Addition of hydrophobic side chains improve the apoptosis inducibility of the human glyoxalase I inhibitor, TLSC702.

Glyoxalase I (GLO I) is a known therapeutic target in cancer. Even though TLSC702, a GLO I inhibitor that we discovered, induces apoptosis in tumor cells, exceptionally higher doses are required compared with those needed to inhibit GLO I activity in vitro. In this work, structure-activity optimization studies were conducted on four sections of the TLSC702 molecule to determine the partial structural features necessary for the inhibition of GLO I. Herein, we found that the carboxy group in TLSC702 was critical for binding with the divalent zinc at the active site of GLO I. In contrast, the side chain substituents in the meta- and para- positions of the benzene ring had little influence on the in vitro inhibition of GLO I. The CLogP values of the TLSC702 derivatives showed a positive correlation with the antiproliferative effects on NCI-H522 cells. Thus, two derivatives of TLSC702, which displayed either high or low lipophilicity due to the types of substituents at the phenyl position, were selected. Even though both derivatives showed comparable inhibitory effects as that of their parent compound, the derivative with the high CLogP value was distinctly more antiproliferative than TLSC702. In contrast, the derivative with the low CLogP value did not decrease cell viability in NCI-H522 and HL-60 cells. These findings suggested that structural improvements, such as the addition of hydrophobic moieties to the phenyl group, enhanced the ability of TLSC702 to induce apoptosis by increasing cell membrane permeability.

Polyborane-encapsulated PEGylated Liposomes Prepared Using Post-insertion Technique for Boron Neutron Capture Therapy.

PEGylated liposomes are one of the useful boron carriers for boron neutron capture therapy (BNCT). Recently, a method of adding PEG after liposome formation (post-insertion) was reported. In this study, we prepared polyborane-encapsulated PEGylated liposomes for BNCT with half the amount of DSPE-PEG of the conventional method using post-insertion technique (post-PEG liposomes), and their usefulness were evaluated in comparison with conventional PEGylated liposomes (pre-PEG liposomes). From the results of physicochemical property measurements, it was confirmed that particle size distributions, surface charge densities, and fixed aqueous layer thicknesses of these liposomes were equivalent. In vitro cytotoxicity and cell uptake tests were also carried out using B16 melanoma and RAW264.7 cells. Polyborane solution and bare liposomes were used for comparison. From the results of these tests, we confirmed that post-PEG liposomes and pre-PEG liposomes have the same influence of PEGylation. To evaluate biodistribution properties at 24 h post-administration, these liposomes and polyborane solution were injected into the tail veins of tumor-bearing mice. Boron concentration and tumor/blood ratios of PEGylated liposomes were 73.2-77.6 µg/g of tumor tissue and 5.5-5.8, respectively. From these results, it was found that by using post-insertion technique, liposomes for BNCT having same effect as the liposome prepared using the conventional method can be prepared with half amount of DSPE-PEG.

Synthesis of 2,2-Dialkyl Chromanes by Intramolecular Ullmann C-O Coupling Reactions toward the Total Synthesis of D-α-Tocopherol.

The complete synthesis of D-α-tocopherol was achieved using our developed-Ullmann C-O coupling reaction as a key reaction. The synthesis of the core structure of D-α-tocopherol, which is a chiral chromane, has never been reported using intramolecular Ullmann C-O coupling reactions owing to the low reactivity of electron-rich iodoarenes with tertiary alcohols. Because the developed intramolecular C-O coupling reactions prefer electron-rich iodoarenes with tertiary alcohols, we successfully synthesized the chiral chromane core and achieved the total synthesis of D-α-tocopherol.

Decarboxylation of indomethacin induced by heat treatment.

As crystalline indomethacin is heated and subsequently cooled, it transforms into glassy indomethacin. While the original crystals are off-white in color, the glass becomes blackish-brown via a yellow intermediate stage. TLC of the components of the glass revealed three bands. The yellow component, which is generated either under hypoxic conditions or in the dark, was elucidated by NMR spectroscopy to be a decarboxylated fragment produced by thermal degradation. The colorless component is proposed to be formed by the opening of the indole ring of indomethacin; the structure of this degradation product was identified by EI-MS to be the same as the oxidative-cleavage product formed upon UV-irradiation, as previously reported. Another band was a blackish-brown pigment whose mobility placed it close to the TLC baseline. This oxidative-cleavage product and the blackish-brown pigment are not generated under hypoxic conditions. However, the extent of indomethacin decarboxylation under hypoxic conditions was found to be dependent on the heating temperature and time. Consequently, we prepared amorphous indomethacin through control of the heating temperature and time; heating at 160 °C for 30 min or less under hypoxic conditions is optimum for obtaining pure amorphous indomethacin.

