Structure-activity relationship studies of benzothiazole-phenyl analogs as multi-target ligands to alleviate pain without affecting normal behavior.

Soluble epoxide hydrolase (sEH) and fatty acid amide hydrolase (FAAH) are potential targets for several diseases. Previous studies have reported that concomitant selective inhibition of sEH and FAAH produced antinociception effects in an animal model of pain. However, the co-administration of a selective sEH inhibitor and a selective FAAH inhibitor might produce serious side effects due to drug-drug interactions that could complicate drug development in the long term. Thus, discovering dual sEH/FAAH inhibitors, single small molecules that can simultaneously inhibit both sEH and FAAH, would be a significant accomplishment in the medicinal chemistry field. Herein, we report the synthesis and biological evaluation of benzothiazole-phenyl-based analogs as potential dual sEH/FAAH inhibitors. This work represents a follow-up structure-activity relationship (SAR) and metabolic-stability studies of our best dual sEH/FAAH inhibitor identified previously, as well as in vivo evaluation of its effects on voluntary locomotor behavior in rats. Our SAR study indicates that trifluoromethyl groups on the aromatic rings are well tolerated by the targeted enzymes when placed at the ortho and para positions; however, they, surprisingly, did not improve metabolic stability in liver microsomes. Our behavioral studies indicate that doses of dual sEH/FAAH inhibitors that alleviate pain do not depress voluntary behavior in naïve rats, which is a common side effect of currently available analgesic drugs (e.g., opioids). Thus, dual sEH/FAAH inhibitors may be a safe and effective approach to treat pain.

Synthesis, kinetic evaluation and molecular docking studies of donepezil-based acetylcholinesterase inhibitors.

In an effort to develop new therapeutic agents to treat Alzheimer's disease, a series of donepezil-based analogs were designed, synthesized using an environmentally friendly route, and biologically evaluated for their inhibitory activity against electric eel acetylcholinesterase (AChE) enzyme. In vitro studies revealed that the phenyl moiety of donepezil can be successfully replaced with a pyridine ring leading to equally potent inhibitors of electric eel AChE. Further kinetic evaluations of the most potent inhibitor showed a dual-binding (mixed inhibition) mode, similar to donepezil. Molecular modeling studies suggest that several additional residues could be involved in the binding of this inhibitor in the human AChE enzyme active site compared to donepezil.

Further exploration of the structure-activity relationship of dual soluble epoxide hydrolase/fatty acid amide hydrolase inhibitors.

Fatty acid amide hydrolase (FAAH) is a membrane protein that hydrolyzes endocannabinoids, and its inhibition produces analgesic and anti-inflammatory effects. The soluble epoxide hydrolase (sEH) hydrolyzes epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatetraenoic acids. EETs have anti-inflammatory and inflammation resolving properties, thus inhibition of sEH consequently reduces inflammation. Concurrent inhibition of both enzymes may represent a novel approach in the treatment of chronic pain. Drugs with multiple targets can provide a superior therapeutic effect and a decrease in side effects compared to ligands with single targets. Previously, microwave-assisted methodologies were employed to synthesize libraries of benzothiazole analogs from which high affinity dual inhibitors (e.g. 3, sEH IC50 = 9.6 nM; FAAH IC50 = 7 nM) were identified. Here, our structure-activity relationship studies revealed that the 4-phenylthiazole moiety is well tolerated by both enzymes, producing excellent inhibition potencies in the low nanomolar range (e.g. 6o, sEH IC50 = 2.5 nM; FAAH IC50 = 9.8 nM). Docking experiments show that the new class of dual inhibitors bind within the catalytic sites of both enzymes. Prediction of several pharmacokinetic/pharmacodynamic properties suggest that these new dual inhibitors are good candidates for further in vivo evaluation. Finally, dual inhibitor 3 was tested in the Formalin Test, a rat model of acute inflammatory pain. The data indicate that 3 produces antinociception against the inflammatory phase of the Formalin Test in vivo and is metabolically stable following intraperitoneal administration in male rats. Further, antinociception produced by 3 is comparable to that of ketoprofen, a traditional nonsteroidal anti-inflammatory drug. The results presented here will help toward the long-term goal of developing novel non-opioid therapeutics for pain management.

Development of multitarget inhibitors for the treatment of pain: Design, synthesis, biological evaluation and molecular modeling studies.

Multitarget-directed ligands are a promising class of drugs for discovering innovative new therapies for difficult to treat diseases. In this study, we designed dual inhibitors targeting the human fatty acid amide hydrolase (FAAH) enzyme and human soluble epoxide hydrolase (sEH) enzyme. Targeting both of these enzymes concurrently with single target inhibitors synergistically reduces inflammatory and neuropathic pain; thus, dual FAAH/sEH inhibitors are likely to be powerful analgesics. Here, we identified the piperidinyl-sulfonamide moiety as a common pharmacophore and optimized several inhibitors to have excellent inhibition profiles on both targeted enzymes simultaneously. In addition, several inhibitors show good predicted pharmacokinetic properties. These results suggest that this series of inhibitors has the potential to be further developed as new lead candidates and therapeutics in pain management.

Quinolinyl-based multitarget-directed ligands with soluble epoxide hydrolase and fatty acid amide hydrolase inhibitory activities: Synthetic studies and pharmacological evaluations.

