Aspects for development of novel antibacterial medicines using a vitamin D3 decomposition product in Helicobacter pylori infection.

A previous study by our group demonstrated that a vitamin D3 decomposition product (VDP1) acts as the selective bactericidal substance on Helicobacter pylori. VDP1 is an indene compound modified with a carbonyl and an alkyl. The alkyl of VDP1 turned out to be a mandatory structure to exert effective bactericidal action on H. pylori. Meanwhile, it still remains to be clarified as to how influence the alteration of the carbonyl in VDP1 has on the anti-H. pylori activity. In this study, we synthesized novel VDP1 derivatives that replaced the carbonyl of VDP1 by various functional groups and investigated the antibacterial action of the VDP1 derivatives on H. pylori. VDP1 derivatives retaining either a hydroxy (VD3-1) or an acetic ester (VD3-3) exhibited more effective bactericidal action to H. pylori than VDP1. The replacement of the carbonyl of VDP1 by either an allyl acetate (VD3-2) or an acrylic acid (VD3-5) provided almost no change to the anti-H. pylori activity. Apart from this, an isomer of VDP1 (VD3-4) slightly improved anti-H. pylori activity of VDP1. Meanwhile, the replacement of the carbonyl of VDP1 by a methyl acrylate (VD3-6) attenuated the anti-H. pylori activity. As with VDP1, its derivatives also were suggested to exert the anti-H. pylori action through the interaction with myristic acid side chains of dimyristoyl-phosphatidylethanolamine, a characteristic membrane lipid constituent of this pathogen. These results indicate that it is capable of developing specific antibacterial medicines for H. pylori targeting the biomembranal dimyristoyl-phosphatidylethanolamine using VDP1 as the fundamental structure.

A novel role of catalase in cholesterol uptake of Helicobacter pylori.

Helicobacter pylori infection is known to be a significant risk factor for the development of gastric cancers in humans. This pathogen exhibits unique biological characteristics in membrane lipid composition. Specifically, H. pylori incorporates exogenous cholesterol into biomembranes and uses cholesterol as the membrane lipid constituents. A previous study by our group demonstrated that phosphatidylethanolamine of H. pylori functions as the cholesterol-binding lipid. It is, however, unclear whether H. pylori is equipped with protein molecules involved in the cholesterol uptake. We, therefore, examined H. pylori proteins that tightly bind to cholesterol. As a consequence, H. pylori catalase (KatA) turned out to be a candidate of the cholesterol uptake-associated protein. In addition, an H. pylori mutant strain that expresses KatA protein lacking catalase activity was significantly lower in total cholesterol contents than the wild-type H. pylori strain. The putative amino acid sequence of KatA found out to contain a number of the cholesterol recognition/interaction amino acid consensus sequence domains (CRAC and CARC domains). These results suggest that H. pylori KatA with normal folding conformation acts as the cholesterol-binding or -storage protein.

A short review, effect of dimethyl-ß-cyclodextrin on the interaction between Helicobacter pylori and steroidal compounds.

The 2,6-di-O-methyl-ß-cyclodextrin (dMßCD) is an amphiphilic annular compound consisting of seven dimethyl-glucose molecules. This compound is well known as a solubilizer of lipophilic compounds. Especially, dMßCD extracts cholesterol from the plasma membrane of mammalian cells and releases the cholesterol to the aqueous solution. The experimental use of dMßCD, therefore, serves to investigate the role of cholesterol in the mammalian cell membrane. It is, however, unclear as to how dMßCD extracts cholesterol incorporated into the glycerophospholipid biomembrane. Meanwhile, dMßCD acts as a beneficial compound for Helicobacter pylori and is used as the standard component for supporting the growth of this bacterium in the serum-free culture. However, the detailed mechanism of dMßCD for supporting the growth of H. pylori is still to be clarified. H. pylori is a Gram-negative microaerophilic bacillus recognized as a pathogen concerned with gastrointestinal diseases in human. Previous studies by our group have successfully obtained the H. pylori strains culturable without dMßCD and demonstrated the distinct effects of dMßCD on the interaction between H. pylori and exogenous steroidal compounds. For instance, dMßCD promotes and inhibits the absorption of cholesterol and several steroidal compounds respectively into the biomembranes of H. pylori. In this study we summarized behaviors of dMßCD toward steroidal compounds relevant to H. pylori.

