Generation of novel radiolabeled opiates through site-selective iodination

Tritiated opioid radioligands have proven valuable in exploring opioid binding sites. However, tritium has many limitations. Its low specific activity and limited counting efficiency makes it difficult to examine low abundant, high affinity sites and its disposal is problematic due to the need to use organic scintillants and its relatively long half-life. To overcome these issues, we have synthesized both unlabeled and carrier-free radioiodinated iodobenzoyl derivatives of 6β-naltrexamine (¹²⁵I-BNtxA, 18), 6β-naloxamine (¹²⁵I-BNalA, 19) and 6β-oxymorphamine (¹²⁵I-BOxyA, 20) with specific activities of 2100 Ci/mmol. To optimize the utility of the radioligand, we designed a synthesis in which the radiolabel is incorporated in the last synthetic step, which required the selective iodination of the benzoyl moiety without incorporation into the phenolic A ring. Competition studies demonstrated high affinity of the unlabelled compounds for opioid receptors in transfected cell lines, as did the direct binding of the ¹²⁵I-ligands to the opioid receptors. The radioligand displayed very high sensitivity, enabling a marked reduction in tissue, as well as excellent signal/noise characteristics. These new ¹²⁵I-radioligands should prove valuable in future studies of opioid binding sites.     

Isolation,identification and characterization of a novel Rhodococcus sp. strain in biodegradation of tetrahydrofuran and its medium optimization using sequential statistics-based experimental designs

Statistics-based experimental designs were applied to optimize the culture conditions for tetrahydrofuran (THF) degradation by a newly isolated Rhodococcus sp. YYL that tolerates high THF concentrations. Single factor experiments were undertaken for determining the optimum range of each of four factors (initial pH and concentrations of K₂HPO₄ · 3H₂O, NH₄Cl and yeast extract) and these factors were subsequently optimized using the response surface methodology. The Plackett–Burman design was used to identify three trace elements (Mg²⁺, Zn²⁺and Fe²⁺) that significantly increased the THF degradation rate. The optimum conditions were found to be: 1.80 g/L NH₄Cl, 0.81 g/L K₂HPO₄ · 3H₂O, 0.06 g/L yeast extract, 0.40 g/L MgSO₄ · 7H₂O, 0.006 g/L ZnSO₄ · 7H₂O, 0.024 g/L FeSO₄ · 7H₂O, and an initial pH of 8.26. Under these optimized conditions, the maximum THF degradation rate increased to 137.60 mg THF h⁻¹ g dry weight in Rhodococcus sp. YYL, which was nearly five times of that by the previously described THF degrading Rhodococcus strain.     

Novel tetrahydrofuran derivatives from Trigonostemon howii with their potential anti-HIV-1 activities

A novel tetrahydrofuran derivative, trigonohowine (1), together with five known tetrahydrofuran derivatives (2–6), were isolated from the stems and leaves of Trigonostemon howii. The structure of 1 was elucidated by extensive spectroscopic methods and the known compounds were identified by comparisons with the data reported in literature. Among them, trigonohowine (1) represents the first example of a new type of tetrahydrofuran derivative, possessing an unprecedented carbon skeleton containing 23 carbon atoms on the carbon skeleton and the known compouds (2–6) are rare tetrahydrofuran derivatives in the plant kingdom with various carbon skeletons. All isolated compounds were evaluated for their anti-HIV-1 activities. Compounds 1–6 showed significant anti-HIV-1 activities with EC₅₀ ranged from 0.08 to 1.03 µM. These findings suggest that the discoveries of these tetrahydrofuran derivatives with significant anti-HIV-1 activities isolated from T. howii could be of great importance to the development of new anti-HIV agents.     

