Cytotoxic Yohimbine-Type Alkaloids from the Leaves of Rauvolfia vomitoria.

Two new yohimbine-type monoterpene indole alkaloids, rauvines A and B, and six known derivatives were obtained from the leaves of R. vomitoria. The structures of rauvines A and B were determined by extensive spectroscopic analyses, 13 C-NMR, and ECD calculations. This is the first time to determine the absolute configurations of yohimbine-type N-oxides by quantum chemistry calculations (13 C-NMR and ECD calculations). All the isolates were tested for their cytotoxicity against five human cancer cell lines. Rauvine B showed moderate cytotoxicity on human MCF-7 breast, SWS80 colon, and A549 lung cancer cell lines with IC50 values of 25.5, 22.6, and 26.0 µM, respectively.

A Fast and Reliable UHPLC-MS/MS-Based Method for Screening Selected Pharmacologically Significant Natural Plant Indole Alkaloids.

Many substances of secondary plant metabolism have often attracted the attention of scientists and the public because they have certain beneficial effects on human health, although the reason for their biosynthesis in the plant remains unclear. This is also the case for alkaloids. More than 200 years have passed since the discovery of the first alkaloid (morphine), and several thousand substances of this character have been isolated since then. Most often, alkaloid-rich plants are part of folk medicine with centuries-old traditions. What is particularly important to monitor for these herbal products is the spectrum and concentrations of the present active substances, which decide whether the product has a beneficial or toxic effect on human health. In this work, we present a fast, reliable, and robust method for the extraction, preconcentration, and determination of four selected alkaloids with an indole skeleton, i.e., harmine, harmaline, yohimbine, and ajmalicine, by ultra-high performance liquid chromatography coupled with tandem mass spectrometry. The applicability of the method was demonstrated for tobacco and Tribulus terrestris plant tissue, the seeds of Peganum harmala, and extract from the bark of the African tree Pausinystalia johimbe.

Yohimbine as a Starting Point to Access Diverse Natural Product-Like Agents with Re-programmed Activities against Cancer-Relevant GPCR Targets.

G protein-coupled receptors (GPCRs) constitute the largest protein superfamily in the human genome. GPCRs play key roles in mediating a wide variety of physiological events including proliferation and cancer metastasis. Given the major roles that GPCRs play in mediating cancer growth, they present promising targets for small molecule therapeutics. One of the principal goals of our lab is to identify complex natural products (NPs) suitable for ring distortion, or the dramatic altering of the inherently complex architectures of NPs, to rapidly generate an array of compounds with diverse molecular skeletal systems. The overarching goal of our ring distortion approach is to re-program the biological activity of select natural products and identify new compounds of importance to the treatment of disease, such as cancer. Described herein are the results from biological screens of diverse small molecules derived from the indole alkaloid yohimbine against a panel of GPCRs involved in various diseases. Several analogues displayed highly differential antagonistic activities across the GPCRs tested. We highlight the re-programmed profile of one analogue, Y7g, which exhibited selective antagonistic activities against AVPR2 (IC50 = 459 nM) and OXTR (IC50 = 1.16 µM). The activity profile of Y7g could correlate its HIF-dependent anti-cancer activity to its GPCR antagonism since these receptors are known to be upregulated in hypoxic cellular environments. Our findings demonstrate that the ring distortion of yohimbine can lead to the identification of new compounds capable of interacting with distinct cancer-relevant targets.

Re-Engineering of Yohimbine's Biological Activity through Ring Distortion: Identification and Structure-Activity Relationships of a New Class of Antiplasmodial Agents.

Select natural products are ideal starting points for ring distortion, or the dramatic altering of inherently complex molecules through short synthetic pathways, to generate an array of novel compounds with diverse skeletal architectures. A major goal of our ring distortion approach is to re-engineer the biological activity of indole alkaloids to identify new compounds with diverse biological activities in areas of significance to human health and medicine. In this study, we re-engineered the biological activity of the indole alkaloid yohimbine through ring rearrangement and ring cleavage synthesis pathways to discover new series of antiplasmodial agents. One new compound, Y7j, was found to demonstrate good potency against chloroquine-resistant Plasmodium falciparum Dd2 cells (EC50 = 0.33 µM) without eliciting cytotoxicity against HepG2 cells (EC50 > 40 µM). Y7j demonstrated stage-specific action against parasites at the late ring/trophozoite stage. A series of analogues was synthesized to gain structure-activity relationship insights, and we learned that both benzyl groups of Y7j are required for activity and fine-tuning of antiplasmodial activities could be accomplished by changing substitution patterns on the benzyl moieties. This study demonstrates the potential for ring distortion to drive new discoveries and change paradigms in chemical biology and drug discovery.