Efficient synthesis of 5-(hydroxymethyl)piperazin-2-ones using automatically prepared chiral bromocarboxylic acid and Garner's aldehyde as versatile building blocks.

An efficient method for the synthesis of substituted 5-(hydroxymethyl)piperazin-2-ones was established by using an automated synthesis process. Thirteen piperazinones were synthesized from chiral α-bromocarboxylic acids and Garner's aldehyde which were prepared by using our originally developed automated synthesizer, ChemKonzert®. The automated method of synthesizing chiral α-bromocarboxylic acids was efficient and safe because the rate of the dropwise addition of the reagent can be controlled using the automated synthesizer. This method is expected to contribute to the synthesis of pharmaceuticals.

Antibacterial effect of indene on Helicobacter pylori correlates with specific interaction between its compound and dimyristoyl-phosphatidylethanolamine.

Recent studies by our group have suggested that the vitamin D3 decomposition product VDP1 [(1R,3aR,7aR)-1-[(1R)-1,5-dimethylhexyl]octahydro-7a-methyl-4H-inden-4-one] confers the potent bactericidal action to Helicobacter pylori by targeting the membranal dimyristoyl-phosphatidylethanolamine (di-14:0 PE). In this study we synthesized a new VDP1 derivative to advance further investigation as for the correlative relationship between VDP1 structure and anti-H. pylori activity or PE vesicle collapse induction activity. The derivative VD3-7 [(1R,7aR)-4-fluoro-7a-methyl-1-((R)-6-methylheptan-2-yl)octahydro-1H-indene] retained a fluorine atom in place of the oxygen atom of VDP1. The fluorination of the carbonyl portion of VDP1 forfeited the effective anti-H. pylori activity. We, therefore, prepared Coomassie brilliant blue (CBB)-containing unilamellar vesicles consisting of various PE molecular species, and examined the vesicle collapse induction activity of either VDP1 or VD3-7 by detecting the CBB eluted from the PE unilamellar vesicles. VDP1 strongly induced CBB elution from the unilamellar vesicles of rectus-PE retaining the same two fatty acid side-chains shorter than carbon numbers 14, indicating that VDP1 specifically disrupted the vesicular conformation of those PE unilamellar vesicles. Meanwhile, VD3-7 had no influence on the structural stability of any PE unilamellar vesicles. This study obtained additional evidence that VDP1 acts as a bactericidal agent on H. pylori by targeting the membranal di-14:0 PE.

Synthesis of 3ß-tert-Butyldimethylsiloxy-22-phenylthio-23,24-bisnorchola-5,9(11)-diene and Reductive Nucleophilic Attack on a Branched Aliphatic Aldehyde.

3ß-tert-Butyldimethylsiloxy-22-phenylthio-23,24-bisnorchola-5,9(11)-diene, which has a double bond between C-9 and C-11 and a phenylsulfenyl group on the terminus of the side chain, is a potential synthetic intermediate for steroids with 9,11-unsaturation or 9,11-seco skeletons. We describe here the synthesis of the title compound from 17-ethylenedioxy-3-acetoxyandrosta-3,5-dien-11-one. The introduction of an ethylene unit to 3ß-tert-butyldimethylsiloxyandrosta-5,9(11)-dien-17-one by the action of ethyltriphenylphosphonium bromide under basic conditions resulted in an inseparable mixture of two stereoisomeric products (5 : 1). However, in the subsequent step, only the (Z)-isomer was susceptible to the Lewis acid-catalyzed ene reaction with formaldehyde, giving a stereochemically pure product with the desired configuration. Within three steps, the ene-product was derivatized to the title compound, with a total yield of 53% over seven steps. Reductive terminal anion formation by treatment with lithium di-tert-butylbiphenyl (LiDBB) and subsequent nucleophilic attack on a branched aliphatic aldehyde was demonstrated, with an eye toward the introduction of side chains, especially for steroids with oxygen functionality at C-23.