Total Synthesis of GKK1032A2 via Direct 13-Membered Macrocyclization Using a Nucleophilic Aromatic Substitution of an (η6 -Arene)Chromium Complex.

The first enantioselective total synthesis of GKK1032A2 has been achieved. The key step is a direct construction of the highly strained 13-membered macrocycle of GKK1032A2 by an intramolecular nucleophilic aromatic substitution (SN Ar) reaction. This is the first successful example of construction of a macrocycle with an aryl ether linkage utilizing an intramolecular SN Ar reaction of an (η6 -arene)chromium complex.

TLSC702, a Novel Inhibitor of Human Glyoxalase I, Induces Apoptosis in Tumor Cells.

Human glyoxalase I (hGLO I) is a rate-limiting enzyme in the pathway for detoxification of apoptosis-inducible methylglyoxal (MG), which is the side product of tumor-specific aerobic glycolysis. GLO I has been reported to be overexpressed in various types of cancer cells, and has been expected as an attractive target for the development of new anticancer drugs. We previously discovered a novel inhibitor of hGLO I, named TLSC702, by our in silico screening method. Here, we show that TLSC702 inhibits the proliferation of human leukemia HL-60 cells and induces apoptosis in a dose-dependent manner. In addition, TLSC702 more significantly inhibits the proliferation of human lung cancer NCI-H522 cells, which highly express GLO I, than that of GLO I lower-expressing human lung cancer NCI-H460 cells. Furthermore, this antiproliferative effect of TLSC702 on NCI-H522 cells is in a dose- and time-dependent manner. These results suggest that TLSC702 can induce apoptosis in tumor cells by GLO I inhibition, which lead to accumulation of MG. Taken together, TLSC702 could become a unique seed compound for the generation of novel chemotherapeutic drugs targeting GLO I-dependent human tumors.

Total synthesis of diaporthichalasin by using the intramolecular Diels-Alder reaction of an α,ß-unsaturated γ-hydroxylactam in aqueous media.

The first total synthesis of diaporthichalasin has been successfully achieved and complete structure elucidation, including the absolute configuration, was also accomplished. The intramolecular Diels-Alder (IMDA) reaction between the diene side chain on the decalin skeleton and α,ß-unsaturated γ-hydroxy-γ-lactam in aqueous media was effectively employed as the key step. From this synthetic study, we found that α,ß-unsaturated γ-hydroxy-γ-lactam is an essential precursor for the construction of the diaporthichalasin-type pentacyclic skeleton. This important finding strongly suggests that this route is involved in the biosynthetic pathway for diaporthichalasin.

Characteristics of amorphous complex formed between indomethacin and lidocaine hydrochloride.

Indomethacin (IM) easily forms an amorphous complex with lidocaine (LC) by heat treatment. To know the mechanism involved in the formation of this complex, we studied temperature-dependent phase changes in mixtures of IM and lidocaine hydrochloride (LH), in which the cationic form of LC forms a salt with Cl(-), in various molar ratios by using DSC and NMR. Although heating of the mixture of IM and LC (IM+LC), formed a eutectic mixture, that of IM and LH (IM+LH) did not, and IM in the IM+LH mixture was dissolved into fused LH. Cooling of the fused IM+LH showed the glass transition in all of the samples containing various amounts of IM, suggesting that fused IM+LH took a homogenous amorphous state (IM/LH) below its glass transition temperature, in contrast to the fused IM+LC, which formed the rubber state and/or glass state depending on the molar content of IM. The results of the NMR study showed that IM in IM/LH caused the electronic structure of LH to change in such a way as to become similar to that of LC, but this effect was limited. Hence, mode of interaction of LH with IM is different from that of LC with IM.

Features of heat-induced amorphous complex between indomethacin and lidocaine.

We studied temperature-dependent phase changes in the mixture of indomethacin (IM) and lidocaine (LC) in various molar ratios by differential scanning calorimetry (DSC). DSC studies were carried out between -40°C and 180°C heating/cooling/heating cycle at a rate of 10°C/min under a nitrogen gas flow. Although LC in the liquid state was crystallized, fused IM showed a glass transition signal upon cooling. Hence, cooling of fused IM caused it to assume the amorphous state below its glass transition temperature. Heating of the mixture of IM and LC resulted in formation of a eutectic mixture between them, and cooling of the fused mixture caused formation of the amorphous state at any molar ratio examined. It is noteworthy that the amorphous solid of the fused complex of IM and LC was in the rubber state and glass state in the mixtures containing IM between 20% and 40%, whereas there was only the glass state in the samples containing 50% IM and higher. In addition, crystallization of LC took place by cooling the fused sample containing 10% IM.

