Construction of Hydroindole Skeleton by Photocatalytic Oxidative Dearomatization and Cyclization Sequence, and a Concise Synthesis of Melodamide A and (±)-Toussaintine C.

A method of synthesizing hydroindole skeletons has been developed by using photocatalytic oxidative dearomatization and an aza-Michael addition sequence. Using this method, optically active hydroindoles, which are often used in natural product synthesis as chiral building blocks, can be easily prepared with >99% ee. Furthermore, the synthesis of melodamide A and (±)-toussaintine C was achieved using this method as a key step.

Synthesis and biological evaluation of coprinoferrin, an acylated tripeptide hydroxamate siderophore.

Coprinoferrin (CPF), originally isolated from a genetically engineered strain (ΔlaeA) of the mushroom fungus Coprinopsis cinerea, is an acylated tripeptide hydroxamate consisting of tandem aligned N5-hexanoyl-N5-hydroxy-L-ornithine with modifications of N-acetyl and C-carboxamide. These unique chemical properties make CPF an iron(III) binder (siderophore), which helps in iron acquisition from the environment and promotes hyphal growth as well as fruiting body formation in C. cinerea. However, CPF's detailed mode of action remains enigmatic. In this study, we have accomplished the synthesis of CPF from N-Boc-L-glutamic acid 5-benzyl ester. The physicochemical characteristics, spectroscopic features, and biological activity observed in the synthetic CPF closely match those of natural CPF. This alignment provides unequivocal confirmation of the proposed chemical structure, facilitating a deeper understanding of its physiological role in nature, particularly in fruiting body formation.

Structure-activity relationship studies on an antitumor marine macrolide using aplyronine a-swinholide A hybrid.

An aplyronine A-swinholide A hybrid, consisting of the macrolactone part of aplyronine A and the side chain part of swinholide A, was designed, synthesized, and biologically evaluated. This hybrid induced protein-protein interactions between two major cytoskeletal proteins actin and tubulin in the same manner as aplyronine A, and exhibited potent cytotoxicity and actin-depolymerizing activity. The importance of the methoxy group in the N,N,O-trimethylserine ester was clarified by the structure-activity relationship studies of the amino acid moiety by using the hybrid analogs. Furthermore, the comparison of the actin-depolymerizing activities between the side chain analogs of aplyronine A and swinholide A showed that the side chain analog of swinholide A had much weaker actin-depolymerizing activity than that of aplyronine A.

Toward the Synthesis of Paspaline-Type Indole-Terpenes: Stereoselective Construction of Core Scaffold with Contiguous Asymmetric Quaternary Carbon Centers.

The core scaffold of paspaline-type indole-terpenes was synthesized by using the House-Meinwald rearrangement as a key step. Rearrangement of the epoxide methyl group in the precursor with methylaluminum bis(4-bromo-2,6-di-tert-butylphenoxide) as a Lewis acid proceeded smoothly to construct contiguous asymmetric quaternary carbon centers by a 1,2-chirality transfer.

Toward the Synthesis of SB-203207: Construction of Four Contiguous Nitrogen-Containing Stereogenic Centers.

SB-203207 is an altemicidin-type alkaloid that potently inhibits isoleucyl tRNA synthetase activity. Its main structural feature is a hexahydro-6-azaindene framework containing a unique ß-hydroxy α,α-disubstituted α-amino acid moiety on the cyclopentane portion. Herein we have established a method for constructing the four contiguous nitrogen-containing stereogenic centers of SB-203207 by using as key steps the stereoselective alkylation of bowl-shaped tricyclic lactone to construct a quaternary carbon at C1, the stereoselective hydroboration-oxidation reaction to install the C2 hydroxy group, and the Curtius rearrangement to introduce a nitrogen atom onto the C1 quaternary carbon.

Second-generation total synthesis of aplyronine A featuring Ni/Cr-mediated coupling reactions.

Second-generation total synthesis of aplyronine A, a potent antitumor marine macrolide, was achieved using Ni/Cr-mediated coupling reactions as key steps. The overall yield of the second-generation synthetic pathway of aplyronine A was 1.4%, obtained in 38 steps based on the longest linear sequence. Compared to our first-generation synthetic pathway of aplyronine A, the second-generation synthesis greatly improved both the yield and number of steps. In particular, we improved the stereoselectivity in the construction of the C13 stereogenic center and the C14-C15 (E)-trisubstituted double bond using the asymmetric Ni/Cr-mediated coupling reaction. Furthermore, we established efficient reaction conditions for the asymmetric Ni/Cr-mediated coupling reaction between the C21-C28 segment and C29-C34 segment. Thus, this coupling reaction proceeded with an equimolar ratio of each segment.