Synthesis of enantiomerically enriched drug precursors and an insect pheromone via reduction of ketones using commercially available carbonyl reductase screening kit "Chiralscreen® OH".

Commercially available "Chiralscreen® OH" starter kit containing five types of carbonyl reductases (E001, E007, E031, E039, and E078) was used for the reduction of several aromatic and aliphatic ketones to obtain enantiomerically enriched drug precursors and an insect pheromone. Almost stereochemically pure secondary alcohols, used in the synthesis of drugs such as (R)-rasagiline mesylate, (S)-rivastigmine, (R)-chlorphenesin carbamate, and (R)-mexiletine, and the insect pheromone (4S,5R)-sitophilure, were conveniently obtained. The enzymes worked well with ketones containing at least one non-bulky substituent at the carbonyl group. The diverse stereochemical preference of the above five carbonyl reductases was clarified.

Formal synthesis of englerin A utilizing regio- and diastereoselective [4+3] cycloaddition.

Englerin A, a guaiane sesquiterpene isolated from Phyllanthus engleri, showed highly potent and selective growth inhibitory activities against renal cancer cell lines. We synthesized the key tricyclic intermediate from commercially available 2,2-dimethyl-1,3-dioxan-5-one via regio- and diastereoselective [4+3] cycloaddition between the formyl enol silyl ether and the disubstituted furan, in 4.8% total yield over 10 steps.

Short-step syntheses of naturally occurring polyoxygenated aromatics based on site-selective transformation.

Wogonin and astringin were synthesized from inexpensive chrysin and piceid in short steps. The key feature of these syntheses is site-selective transformation. The target molecules were obtained in 27 and 62% yields from the starting materials, respectively.

Synthesis of Linezolid Metabolites PNU-142300 and PNU-142586 toward the Exploration of Metabolite-Related Events.

Linezolid (1) is an oxazolidinone antibiotic that is partially metabolized in vivo via ring cleavage of its morpholine moiety to mainly form two metabolites, PNU-142300 (2) and PNU-142586 (3). It is supposed that accumulation of 2 and 3 in patients with renal insufficiency may cause thrombocytopenia, one of the adverse effects of linezolid. However, the poor availability of 2 and 3 has hindered further investigation of the clinical significance of the accumulation of these metabolites. In this paper, we synthesized metabolites 2 and 3 via a common synthetic intermediate, 4; this will encourage further exploration of events related to these metabolites and lead to improved clinical use of linezolid.

Simple Synthesis of Sakuranetin and Selinone via a Common Intermediate, Utilizing Complementary Regioselectivity in the Deacetylation of Naringenin Triacetate.

Sakuranetin and selinone were successfully synthesized utilizing the regioselective deacetylation of naringenin triacetate. Deacetylation of the latter at C-7 with imidazole in 1,4-dioxane at 40°C furnished the corresponding diacetate in 80% yield. Methylation of the obtained free hydroxy group and subsequent removal of the remaining two acetyl groups gave sakuranetin, which was previously isolated as a phytoalexin against rice blast disease fungus, Pyricularia oryzae, in 71% overall yield. The same intermediate, naringenin triacetate, was subjected to transesterification with 2-propanol in tetrahydrofuran, catalyzed by Candida antarctica lipase B. A contrasting regioselective preference for C-4' deacetylation was observed, giving an isomeric diacetate in 82% yield. Prenylation of the free hydroxy group under Mitsunobu conditions and subsequent deprotection furnished selinone, which was previously isolated from Monotes engleri and exhibits antifungal activity against Candida albicans, in 55% overall yield.

