[Stereo-divergent Synthesis of Nonproteinogenic Amino Acids and Synthetic Study for Biologically Active Cyclopeptides].

Naturally occurring cyclopeptides are potential middle-molecule drug candidates beyond Lipinski's rule of five. This paper focuses on the structural determination and structure-activity relationship (SAR) study of two cyclopeptides: asperterrestide A and decatransin. The proposed asperterrestide A was synthesized by solution-phase peptide elongation, followed by macrolactamization. NMR analysis and molecular modeling studies revealed the stereochemistry at the two α-positions of amino acid residues as opposite to each other. This was further confirmed by the total synthesis of the revised asperterrestide A. SAR study of synthetic products revealed that the ß-hydroxy group in the nonproteinogenic amino acid residue was not essential for its cytotoxicity. In addition, N-alkyl-enriched peptide fragments of decatransin were synthesized in solution-phase without diketopiperadine formation. The putative candidates of decatransin was synthesized by convergent peptide coupling, followed by macrocyclization under modified Mitsunobu conditions. The structure of the natural decatransin, including its absolute configuration, was determined through a comparison of spectral data and the cytotoxicity exhibited by the synthetic products.

Synthesis and Biological Evaluation of Lysophosphatidic Acid Analogues Using Conformational Restriction and Bioisosteric Replacement Strategies.

Lysophosphatidic acid (LPA) is a key player in many physiological and pathophysiological processes. The biological activities of LPA are mediated through interactions with-at least-six subtypes of G-protein-coupled receptors (GPCRs) named LPA1-6. Developing a pharmacological tool molecule that activates LPA subtype receptors selectively will allow a better understanding of their specific physiological roles. Here, we designed and synthesized conformationally restricted 25 1-oleoyl LPA analogues MZN-001 to MZN-025 by incorporating its glycerol linker into dihydropyran, tetrahydropyran, and pyrrolidine rings and variating the lipophilic chain. The agonistic activities of these compounds were evaluated using the TGFα shedding assay. Overall, the synthesized analogues exhibited significantly reduced agonistic activities toward LPA1, LPA2, and LPA6, while demonstrating potent activities toward LPA3, LPA4, and LPA5 compared to the parent LPA. Specifically, MZN-010 showed more than 10 times greater potency (EC50 = 4.9 nM) than the standard 1-oleoyl LPA (EC50 = 78 nM) toward LPA5 while exhibiting significantly lower activity on LPA1, LPA2, and LPA6 and comparable potency toward LPA3 and LPA4. Based on the MZN-010 scaffold, we synthesized additional analogues with improved selectivity and potency toward LPA5. Compound MZN-021, which contains a saturated lipophilic chain, exhibited 50 times more potent activity (EC50 = 1.2 nM) than the natural LPA against LPA5 with over a 45-fold higher selectivity when compared to those of other LPA receptors. Thus, MZN-021 was found to be a potent and selective LPA5 agonist. The findings of this study could contribute to broadening the current knowledge about the stereochemical and three-dimensional arrangement of LPA pharmacophore components inside LPA receptors and paving the way toward synthesizing other subtype-selective pharmacological probes.

Total Synthesis and Structural Determination of Cyclodepsipeptide Decatransin.

The structure determination of the 30-membered cyclodepsipeptide decatransin was demonstrated on the basis of total synthesis. Both (R)- and (S)-2-hydroxy-5-methylhexanoic acid derivatives were prepared via the Evans asymmetric alkylation. N-Alkyl-enriched peptide fragments were synthesized by the Cbz strategy in the solution phase without formation of diketopiperazine and epimerization. The synthesis of putative candidates was achieved by convergent peptide coupling of three peptide fragments, followed by macrocyclization under the Mitsunobu conditions.

A Phenylfurocoumarin Derivative Reverses ABCG2-Mediated Multidrug Resistance In Vitro and In Vivo.

