Ynolates are carbanions having a triple bond in place of the double bond in enolate anions. Ynolates are ketene anion equivalents, thus ynolates introduce a ketene unit into substrates and the resulting products possess high reactivity. This allows ynolates to undergo unique reaction sequences. For the past 20 years, several methods for the generation of ynolates and their reactions have been developed. Recently, we have developed a novel efficient method for their generation via cleavage of ester dianions. Starting from this success, we have found new reactions of ynolate anions. Ynolate anions react with carbonyl compounds to give β-lactone enolates, which are converted into olefins with high E-selectivity. It is noteworthy that high E-selectivity was achieved in the synthesis of tetrasubstituted olefins. Utilizing the strong nucleophilicity of the β-lactone enolates, we have succeeded in the first tandem [2+2] cycloaddition-Dieckmann condensation to lead synthetically useful 2, 3-disubstituted-2-cycloalkenones in good yields. Ynolate anions were found to react with N-sulfonyl aldimines to give β-lactams. N-2-methoxyphenyl aldimines efficiently activate their cycloaddition of lithium ynolates via chelation to give β-lactams and α, β-unsaturated amides. We have demonstrated the high functionality of ynolate anions. Ynolate chemistry has begun and much remains to be discovered.
In our study, we focused on the fact that platelets play a significant role in thrombus formation in the arterial vessels, and started exploratory research on the antiplatelet agent with a vasodilating action in order to discover a more effective drug for arterial thrombosis. We synthesized many 2(1H)-quinolinone derivatives and evaluated their inhibition of platelet aggregation and their vasodilating activities. First we found cilostamide, which has an amide moiety in the side chain. This compound possessed desired activities, but it caused a side effect of tachycardia, and so, unfortunately, we were unable to pursue its development. After many efforts to modify the side chain moiety to eliminate this side effect, we finally invented cilostazol (OPC-13013), a 2(1H)-quinolinone derivative with a tetrazol ring in the side chain. Cilostazol inhibited human platelet aggregation induced by various stimuli including shear stress in vitro and showed potent antiplatelet effects both in vitro and ex vivo. It was also shown that the drug has antithrombotic effects in experimental thrombus models and a vasodilating activity of the femoral artery and vertebral artery. The mechanism of the action for cilostazol is specific inhibition of cyclic nucleotide phosphodiesterase type 3 (PDE3). Cilostazol was marketed first in Japan in 1988 and later in seven other countries for the treatment of chronic arterial occlusion. It was launched in the U.S. in 1999, and approved in United Kingdom for the treatment of intermittent claudication. More recently, cilostazol was shown to be effective in a clinical prevention study on recurrence of cerebral infarction, and has been applied to the approval of the indication in Japan.
Blockade of the action of angiotensin II (AII) has long been a target for the development of novel antihypertensive agents. We recently discovered a novel class of potent nonpeptide AII receptor antagonists, benzimidazole-7-carboxylic acids including candesartan. Candesartan is a highly potent and insurmountable AII type-1 receptor (AT1)-selective antagonist. Structure-activity relationship (SAR) studies revealed that the adjacent arrangement of a lipophilic substituent, a tetrazolylbiphenylmethyl moiety and a carboxyl group was the important structural requirement for potent AII antagonistic activity. Especially, the presence of a carboxyl group at the 7-position was found to be essential for insurmountable antagonism. To improve bioavailability of candesartan, chemical modification was examined to yield candesartan cilexetil, a prodrug of candesartan. Candesartan cilexetil is a potent and long-acting blocker that, when given once a day to patients, provides effective 24 hr blood pressure control.
