Discovery of DS34942424: An orally potent analgesic without mu opioid receptor agonist activity.

We identified (5'S)-10'-fluoro-6'-methyl-5',6'-dihydro-3'H-spiro[cyclopropane-1,4'-[2,6]diaza[2,5]methano[2,6]benzodiazonin]-7'(1'H)-one, 22b (DS34942424) with a unique and original bicyclic skeleton. 22b showed an orally potent analgesic in the acetic acid-induced writhing test and formalin test in ddY mice without sedation. Moreover, 22b did not exhibit mu opioid receptor agonist activity.

Discovery of EST73502, a Dual µ-Opioid Receptor Agonist and σ1 Receptor Antagonist Clinical Candidate for the Treatment of Pain.

The synthesis and pharmacological activity of a new series of 4-alkyl-1-oxa-4,9-diazaspiro[5.5]undecane derivatives as potent dual ligands for the σ1 receptor (σ1R) and the µ-opioid receptor (MOR) are reported. A lead optimization program over the initial 4-aryl analogues provided 4-alkyl derivatives with the desired functionality and good selectivity and ADME profiles. Compound 14u (EST73502) showed MOR agonism and σ1R antagonism and a potent analgesic activity, comparable to the MOR agonist oxycodone in animal models of acute and chronic pain after single and repeated administration. Contrary to oxycodone, 14u produces analgesic activity with reduced opioid-induced relevant adverse events, like intestinal transit inhibition and naloxone-precipitated behavioral signs of opiate withdrawal. These results provide evidence that dual MOR agonism and σ1R antagonism may be a useful strategy for obtaining potent and safer analgesics and were the basis for the selection of 14u as a clinical candidate for the treatment of pain.

Evaluation of heritability of ß-eudesmol/hinesol content ratio in Atractylodes lancea De Candolle.

BACKGROUND: Atractylodes lancea De Candolle is a medicinal plant distributed in East Asia. Its rhizome has been used as an important crude drug in traditional Chinese and Japanese medicines for the treatment of numerous diseases and disorders. In recent years, the demand for mass production of the crude drug with a stable quality has increased. Its major active compounds are sesquiterpenoids, such as ß-eudesmol and hinesol that have closely related chemical structures with each other. As the criteria for evaluating the quality of A. lancea, the ß-eudesmol/hinesol content ratio is considered important. In A. lancea, the ratio could be considered to be influenced by genetic factors, geographical environment factors and these interactions. Few studies of a detail genetic analyses for ß-eudesmol/hinesol content ratio have been reported. Therefore, we evaluated the heritability and genotype-environment interaction on the ß-eudesmol/hinesol content ratio in A. lancea using clonal lines propagated with division of rhizome. RESULTS: The heritability of the ß-eudesmol/hinesol content ratio in A. lancea was evaluated through the cultivation of clonal lines of A. lancea in both different years (2016, 2017) and locations (Hokkaido, Ibaraki). Correlations between ß-eudesmol and hinesol contents were identified in all clonal lines, with high correlation coefficients (r = 0.73-0.99). The broad-sense heritability of the ß-eudesmol/hinesol content ratio was revealed to be high at 0.92. The effects of cultivation year were smaller than that of genotype, and few genotype-environment interactions were observed. In addition, the influence of cultivation location was also smaller than that of genotype, and the correlation between the two cultivation locations on the ß-eudesmol/hinesol content ratio was high. The results suggested that the ß-eudesmol/hinesol content ratio in A. lancea is highly dependent on genetic factors. CONCLUSION: We demonstrate that the heritability of ß-eudesmol/hinesol content ratio is high and that the effects of genetic factors were stronger than that of environmental factors such as cultivation location and year. Our findings suggested that selective breeding and clonal propagation are effective strategies for the production of A. lancea with stable qualities for use in the production of crude drugs.

Microbial transformation of diosgenin to diosgenone by Wickerhamomyces anomalus JQ-1 obtained from Naxi traditional Jiu Qu.