Total synthesis of natural derivatives and artificial analogs of 13-oxyingenol and their biological evaluation.

We have established an efficient synthetic methodology for the 13-oxyingenol natural derivative (13-oxyingenol-13-dodecanoate-20-hexanoate), featuring a ring-closing olefin metathesis reaction for the "direct" construction of a highly strained inside-outside framework and a Mislow-Evans-type [2,3]-sigmatropic rearrangement for the stereoselective introduction of the hydroxy group at C5. We also synthesized artificial analogs of 13-oxyingenol and ingenol by using our synthetic strategy. In vitro activation assays of protein kinase C (PKC) α and δ revealed that the dodecanoyl group at O13 on 13-oxyingenol analogs had a significant role in PKCδ activation. The PKCα- or PKCδ-activating 13-oxyingenol and ingenol analogs induced both distinct morphological changes and increases of CD11b expression in HL-60 cells, which would be typical signs of HL-60 cell differentiation to macrophage-like cells, as expected by previous reports. Intriguingly, however, similar differentiation phenotypes were observed with the use of 13-oxyingenol natural derivatives and 13-oxyingenol-13-dodecanoate showing a remarkably less potent PKCα or PKCδ activation ability, which the PKC inhibitor Gö6983 diminished. This indicated the involvement of other PKC isozymes or related kinase activities. 13-Oxyingenol analogs, which induced HL-60 cell differentiation, also induced HL-60 cell death, similar to the action of a phorbol ester, a strong PKC activator.

Discovery of O6-benzyl glaziovianin A, a potent cytotoxic substance and a potent inhibitor of α,ß-tubulin polymerization.

We have discovered O6-benzyl glaziovianin A, which showed stronger inhibition of microtubule polymerization (IC50=2.1µM) than known α,ß-tubulin inhibitors, such as colchicine and glaziovianin A. Also, we performed competition binding experiments of O6-benzyl glaziovianin A and revealed that O6-benzyl glaziovianin A binds to the colchicine binding site with high affinity. It is interesting that glaziovianin A derivatives change their mode of action in benzylation at the O6 (α,ß-tubulin inhibitor) or O7 (γ-tubulin-specific inhibitor) position.

Synthesis and structure-activity relationships for cytotoxicity and apoptosis-inducing activity of (+)-halichonine B.

Halichonine B is a sesquiterpene alkaloid isolated from the marine sponge Halichondria okadai Kadota. Halichonine B has exhibited cytotoxicity against mammalian cancer cells and induced apoptosis in the human leukemia cell line HL60. Here we established a practical route for the synthesis of halichonine B and its analogues, and we evaluated their biological activities. It was revealed that the secondary amino groups in the side chain portion are important for the strong cytotoxicity of halichonine B and that the N(11)-prenyl group is unimportant. Halichonine B and its analogues were also observed to induce apoptosis in HL60 cells.

Design, synthesis, and biological evaluation of the analogues of glaziovianin A, a potent antitumor isoflavone.

Various analogues of glaziovianin A, an antitumor isoflavone, were synthesized, and their biological activities were evaluated. O(7)-modified glaziovianin A showed strong cytotoxicity against HeLa S(3) cells. Compared to glaziovianin A, the O(7)-benzyl and O(7)-propargyl analogues were more cytotoxic against HeLa S(3) cells and more potent M-phase inhibitors. Furthermore, O(7)-modified molecular probes of glaziovianin A were synthesized for biological studies.

Total synthesis of (-)-13-oxyingenol and its natural derivative.

Ring functionalization: the total synthesis of a natural derivative of (-)-13-oxyingenol, a potent anti-HIV diterpenoid, is reported. The key steps in this synthesis include a ring-closing olefin metathesis and a Mislow-Evans-type [2,3]-sigmatropic rearrangement. This synthesis provides access to (-)-13-oxyingenol and its natural derivative in 21 steps from a synthetic intermediate previously prepared by Kigoshi and co-workers.

DS-7300a, a DNA Topoisomerase I Inhibitor, DXd-Based Antibody-Drug Conjugate Targeting B7-H3, Exerts Potent Antitumor Activities in Preclinical Models.