We investigated the effects of drugs, especially anti-pulmonary disease agents, on the production of cytokines from human peripheral blood mononuclear cells (PBMC). Roxithromycin (RXM), a macrolide antibiotic with the structure of 14-member macrocycline ring increased adherent cells (monocyte/macrophages), whereas it suppressed the proliferation of PBMC stimulated with phytohemagglutinin (PHA). RXM suppressed the production of IL-1β and TNF-α from lipopolysaccharide (LPS)-stimulated PBMC in a dose-dependent manner. Levofloxacin, a fluorinated quinolone, increased IL-2 production by PBMC stimulated with PHA. The production of GM-CSF and soluble IL-2 receptor was suppressed at high concentrations of LVFX. LVFX suppressed IL-1β production, but did not the production of TNF-α and IL-8 production. A β-adrenoceptor agonists (β-agonist), procaterol, clenbuterol, fenoterol and terbutaline suppressed the production of TNF- and IL-1β. TNF-α production was almost completely suppressed by dibutyryl cyclic AMP (dbcAMP), whereas IL-1β production appeared to be partially refractory even at the highest concentration examined. Both procaterol and theophylline elevated cAMP levels in LPS-stimulated PMBC, but the effect of procaterol was limited. The inhibition of the production of TNF-α and IL-1β by procaterol was additively potentiated with theophylline. Of examined phosphodiesterase (PDE) isozyme inhibitors type IV PDE inhibitors were more effective in inhibiting the production of TNF-α and IL-1β by LPS-stimulated PBMC than a nonselective, type III or type III/IV inhibitor. The addition of the β-agonist increased the inhibitory effect of tested PDE inhibitors on the production of TNF-α and IL-1β Type IV, type III and nonselective PDE inhibitors were effective in inhibiting the production of IFN-γ and IL-2 in a dose-dependent manner. In contrast, the production of IL-4 and IL-5 was inhibited by only the highest concentration of type IV inhibitor, and other agents had no effect on the production. Similarly, dbcAMP inhibited the production of IFN-γ and IL-2 more potently than that of IL-4 and IL-5. The addition of the β-agonist increased the inhibitory effect of tested PDE inhibitors on the production of IFN-γ and IL-2 production. These findings indicate that these agents have an immunodulatory action on the production of cytokines by PBMC and also indicate that they could be potent pharmacological agents for the treatment of diseases in which several cytokines are important etiological factors.
This review summarizes our studies using pharmacological, neurochemical and molecular biological methods on the nociception in the CNS and opioid receptors (OPRs). We designed an in vitro fluorometric on-line monitoring system including an immobilized glutamate dehydrogenase column, and for the first time actually demonstrated that capsaicin induced the release of glutamate from rat dorsal horn slices containing the terminal area of primary afferents, in concentration-dependent, extracellular Ca2+-dependent and tetrodotoxin-resistant manners. Further, such a release was shown to be inhibited through μ- and δ-opioid receptors and α2-adrenoceptors. On the other hand, we found that intracerebroventricular injections of interleukin (IL)-1β in rats produced biphasic effects on the mechanical nociception in rats (hyperalgesia in lower concentrations but analgesia in higher ones) and that similar injections of cytokine-induced neutrophil chemoattractant-1 (CINC-1) facilitated mechanical nociception in rats. The above described facts suggest that glutamate and some sorts of cytokines (IL-1β and CINC-1) contribute to nociception at least from the primary afferents to the spinal dorsal horn neurons and in higher brain, respectively. We have cloned rat κ- and μ-opioid receptors. Using cloned cDNA for OPRs, we demonstrated (1) the distribution of mRNAs for OPRs in the rat central nervous system, (2) coexistence of each type of mRNA for μ-, δ- and κ-OPRs and pre-protachykinin A mRNA in the dorsal root ganglion neurons, (3) an increased expression of μ- and κ-OPR mRNAs in the I-II layers of rat lumbar dorsal horn with an adjuvant arthritis in the hind limb, (4) the inhibitions of N- and Q-types of Ca2+ channels by μ-and κ-OPR agonists and (5) cross-desensitization of the inhibition through a common intracellular phosphorylation-independent mechanism, (6) pharmacological characterization of "antagonist analgesics" as partial agonists at every type of OPRs, and (7) the key-structure(s) of OPRs for discriminative binding of DAMGO to μ-OPR.