Discovery and evaluation of nNav1.5 sodium channel blockers with potent cell invasion inhibitory activity in breast cancer cells

Voltage-gated sodium channels (VGSC) are a well-established drug target for anti-epileptic, anti-arrhythmic and pain medications due to their presence and the important roles that they play in excitable cells. Recently, their presence has been recognized in non-excitable cells such as cancer cells and their overexpression has been shown to be associated with metastatic behavior in a variety of human cancers. The neonatal isoform of the VGSC subtype, Na_v1.5 (nNa_v1.5) is overexpressed in the highly aggressive human breast cancer cell line, MDA-MB-231. The activity of nNa_v1.5 is known to promote the breast cancer cell invasion in vitro and metastasis in vivo, and its expression in primary mammary tumors has been associated with metastasis and patient death. Metastasis development is responsible for the high mortality of breast cancer and currently there is no treatment available to specifically prevent or inhibit breast cancer metastasis. In the present study, a 3D-QSAR model is used to assist the development of low micromolar small molecule VGSC blockers. Using this model, we have designed, synthesized and evaluated five small molecule compounds as blockers of nNa_v1.5-dependent inward currents in whole-cell patch-clamp experiments in MDA-MB-231 cells. The most active compound identified from these studies blocked sodium currents by 34.9?±?6.6% at 1?μM. This compound also inhibited the invasion of MDA-MB-231 cells by 30.3?±?4.5% at 1?μM concentration without affecting the cell viability. The potent small molecule compounds presented here have the potential to be developed as drugs for breast cancer metastasis treatment.     

A new cleavable reagent for cross-linking and reversible immobilization of proteins.

We have prepared a new bifunctional reagent for the cross-linking and reversible immobilization of proteins through their amine groups. This compound, ethylene glycolyl bis(succinimidyl succinate), reacts rapidly with proteins, at pH 7 and at high dilution. The resulting protein cross-links are readily cleaved at pH 8.5 using hydroxylamine for 3–6 hr. at 37°C. Substantial enzymatic activity was observed with lactic dehydrogenase after such reversible cross-linking. Trypsin immobilized on agarose using this reagent retains full specific activity, is stable for weeks in the cold, and may be released with hydroxylamine at 25°C. This compound appears suitable for studies involving proteins with essential disulfide linkages.     

Isolation,structure elucidation and in-vitro evaluation of new trisubstituted Tetrahydrofuran Lignans from Rabdosia lophanthoides var. gerardiana as anti-inflammatory agents

Four new trisubstituted tetrahydrofuran lignans, named (−)-sesaminone-rutinoside (1), (+)-episesaminone-rutinoside (2) and rabdosiacosides B (3) and C (4) were isolated from the air-dried whole herbs of Rabdosia lophanthoides var. gerardiana together with two known compounds, (+)− 1-hydroxypinoresinol-1-β-d-glucoside (5) and rosmarinic acid (6). The structures of these new lignans were elucidated by a combination of mass spectrometry, 1D and 2D NMR experiments including distortionless enhancement by polarization transfer, ¹H–¹H correlation, heteronuclear single quantum coherence, heteronuclear multiple bond correlation, and nuclear Overhauser effect spectroscopies. Both the cell viability and their anti-inflammatory effects of the lignans were further studied. Among them, no significant cytotoxic activity was exhibited. Compound 4 dramatically suppressed LPS-induced nitric oxide release and reduced the release of the pro-inflammatory cytokines TNF-α and IL-6 in a dose-dependent manner. Overall, our findings indicate that compound 4 was noteworthy in reducing LPS-induced inflammatory responses in RAW264.7 macrophages.     

Recombinant expression, purification, and characterization of ThmD, the oxidoreductase component of tetrahydrofuran monooxygenase

Tetrahydrofuran monooxygenase (Thm) catalyzes the NADH-and oxygen-dependent hydroxylation of tetrahydrofuran to 2-hydroxytetrahydrofuran. Thm is composed of a hydroxylase enzyme, a regulatory subunit, and an oxidoreductase named ThmD. ThmD was expressed in Escherichia coli as a fusion to maltose-binding protein (MBP) and isolated to homogeneity after removal of the MBP. Purified ThmD contains covalently bound FAD, [2Fe-2S] center, and was shown to use ferricyanide, cytochrome c, 2,6-dichloroindophenol, and to a lesser extent, oxygen as surrogate electron acceptors. ThmD displays 160-fold preference for NADH over NADPH and functions as a monomer. The flavin-binding domain of ThmD (ThmD-FD) was purified and characterized. ThmD-FD displayed similar activity as the full-length ThmD and showed a unique flavin spectrum with a major peak at 463 nm and a small peak at 396 nm. Computational modeling and mutagenesis analyses suggest a novel three-dimensional fold or covalent flavin attachment in ThmD.     

