Total Synthesis of 19-Nordigitoxigenin, An Antiaroside Y Aglycon.

The first total synthesis of 19-nordigitoxigenin, an aglycon of antiroside Y, has been achieved. The key steps of our synthesis are (i) construction of the 19-norsteroid ring system via a Mizoroki-Heck reaction between a bromoanisole corresponding to the A-ring and cyclic alkene incorporating the CD-rings, followed by a Friedel-Crafts-type cyclodehydration, and (ii) incorporation of the butenolide moiety at C17 via a silyl-tethered radical cyclization and subsequent ozone oxidation.

Structural verification of petromyzestrosterol by total syntheses of both C14-epimers of its 3-O-methyl derivative.

The structure of petromyzestrosterol, a pheromonal steroid of the sea lamprey, was verified by total syntheses of both C14-epimers of its 3-O-methyl derivative. The key features of our synthesis involve (1) a highly stereoselective Mizoroki-Heck reaction to unite the A- and CD-ring segments and (2) Friedel-Crafts-type cyclodehydration to construct the B-ring. Petromyzestrosterol is concluded to bear an α-configured C14 hydroxy group based on a comparison of NMR data of both the synthesized C14-epimers of the 3-O-methyl derivative with those of the natural petromyzestrosterol. The downfield shifts of C9 and C12 via the γ-gauche effect in the 14ß-isomer would enable the structural elucidation of C14 in the 14-hydroxy estrogenic steroids.

Total synthesis and biological evaluation of oscillatoxins D, E, and F.

Oscillatoxins (OTXs) and aplysiatoxins are biosynthetically related polyketides produced by marine cyanobacteria. We previously developed a synthetic route to phenolic O-methyl analogs of OTX-D and 30-methyl-OTX-D during collective synthesis of these natural products. According to our synthetic strategy, we achieved total synthesis of OTX-D, 30-methyl-OTX-D, OTX-E, and OTX-F by deprotecting the O-methyl group in an earlier intermediate, and determined their biological activities. Although OTX-D and 30-methyl-OTX-D have been reported to show antileukemic activity against L1210 cell line, we found that their cytotoxicity in vitro against this cell line is relatively weak (IC50: 29-52 µm). In contrast, OTX-F demonstrated cell line-selective antiproliferative activity against DMS-114 lung cancer cells, which implies that OTXs target as yet unknown target molecules as part of this unique activity.

Total Synthesis of the Cardiotonic Steroid (+)-Cannogenol.

The total synthesis of (+)-cannogenol, an aglycon common to various biologically important cardiotonic glycosides, has been achieved. Synthesis of the versatile intermediate involves Mizoroki-Heck and intramolecular Diels-Alder reactions from the enantiomerically pure CD-ring segment, newly prepared in a multidecagram scale this time. Total synthesis by the site-selective transformations of the versatile intermediate demonstrated the applicability of our synthetic approach.

Biomimetic Synthesis of the CDE Ring Moiety of Physalins, Complex 13,14-Secosteroids.

Physalins are a structurally complex family of 13,14-secosteroids isolated from the genus Physalis. We disclose a two-step construction of the CDE ring moiety of the physalins from a steroidal compound bearing 14-OH, 18-COOMe, and 17, 20-α-epoxide based on our biosynthetic proposal. C13-C14 bond cleavage by an alkoxy radical at C-14 and spontaneous epoxide ring opening gave a compound having a cyclononene and γ-lactone. Diastereoselective dihydroxylation of the resulting alkene with OsO4 provided the CDE ring moiety of physalin.

Biosynthesis of Indole Diterpene Lolitrems: Radical-Induced Cyclization of an Epoxyalcohol Affording a Characteristic Lolitremane Skeleton.

Lolitrems are tremorgenic indole diterpenes that exhibit a unique 5/6 bicyclic system of the indole moiety. Although genetic analysis has indicated that the prenyltransferase LtmE and the cytochrome P450 LtmJ are involved in the construction of this unique structure, the detailed mechanism remains to be elucidated. Herein, we report the reconstitution of the biosynthetic pathway for lolitrems employing a recently established genome-editing technique for the expression host Aspergillus oryzae. Heterologous expression and bioconversion of the various intermediates revealed that LtmJ catalyzes multistep oxidation to furnish the lolitrem core. We also isolated the key reaction intermediate with an epoxyalcohol moiety. This observation allowed us to establish the mechanism of radical-induced cyclization, which was firmly supported by density functional theory calculations and a model experiment with a synthetic analogue.

Studies toward the Synthesis of Chartelline C.

Chartelline C, a marine alkaloid, possesses a unique core scaffold including indolenine ß-lactam and imidazole moieties linked by an unsaturated 10-membered ring. A new synthetic approach for the construction of the indolenine ß-lactam was planned, based on the inherent reactivity of chartelline A with NaOMe, triggered by bromination of bromoenamide, and proceeding through an N-acyl imine. However, the N-acyl imine intermediate was not observed. Instead, the corresponding bromoindolenine was obtained, which led to the desired indolenine ß-lactam in 92% yield.