B7-H3 is overexpressed in various solid tumors and has been considered as an attractive target for cancer therapy. Here, we report the development of DS-7300a, a novel B7-H3-targeting antibody-drug conjugate with a potent DNA topoisomerase I inhibitor, and its in vitro profile, pharmacokinetic profiles, safety profiles, and in vivo antitumor activities in nonclinical species. The target specificity and species cross-reactivity of DS-7300a were assessed. Its pharmacologic activities were evaluated in several human cancer cell lines in vitro and xenograft mouse models, including patient-derived xenograft (PDX) mouse models in vivo. Pharmacokinetics was investigated in cynomolgus monkeys. Safety profiles in rats and cynomolgus monkeys were also assessed. DS-7300a specifically bound to B7-H3 and inhibited the growth of B7-H3-expressing cancer cells, but not that of B7-H3-negative cancer cells, in vitro. Additionally, treatment with DS-7300a and DXd induced phosphorylated checkpoint kinase 1, a DNA damage marker, and cleaved PARP, an apoptosis marker, in cancer cells. Moreover, DS-7300a demonstrated potent in vivo antitumor activities in high-B7-H3 tumor xenograft models, including various tumor types of high-B7-H3 PDX models. Furthermore, DS-7300a was stable in circulation with acceptable pharmacokinetic profiles in monkeys, and well tolerated in rats and monkeys. DS-7300a exerted potent antitumor activities against B7-H3-expressing tumors in in vitro and in vivo models, including PDX mouse models, and showed acceptable pharmacokinetic and safety profiles in nonclinical species. Therefore, DS-7300a may be effective in treating patients with B7-H3-expressing solid tumors in a clinical setting.

Structure-activity relationship study of glaziovianin A against cell cycle progression and spindle formation of HeLa S3 cells.

Various derivatives of glaziovianin A, an antitumor isoflavone, were synthesized, and the cytotoxicity of each against HeLa S3 cells was investigated. Compared to glaziovianin A, the O7-allyl derivative was found to be more cytotoxic against HeLa S3 cells and a more potent M-phase inhibitor.

Quantitative analysis of the Tricholoma ustale-derived toxin, ustalic acid, in mushroom and food samples by LC-MS/MS.

Tricholoma ustale, a poisonous member of the Tricholomataceae family, causes gastrointestinal symptoms such as diarrhea and vomiting. In Japan, 86 cases (affecting a total of 347 patients) of poisoning with Tricholoma ustale have been reported between 1989 and 2010. Ustalic acid is one of the primary toxic components in Tricholoma ustale. In the present study, the quantitative analysis of the ustalic acid content in mushroom and food samples was conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mushroom and food samples were extracted using methanol containing 0.5% formic acid and 50% aqueous methanol, respectively. Purification using SAX solid-phase extraction (SPE) was conducted prior to LC-MS/MS analysis, which was performed in the ESI negative mode using a C18 column. The method developed for the LC-MS/MS analysis of ustalic acid was extremely sensitive. The limits of quantitation calculated at a signal-to-noise ratio of 10 were 10ng/g (shiitake mushroom) and 0.40ng/g (miso soup). The accuracies of quantitation in the shiitake mushroom and miso soup samples ranged from 99.8%-105% and 98.8%-102%, respectively. This method was applied to leftover mushroom samples from a food poisoning case; here, ustalic acid was detected at 0.57, 3.7µg/g. This analytical method using LC-MS/MS could be useful in food poisoning cases involving mushrooms. This is the first report in which the ustalic acid content was determined using the leftovers of a food poisoning case.

Dual Inhibition of γ-Tubulin and Plk1 Induces Mitotic Cell Death.

α/ß-Tubulin inhibitors that alter microtubule (MT) dynamics are commonly used in cancer therapy, however, these inhibitors also cause severe side effects such as peripheral neuropathy. γ-Tubulin is a possible target as antitumor drugs with low side effects, but the antitumor effect of γ-tubulin inhibitors has not been reported yet. In this study, we verified the antitumor activity of gatastatin, a γ-tubulin specific inhibitor. The cytotoxicity of gatastatin was relatively weak compared with that of the conventional MT inhibitors, paclitaxel and vinblastine. To improve the cytotoxicity, we screened the chemicals that improve the effects of gatastatin and found that BI 2536, a Plk1 inhibitor, greatly increases the cytotoxicity of gatastatin. Co-treatment with gatastatin and BI 2536 arrested cell cycle progression at mitosis with abnormal spindles. Moreover, mitotic cell death induced by the combined treatment was suppressed by the Mps1 inhibitor, reversine. These findings suggest that co-treatment with Plk1 and γ-tubulin inhibitors causes spindle assembly checkpoint-dependent mitotic cell death by impairing centrosome functions. These results raise the possibility of Plk1 and γ-tubulin inhibitor co-treatment as a novel cancer chemotherapy.

