Synthesis of Anhydroryanodol.

A stereoselective entry to ryanoids is described that culminates in the synthesis of anhydroryanodol and thus the formal total synthesis of ryanodol. The pathway described features an annulation reaction conceived to address the uniquely complex and highly oxygenated polycyclic skeleton common to members of this natural product class. It is demonstrated that metallacycle-mediated intramolecular coupling of an alkyne and a 1,3-diketone can proceed with a highly functionalized enyne and with outstanding levels of stereoselection. Furthermore, the first application of this technology in natural product synthesis is demonstrated here. More broadly, the advances described demonstrate the value that programs in natural product total synthesis have in advancing organic chemistry, here through the design and realization of an annulation reaction that accomplishes what previously established reactions do not.

Asymmetric Total Synthesis of (+)-Ryanodol and (+)-Ryanodine.

(+)-Ryanodine (1) is the ester derivative of 1H-pyrrole-2-carboxylic acid and the complex terpenoid (+)-ryanodol (2), which possesses eleven contiguous stereogenic centers on the ABCDE-ring system. Compound 1 is known to be a potent modulator of intracellular calcium release channels, whereas the activity of 2 is significantly weaker. To chemically construct 1, the multiple oxygen functional groups must be installed on the fused pentacycle in stereoselective fashions and the extremely hindered C3-hydroxy group must be acylated in a site-selective manner. First, the total synthesis of 2 was accomplished by introducing the five stereocenters from the previously prepared enantiopure ABDE-ring 7. Stereoselective construction of the C3-secondary, C2- and C6-tertiary alcohols was achieved by three nucleophilic reactions. The C9- and C10-trisubstituted carbon centers were regio- and stereoselectively introduced by hydroboration/oxidation of the six-membered C-ring, which was formed by the ring-closing metathesis reaction. Direct esterification of the C3-alcohol with pyrrole-2-carboxylic acid proved unsuccessful; therefore, we developed a new, two-step protocol for attachment of the pyrrole moiety. The C3-hydroxy group was first converted into the less sterically cumbersome glycine ester, which was then transformed into the pyrrole ring through condensation with 1,3-bis(dimethylamino)allylium tetrafluoroborate. This procedure resulted in the first total synthesis of 1.

Symmetry-Driven Strategy for the Assembly of the Core Tetracycle of (+)-Ryanodine: Synthetic Utility of a Cobalt-Catalyzed Olefin Oxidation and α-Alkoxy Bridgehead Radical Reaction.

Ryanodine (1) is a potent modulator of intracellular calcium release channels, designated as ryanodine receptors. The exceptionally complex molecular architecture of 1 comprises a highly oxygenated pentacyclic system with eleven contiguous stereogenic centers, which makes it a formidable target for organic synthesis. We identified the embedded C2 -symmetric tricyclic substructure within 1. This specific recognition permitted us to design a concise synthetic route to enantiopure tricycle 9 by utilizing a series of pairwise functionalizations. The four tetrasubstituted carbon centers of 9 were effectively constructed by three key reactions, a dearomatizing Diels-Alder reaction, the kinetic resolution of the obtained racemic 14 through asymmetric methanolysis, and the transannular aldol reaction of the eight-membered diketone 10. A new combination of cobalt-catalyzed hydroperoxidation and NfF-promoted elimination enabled conversion of the hindered olefin of 9 into the corresponding ketone, thus realizing the desymmetrization. Finally, the tetrasubstituted carbon was stereospecifically installed by utilizing the α-alkoxy bridgehead radical to deliver the core tetracycle 7 with the six contiguous tetrasubstituted carbon centers. Consequently, the present work not only accomplishes efficient assembly of four out of the five fused rings of 1, but also develops two new powerful methodologies: two-step ketone formation and bridgehead radical reaction.

Chemical Conversion of Ryanodol to Ryanodine.

Ryanodine (1) is a plant-derived natural product with powerful pharmacological and insecticidal action, and is a potent modulator of intracellular calcium release channels. Compound 1 possesses a 1H-pyrrole-2-carboxylate ester at the C3-position of heptahydroxylated terpenoid ryanodol (2). Whereas 2 was readily obtained from 1 by basic hydrolysis, 1 has never been synthesized from 2, due to the extreme difficulty in selectively introducing the bulky pyrrole moiety at the severely hindered C3-hydroxyl group of heptaol 2. Here we report chemical conversion of 2 to 1 for the first time. The derivatization was realized through the use of a new protective group strategy and the application of on-site construction of the pyrrole-2-carboxylate ester from the glycine ester and 1,3-bis(dimethylamino)allylium tetrafluoroborate.

Total synthesis of ryanodol.

Ryanodol (1) exists in nature in the form of the 1H-pyrrole-2-carboxylate ester derivative known as ryanodine, which is a potent modulator of the calcium release channel. The pentacyclic ABCDE-ring system of 1 is fabricated with eight oxy groups, three methyl groups, and one isopropyl group. All the eight tetrasubstituted stereocenters are concentrated within the 10-carbon ABDE framework. The total synthesis of this exceptionally complex molecule was achieved in 22 steps from the simple C2-symmetric tricycle 8. The synthetic route is based on installation of the seven stereogenic centers and formation of the four C-C bonds within the highly congested multicyclic format. The novel and flexible strategy developed here will enable the generation of chemical derivatives with different functional properties toward calcium release channels.