High efficiency degradation of tetrahydrofuran (THF) using a membrane bioreactor: identification of THF-degrading cultures of<Emphasis Type="Italic"> Pseudonocardia</Emphasis> sp. strain M1 and<Emphasis Type="Italic"> Rhodococcus ruber</Emphasis> isolate M2

A mixed microbial culture capable of growing aerobically on tetrahydrofuran (THF) as a sole carbon and energy source was used as the inoculum in a 10 l working volume membrane bioreactor. Following start-up, the reactor was operated in batch mode for 24 h and then switched to continuous feed with 100% biomass recycle. On average, greater than 96% of THF fed to the reactor was removed during the 8-month study. THF loading rates ranged from 0.62 to 9.07 g l^–1 day^–1 with a hydraulic retention time of 24 h. THF concentrations as high as 800 mg/l were tolerated by the culture. Biomass production averaged 0.28 kg total suspended solids/kg chemical oxygen demand removed, i.e., comparable to a conventional wastewater treatment process. Periodic batch wasting resulted in a solids retention time of 7–14 days. Reactor biomass typically ranged from 4 to 10 g/l volatile suspended solids and the effluent contained no solids. Pure THF-degrading cultures were isolated from the mixed culture based on morphological characteristics, Gram-staining and THF degradation. Based on 16S rDNA analysis the isolates were identified as Pseudonocardia sp. M1 and Rhodococcus ruber M2.     

Neuropharmacological profile of tetrahydrofuran in mice

Since the regulation of illicit gamma-hydroxybutyric acid (GHB) as a Federal Schedule I drug, the use of substitute chemical precursors such as gamma-butyrolactone (GBL) and 1,4-butanediol have emerged. Most recently there have been concerns about another potential analog of GHB, namely tetrahydrofuran (THF). While there is some suggestion that THF can be converted to GHB or GBL, little is known about the pharmacology of THF. Various doses of THF and GBL were studied in neurobehavioral tests to better characterize the pharmacology of THF. The TD(50)'s (with 95% confidence intervals) of THF for loss of the righting reflex and failure of performance on the rotarod test were 15.18 (11.88-19.39) and 7.00 (5.22-9.40) mmol/kg, respectively. These values were significantly greater (p<0.05) than those determined for GBL: 4.60 (3.25-6.51), and 0.85 (0.52-1.38) mmol/kg, respectively. The effects of THF on the impairment of motor function in the rotarod test were antagonized by pretreatment with the GABA(B) receptor antagonist CGP-35348 (200 mg/kg, i.p.).While both THF and GBL had depressant effects on open-field locomotor activity, the pattern of activity at the lower doses of THF and GBL were dissimilar. Chronic treatment with low dose THF (5 or 10 mmol/kg, i.p.) followed by acute challenge with THF (15 mmol/kg, i.p.) demonstrated tolerance to the observed sedative effects. While some of the mechanisms of the THF actions on the central nervous system appear likely to involve direct or indirect interactions with the GABA(B) receptor, some differences in its qualitative and quantitative pharmacology suggests other mechanisms are also likely involved in the observed neurobehavioral effects of these selected doses of THF in mice.     

Trypanocidal tetrahydrofuran lignans from Peperomia blanda

Five tetrahydrofuran lignans and two known flavones were isolated from the aerial parts of Peperomia blanda. The structures of the isolated lignans were elucidated by interpretation of their spectroscopic data, including by gHMQC and gHMBC. The relative and absolute configurations of the isolates were determined from NOESY interactions and optical properties, respectively. Four of the lignans were diastereomeric whilst one was of mixed biosynthetic origin. All but one of the lignans exhibited high in vitro trypanocidal activity when assayed against epimastigotes of Trypanosoma cruzi strain Y.