The ATP-binding cassette subfamily G member 2 (ABCG2) transporter is involved in the development of multidrug resistance in cancer patients. Many inhibitors of ABCG2 have been reported to enhance the chemosensitivity of cancer cells. However, none of these inhibitors are being used clinically. The aim of this study was to identify novel ABCG2 inhibitors by high-throughput screening of a chemical library. Among the 5812 compounds in the library, 23 compounds were selected in the first screening, using a fluorescent plate reader-based pheophorbide a (PhA) efflux assay. Thereafter, to validate these compounds, a flow cytometry-based PhA efflux assay was performed and 16 compounds were identified as potential inhibitors. A cytotoxic assay was then performed to assess the effect these 16 compounds had on ABCG2-mediated chemosensitivity. We found that the phenylfurocoumarin derivative (R)-9-(3,4-dimethoxyphenyl)-4-((3,3-dimethyloxiran-2-yl)methoxy)-7H-furo [3,2-g]chromen-7-one (PFC) significantly decreased the IC50 of SN-38 in HCT-116/BCRP colon cancer cells. In addition, PFC stimulated ABCG2-mediated ATP hydrolysis, suggesting that this compound interacts with the substrate-binding site of ABCG2. Furthermore, PFC reversed the resistance to irinotecan without causing toxicity in the ABCG2-overexpressing HCT-116/BCRP cell xenograft mouse model. In conclusion, PFC is a novel inhibitor of ABCG2 and has promise as a therapeutic to overcome ABCG2-mediated MDR, to improve the efficiency of cancer chemotherapy.

Stereoselective Synthesis of 1-Aminocyclopropanecarboxylic Acid Carnosadines via Inter-intramolecular Double Alkylation with Optically Active 2-Methylaziridine Derivatives.

The stereoselective and short-step synthesis of N-protected allo-carnosadine, ent-carnosadine, and carnosadine lactam was accomplished from a common cyclopropane intermediate. The inter-intramolecular double alkylation of diethyl malonate with an optically active 2-methylaziridine derivative gave the key cyclopropane in excellent yield and optical purity. The following monohydrolysis of the diester moiety using different reaction conditions provided both diastereomers of monoacids, which were converted to three carnosadine derivatives in 5-6 steps from the common diester.

Gold-Catalyzed Amide/Carbamate-Linked N,O-Acetal Formation with Bulky Amides and Alcohols.

A gold-catalyzed N,O-acetal formation was established to construct an amide/carbamate-linked N,O-acetal substructure with bulky alcohols. The acyliminium cation species generated from o-alkynylbenzoic acid ester in the presence of a gold catalyst is highly reactive and underwent nucleophilic attack of various bulky alcohols and phenols at room temperature under neutral conditions, leading to the corresponding N,O-acetals in yields of 34-89% with good functional group tolerance.

C-Methylation of S-adenosyl-L-Methionine Occurs Prior to Cyclopropanation in the Biosynthesis of 1-Amino-2-Methylcyclopropanecarboxylic Acid (Norcoronamic Acid) in a Bacterium.

Many pharmacologically important peptides are bacterial or fungal in origin and contain nonproteinogenic amino acid (NPA) building blocks. Recently, it was reported that, in bacteria, a cyclopropane-containing NPA 1-aminocyclopropanecarboxylic acid (ACC) is produced from the L-methionine moiety of S-adenosyl-L-methionine (SAM) by non-canonical ACC-forming enzymes. On the other hand, it has been suggested that a monomethylated ACC analogue, 2-methyl-ACC (MeACC), is derived from L-valine. Therefore, we have investigated the MeACC biosynthesis by identifying a gene cluster containing bacterial MeACC synthase genes. In this gene cluster, we identified two genes, orf29 and orf30, which encode a cobalamin (B12)-dependent radical SAM methyltransferase and a bacterial ACC synthase, respectively, and were found to be involved in the MeACC biosynthesis. In vitro analysis using their recombinant enzymes (rOrf29 and rOrf30) further revealed that the ACC structure of MeACC was derived from the L-methionine moiety of SAM, rather than L-valine. In addition, rOrf29 was found to catalyze the C-methylation of the L-methionine moiety of SAM. The resulting methylated derivative of SAM was then converted into MeACC by rOrf30. Thus, we demonstrate that C-methylation of SAM occurs prior to cyclopropanation in the biosynthesis of a bacterial MeACC (norcoronamic acid).

Total Synthesis and Structural Revision of Cyclotetrapeptide Asperterrestide A.

The structural revision of cyclotetrapeptide asperterrestide A has been achieved based on total synthesis and molecular modeling. For these studies, (2 R,3 S)-MePhe(3-OH) and (2 S,3 S)-MePhe(3-OH) suitably protected for peptide synthesis were prepared via a stereoselective reduction of a ketone precursor derived from L- or d-serine, using L-selectride or DIBAL-H. The synthesis of the proposed structure of asperterrestide A (1a) was accomplished by solution-phase synthesis of a linear precursor followed by macrolactamization. The NMR spectra of our synthetic 1a were not identical to those reported for the natural compound. Molecular modeling studies suggested that the correct structure 1b was the one in which the stereochemistry at the α-positions of the Ala and MePhe(3-OH) residues is the opposite to that of the proposed structure. This was confirmed by the total synthesis of 1b and its subsequent structural characterization.