Quantitative analysis of oxidized vitamin B1 metabolites generated by hypochlorous acid.

Thiamine, a water-soluble essential vitamin known as vitamin B1, acts as an important cofactor in various cellular processes, such as metabolism and energy production. Thiamine is also thought to have antioxidant effects as a singlet oxygen scavenger and a lipid peroxidation inhibitor. However, the oxidation mechanism and oxidized metabolites of thiamine are not completely established. In the present study, we investigated the oxidative reactivity of thiamine and found that three products were formed upon the reaction of thiamine with hypochlorous acid (HOCl). Based on the NMR and high resolution mass spectrometric analysis, the HOCl-oxidized metabolites of thiamine were identified as formylaminopyrimidine (FAP), thiamine sulfonic acid (TSA), and thiamine sulfinic ester (TSE). To evaluate the formation of these oxidized metabolites in vivo, we established a specific method for quantification of the oxidized thiamine metabolites using liquid chromatography-tandem mass spectrometry coupled with a stable isotope dilution method. Using this method, it was shown that the oxidized thiamine metabolites were generated in the culture media of phorbol-12-myristate-acetate-treated neutrophil-like cells in a myeloperoxidase-dependent manner. Moreover, significantly higher amounts of FAP and TSE were detected in the lung tissues of the lipopolysaccharide-treated mice compared to the controls. These findings provide not only insights into the oxidative metabolism of thiamine, but also the possibility that the oxidized thiamine metabolites may be potential biomarkers for HOCl-related oxidative stress.

Biomimetic Synthesis of Chaxine and its Related Compounds.

The highly oxidized natural products chaxine B and BB have been synthesized from ergosterol in eight steps according to a route inspired by their proposed biosynthesis; key steps were an oxidative cascade from a furan intermediate to an enol ester using m-chloroperbenzoic acids (MCPBA), followed by diastereoselective epoxidation and acyloxy migration. This concise synthesis resulted in the revision of the structures of chaxine B and its naturally occurring analogs and syntheses of the unnatural analogues of these natural products for biological investigations.

Synthesis of Oxy-Functionalized Steroidal Skeletons via Mizoroki-Heck and Intramolecular Diels-Alder Reactions.

Estrogenic and cardiotonic steroidal skeletons were concisely constructed via Mizoroki-Heck and intramolecular Diels-Alder (IMDA) reactions. Simple modification of the dienophile unsaturation of the IMDA precursor enabled representative AB-ring systems of both steroid classes to be accessed from the same intermediate. The diastereoselectivity of the IMDA reaction used to access the cardiotonic steroidal skeleton was found to be significantly enhanced by performing the reaction in water.

Asymmetric Synthesis of the Aromatic Fragment of Sespendole.

Sespendole is an indole sesquiterpene alkaloid bearing two isoprenyl groups, one of which is highly oxidized. Herein, we disclose an eight-step synthesis of the aromatic fragment of sespendole in an optically pure form, starting from 4-bromo-2-fluoronitrobenzene. The key steps were a Claisen rearrangement at room temperature for introduction of the prenyl group and a coupling between the dianion generated from prenylated bromo-N-tosylanilide and a chiral epoxy aldehyde.

Toward a Synthesis of Fawcettimine-Type Lycopodium Alkaloids: Stereocontrolled Synthesis of a Functionalized Azaspirocycle Precursor.

The stereocontrolled synthesis of a new azaspirocycle precursor of the fawcettimine-type Lycopodium alkaloids is described. Our approach provides an efficient entry to the azaspirocycle via a cascade Wacker-allylation sequence followed by a highly stereoselective Claisen rearrangement. This azaspirocycle, bearing all of the requisite functionality with pivotal stereogenic centers, is considered to be a versatile precursor useful for the fawcettimine-type Lycopodium alkaloids.

Semi-synthesis and Structure-Activity Relationship of Neuritogenic Oleanene Derivatives.

(3S,4R)-23,28-Dihydroxyolean-12-en-3-yl (2E)-3-(3,4-dihydroxyphenyl)acrylate (1 a), which possesses significant neuritogenic activity, was isolated from the traditional Chinese medicine (TCM) plant, Desmodium sambuense. To confirm the structure and to assess biological activity, we semi-synthesized 1 a from commercially available oleanolic acid. A series of novel 1 a derivatives was then designed and synthesized for a structure-activity relationship (SAR) study. All synthetic derivatives were characterized by analysis of spectral data, and their neuritogenic activities were evaluated in assays with PC12 cells. The SAR results indicate that the number and position of the hydroxy groups on the phenyl ring and the triterpene moiety, as well as the length of the (saturated or unsaturated) alkyl chain that links the phenyl ring with the triterpene critically influence neuritogenic activity. Among all the tested compounds, 1 e [(3S,4R)-23,28-dihydroxyolean-12-en-3-yl (2E)-3-(3,4,5-trihydroxyphenyl)acrylate] was found to be the most potent, inducing significant neurite outgrowth at 1 µm.