Structure Optimization of Gatastatin for the Development of γ-Tubulin-Specific Inhibitor.

Gatastatin (O 7-benzyl glaziovianin A) is a γ-tubulin-specific inhibitor that is used to investigate γ-tubulin function in cells. We have previously reported that the unsubstituted phenyl ring of the O 7-benzyl group in gatastatin is important for γ-tubulin inhibition. To obtain further structural information regarding γ-tubulin inhibition, we synthesized several gatastatin derivatives containing a fixed O 7-benzyl moiety. Modifications of the B-ring resulted in drastic decrease in cytotoxicity, abnormal spindle formation activity, and inhibition of microtubule (MT) nucleation. In contrast, various O 6-alkylated gatastatin derivatives showed potent cytotoxicity, induced abnormal spindle formation, and inhibited MT nucleation. We had previously reported that O 6-benzyl glaziovianin A is a potent α/ß-tubulin inhibitor; thus, these new results suggest that the O 6-position restricts affinity for α/ß- and γ-tubulin. Considering that an O 7-benzyl group increases specificity for γ-tubulin, more potent and specific γ-tubulin inhibitors can be generated through O 6-modifications of gatastatin.

Total synthesis and cytotoxicity of haterumalides NA and B and their artificial analogues.

The total synthesis of haterumalides NA and B, potent cytotoxic marine macrolides, was achieved by using B-alkyl Suzuki-Miyaura coupling and Nozaki-Hiyama-Kishi coupling as key steps. Compared to our first-generation approach for ent-haterumalide NA methyl ester, this second-generation synthesis yielded much more of the key intermediate. This synthesis established the relative stereochemistry of haterumalide B. Furthermore, the structure-cytotoxicity relationships of haterumalides were investigated. The combination of macrolide and side chain parts proved to be important to the cytotoxicity.

Catalytic enantioselective Hosomi-Sakurai reaction of α-ketoesters promoted by chiral copper(ii) complexes.

A catalytic enantioselective Hosomi-Sakurai reaction of α-ketoesters has been developed. A copper(ii) complex with a chiral bis(oxazoline) ligand bearing methanesulfonamide groups shows excellent catalytic activity to give α,α-disubstituted α-hydroxyesters in high yields with high enantioselectivities. This is the first successful method for the catalytic enantioselective 1,2-addition of α-ketoesters with allylic silanes.

Toward the Synthesis of Yuzurimine-Type Alkaloids: Stereoselective Construction of the Heterocyclic Portions of Deoxyyuzurimine and Macrodaphnine.

The heterocyclic portions of yuzurimine-type alkaloids, such as deoxyyuzurimine and macrodaphnine, were synthesized by using a stereoselective hydroboration-oxidation reaction to install the C20 methyl group, the intramolecular Mitsunobu reaction to construct the E-ring portion, and the intramolecular SN2 reaction to construct the F-ring portion as key steps.

A Novel HER3-Targeting Antibody-Drug Conjugate, U3-1402, Exhibits Potent Therapeutic Efficacy through the Delivery of Cytotoxic Payload by Efficient Internalization.

PURPOSE: HER3 is a compelling target for cancer treatment; however, no HER3-targeted therapy is currently clinically available. Here, we produced U3-1402, an anti-HER3 antibody-drug conjugate with a topoisomerase I inhibitor exatecan derivative (DXd), and systematically investigated its targeted drug delivery potential and antitumor activity in preclinical models. EXPERIMENTAL DESIGN: In vitro pharmacologic activities and the mechanisms of action of U3-1402 were assessed in several human cancer cell lines. Antitumor activity of U3-1402 was evaluated in xenograft mouse models, including patient-derived xenograft (PDX) models. Safety assessments were also conducted in rats and monkeys. RESULTS: U3-1402 showed HER3-specific binding followed by highly efficient cancer cell internalization. Subsequently, U3-1402 was translocated to the lysosome and released its payload DXd. While U3-1402 was able to inhibit HER3-activated signaling similar to its naked antibody patritumab, the cytotoxic activity of U3-1402 in HER3-expressing cells was predominantly mediated by released DXd through DNA damage and apoptosis induction. In xenograft mouse models, U3-1402 exhibited dose-dependent and HER3-dependent antitumor activity. Furthermore, U3-1402 exerted potent antitumor activity against PDX tumors with HER3 expression. Acceptable toxicity was noted in both rats and monkeys. CONCLUSIONS: U3-1402 demonstrated promising antitumor activity against HER3-expressing tumors with tolerable safety profiles. The activity of U3-1402 was driven by HER3-mediated payload delivery via high internalization into tumor cells.