Simultaneous inhibition of soluble epoxide hydrolase (sEH) and fatty acid amide hydrolase (FAAH) with a single small molecule represents a novel therapeutic approach in treating inflammatory pain, since both targets are involved in pain and inflammation processes. In this study using multi-target directed ligands methodology we designed and synthesized 7 quinolinyl-based dual sEH/FAAH inhibitors, using an optimized microwave-assisted Suzuki-Miyaura coupling reaction and tested their potency in human FAAH and human, rat, and mouse sEH inhibition assays. The structure-activity relationship study showed that quinolinyl moiety is well tolerated in the active sites of both enzymes, yielding several very potent dual sEH/FAAH inhibitors with the IC50 values in the low nanomolar range. The most potent dual inhibitor 4d was further evaluated in stability assay in human and rat plasma where it performed better than the standard Warfarin while in vivo study revealed that 1 mg/kg 4d can inhibit acute inflammatory pain in male rats to a similar degree as the traditional nonsteroidal anti-inflammatory drug ketoprofen (30 mg/kg) after intraperitoneal injection. ADMET prediction studies for this dual inhibitor show favorable pharmacokinetic properties which will guide the future in vivo evaluations.

Deliquescence phase transition measurements by quartz crystal microbalance frequency shifts.

Measurements of the hygroscopic properties of aerosols are needed to better understand the role of aerosols as cloud condensation nuclei. Several techniques have been used to measure deliquescence (solid to liquid) phase transitions in particular. In this study, we have measured the deliquescence relative humidity (DRH) of organic and inorganic salts, organic acids (glutaric and succinic acid), and mixtures of organic acids with ammonium sulfate using a quartz crystal microbalance (QCM). The QCM allows for measurement of the deliquescence phase transition due to inherent measurement differences between solids and liquids in the oscillation frequency of a quartz crystal. The relative humidity dependent frequency measurements can be used to identify compounds that adsorb monolayer amounts of water or form hydrates prior to deliquescence (e.g., lithium chloride, potassium and sodium acetate). Although the amount of water uptake by a deliquescing material cannot be quantified with this technique, deliquescence measurements of mixtures of hygroscopic and nonhygroscopic components (e.g., ammonium sulfate and succinic acid (DRH > 95%)) show that the mass fraction of the deliquescing portion of the sample can be quantitatively determined from the relative change in oscillation frequency at deliquescence. The results demonstrate the use of this technique as an alternative method for phase transition measurements and as a direct measurement of the mass fraction of a sample that undergoes deliquescence. Further, deliquescence measurements by the QCM may provide improved understanding of discrepancies in atmospheric particle mass measurements between filter samples and the tapered element oscillating microbalance given the similar measurement principle employed by the QCM.

A spectroscopic study of atmospherically relevant concentrated aqueous nitrate solutions.

Concentrated aqueous nitrate aerosols are present in the Earth's atmosphere as a result of heterogeneous reactions of sea salt and mineral dust aerosol with nitrogen oxides (e.g., NO2, NO3, HNO3 and N2O5). Because the water content of these aerosols depends on relative humidity (RH), the composition and nitrate ion concentration will also depend on RH. Unlike the original aerosols, aqueous nitrate aerosols are photochemically active at solar wavelengths. To gain a better understanding of the nitrate ion chromophore in concentrated aqueous nitrate aerosols, we have measured the ATR-FTIR and UV/vis spectra of concentrated nitrate solutions over a large concentration range. Both ATR-FTIR and UV/vis spectroscopy show changes in the nitrate ion spectra with increasing concentration. Ab initio calculations are used to aid in the assignment and interpretation of these spectra. From these data, we predict that the photoreactivity of aqueous nitrate aerosols will strongly depend on relative humidity as the molecular and electronic structure of the nitrate ion becomes increasingly perturbed from that of the isolated ion in highly concentrated atmospherically relevant solutions.

Physicochemical properties of nitrate aerosols: implications for the atmosphere.

As aerosols, such as sea salt and mineral dust, are transported through the atmosphere they undergo heterogeneous reactions with nitrogen oxides to form nitrate salts. The nitrate salt can have quite different physicochemical properties than the original aerosol, resulting in an aerosol that will markedly differ in its climate impact, heterogeneous chemistry, and photoactivity. In this Feature Article, we will review some aspects of the importance of aqueous nitrate aerosols as well as describe a new multi-analysis aerosol reactor system (MAARS) that is used to measure the physicochemical properties of these atmospherically relevant aerosols. Here we show measurements of the hygroscopic properties, cloud condensation nuclei activity, and FTIR extinction of nitrate salt aerosol. In particular, we have measured the hygroscopic growth of 100 nm size-selected nitrate particles including NaNO3, Ca(NO3)2, Mg(NO3)2, and a 1:1 mixture of Ca(NO3)2 and Mg(NO3)2 as a function of relative humidity (RH) at 298 K. Using Köhler theory, we have quantified the water content of these particles with increasing RH. FTIR extinction measurements of the full size distribution of each of the nitrate aerosols are analyzed to yield information about the local solvation environment of the nitrate ions and the long-wavelength light scattering of the particles at different RH. Furthermore, we have measured and compared the cloud condensation nuclei (CCN) activity of CaCO3, a large component of mineral dust aerosol, and Ca(NO3)2, a product of atmospherically aged CaCO3 through reaction with nitrogen oxides, at supersaturations from 0.1% to 0.9%. These quantitative physicochemical data are needed if we are to better understand the chemistry as well as the climate effects of atmospheric aerosols as they are entrained, transported, reacted, and aged in the atmosphere. Our studies here focus on aqueous nitrate salts, the products of the reaction of nitrogen oxides with sea salt and mineral dust aerosol.