Development of a novel sulfonate ester-based prodrug strategy.

A self-immolative γ-aminopropylsulfonate linker was investigated for use in the development of prodrugs that are reactive to various chemical or biological stimuli. To demonstrate their utility, a leucine-conjugated prodrug of 5-chloroquinolin-8-ol (5-Cl-8-HQ), which is a potent inhibitor against aminopeptidase from Aeromonas proteolytica (AAP), was synthesized. The sulfonate prodrug was considerably stable under physiological conditions, with only enzyme-mediated hydrolysis of leucine triggering the subsequent intramolecular cyclization to simultaneously release 5-Cl-8-HQ and form γ-sultam. It was also confirmed that this γ-aminopropylsulfonate linker was applicable for prodrugs of not only 8-HQ derivatives but also other drugs bearing a phenolic hydroxy group.

New synthesis of artepillin C, a prenylated phenol, utilizing lipase-catalyzed regioselective deacetylation as the key step.

We have synthesized artepillin C, a diprenylated p-hydroxycinnamate originally isolated from Brazilian propolis and exhibiting antioxidant and antitumor activities, from 2,6-diallylphenol. Replacement of the terminal vinyl with 2,2-dimethylvinyl group by olefin cross-metathesis and subsequent transformation yielded 2,6-diprenyl-1,4-hydroquinone diacetate. Candida antarctica lipase B-catalyzed deacetylation in 2-propanol regioselectively removed the less hindered acetyl group to give 2,6-diprenyl-1,4-hydroquinone 1-monoacetate. After triflation of the liberated 4-hydroxy group, a three-carbon side chain was introduced by palladium-mediated alkenylation with methyl acrylate. Final hydrolysis of the esters furnished artepillin C.

Synthesis and biological comparison of enantiomers of mepenzolate bromide, a muscarinic receptor antagonist with bronchodilatory and anti-inflammatory activities.

Chronic obstructive pulmonary disease (COPD) is characterized by abnormal inflammatory responses and airflow limitations. We recently proposed that the muscarinic antagonist mepenzolate bromide (mepenzolate) would be therapeutically effective against COPD due to its muscarinic receptor-dependent bronchodilatory activity as well as anti-inflammatory properties. Mepenzolate has an asymmetric carbon atom, thus providing us with the opportunity to synthesize both of its enantiomers ((R)- and (S)-mepenzolate) and to examine their biochemical and pharmacological activities. (R)- or (S)-mepenzolate was synthesized by condensation of benzilic acid with (R)- or (S)-alcohol, respectively, followed by quaternization of the tertiary amine. As predicted by computational simulation, a filter-binding assay in vitro revealed that (R)-mepenzolate showed a higher affinity for the muscarinic M3 receptor than (S)-mepenzolate. In vivo, the bronchodilatory activity of (R)-mepenzolate was superior to that of (S)-mepenzolate, whereas anti-inflammatory activity was indistinguishable between the two enantiomers. We confirmed that each mepenzolate maintained its original stereochemistry in the lung when administered intratracheally. These results suggest that (R)-mepenzolate may have superior properties to (S)-mepenzolate as a drug to treat COPD patients given that the former has more potent bronchodilatory activity than the latter.

Structure-activity relationship of celecoxib and rofecoxib for the membrane permeabilizing activity.

Non-steroidal anti-inflammatory drugs (NSAIDs) achieve their anti-inflammatory effect by inhibiting cyclooxygenase activity. We previously suggested that in addition to cyclooxygenase-inhibition at the gastric mucosa, NSAID-induced gastric mucosal cell death is required for the formation of NSAID-induced gastric lesions in vivo. We showed that celecoxib exhibited the most potent membrane permeabilizing activity among the NSAIDs tested. In contrast, we have found that the NSAID rofecoxib has very weak membrane permeabilizing activity. To understand the membrane permeabilizing activity of coxibs in terms of their structure-activity relationship, we separated the structures of celecoxib and rofecoxib into three parts, synthesized hybrid compounds by substitution of each of the parts, and examined the membrane permeabilizing activities of these hybrids. The results suggest that the sulfonamidophenyl subgroup of celecoxib or the methanesulfonylphenyl subgroup of rofecoxib is important for their potent or weak membrane permeabilizing activity, respectively. These findings provide important information for design and synthesis of new coxibs with lower membrane permeabilizing activity.