Synthesis and biological evaluation of thielocin B1 analogues as protein-protein interaction inhibitors of PAC3 homodimer.

The synthesis and biological evaluation of thielocin B1 analogues have been demonstrated. Fourteen analogues modified in the central core and terminal carboxylic acid moiety were concisely synthesized by simple esterification or etherification reaction. The evaluation of synthetic analogues as inhibitors of proteasome assembling chaperone (PAC) complexes (the PAC3 homodimer and PAC1/PAC2) revealed that the natural product-like bending structure and terminal carboxylic acid groups were crucial for its biological activity. Moreover, SAR and in silico docking studies indicated that all methyl groups on the diphenyl ether moiety of thielocin B1 contribute to the potent and selective inhibition of the PAC3 homodimer via hydrophobic interactions.

Novel Potent ABCB1 Modulator, Phenethylisoquinoline Alkaloid, Reverses Multidrug Resistance in Cancer Cell.

ATP-binding cassette (ABC) transporters, which are concerned with the efflux of anticancer drugs from cancer cells, have a pivotal role in multidrug resistance (MDR). In particular, ABCB1 is a well-known ABC transporter that develops MDR in many cancer cells. Some ABCB1 modulators can reverse ABCB1-mediated MDR; however, no modulators with clinical efficacy have been approved. The aim of this study was to identify novel ABCB1 modulators by using high-throughput screening. Of the 5861 compounds stored at Tohoku University, 13 compounds were selected after the primary screening via a fluorescent plate reader-based calcein acetoxymethylester (AM) efflux assay. These 13 compounds were validated in a flow cytometry-based calcein AM efflux assay. Two isoquinoline derivatives were identified as novel ABCB1 inhibitors, one of which was a phenethylisoquinoline alkaloid, (±)-7-benzyloxy-1-(3-benzyloxy-4-methoxyphenethyl)-1,2,3,4-tetrahydro-6-methoxy-2-methylisoquinoline oxalate. The compound, a phenethylisoquinoline alkaloid, was subsequently evaluated in the cytotoxicity assay and shown to significantly enhance the reversal of ABCB1-mediated MDR. In addition, the compound activated the ABCB1-mediated ATP hydrolysis and inhibited the photolabeling of ABCB1 with [125I]-iodoarylazidoprazosin. Furthermore, the compound also reversed the resistance to paclitaxel without increasing the toxicity in the ABCB1-overexpressing KB-V1 cell xenograft model. Overall, we concluded that the newly identified phenethylisoquinoline alkaloid reversed ABCB1-mediated MDR through direct interaction with the substrate-binding site of ABCB1. These findings may contribute to the development of more potent and less toxic ABCB1 modulators, which could overcome ABCB1-mediated MDR.

Discovery of Novel Cinchona-Alkaloid-Inspired Oxazatwistane Autophagy Inhibitors.

The cinchona alkaloids are a privileged class of natural products and are endowed with diverse bioactivities. However, for compounds with the closely-related oxazatricyclo[4.4.0.0]decane ("oxazatwistane") scaffold, which are accessible from cinchonidine and quinidine by means of ring distortion and modification, biological activity has not been identified. We report the synthesis of an oxazatwistane compound collection through employing state-of-the-art C-H functionalization, and metal-catalyzed cross-coupling reactions as key late diversity-generating steps. Exploration of oxazatwistane bioactivity in phenotypic assays monitoring different cellular processes revealed a novel class of autophagy inhibitors termed oxautins, which, in contrast to the guiding natural products, selectively inhibit autophagy by inhibiting both autophagosome biogenesis and autophagosome maturation.

A direct and mild formylation method for substituted benzenes utilizing dichloromethyl methyl ether-silver trifluoromethanesulfonate.

A silver trifluoromethanesulfonate (AgOTf)-promoted direct and mild formylation of benzenes has been developed. The reaction utilizing dichloromethyl methyl ether (Cl2CHOMe) and AgOTf powerfully formylated various substituted benzenes under temperature conditions as low as -78 °C without losing the protecting groups on the phenolic hydroxyl group.