Studies on the biosynthesis of paraherquamide A and the total synthesis of (±) VM55599

2001 Summer.Includes bibliographical references.Studies that partially elucidate the biosynthetic pathway of the heptacyclic fungal metabolite, Paraherquamide A are presented. Through biosynthetic feeding experiments with stable isotope labeled compounds, it was determined that the carbon skeleton of paraherquamide A consists of three amino acids and two equivalents of dimethylallyl pyrophosphate. L-methionine was determined to be the precursor to C-29, L-tryptophan was determined to be the precursor to the oxindole moiety and L-isoleucine was determined to be the precursor to the β-hydroxy- β-methylproline ring of paraherquamide A. A subsequent feeding experiment with [5-13C3H2]-L-isoleucine indicated that L-isoleucine is converted to β-methyl-proline via a 4-electron oxidation and that the pro-S hydrogen of C-5 is retained in the oxidative cyclization. The incorporation of [1-13C]-3(S)-methyl-L-proline also indicated that L-isoleucine undergoes oxidative cyclization prior to coupling to L-tryptophan. The two dimethylallyl pyrophosphate-derived portions of paraherquamide A were found to arise via the well-known mevalonic acid pathway. In addition, it was determined that P. fellutanum, a paraherquamide A producing fungus, installs theses two dimethylallyl groups in two stereo-facially distinct manners. The C5 unit (C-19~C-23) leading to the bicyclo[2.2.2]diazaoctan ring is formed in an entirely non-stereospecific manner, while the C-5 unit (C-24~C-28) leading to the dioxepin ring is installed in a completely stereospecific manner. Only the latter observation is consistent with the mechanism of known prenylase enzymes. The first total synthesis of VM55599, a proposed precursor of paraherquamide A, was completed. Feeding experiments with doubly 13C-labeled racemic VM55599, its diastereomer and the oxidized forms of these compounds were performed on P. fellutanum. Only the diasteromer of VM55599 (244a) was incorporated. The incorporation of the bicyclic intermediate signifies that the dimethylallyl group leading to C-19~C-23 of paraherquamide A is installed prior to the dimethylallyl group leading to C-24~C-28. In addition, the incorporation 244a indicates that the oxidations of the indole ring leading to the spirooxindole and the dioxepin ring occur after the formation of the bicyclic ring system

In Vitro Pharmacological Characterization of RXFP3 Allosterism: An Example of Probe Dependency

Recent findings suggest that the relaxin-3 neural network may represent a new ascending arousal pathway able to modulate a range of neural circuits including those affecting circadian rhythm and sleep/wake states, spatial and emotional memory, motivation and reward, the response to stress, and feeding and metabolism. Therefore, the relaxin-3 receptor (RXFP3) is a potential therapeutic target for the treatment of various CNS diseases. Here we describe a novel selective RXFP3 receptor positive allosteric modulator (PAM), 3-[3,5-Bis(trifluoromethyl)phenyl]-1-(3,4-dichlorobenzyl)-1-[2-(5-methoxy-1H-indol-3-yl)ethyl]urea (135PAM1). Calcium mobilization and cAMP accumulation assays in cell lines expressing the cloned human RXFP3 receptor show the compound does not directly activate RXFP3 receptor but increases functional responses to amidated relaxin-3 or R3/I5, a chimera of the INSL5 A chain and the Relaxin-3 B chain. 135PAM1 increases calcium mobilization in the presence of relaxin-3NH2 and R3/I5NH2 with pEC50 values of 6.54 (6.46 to 6.64) and 6.07 (5.94 to 6.20), respectively. In the cAMP accumulation assay, 135PAM1 inhibits the CRE response to forskolin with a pIC50 of 6.12 (5.98 to 6.27) in the presence of a probe (10 nM) concentration of relaxin-3NH2. 135PAM1 does not compete for binding with the orthosteric radioligand, [125I] R3I5 (amide), in membranes prepared from cells expressing the cloned human RXFP3 receptor. 135PAM1 is selective for RXFP3 over RXFP4, which also responds to relaxin-3. However, when using the free acid (native) form of relaxin-3 or R3/I5, 135PAM1 doesn't activate RXFP3 indicating that the compound's effect is probe dependent. Thus one can exchange the entire A-chain of the probe peptide while retaining PAM activity, but the state of the probe's c-terminus is crucial to allosteric activity of the PAM. These data demonstrate the existence of an allosteric site for modulation of this GPCR as well as the subtlety of changes in probe molecules that can affect allosteric modulation of RXFP3

135PAM1 increases the intracellular Ca²⁺ response to amidated, but not free acid RXFP3 agonists in cells coexpressing RXFP3 and G_qI5.

<p>Intracellular Ca²⁺ responses by HEK-293 cells coexpressing RXFP3 and G_qI5 were measured in response to escalating concentrations of 135PAM1 using probe (EC₂₀) concentrations of Relaxin-3_NH2 (A), Relaxin-3_OH (B), R3/I5_NH2 (C), or R3/I5_OH (D).</p

135PAM1 shifts the concentration response curves of Relaxin-3_NH2 and R3/I5_NH2.

<p>HEK-293 cells coexpressing RXFP3 and G_qI5 were incubated with fixed concentrations of 135PAM1 (0, 0.2, 2 and 20 µM) 10 min before the addition of increasing concentrations of Relaxin-3_NH2 (A), R3I5_NH2 (B), Relaxin-3_OH (C) or R3I5_OH (D).</p

135PAM1 lacks affinity at the orthosteric binding site of RXFP3 receptor.

<p>135PAM1 did not displace [125I] R3/I5_NH2 at concentrations of up to 20 µM, but instead increased total binding. R3/I5_NH2 displaced the tracer with a pIC₅₀ of 8.76 (8.91 to 8.61).</p