This review summarizes novel stereoselective syntheses of 3, 4-cis- and 3, 4-trans-3-alkyl-4-silyloxycyclopentanones using a dirhodium(II)-catalyzed intramolecular C-H insertion reaction as a key reaction. Treatment of diazoketoesters (20a-e) with 1 mol % of dirhodium(II) tetraacetate gave 2, 3-trans-3, 4-cis-cyclopentanones (21a-e) as major products. The presence of both the keto and ester groups in the precursors was found to be essential for this chemo- and stereoselective intramolecular C-H insertion reaction to take place. A possible interpretation for the observed stereoselectivity is presented. Optically active Corey lactone (38) was synthesized using a similar reaction of chiral α-diazo-β-ketoester (33). Next, reactions of 5, 6-bisoxygenated diazoketones with Rh2(OAc)4 were investigated. An acetonide derivative (39), upon treatment with Rh2(OAc)4, gave 3, 8-dioxabicyclo[3.2.1]octane (42) via oxonium ylide formation/1, 2-shift. On the other hand, similar treatment of 5, 6-bis(tert-butyldimethylsilyloxy) derivative (47) gave a C-H insertion product (51) which was purified by silica gel column chromatography to give 4-silyloxycyclopentenone (48). Direct reduction of 51 with lithium aluminum hydride gave stereoselectively diol (52) in 52% yield from 47. Reaction of 2-methoxycarbonylcyclopentenone (48) and a 2-benzenesulfonyl congener (50) with R2CuLi or RMgBr-CuI stereoselectively gave 2, 3-trans-3, 4-trans-cyclopentanones (22, 57) as a major diastereoisomer. On the other hand, reaction with R3 Al in toluene exclusively gave the corresponding 3, 4-cis-adducts (21 and 58).
The synthetic potential of some simple five-membered heterocycles, including 2-oxazolone, 1, 3-dihydro-2-imidazolone and 2-thiazolone as building blocks for chiral polyfunctional compounds as well as chiral heterocyclic auxiliaries for asymmetric synthesis is reviewed. The stereodefined introduction of easily replaceable groups to the 4, 5-olefinic moiety of the 2-oxazolone ring to give versatile chiral synthons, followed by stereospecific and stepwise substitution, provides a working and versatile strategy for achieving a chiral synthesis, which leads to the preparation of a wide variety of 2-amino alcohols of biological interest. Sterically constrained chiral 2-oxazolidinones and the derived conformationally rigid amino alcohols, which are derived from cycloaddition reactions of the 2-oxazolone to cyclic dienes such as 9, 10-dimethylanthracene and hexamethylcyclopentadiene, represent excellent chiral auxiliaries and chiral ligands which are of general use in asymmetric synthesis. The strategy developed using 2-oxazolone can also be used in the cases of the structurally similar 1, 3-dihydro-2-imidazolone and 2-thiazolone derivatives.
The mechanochemical polymerization of solid-state monomer was first reported in 1959. Nevertheless, relatively little work has been done. We reported the first in-depth study of the mechanochemical polymerization of specially synthesized solid-state monomers, methacryloyl derivatives of bioactive compounds including detailed mechanistic implications. There are many advantages for this reaction. One of the most striking properties observed in the resulting polymers is that these polymeric prodrugs are of very low heterogeneity (narrow molecular weight distribution) which is of great value in pharmaceuticals for highly functionalized polymeric prodrugs. Thus, the present reaction provides a novel and simple methodology for the syntheses of highly functionalized polymeric prodrugs through a totally dry process. The nature of drug release from many kinds of polymeric prodrugs prepared by mechanochemical polymerization was also investigated. The rate of drug release from the polymeric prodrugs can be controlled by the property of comonomer and the structure of spacer between the main chain of polymer and drug. Several applications of polymeric prodrugs (chemoembolization and hybrid polymeric prodrugs) were also described. We have carried out the mechanochemical polymerization in the presence of pharmaceutical aids to exploit the features of this reaction. If one takes the physicochemical property of pharmaceutical aids into consideration, novel composite polymeric prodrugs possessing a variety of rates of drug release can be synthesized simultaneously with mixing.