Diosgenone [(20S,22R,25R)-spirost-4-en-3-one, C27H40O3] has been considered as a potential therapeutic alternative remedy for malaria. An efficient and economical approach of microbial transformation with diosgenin to diosgenone by the yeast strain Wickerhamomyces anomalus JQ-1 from Naxi traditional Jiu Qu was developed in this study. Chromatographic analysis confirmed that 85% of 0.1 mM diosgenin was transformed to diosgenone within 72 h. This research demonstrates that diosgenin could be converted to diosgenone through two-step pathway including 3ß-hydroxyl oxidation and double bond isomerization rather than through one-step pathway, which prompted a further inference that the oxidation activity in W. anomalus JQ-1 has the same function with the Oppenauer-type oxidation which can convert diosgenin into diosgenone. Gaining specific functional strains from traditional fermented products will be a potential direction and ethnobotanical researches could provide helps with discovery and utilization of microbial resources.

Repeated evolution of cytochrome P450-mediated spiroketal steroid biosynthesis in plants.

Diosgenin is a spiroketal steroidal natural product extracted from plants and used as the single most important precursor for the world steroid hormone industry. The sporadic occurrences of diosgenin in distantly related plants imply possible independent biosynthetic origins. The characteristic 5,6-spiroketal moiety in diosgenin is reminiscent of the spiroketal moiety present in anthelmintic avermectins isolated from actinomycete bacteria. How plants gained the ability to biosynthesize spiroketal natural products is unknown. Here, we report the diosgenin-biosynthetic pathways in himalayan paris (Paris polyphylla), a monocot medicinal plant with hemostatic and antibacterial properties, and fenugreek (Trigonella foenum-graecum), an eudicot culinary herb plant commonly used as a galactagogue. Both plants have independently recruited pairs of cytochromes P450 that catalyze oxidative 5,6-spiroketalization of cholesterol to produce diosgenin, with evolutionary progenitors traced to conserved phytohormone metabolism. This study paves the way for engineering the production of diosgenin and derived analogs in heterologous hosts.

Stereoselective Metabolism of the Sterol Biosynthesis Inhibitor Fungicides Fenpropidin, Fenpropimorph, and Spiroxamine in Grapes, Sugar Beets, and Wheat.

Metabolism of chiral pesticides in crops is typically studied using achiral analytical methods and, consequently, the stereoisomer composition of residues is unknown. In this study, we developed an enantioselective GC-MS/MS method to quantify residues of the fungicides fenpropidin, fenpropimorph, and spiroxamine in plant matrices. In field trials, the fungicides were applied to grapevines, sugar beets, or wheat. Fenpropidin was metabolized with no or only weak enantioselectivity. For fenpropimorph, slightly enantioselective metabolism was observed in wheat but more pronounced in sugar beets. This enantioselectivity was due to different rates of metabolism and not due to interconversion of enantiomers. The four stereoisomers of spiroxamine were also metabolized at different rates, but selectivity was only found between diastereomers and not between enantiomers. trans-Spiroxamine was preferentially degraded in grapes and cis-spiroxamine in wheat. These findings may affect the consumer dietary risk assessment because toxicological end points were determined using racemic test substances.

Synthesis and biological evaluation of spirocyclic antagonists of CCR2 (chemokine CC receptor subtype 2).