A Tryptoline Ring-Distortion Strategy Leads to Complex and Diverse Biologically Active Molecules from the Indole Alkaloid Yohimbine.

High-throughput screening (HTS) is the primary driver to current drug-discovery efforts. New therapeutic agents that enter the market are a direct reflection of the structurally simple compounds that make up screening libraries. Unlike medically relevant natural products (e.g., morphine), small molecules currently being screened have a low fraction of sp3 character and few, if any, stereogenic centers. Although simple compounds have been useful in drugging certain biological targets (e.g., protein kinases), more sophisticated targets (e.g., transcription factors) have largely evaded the discovery of new clinical agents from screening collections. Herein, a tryptoline ring-distortion strategy is described that enables the rapid synthesis of 70 complex and diverse compounds from yohimbine (1); an indole alkaloid. The compounds that were synthesized had architecturally complex and unique scaffolds, unlike 1 and other scaffolds. These compounds were subjected to phenotypic screens and reporter gene assays, leading to the identification of new compounds that possessed various biological activities, including antiproliferative activities against cancer cells with functional hypoxia-inducible factors, nitric oxide inhibition, and inhibition and activation of the antioxidant response element. This tryptoline ring-distortion strategy can begin to address diversity problems in screening libraries, while occupying biologically relevant chemical space in areas critical to human health.

Characterization of a prawn OA/TA receptor in Xenopus oocytes suggests functional selectivity between octopamine and tyramine.

Here we report the characterization of an octopamine/tyramine (OA/TA or TyrR1) receptor (OA/TAMac) cloned from the freshwater prawn, Macrobrachium rosenbergii, an animal used in the study of agonistic social behavior. The invertebrate OA/TA receptors are seven trans-membrane domain G-protein coupled receptors that are related to vertebrate adrenergic receptors. Behavioral studies in arthropods indicate that octopaminergic signaling systems modulate fight or flight behaviors with octopamine and/or tyramine functioning in a similar way to the adrenalins in vertebrate systems. Despite the importance of octopamine signaling in behavioral studies of decapod crustaceans there are no functional data available for any of their octopamine or tyramine receptors. We expressed OA/TAMac in Xenopus oocytes where agonist-evoked trans-membrane currents were used as readouts of receptor activity. The currents were most effectively evoked by tyramine but were also evoked by octopamine and dopamine. They were effectively blocked by yohimbine. The electrophysiological approach we used enabled the continuous observation of complex dynamics over time. Using voltage steps, we were able to simultaneously resolve two types of endogenous currents that are affected over different time scales. At higher concentrations we observe that octopamine and tyramine can produce different and opposing effects on both of these currents, presumably through the activity of the single expressed receptor type. The pharmacological profile and apparent functional-selectivity are consistent with properties first observed in the OA/TA receptor from the insect Drosophila melanogaster. As the first functional data reported for any crustacean OA/TA receptor, these results suggest that functional-selectivity between tyramine and octopamine is a feature of this receptor type that may be conserved among arthropods.

Broad spectrum chemokine inhibitors related to NR58-3.14.3.

The chemokine family consists of more than 50 structurally-related small proteins which signal through type 1 G-protein coupled receptors (GPCRs) to regulate a range of immune functions, with particular focus on regulating leukocyte trafficking. They have been implicated both in normal physiological leukocyte traffic, and in recruitment of leukocytes to sites of pathological inflammation. As a result, chemokine inhibitors may have useful anti-inflammatory therapeutic properties in vivo. Compounds with chemokine-inhibitory properties that have been described to date, fall into two broad categories: receptor-specific antagonists which block the action of one or a small number of related chemokines, and broad-spectrum chemokine inhibitors (BSCIs) which block leukocyte migration in response to many, if not all, chemokines simultaneously. Since many chemokines apparently show functional redundancy in vivo, the BSCI class are attractive candidates for development as anti-inflammatory therapies. Here, we review the development of BSCIs, with particular focus on the design and characterisation of non-peptide compounds. The key structural requirements for BSCI activity are discussed, together with their implications for the mechanism of BSCI action.

Identification and characterization of the imidazoline I2b-binding sites in the hamster brown adipose tissue as a study model for imidazoline receptors.