Synthetic Route to Oscillatoxin D and Its Analogues.

O-Methyloscillatoxin D and its analogues were concisely synthesized by a bioinspired intramolecular Mukaiyama aldol reaction as a key step, which involves the construction of a novel spiro-ether moiety.

De Novo Synthesis of Possible Candidates for the Inagami-Tamura Endogenous Digitalis-like Factor.

De novo synthesis of possible candidates for the Inagami-Tamura endogenous digitalis-like factor (EDLF) was achieved to validate a previously proposed structure. Our synthetic approach involves a highly regio- and diastereoselective Mizoroki-Heck reaction and a Friedel-Crafts-type cyclodehydration to construct steroidal tetracycle 14 as a versatile common intermediate leading to seven 2,14ß-dihydroxyestradiol analogues 1a-c, 2a-c, and 3 as possible candidates. By comparing the potency of inhibitory activity against Na+/K+-ATPase between the synthesized candidates and the EDLF, it was found that the proposed structure is not likely to be a true structure of the Inagami-Tamura EDLF.

Inhibition of veratridine-induced delayed inactivation of the voltage-sensitive sodium channel by synthetic analogs of crambescin B.

Crambescin B carboxylic acid, a synthetic analog of crambescin B, was recently found to inhibit the voltage-sensitive sodium channels (VSSC) in a cell-based assay using neuroblastoma Neuro 2A cells. In the present study, whole-cell patch-clamp recordings were conducted with three heterologously expressed VSSC subtypes, Nav1.2, Nav1.6 and Nav1.7, in a human embryonic kidney cell line HEK293T to further characterize the inhibition of VSSC by crambescin B carboxylic acid. Contrary to the previous observation, crambescin B carboxylic acid did not inhibit peak current evoked by depolarization from the holding potential of -100mV to the test potential of -10mV in the absence or presence of veratridine (VTD). In the presence of VTD, however, crambescin B carboxylic acid diminished VTD-induced sustained and tail currents through the three VSSC subtypes in a dose-dependent manner, whereas TTX inhibited both the peak current and the VTD-induced sustained and tail currents through all subtypes of VSSC tested. We thus concluded that crambescin B carboxylic acid does not block VSSC in a similar manner to TTX but modulate the action of VTD, thereby causing an apparent block of VSSC in the cell-based assay.

Biomimetic Synthesis and Structural Revision of Chaxine B and Its Analogues.

Chaxine B and its analogues were synthesized from ergosterol in eight steps on the basis of our proposed biosynthetic pathway, which includes a highly site-selective and regioselective Baeyer-Villiger oxidation as the key step. This synthesis enabled the revision of the structures of chaxine B and its analogues.

Palladium-Catalyzed Cascade Wacker/Allylation Sequence with Allylic Alcohols Leading to Allylated Dihydropyrones.

We describe a cascade Wacker/allylation sequence of ß-hydroxy ynones by directly using simple allylic alcohols. This palladium(II)-catalyzed reaction occurs under mild conditions (0 °C to room temperature) and provides a new and efficient synthetic method for the preparation of allylated dihydropyrones. The regiochemical outcomes are consistent with a reaction pathway that includes an insertion/ß-OH elimination sequence to form the allylic moiety. A remarkable changeover from allyl to formylethyl products occurs under the simple action of LiBr.

A Synthetic Strategy for Saxitoxin Skeleton by a Cascade Bromocyclization: Total Synthesis of (+)-Decarbamoyl-α-saxitoxinol.

A new synthetic strategy for the formation of the ABC tricyclic framework of saxitoxin was developed. The BC ring moiety, including a spiro-aminal structure, was first constructed stereoselectively by a newly designed cascade bromocyclization of a readily available internal alkyne bearing guanidine and urea. The A ring was then synthesized by a guanylation of a cyclic urea, easily prepared via the oxidative cleavage of the diol of the cascade product, followed by addition of cyanide. This strategy enables the concise stereocontrolled total synthesis of (+)-decarbamoyl-α-saxitoxinol, which is a naturally occurring saxitoxin analogue.

A Divergent Approach to the Diastereoselective Synthesis of 3,3-Disubstituted Oxindoles from Atropisomeric N-Aryl Oxindole Derivatives.

3,3-Disubstituted oxindoles were divergently synthesized by diastereoselective transformations including nucleophilic addition, alkylation, and cycloaddition using common, axially chiral N-aryl oxindoles. Notably, high diastereoselectivities (up to >95:5) were observed with ortho-monosubstituted N-aryl oxindoles to give various oxindole scaffolds, and facile removal of the p-(benzyloxy)aryl moiety in axially twisted amides was achieved by a mild, two-step sequence.