Formal synthesis of (+)-madindoline A, a potent IL-6 inhibitor, utilizing enzymatic discrimination of quaternary carbon.

A formal synthesis of (+)-madindoline A was achieved. The Sunazuka's key intermediate, (4R,5S)-(-)-3-butyl-4-(tert-butyldimethylsiloxy)-5-methoxycarbonyl-2,5-dimethyl-2-cyclopentenone, was synthesized from easily available (2S,3S)-3-acetoxy-2-ethenyl-2-methylcyclopentanone. The starting material was reliably supplied by a chemo-enzymatic procedure in enantiomerically pure form. The synthesis was performed by straightforward transformations involving enone formation and regioselective introduction of the two alkyl side chains.

Synthesis of okicamelliaside, a glucoside of ellagic acid with potent anti-degranulation activity.

Okicamelliaside, a glucoside of ellagic acid with potent anti-degranulation activity, was synthesized from ellagic acid. The regioselectivity, solubility, and high reactivity of the intermediates throughout the synthesis were obtained by the complementary use of triisopropylsilyl (TIPS) and methoxyethoxymethyl (MEM) protective groups on the aglycone skeleton.

(-)-Benzyl 2,3-dide-oxy-ß-d-erythro-hex-2-eno-pyran-oside.

In the title compound, C13H16O4, the six-membered ring of the sugar moiety shows a half-chair conformation. In the crystal, mol-ecules are connected via O-H⋯O hydrogen bonds, forming columns around twofold screw axes along the b-axis direction. There is a disorder of the benz-yloxy group, which has two possible orientations with the phenyl group lying on a common plane [site-occupancy factors = 0.589 (9) and 0.411 (9)].

Triazoyl-phenyl linker system enhancing the aqueous solubility of a molecular probe and its efficiency in affinity labeling of a target protein for jasmonate glucoside.

In methods employing molecular probes to explore the targets of bioactive small molecules, long or rigid linker moieties are thought to be critical factors for efficient tagging of target protein. We previously reported the synthesis of a jasmonate glucoside probe with a highly rigid linker consisting of a triazoyl-phenyl (TAzP) moiety, and this probe demonstrated effective target tagging. Here we compare the TAzP probe with other rigid or flexible probes with respect to target tagging efficiency, hydrophobic parameters, aqueous solubility, and dihedral angles around the biaryl linkage by a combination of empirical and calculation methods. The rigid biaryl linkage of the TAzP probe has a skewed conformation that influences its aqueous solubility. Such features that include rigidness and good aqueous solubility resulted in highly efficient target tagging. These findings provide a promising guideline toward designing of better linkers for improving molecular probe performance.

A chemo-enzymatic expeditious route to racemic dihexanoyl (2R*,3R*,4R*)-dehydroxymethylepoxyquinomycin (DHMEQ), the precursor for lipase-catalyzed synthesis of the potent nuclear factor-κB inhibitor, (2S,3S,4S)-DHMEQ.

In order to synthesize the potent nuclear factor (NF)-κB inhibitor, (2S,3S,4S)-dehydroxymethylepoxyquinomycin (DHMEQ), in a large scale, a new route for its corresponding racemic precursor, dihexanoyl (2R*,3R*,4R*)-DHMEQ, was developed. By employing both hydroquinone and benzoquinone intermediates, the total yield, reproducibility, and synthetic steps were improved and the synthetic cost was reduced.