Rational design of 2-benzylsulfinyl-benzoxazoles as potent and selective indoleamine 2,3-dioxygenase 1 inhibitors to combat inflammation.

Mimicking the transition state of tryptophan (Trp) and O2 in the enzymatic reaction is an effective approach to design indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors. In this study, we firstly assembled a small library of 2-substituted benzo-fused five membered heterocycles and found 2-sulfinyl-benzoxazoles with interesting IDO1 inhibitory activities. Next the inhibitory activity toward IDO1 was gradually improved. Several benzoxazoles showed potent IDO1 inhibitory activity with IC50 of 82-91 nM, and exhibited selectivity between IDO1 and tryptophan 2,3-dioxygenase (TDO2). Enzyme binding studies showed that benzoxazoles are reversible type II IDO1 inhibitors, and modeling studies suggested that the oxygen atom of the sulfoxide in benzoxazoles interacts with the iron atom of the heme group, which mimics the transition state of Fe-O-O-Trp complex. Especially, 10b can effectively inhibit the NO production in lipopolysaccharides (LPS) stimulated RAW264.7 cells, and it also shows good anti-inflammation effect on mice acute inflammation model of croton oil induced ear edema.

Formation and Disproportionation of Xanthenols to Xanthenes and Xanthones and Their Use in Synthesis.

A facile and versatile strategy employing TiCl4-mediated cyclization followed by a Cannizzaro reaction has been developed for the synthesis of various xanthene derivatives. The reaction proceeded smoothly to afford both xanthenes/xanthones or their sulfur derivatives and tolerated a wide range of electronically diverse substrates. Using this methodology, pranoprofen was synthesized in three steps in 59% overall yield from commercially available starting materials.

S1P1-selective agonist prodrug IMMH002 is phosphorylated in rats to form an S-configured enantiomer: Synthesis, verification, and biological activity of the in vivo active metabolite.

IMMH002 (1), a prodrug for a sphingosine-1-phosphate receptor 1 (S1P1) agonist, is converted to the monophosphate ester, which has an immunomodulatory effect. Starting from prochiral amino alcohol 1, racemic and enantiomerically pure phosphates of 1 were synthesized. Pure enantiomers were obtained after the chiral resolution of the key intermediate by chiral high-performance liquid chromatography and the absolute configuration was determined by circular dichroism. In the in vitro homogeneous time-resolved fluorescence-IP1 functional assay, the (S)-enantiomer showed much higher S1P1 activity and selectivity than the (R)-enantiomer. In the pharmacokinetic study, the ex vivo o-phthaldialdehyde derivatization protocol showed that the phosphate of 1 in rats was the S-configured enantiomer with >99% enantiomeric excess.

Design and synthesis of analogues of the sphingosine-1-phosphate receptor 1 agonist IMMH001 with improved phosphorylation rate in human blood.

IMMH001, which is a prodrug for sphingosine-1-phosphate receptor 1 (S1P1) agonist, is converted to the active form, its monophosphate ester (S)-IMMH001-P, by sphingosine kinase 1 (SphK1) and sphingosine kinase 2 (SphK2) in vivo. In this study, we designed head-piece-modified analogues of IMMH001 based on structural information and prepared them with an efficient modular synthetic strategy. The analogues showed higher phosphorylation rates in human blood than the parent compound. These results indicated that the pro-R hydroxymethyl in the head-piece-moiety of IMMH001 prevents the pro-S hydroxymethyl from being phosphorylated by the kinase and ATP. The analogues may have better therapeutic potential.

In Vitro and In Vivo Activities of the Riminophenazine TBI-166 against Mycobacterium tuberculosis.

The riminophenazine agent clofazimine (CFZ) is repurposed as an important component of the new short-course multidrug-resistant tuberculosis regimen and significantly shortens first-line regimen for drug-susceptible tuberculosis in mice. However, CFZ use is hampered by its unwelcome skin discoloration in patients. A new riminophenazine analog, TBI-166, was selected as a potential next-generation antituberculosis riminophenazine following an extensive medicinal chemistry effort. Here, we evaluated the activity of TBI-166 against Mycobacterium tuberculosis and its potential to accumulate and discolor skin. The in vitro activity of TBI-166 against both drug-sensitive and drug-resistant M.tuberculosis is more potent than that of CFZ. Spontaneous mutants resistant to TBI-166 were found at a frequency of 2.3 × 10-7 in wild strains of M. tuberculosis TBI-166 demonstrates activity at least equivalent to that of CFZ against intracellular M. tuberculosis and in low-dose aerosol infection models of acute and chronic murine tuberculosis. Most importantly, TBI-166 causes less skin discoloration than does CFZ despite its higher tissue accumulation. The efficacy of TBI-166, along with its decreased skin pigmentation, warrants further study and potential clinical use.