The imidazoline-type compound, MPV-1743, has been found to activate nonshivering thermogenesis (NST) in brown adipose tissue (BAT) of the genetically obese Zucker rats. The regulation of NST in BAT is linked to the catecholamine metabolism, and the imidazoline I2-binding sites have been found on the monoamine oxidase, a catecholamine metabolising enzyme. In this study, the I2-binding sites of hamster BAT have been characterised using a receptor binding assay with 3H-idazoxan as a radioligand, and the interaction of MPV-1743 with these I2-binding sites has been studied using the enantiomers of MPV 1743, that is, MPV 2088 and MPV 2089. Cirazoline was used to determine the specific binding of 3H-idazoxan to the imidazoline I2-binding sites. Rauwolscine was added in the 3H-idazoxan binding assay in order to inhibit any binding to potential alpha2-adrenergic sites. In the presence of rauwolscine mask 3H-Idazoxan labelled a population of non-adrenergic binding sites expressing the properties of the imidazoline I2b-receptor subtype similar to that found in the rat liver (cirazoline >> guanabenz = amiloride >> clonidine). The binding of 3H-idazoxan to the I2b-binding sites could be displaced by the imidazole compounds with the following affinities: detomidine (KiHigh 9.2 nM; KiLow 3200 nM), MPV-2088 (KiHigh 19 nM; IKiLow 760 nM) and MPV-2089 (KiHigh 190 nM; KiLow 1300 nM), atipamezole (3500 nM) and dexmedetomidine (Ki 8400 nM). These results have shown that the hamster BAT contains the imidazoline I2b-binding sites with heterogeneous binding properties for some test compounds. In addition, the enantiomers of MPV 1743, that is, MPV 2088 and MPV 2089, had high affinity to these BAT imidazoline I2b-binding sites. Therefore, it is suggested that the regulation of NST in the hamster BAT may be an attractive model to study the role of imidazoline I2b-binding sites.

Determination of relative triplet sublevel populating rates during optical pumping using ODMR.

A method is introduced, based on optical detection of triplet state magnetic resonance (ODMR), to determine the relative populating rates of photoexcited triplet state sublevels during optical pumping. Phosphorescence transients induced by microwave rapid passage during optical pumping are analyzed globally utilizing kinetic parameters obtained from separate microwave-induced delayed phosphorescence measurements to obtain relative sublevel populating rates. Results are unaffected by phosphorescence from triplet populations that do not yield an ODMR response. The method is applied to the triplet state of the indole chromophore in various environments to reveal the effects of local interactions on the pattern of intersystem crossing. Enhanced spin--orbit coupling effects are attributed to interactions that reduce the planar symmetry of the indole chromophore.

A structure-function relationship among reserpine and yohimbine analogues in their ability to increase expression of mdr1 and P-glycoprotein in a human colon carcinoma cell line.

We previously showed that there is a structure-function relationship among reserpine and yohimbine analogues in their ability to inhibit the function of P-glycoprotein (P-gp) and reverse multidrug resistance (MDR). Because some P-gp inhibitors (e.g., verapamil and nifedipine) can increase mdr1 and P-gp expression in human colon carcinoma cell lines, we used our reserpine/yohimbine analogues to determine whether there was a structural requirement for this induction. We found that 10 microM reserpine increased both mdr1 and P-gp expression by 4-10-fold in 48 hr in a human colon carcinoma cell line that expresses moderate levels of mdr1 (LS180-Ad50) but not in several other cell lines that expressed no mdr1. The reserpine/yohimbine analogues rescinnamine, trimethoxybenzoylyohimbine, and LY191401 (compound G), all of which contain the three structural elements used to describe the MDR pharmacophore, also increased both mdr1 and P-gp expression significantly. Despite some exceptions, we found that there was a good association between the ability of these analogues to induce mdr1 and P-gp expression and their ability to reverse vinblastine and doxorubicin resistance, revealing a structure-function relationship for this phenomenon. The increased P-gp expressed by these cells appeared to be functional, as determined by flow cytometric detection of rhodamine 123 retention. The increased expression was suppressed by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, an RNA synthesis inhibitor, whereas the protein synthesis inhibitor cycloheximide enhanced the expression several-fold, suggesting that induction of mdr1 by these analogues is regulated at both the transcriptional and post-transcriptional levels.(ABSTRACT TRUNCATED AT 250 WORDS)