SUTAF, a novel ß-methoxyacrylate derivative, promotes neurite outgrowth with extracellular signal-regulated kinase and c-jun N-terminal kinase activation.

ß-Methoxyacrylate antibiotics are well known to inhibit the fungal and yeast mitochondrial respiratory chain. In addition, ß-methoxyacrylates are reported to suppress the proliferation of mammalian cancer cells. Differentiation and cell-cycle arrest are closely related. The cell cycle of proliferating cells is suppressed before differentiation. In this study, we synthesized a ß-methoxyacrylate analog and treated neuronal differential model cells with it. We then estimated ß-methoxyacrylate's neurotrophic effect by inhibiting cell proliferation so as to orient neuronal differentiation. SUTAF-027-a novel ß-methoxyacrylate derivative, arrested the cell cycle and thereby suppressed the proliferation of PC12 rat pheochromocytoma cells and mouse neuroblastoma Neuro2a cells at very low treatment doses, as low as 1nM. However, a single SUTAF-027 treatment did not affect neuritogenesis. Surprisingly, however, co-treatment of SUTAF-027 and nerve growth factor (NGF) significantly augmented the NGF-induced neurite outgrowth of PC12. On the other hand, a single treatment of 1nM SUTAF-027 induced neurite outgrowth in Neuro2a cells. Further signal transduction mechanism studies revealed that SUTAF-027 induced the phosphorylation of extracellular signal-regulated kinase (ERK) and slight phosphorylation of c-jun N-terminal kinase (JNK). Moreover, inhibition of ERK and JNK blocked SUTAF-027-augmented neurite outgrowth. These results suggested that the novel ß-methoxyacrylate analog SUTAF-027 augmented neurite outgrowth by arresting the cell cycle and activating the ERK and JNK pathways.

A novel convergent method for the synthesis of α-acyl-γ-hydroxylactams and its application in the total synthesis of PI-090 and 091.

A novel convergent method for the synthesis of α-acyl-γ-hydroxylactams utilizing the aldol reaction of N-Boc-protected γ-methoxylactams was developed. As the first application of this method for the synthesis of biologically active natural products, the total synthesis of platelet aggregation inhibitors PI-090 and PI-091 were also investigated and successfully achieved.

Total synthesis of hirsutellone B via Ullmann-type direct 13-membered macrocyclization.

Total synthesis of Hirsutellone B has been achieved by a convergent synthetic strategy. This synthesis features direct construction of the highly strained 13-membered macrocycle of Hirsutellone B utilizing the Ullmann-type reaction. To the best of our knowledge, this is the first application of macrocyclization utilizing an intramolecular Ullmann-type reaction between an aliphatic alcohol and aryl halide.

Discovery of a new type inhibitor of human glyoxalase I by myricetin-based 4-point pharmacophore.

The human glyoxalase I (hGLO I), which is a rate-limiting enzyme in the pathway for detoxification of apoptosis-inducible methylglyoxal (MG), has been expected as an attractive target for the development of new anti-cancer drugs. We have previously identified a natural compound myricetin as a substrate transition-state (Zn(2+)-bound MG-glutathione (GSH) hemithioacetal) mimetic inhibitor of hGLO I. Here, we constructed a hGLO I/inhibitor 4-point pharmacophore based on the binding mode of myricetin to hGLO I. Using this pharmacophore, in silico screening of chemical library was performed by docking study. Consequently, a new type of compound, which has a unique benzothiazole ring with a carboxyl group, named TLSC702, was found to inhibit hGLO I more effectively than S-p-bromobenzylglutathione (BBG), a well-known GSH analog inhibitor. The computational simulation of the binding mode indicates the contribution of Zn(2+)-chelating carboxyl group of TLSC702 to the hGLO I inhibitory activity. This implies an important scaffold-hopping of myricetin to TLSC702. Thus, TLSC702 may be a valuable seed compound for the generation of a new lead of anti-cancer pharmaceuticals targeting hGLO I.

Delphinidin, a dietary anthocyanidin in berry fruits, inhibits human glyoxalase I.