One-Pot Synthesis of O-Heterocycles or Aryl Ketones Using an InCl3/Et3SiH System by Switching the Solvent.

An efficient one-pot synthesis of O-heterocycles or aryl ketones has been achieved using Et3SiH in the presence of InCl3 via a sequential ionic hydrogenation reaction by switching the solvent. This methodology can be used to construct C-O bonds and to prepare conjugate reduction products, including chromans, tetrahydrofurans, tetrahydropyrans, dihydroisobenzofurans, dihydrochalcones, and 1,4-diones in a facile manner. In addition, a novel plausible mechanism involving a conjugate reduction and a tandem reductive cyclization was verified by experimental investigations.

A Computational Approach to the Study of the Binding Mode of S1P1R Agonists Based on the Active-Like Receptor Model.

Sphingosine-1-phosphate receptor 1 (S1P1R), a member of the G protein-coupled receptor (GPCR) family, is an attractive protein target for the treatment of autoimmune diseases, and a diverse array of S1P1R agonists have been developed. Rational drug design based on S1P1R remains challenging due to the limited information available on the binding mode between S1P1R and its agonists. In this work, the active-like state of S1P1R was modeled via Gaussian accelerated molecular dynamics (GaMD) based on its inactive form, which was further validated by docking studies with two representative S1P1R agonists. Moreover, with the usage of the induced active-like state, the binding mode between S1P1R and its agonists was studied through molecular dynamics simulations and MM-GBSA calculations. The results of those studies indicated that four groups of binding site residues were the major contributors to the ligand and receptor interactions. In addition, this model was verified by five chemically similar compounds synthesized in-house and 1145 known S1P1R agonists collected from the BindingDB database. The elucidation of the key binding characteristics will further complete the cognition of S1P1R, which can guide the rational design of novel S1P1R agonists.

Identification and Structure-Activity Relationship (SAR) of potent and selective oxadiazole-based agonists of sphingosine-1-phosphate receptor (S1P1).

Agonism of S1P1 receptor has been proven to be responsible for peripheral blood lymphopenia and elicts the identification of various S1P1 modulators. In this paper we described a series of oxadiazole-based S1P1 direct-acting agonists disubstituted on terminal benzene ring, with high potency for S1P1 receptor and favorable selectivity against S1P3 receptor. In addition, two representative agents named 16-3b and 16-3g demonstrated impressive efficacy in lymphocyte reduction along with reduced effect on heart rate when orally administered. Furthermore, these compounds have been shown to possess desired pharmacokinetic (PK) and physicochemical profiles. The binding mode between 16-3b and the activated S1P1 model was also studied.

Diversity-Oriented Synthesis of Heterocycles: Al(OTf)3-Promoted Cascade Cyclization and Ionic Hydrogenation.

An efficient and facile method has been developed for the diversity-oriented synthesis of heterocycles. Hexahydrophenoxazines, tetrahydroquinolines, indolines, hexahydrocarbazoles, and lactones were conducted via Al(OTf)3-promoted cascade cyclization and ionic hydrogenation. Furthermore, this protocol was utilized to smoothly prepare piracetam and its key intermediate as well.

A Suzuki-Miyaura method for labelling proliferating cells containing incorporated BrdU.

The 5-bromo-2'-deoxyuridine (BrdU) incorporation cell proliferation assay is the most commonly used method for assessing DNA replication. The current detection of BrdU in cells relies on antibody immunostaining, but has various limitations including low antibody specificity and poor tissue penetration. In this study, we utilised a Suzuki-Miyaura reaction to develop a chemical method to label cellular BrdU with fluorescent boronic acid probes. The coupling conditions were optimised for complex cellular environments, and the key observation was the need to use oxygen scavengers and zerovalent palladium to prevent side reactions and increase the rate of coupling. The reliability and specificity of the BrdU Suzuki-Miyaura labelling method were verified under various biological conditions. The applicability of the BrdU Suzuki-Miyaura labelling methodology was also investigated, and we show that labelling cellular BrdU is highly sensitive and reliable, which is comparable to the ideal performance of BrdU immunostaining. Moreover, the Suzuki-Miyaura reaction protocol provides high BrdU recognition specificity. Taken together, the BrdU Suzuki-Miyaura labelling protocol provides an attractive alternative to the more traditional cell proliferation assay.

17ß-estradiol (E2) in membranes: Orientation and dynamic properties.

