetration (rat brain/plasma K p = 0.28, K p,uu = 0.32; mouse K p = 0.16, K p,uu = 0.18), and no cholinergic adverse events (AEs, e.g., seizures). This work demonstrates that within a chemical series prone to robust M1 ago-PAM activity, SAR can result, which affords pure M1 PAMs, devoid of cholinergic toxicity/seizure liability.

Transforming Sphingosine Kinase 1 Inhibitors into Dual and Sphingosine Kinase 2 Selective Inhibitors: Design, Synthesis, and in Vivo Activity.

Sphingosine 1-phosphate (S1P) is a pleiotropic signaling molecule that interacts with its five G-protein coupled receptors (S1P1-5) to regulate cell growth and survival and has been implicated in a variety of diseases including cancer and sickle cell disease. As the key mediators in the synthesis of S1P, sphingosine kinase (SphK) isoforms 1 and 2 have attracted attention as viable targets for pharmaceutical inhibition. In this article, we describe the design, synthesis, and biological evaluation of aminothiazole-based guanidine inhibitors of SphK. Surprisingly, combining features of reported SphK1 inhibitors generated SphK1/2 dual inhibitor 20l (SLC4011540) (hSphK1 Ki = 120 nM, hSphK2 Ki = 90 nM) and SphK2 inhibitor 20dd (SLC4101431) (Ki = 90 nM, 100-fold SphK2 selectivity). These compounds effectively decrease S1P levels in vitro. In vivo administration of 20dd validated that inhibition of SphK2 increases blood S1P levels.

The impact of sphingosine kinase inhibitor-loaded nanoparticles on bioelectrical and biomechanical properties of cancer cells.

Cancer progression and physiological changes within the cells are accompanied by alterations in the biophysical properties. Therefore, the cell biophysical properties can serve as promising markers for cancer detection and physiological activities. To aid in the investigation of the biophysical markers of cells, a microfluidic chip has been developed which consists of a constriction channel and embedded microelectrodes. Single-cell impedance magnitudes at four frequencies and entry and travel times are measured simultaneously during their transit through the constriction channel. This microchip provides a high-throughput, label-free, automated assay to identify biophysical signatures of malignant cells and monitor the therapeutic efficacy of drugs. Here, we monitored the dynamic cellular biophysical properties in response to sphingosine kinase inhibitors (SphKIs), and compared the effectiveness of drug delivery using poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) loaded with SphKIs versus conventional delivery. Cells treated with SphKIs showed significantly higher impedance magnitudes at all four frequencies. The bioelectrical parameters extracted using a model also revealed that the highly aggressive breast cells treated with SphKIs shifted electrically towards that of a less malignant phenotype; SphKI-treated cells exhibited an increase in cell-channel interface resistance and a significant decrease in specific membrane capacitance. Furthermore, SphKI-treated cells became slightly more deformable as measured by a decrease in their channel entry and travel times. We observed no significant difference in the bioelectrical changes produced by SphKI delivered conventionally or with NPs. However, NPs-packaged delivery of SphKI decreased the cell deformability. In summary, this study showed that while the bioelectrical properties of the cells were dominantly affected by SphKIs, the biomechanical properties were mainly changed by the NPs.