Glyoxalase I (GLO I) is the rate-limiting enzyme for detoxification of methylglyoxal (MG), a side-product of glycolysis, which is able to induce apoptosis. Since GLO I is known to be highly expressed in the most tumor cells and little in normal cells, inhibitors of this enzyme has been expected to be new anticancer drugs. Here, we examined the inhibitory abilities to the human GLO I of anthocyanidins, such as delphinidin, cyanidin and pelargonidin. Among them, delphinidin was found to have the most potent inhibitory effect on human GLO I. Also, only delphinidin-induced apoptosis in HL-60 cells in a dose- and time-dependent manner. Furthermore, we determined a pharmacophore for delphinidin binding to the human GLO I by computational simulation analyses of the binding modes of delphinidin, cyanidin and pelargonidin to the enzyme hot spot. These results suggest that delphinidin could be a useful lead compound for the development of novel GLO I inhibitory anticancer drugs.

The first example of the stereoselective synthesis of 7 beta-carbamoyl-4,5alpha-epoxymorphinan via a novel and reactive gamma-lactone.

7beta-Carbamoyl-4,5alpha-epoxymorphinans 5 were stereoselectively synthesized from the 7alpha-carboxylate intermediate 3 in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and amines under reflux conditions in mesitylene via a novel and reactive gamma-lactone 7. These were the first examples of the stereoselective syntheses of 7beta-substituted 4,5alpha-epoxymorphinans. The mechanism of the reaction process was elucidated as follows: 1) epimerization of 7alpha-carboxylate 3, 2) intramolecular lactonization of 7beta-carboxylate 6, and 3) aminolysis of the resultant gamma-lactone 7. The aminolysis of the isolated reactive gamma-lactone 7 with allylamine and the alcoholysis with MeOH in the presence of NaBH(4) proceeded at room temperature. The gamma-lactone 7 can be a useful intermediate for the preparation of 7beta-substituted 4,5alpha-epoxymorphinans that would be potent selective delta opioid receptor ligands. The stereoselective syntheses of the 7alpha-carbamoyl-4,5alpha-epoxymorphinans 9 from 7alpha-carboxylate 3 via 7alpha-carboxylic acid were also successful.

Remarkable influence of the aromatic substructure in 9-methoxystrobilurin derivatives on their antifungal activity.

9-Methoxystrobilurin-type beta-methoxyacrylate antibiotics (MOSBs) having various aromatic substructures were synthesized. The antifungal activity of the synthesized MOSBs against pathogenic and non-pathogenic fungi was examined, and the obtained results revealed that the antifungal activity of MOSBs was highly dependent on the aromatic substructures. However, no significant correlation was observed between cytotoxicity against human fibroblasts-like cell line and their structural properties. In addition, our results suggested that the strong growth-inhibitory activity of 9-methoxystrobilurin K against human-derived cell lines should be related to its hindered ether-type substructure.

The first synthesis and antifungal activities of 9-methoxystrobilurin-type beta-substituted beta-methoxyacrylate.

The first synthesis of 9-methoxystrobilurin-type beta-substituted MOAs was successfully achieved. A chiral oudemansin-type beta-substituted MOA was also synthesized utilizing Mukaiyama's asymmetric aldol reaction. Antifungal activities of the synthesized compounds against several representative fungi were examined by disk-diffusion assay. As a result, unique and superior antifungal properties of 9-methoxystrobilurin-type beta-substituted MOAs compared with those of oudemansin-type analogue were clearly revealed.

A novel lipid compound, epolactaene, induces apoptosis: its action is modulated by its side chain structure.

A novel lipid compound, epolactaene, was isolated from the culture supernatant of Penicillium sp. 1689-P and it has already been reported that it induced neurite outgrowth in a human neuroblastoma cell line. In this study, we first investigated the effects of epolactaene on a human leukemia B-cell line, BALL-1 cells, and clarified that epolactaene induces apoptosis in BALL-1 cells in a dose- and time-dependent manner. Furthermore, we focused on the side chain structure of epolactaene, and chemically synthesized epolactaene derivatives. One derivative, which has a straight long alkyl chain as its side chain, induced apoptosis more effectively than epolactaene. On the other hand, other derivatives with a short alkyl side chain had weaker apoptosis-inducing actions. A good correlation was found between the apoptosis-inducing action of these compounds and their octanol/water partition coefficients (log P). These results suggested that the apoptosis-inducing activities of epolactaene and its derivatives were related to the hydrophobicity of these compounds; so that side chain structure of epolactaene is very important for its apoptosis-inducing activities. These apoptosis-inducing actions of epolactaene and its derivatives were also observed in various blood tumor cell lines and normal lymphocytes.