Toward the Total Synthesis of Ryanodol via Oxidative Alkyne-1,3-Diketone Annulation: Construction of a Ryanoid Tetracycle.

A synthetic strategy conceived with the intent of establishing a novel approach to the de novo construction of ryanoids is described that is based on a recently developed metallacycle-mediated intramolecular oxidative alkyne-1,3-diketone coupling reaction. In short, a one-pot annulation/oxidation sequence is shown to be capable of establishing a densely oxygenated polycyclic intermediate that could be converted to a composition of matter that contains the ABCD tetracyclic ring system present in the ryanoid family of natural products.

A 15-step synthesis of (+)-ryanodol.

(+)-Ryanodine and (+)-ryanodol are complex diterpenoids that modulate intracellular calcium-ion release at ryanodine receptors, ion channels critical for skeletal and cardiac muscle excitation-contraction coupling and synaptic transmission. Chemical derivatization of these diterpenoids has demonstrated that certain peripheral structural modifications can alter binding affinity and selectivity among ryanodine receptor isoforms. Here, we report a short chemical synthesis of (+)-ryanodol that proceeds in only 15 steps from the commercially available terpene (S)-pulegone. The efficiency of the synthesis derives from the use of a Pauson-Khand reaction to rapidly build the carbon framework and a SeO2-mediated oxidation to install three oxygen atoms in a single step. This work highlights how strategic C-O bond constructions can streamline the synthesis of polyhydroxylated terpenes by minimizing protecting group and redox adjustments.

High affinity C10-Oeq ester derivatives of ryanodine. Activator-selective agonists of the sarcoplasmic reticulum calcium release channel.

The plant alkaloids ryanodine and dehydroryanodine are specific and potent modulators of the sarcoplasmic reticulum calcium release channel. In the present study, acidic, basic, and neutral side chains esters of these diterpene compounds were prepared and their pharmacologic activities were assessed. Binding affinities of the novel C10-Oeq ester derivatives for the sarcoplasmic reticulum Ca2+ release channel were evaluated with sarcoplasmic reticular vesicles prepared from rabbit skeletal muscle. Kd values of the derivatives varied 500-fold, ranging from 0.5 to 244 nM. In comparison, Kd values for ryanodine and dehydroryanodine were 4.4 nM and 5.4 nM, respectively. Basic substituents at the C10-Oeq side chain terminus produced the highest affinity derivatives (Kd values from 0.5 to 1.3 nM). Neutral and/or hydrophobic side chain derivatives exhibited intermediate affinities for the high affinity ryanodine receptor site (Kd values from 2.5 to 39 nM), whereas a derivative with a terminal acidic group had the lowest affinity (Kd value > 100 nM). Certain of the higher affinity C10-Oeq derivatives were evaluated more extensively for their pharmacologic activity on the sarcoplasmic reticular Ca2+ release channel. Both channel activating (opening) and deactivating (closing) actions were assessed from the ability of the ryanoids to alter Ca2+ efflux rates from skeletal junctional sarcoplasmic reticular vesicles that had been passively loaded with Ca2+. The natural Ryania secondary metabolites ryanodine, dehydroryanodine and esters E and F, all exhibit antithetical concentration-effect curves, indicating both activator and deactivator actions. In contrast, the semi-synthetic C10-Oeq esters selectively activate the Ca2+ release channel. Half-maximal concentrations for such activation (EC50 act) ranged from 0.87 microM to 4.2 microM, compared with an EC50 act of 1.3 microM for ryanodine. These derivatives were also evaluated for their ability to augment ATP-dependent CA2+ accumulation by cardiac junctional sarcoplasmic reticular vesicles, an effect that results from deactivation of the Ca2+ release channels. None of the derivatives tested was able to significantly augment Ca2+ accumulation, further substantiating their inability to deactivate the sarcoplasmic reticular Ca2+ release channel. Additionally, these derivatives functionally antagonized the action of ryanodine to close the Ca2+ release channel. The results presented demonstrate that these C10-Oeq ester derivatives of ryanodine and dehydroryanodine bind specifically to the SR Ca2+ release channel, selectively activate the channel, and, although they fail to effect channel closure, they nevertheless functionally compete with ryanodine at its low affinity (deactivator) site(s).

Radioimmunoassay for the calcium release channel agonist ryanodine.

A novel photo-activatable derivative of ryanodine, 9-hydroxy-21-(4-azidobenzoyloxy)-9-epiryanodine, has been synthesized and conjugated to keyhole limpet hemocyanin for the production of antibodies with high affinity and specificity to ryanodine. The anti-ryanodine antibodies reacted specifically on immunoblots with the azido-ryanodine compound covalently conjugated to bovine serum albumin. A radioimmunoassay specific for ryanodine was developed using the anti-ryanodine antibodies, and a dissociation constant for ryanodine of 1 nM was determined. Half-maximal inhibition constants (IC50) for various ryanodine derivatives were found to range between 3.2 and 200 nM. These IC50 values correlated very well with the IC50 values obtained for the compounds binding to the skeletal muscle membrane receptor. These antibodies should be useful for the characterization of the ryanodine binding site on the sarcoplasmic reticulum Ca2+ release channel.