Non-genomic membrane effects of estrogens are of great interest because of the diverse biological activities they may elicit. To further our understanding of the molecular features of the interaction between estrogenic hormones and membrane bilayers, we have determined the preferred orientation, location, and dynamic properties of 17ß-estradiol (E2) in two different phospholipid membrane environments using (2)H-NMR and 2D (1)H-(13)C HSQC in conjunction with molecular dynamics simulations. Unequivocal spectral assignments to specific (2)H labels were made possible by synthesizing six selectively deuterated E2 molecules. The data allow us to conclude that the E2 molecule adopts a nearly "horizontal" orientation in the membrane bilayer with its long axis essentially perpendicular to the lipid acyl-chains. All four rings of the E2 molecule are located near the membrane interface, allowing both the E2 3-OH and the 17ß-OH groups to engage in hydrogen bonding and electrostatic interactions with polar phospholipid groups. The findings augment our knowledge of the molecular interactions between E2 and membrane bilayer and highlight the asymmetric nature of the dynamic motions of the rigid E2 molecule in a membrane environment.

A Morphological identification cell cytotoxicity assay using cytoplasm-localized fluorescent probe (CLFP) to distinguish living and dead cells.

Cell cytotoxicity assays include cell activity assays and morphological identification assays. Currently, all frequently used cytotoxicity assays belong to cell activity assays but suffer from detection limitations. Morphological identification of cell death remains as the gold standard, although the method is difficult to scale up. At present there is no generally accepted morphological identification based cell cytotoxicity assay. In this study, we applied previous developed cell cytoplasm-localized fluorescent probe (CLFP) to display cell morphologies. Under fluorescence microscopy, the fluorescence morphology and intensity of living cells are distinct from dead cells. Based on these characters we extracted the images of living cells from series of samples via computational analysis. Thus, a novel cell morphological identification cytotoxicity assay (CLFP assay) is developed. The performance of the CLFP assay was similar to cell activity assay (MTT assay), but the accuracy of the CLFP assay was superior when measuring the cytotoxicity of active compounds.

Use of the Putamen as a Surrogate Anatomical Marker for the Internal Segment of the Globus Pallidus in Deep Brain Stimulation Surgery.

OBJECTIVE: The success of deep brain stimulation (DBS) of the internal segment of the globus pallidus (GPi) depends on accurately placing the electrode into the GPi motor territory. Direct targeting can be difficult as GPi laminar borders are not always clearly identifiable on MRI. Here, we report a method for using the putamen (PUT) as a surrogate anatomical marker to target the GPi. METHODS: We developed a PUT-based GPi targeting using the FGATIR (fast gray matter acquisition T1-weighted inversion recovery) MRI sequence and compared it with consensus coordinate-based indirect targeting. Stereotactic target coordinates were obtained and analyzed. RESULTS: In our GPi DBS case sequences, GPi borders were unresolvable on T2-weighted MRI. However, in all cases, application of the PUT-based method resulted in consistently localized GPi targets, which were confirmed by merging the T2-weighted MRI with the FGATIR MRI. Significant differences were noted in the target coordinates between the PUT-based method and indirect targeting based on both the distance from the anterior commissure and the distance from the intercommissural plane. The mean differences for mediolateral distance and anteroposterior distance were 1.4 and 1.42 mm, respectively. In addition, the PUT-based method estimated a target that was closer to the nearest implanted electrode. CONCLUSION: Our PUT-based method allows consistent and precise patient-specific GPi targeting. Further study is planned to correlate PUT-based GPi targeting with microelectrode recording, location of active contact of the DBS electrode and clinical outcome.

Optic Radiation Tractography and Visual Field Deficits in Laser Interstitial Thermal Therapy for Amygdalohippocampectomy in Patients with Mesial Temporal Lobe Epilepsy.

BACKGROUND/AIMS: Laser interstitial thermal therapy (LITT) has become an alternative to open-resective surgery for refractory mesial temporal lobe epilepsy (MTLE). Occurrence of visual field defects (VFDs) following open surgery for MTLE is reported at 52-100%. We examined the rate of VFDs following LITT for amygdalohippocampectomy (AHE) and correlated the occurrence of VFDs with damage to the optic radiations, assessed by diffusion tensor tractography (DTI). METHODS: We performed a retrospective analysis of 5 patients who underwent LITT-AHE for medically refractory MTLE. We examined the association between VFDs and optic radiation damage by correlating postprocedural visual field testing with qualitative assessment of optic radiation fiber tracts. RESULTS: Postoperative assessments showed that 4 patients had normal visual field testing, and 1 had a right superior quadrantanopsia (20%). We performed 3-dimensional reconstruction of the optic radiation, laser probe trajectory, and ablation volume. Damage to Meyer's loop was determined consistent with the VFD. CONCLUSIONS: Short-term follow-up in our series suggests that laser ablation AHE may be associated with a lower rate of VFD than has been reported for open AHE. Our results suggest that incorporating optic radiation mapping through DTI may preoperatively help to minimize the risk of VFD following laser ablation AHE.