Multiple forms of cytochrome P450 (P450 or CYP) enzymes play important roles in the oxidation of structurally diverse xenobiotics and endobiotics. Interindividual variations in the level and activity of P450 enzymes were investigated in the human liver microsomes. Although the total P450 content was higher in Caucasian samples than in Japanese ones, the relative levels (percentage of total P450) of individual forms of P450 determined immunochemically were not very different. CYP3A (about 30% of total P450) and CYP2C (about 20%) enzymes were major forms. Different P450 enzymes in the human liver play major roles in a variety of drug oxidations and the hepatic contents of these P450 forms could be affective to determine which P450 enzymes play major roles in drug metabolism in individual humans. Recently recombinant P450 enzymes from different sources, e.g., microsomes of human lymphoblastoid cells, of yeast, and insect cells infected with baculovirus systems, and Escherichia coli membranes containing coexpressed P450 and reductase, have been widely used for drug metabolism research. However, the marker activities or kinetic parameters of human P450 enzymes reported are not always similar. Cytochrome b5 can enhance the activities of recombinant P450 systems in some cases using different mechanisms. These differences in activities may be a critical factor for understanding the roles of human P450 enzymes involved in drug metabolism. This review provides useful information for the study of drug biotransformation in humans and for the basis of drug toxicities and carcinogenesis.
Four hundred samples of Conocephalum conicum, collected in various places have been analyzed by GC-MS. This resulted in the presence of three chemo-types of C. conicum. One of them elaborate (-)-sabinene (type-I) as a major compound. The other two types characteristically contained (+)-bornyl acetate (type-II) and methyl cinnamate (type-III) as major constituent respectively. The structures of three new monoterpenic esters, isolated from the ether extract of C. conicum (type-I and/or II) have been established by chemical and spectral means. They were shown to be monoterpenic esters of (+)-borneol and p-coumaric acid derivatives. The ether extract of the liverwort Chiloscyphus polyanthos afforded sesquiterpenoids which are enantiomeric to those found in another liverwort Lepidozia vitrea. The absolute configurations of the sesquiterpenoids found in the C. polyanthos were determined by spectroscopic evidence, chemical derivatization and/or x-ray crystallographic analysis. The ether extract of the liverwort Porella perrottetiana afforded (-)-α-eudesmol, which showed an opposite sign of the optical rotation to that found in higher plants. Present work on the absolute configuration and an optical purity of (-)-α-eudesmol strongly suggested that the positive values (e.g.+28.5°) described in many previous papers should be revised. Since the absolute configuration of (-)-α-eudesmol was identical to that (+)-β-eudesmol found in the higher plants, it was apparent that the expression of the positive sign might be revised to (-)-α-eudesmol. The optical purity, reconfirmation of the absolute configuration and synthesis of (-)-α-eudesmol will be discussed.
One of the major goals of a series of our studies is to explore the availability of a method for anti-genotoxic modification of carcinogens by fluorine-substitution. Quinoline, a hepatocarcinogen, mutates bacterial tester strains in the presence of rat liver microsomal enzymes and induces GST-P (placental glutathione S-transferase)-positive foci in a medium-term bioassay system for hepatocarcinogenesis. On the other hand, 3-fluorinated quinoline (3-FQ) was neither mutagenic nor carcinogenic in the same assay system, whereas 5-fluoroquinoline (5-FQ) was mutagenic and carcinogenic. Quinoline, 3-FQ, and 5-FQ were also tested in an in vivo mutagenicity assay system using a lacZ-transgenic mouse (MutaTMMouse). Mutation was induced by quinoline and 5-FQ only in the liver, the target organ of carcinogenesis by quinoline, but not in the other organs examined. 3-FQ was non-mutagenic in all of the organs. The results strongly indicate that fluorine-substitution at the position-3 of quinoline could be an anti-genotoxic structural modification of quinoline in a wide range of its genotoxic end-points. Additionally, seventeen mono- and di-fluorinated derivatives of 1, 7-phenanthroline, 1, 10-phenanthroline, benzo[h]quinoline, and benzo[f]quinoline were subjected to analysis of their structure-mutagenicity relationship. The results support that the enamine epoxide structure of the pyridine moiety, as well as the bay-region epoxide structure, is responsible for mutagenicity. These results suggest that the introduction of a fluorine atom to the molecule in question may be a useful tool to modify their mutagenic potency and to better understand the mechanism of mutation.
We examined the role of protein kinase C (PKC) in the phosphorylation of a p53 protein. Exposure to a protein kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7), increased the phosphorylation of the wild type p53 protein, whereas exposure to a tumor promoter phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), decreased it in vivo after incubation with mouse epidermal JB6 cells for 3h. Exposure to a cAMP dependent protein kinase (PKA) activator, forskolin, did not decrease the phosphorylation of p53 protein. In the transient transfection/luciferase reporter transactivation assay, H7 slightly increased the mouse double minute (MDM) 2 reporter transactivation activity of the p53 protein after treatment for 24h, whereas TPA completely blocked it. Exposure to H7 and a specific PKC inhibitor, bisindolylmaleimide (bis), dose-dependently reduced the lung-colonizing potential of highly metastatic B16-F10 mouse melanoma cells in syngeneic mice. These results suggest that the phosphorylation of the wild type p53 protein is inversely related to PKC activation, and also suggest that the phosphorylation of the p53 protein is involved in the function of its transcription factor. The PKC inhibitor may exhibit a potent anti-metastatic effect through the phosphorylation of wild type p53 protein and the activation of its function.
The kidney contributes to cardiovascular homeostasis through Na+ and water excretion and renin secretion. Changes in renal functions, therefore, have a close relationship to pathophysiology of cardiovascular diseases and to drug efficacy for them. The functions of the kidney are controlled by the sympathetic nervous system and various kinds of humoral factors and by their complicated interactions. Studies in the intact and working kidney in vivo have been providing physiologically significant information on the renal functions and drug actions. This review, by demonstrating data obtained in our laboratory with experiments in anesthetized dogs in vivo, refers mainly to the neural control of renal functions and renal actions of atrial natriuretic peptide (ANP) and an adenylate cyclase activator NKH477, either of which could be used for the treatment of congestive heart failure. Electrical stimulation of the renal nerves, which could mimic the events during elevation of renal sympathetic nerve activity, induces frequency-dependent renal norepinephrine release, renal vasoconstriction, antinatriuresis and renin secretion. ANP causes potent natriuresis and suppresses the nerve stimulation-induced renin secretion and renal vasoconstriction without affecting the norepinephrine release. Effects of ANP on other vasoconstrictive and antinatriuretic stimuli such as angiotensin II and endothelin are also demonstrated. Renal actions of NKH477 had been unknown, but we revealed that NKH477 elevates renal cAMP level and causes vasodilation and natriuresis. NKH477 also suppresses the nerve stimulation-induced renal vasoconstriction, and thereby blunts the antinatriuresis. The renal actions of these drugs clarified in our study may contribute to their curative effects on congestive heart failure.
Molecular mechanisms of DNA recognition and functional expression by bioactive compounds such as bleomycin, enediyne antibiotics, and zinc finger proteins are an important research subject in the pharmaceutical scientific field. Cleavage of cellular DNA by bleomycin substantially contributes to the antitumor activity of this drug. Some physicochemical data have clearly demonstrated that the bleomycin-iron (II) complex forms a dioxygen adduct species. Of special interest is the fact that the bleomycin-iron complex and cytochrome P450 have a similar dioxygen activation cycle. Probably, the site-specific iron-peroxide species of bleomycin accounts for the action mechanism of selective DNA cleavage by bleomycin. The enediyne antibiotics possess an unprecedented chemical structure, potent anticancer activity, and a fascinating mode of biological action. A new compound, C-1027, consists of a carrier apoprotein and an enediyne chromophore. The high-resolution NMR experiments clarified that novel chromophore interacts with DNA through its benzoxazolinate and aminosugar moiety, and also with apoprotein through the benzoxazolinate and macrocyclic moiety. The results provide a molecular basis for the host-recognition mode, the reaction mechanism, and the drug delivery system of chromoprotein C-1027. In addition, we found unique reactivity of C-1027 chromophore toward a tRNAPhe. RNA as well as DNA might be also a potent biological target of the enediyne antibiotics. On the basis of characteristic DNA binding mode of Cys2His2-type zinc finger motif, artificial zinc finger proteins have been created, and new functions such as DNA cleavage, long sequence binding, and DNA bending are produced. The first artificial His4-type zinc finger protein is also made from Cys→His mutations of the Cys2His2-type transcription factor Sp1. Such novel zinc finger proteins may be useful as a gene therapeutic agent and a tool for genetic engineering.