N'-[(3-[benzyloxy]benzylidene]-3,4,5-trihydroxybenzohydrazide (1) protects mice against colitis induced by dextran sulfate sodium through inhibiting NFκB/IL-6/STAT3 pathway.

IBD has attracted much attention for its negative influence on the quality of life and increased risk of colorectal cancer. In this study, we discovered the inhibitory activity of the polyphenol compound (1) in DSS induced colitis in mice by targeting NFκB/IL-6/STAT3 pathway. This compound effectively protected against body weight loss and colon length shortening induced by DSS. Additionally, 1 inhibited DSS induced damage in colon, notably decreasing the severity of inflammation, the extent of inflammation, crypt damage and percent involvement. The production of inflammatory mediators of IL-6 and COX-2 was also significantly attenuated when treated with 1. It may be attributed to inhibiting NFκB signaling. Moreover, this polyphenol suppressed p-STAT3 production as well as its downstream proteins response for apoptosis, such as Bcl-2 and Bax. In summary, the study not only afforded our understanding involved in colitis, but also provided the possible therapy for human with IBD.

A high-resolution method to assess cell multinucleation with cytoplasm-localized fluorescent probes.

Cell multinucleation is closely related to chromosomal instability. We report a simple, convenient method to assess cell multinucleation with cytoplasm-localized fluorescent probes (CLFP) which is superior to conventional nuclear staining methods. The CLFP method provides high-resolution images that enable the accurate calculation of the number of nuclear fragments.

Development of hydroxy-based sphingosine kinase inhibitors and anti-inflammation in dextran sodium sulfate induced colitis in mice.

Sphingosine kinase (SphK)-catalyzed production of sphingosine-1-phosphate (S1P) regulates cell growth, survival and proliferation as well as inflammatory status in animals. In recent study we reported the N'-(3-(benzyloxy)benzylidene)-3,4,5-trihydroxybenzohydrazide scaffold as a potent SphK inhibitor. As a continuation of these efforts, 51 derivatives were synthesized and evaluated by SphK1/2 inhibitory activities for structure-activity relationship (SAR) study. Among them, 33 was identified as the most potent SphK inhibitor. Potency of 33 was also observed to efficiently decrease SphK1/2 expression in human colorectal cancer cells (HCT116) and significantly inhibit dextran sodium sulfate (DSS)-induced colitis as well as the decreased expression of interleukin (IL)-6 and cyclooxygenase-2 (COX-2) in mouse models. Collectively, 33 was validated as an effective SphK inhibitor, which can be served as anti-inflammatory agent to probably treat inflammatory bowel diseases in human.

Synthesis, identification, and biological activity of metabolites of two novel selective S1P1 agonists.

SYL927 and SYL930 are selective S1P1 agonists under preclinical development. However, during their pharmacokinetic studies we detected two metabolites in rat blood that were tentatively identified as monohydroxylated metabolites of SYL927 and SYL930 based on LC-MS/MS data. In this study, we designed and synthesized possible monohydroxylated products 6a-e and used them as references to confirm the structures of the two metabolites detected by LC-MS/MS. We also evaluated the in vitro and in vivo biological activities of these two metabolites.

Interventional magnetic resonance imaging-guided cell transplantation into the brain with radially branched deployment.

Intracerebral cell transplantation is being pursued as a treatment for many neurological diseases, and effective cell delivery is critical for clinical success. To facilitate intracerebral cell transplantation at the scale and complexity of the human brain, we developed a platform technology that enables radially branched deployment (RBD) of cells to multiple target locations at variable radial distances and depths along the initial brain penetration tract with real-time interventional magnetic resonance image (iMRI) guidance. iMRI-guided RBD functioned as an "add-on" to standard neurosurgical and imaging workflows, and procedures were performed in a commonly available clinical MRI scanner. Multiple deposits of super paramagnetic iron oxide beads were safely delivered to the striatum of live swine, and distribution to the entire putamen was achieved via a single cannula insertion in human cadaveric heads. Human embryonic stem cell-derived dopaminergic neurons were biocompatible with the iMRI-guided RBD platform and successfully delivered with iMRI guidance into the swine striatum. Thus, iMRI-guided RBD overcomes some of the technical limitations inherent to the use of straight cannulas and standard stereotactic targeting. This platform technology could have a major impact on the clinical translation of a wide range of cell therapeutics for the treatment of many neurological diseases.

Ti(IV)-catalyzed cascade synthesis of tetrahydrofuro[3,2-d]oxazole from arene-1,4-diones.

A tetrahydrofuro[3,2-d]oxazole scaffold was synthesized efficiently and stereoselectively. The tandem ionic hydrogenation, ketalization, and intramolecular cyclization of arene-1,4-diones with a combination of TiCl4/Et3SiH give facile access to tetrahydrofuro[3,2-d]oxazole derivatives in good yields at room temperature.