Exocytosis is a common process for the secretion of physiologically active substances such as neurotransmitter, hormone, and inflammatory mediators. Exocytosis is triggered by an increase in intracellular calcium ion concentration. At the nerve terminal, voltage dependent calcium channels (VDCCs) are responsible for this calcium increase. There are several types of VDCC which are different from each other in their electro-physiological and pharmacological characteristics. In order to identify the types of VDCC at the cholinergic nerve terminal, acetylcholine (ACh) release from the electric organ synaptosomes was measured in the presence of type-specific channel blockers. At least three types of VDCC were involved in the ACh release, and N-and P/Q-type VDCC had a major contribution. Adenosine receptor A1 was coupled with N-type VDCC and had negative feedback regulation of ACh release, while A2 receptor coupled with P/Q-type VDCC enhanced the ACh release. Investigation of the inhibitory effects of antibodies from patients of autoimmune disease Lambert-Eaton syndrome on ACh release revealed that P/Q-type channel was a target for the autoantibodies. Unlike the nerve terminal, little is known about the mechanism and molecules involved in the exocytosis of immune cells. Ion channel activities of secretory granule proteins of mast cells were observed. The calcium dependency of the open probability of the channel was similar to that of histamine release from mast cells. We also showed the expression of some SNARE proteins in RBL-2H3 cells. Localization and dynamics of VAMP-7 and syntaxin-3 after antigen stimulation suggested the involvement of SNARE proteins in the exocytosis of mast cells.
Alkyl p-hydroxybenzoates such as isobutyl p-hydroxybenzoate (PHBA-iBu), butyl p-hydroxybenzoate (PHBA-nBu), isopropyl p-hydroxybenzoate (PHBA-iPr), propyl p-hydroxybenzoate (PHBA-nPr), ethyl p-hydroxybenzoate (PHBA-Et), and methyl p-hydroxybenzoate (PHBA-Me) are widely used as preservatives, stabilizers and antiseptics for medical supplies, cosmetics, foodstuffs etc. We determined the binding affinity of alkyl p-hydroxybenzoates to human estrogen receptor α (ERα) and β (ERβ) by non-RI receptor binding assays. PHBA-iBu had a high binding affinity for ERα (IC50 : 6.0×10-6M, the relative binding affinity (RBA) : 0.267) and ERβ (IC50 : 5.0×10-6M, RBA : 0.340). These IC50 values and RBA were almost the same as those of bisphenol A. The ranking of the estrogenic potency of alkyl p-hydroxybenzoates for both ERs is different; that is, PHBA-iBu>PHBA-nBu≒PHBA-iPr≒PHBA-nPr>PHBA-Et»PHBA-Me. Alkyl p-hydroxybenzoates bound with equal relative affinity to both ERα and β proteins. Alkyl p-hydroxybenzoate having a long alkyl side-chain showed a high affinity for ERα and β. These findings suggest that p-hydroxybenzoates may be endocrine disruptors.
Ministry Health Welfare of Japan announced the caution for drug interaction of St. John's Wort (SJW), a herbal supplement occasionally used for depression, on May, 2000. Immediately after the announcement, we conducted drug consultation for outpatients prescribed the medicines potentially interacing with SJW. We provided information concerning possible drug interaction with SJW for 741 outpatients (except for pediatrics) during the period of May 22-June 16, 2000. The potential drugs prescribed frequently were warfarin (28.0%), theophylline (19.7%), digitalis (18.4%), carbamazepine (7.2%), disopyramide (6.9%) and cyclosporin (6.3%). Of the patients, 401 subjects were surveyed by collecting the questionnaires to clarify the background of SJW drug interaction. Twenty-two subjects (5.5%) have known commercially available SJW products, 5 subjects (1.2%) have ever taken SJW products before and 2 subjects (0.5%) have taken SJW products concomitant with prescribed medicines. Gender difference was observed in paying attention to SJW products; female subjects (8.6%) tended to have more interest in SJW products than male subjects (2.8%). Two subjects taking SJW have realized for the first time that the supplements they took were SJW products when their package photographs were shown at the consultation. Showing the package photographs might be helpful for making the patients easy to identify the SJW products, because most patients do not pay attention to whether the supplements contain SJW or not. It is recommended that drug consultation should be provided to avoid serious drug interaction with SJW while the outpatients are taking potential medicines prescribed.