Activation of chemokine CC receptors subtype 2 (CCR2) plays an important role in chronic inflammatory processes such as atherosclerosis, multiple sclerosis and rheumatoid arthritis. A diverse set of spirocyclic butanamides 4 (N-benzyl-4-(3,4-dihydrospiro[[2]benzopyran-1,4'-piperidin]-1'-yl)butanamides) was prepared by different combination of spirocyclic piperidines 8 (3,4-dihydrospiro[[2]benzopyran-1,4'-piperidines]) and γ-halobutanamides 11. A key step in the synthesis of spirocyclic piperidines 8 was an Oxa-Pictet-Spengler reaction of ß-phenylethanols 5 with piperidone acetal 6. The substituted γ-hydroxybutanamides 11c-e were prepared by hydroxyethylation of methyl acetates 13 with ethylene sulfate giving the γ-lactones 14c and 14e. Aminolysis of the γ-lactones 14c and 14e with benzylamines provided the γ-hydroxybutanamides 15c-e, which were converted into the bromides 11c-e by an Appel reaction using polymer-bound PPh3. In radioligand binding assays the spirocyclic butanamides 4 did not displace the iodinated radioligand (125)I-CCL2 from the human CCR2. However, in the Ca(2+)-flux assay using human CCR2 strong antagonistic activity of butanamides 4 was detected. Analysis of the IC50-values led to clear relationships between the structure and the inhibition of the Ca(2+)-flux. 4g (4-(3,4-dihydrospiro[[2]benzopyran-1,4'-piperidin]-1'-yl)-N-[3,5-bis(trifluoromethylbenzyl)]-2-(4-fluorophenyl)butanamide) and 4o (N-[3,5-bis(trifluoromethyl)benzyl]-2-cyclopropyl-4-(3,4-dihydrospiro[[2]benzopyran-1,4'-piperidin]-1'-yl)butanamide) represent the most potent CCR2 antagonists with IC50-values of 89 and 17nM, respectively. Micromolar activities were found in the ß-arrestin recruitment assay with murine CCR2, but the structure-activity-relationships detected in the Ca(2+)-flux assay were confirmed.

Highly enantioselective catalytic synthesis of neurite growth-promoting secoyohimbanes.

Natural products endowed with neuromodulatory activity and their underlying structural scaffolds may inspire the synthesis of novel neurotrophic compound classes. The spirocyclic secoyohimbane alkaloid rhynchophylline is the major component of the extracts of Uncaria species used in Chinese traditional medicine for treatment of disorders of the central nervous system. Based on the structure of rhynchophylline, a highly enantioselective and efficient organocatalyzed synthesis method was developed that gives access to the tetracyclic secoyohimbane scaffold, embodying a quaternary and three tertiary stereogenic centers in a one-pot multistep reaction sequence. Investigation of a collection of the secoyohimbanes in primary rat hippocampal neurons and embryonal stem cell-derived motor neurons led to discovery of compounds that promote neurite outgrowth and influence the complexity of neuronal network formation.

Pre-clinical validation of a novel alpha-7 nicotinic receptor radiotracer, [(3)H]AZ11637326: target localization, biodistribution and ligand occupancy in the rat brain.

The alpha-7 neuronal nicotinic receptor is a novel pharmacological target for psychiatric and cognitive disorders. Selective radiotracer tools for pre-clinical receptor occupancy can facilitate the interpretation of the biological actions of small molecules at a target receptor. We discovered a high affinity nicotinic alpha-7 subtype-selective ligand, AZ11637326, with physical-chemical and pharmacokinetic properties suitable for an in vivo radioligand tool. [(3)H]AZ11637326 synthesis by tritiodehalogenation of the corresponding tribromide precursor yielded a high specific activity radiotracer with high affinity alpha-7 receptor binding in the rat hippocampus determined by autoradiography (Kd = 0.2 nM). When [(3)H]AZ11637326 was administered to rats by intravenous bolus, rapid uptake was measured in the brain followed by a 3-4 fold greater specific binding in regions containing the alpha-7 receptor (frontal cortex, hippocampus, hypothalamus and midbrain) when compared to non-target regions (striatum and cerebellum). Systemic administration of the high affinity alpha-7 receptor antagonist, methyllycaconitine (MLA), or pretreatment with alpha-7 selective agonists (AR-R17779, PyrQTC, DBCO-4-POM, and DBCO-3-POM) significantly blocked the alpha-7 specific binding of [(3)H]AZ11637326 in the rat brain. The rank order of ligand ED(50) values for in vivo alpha-7 receptor occupancy in rat hippocampus was: DBCO-4-POM > DBCO-3-POM âˆ¼ MLA > PyrQTC > AR-R17779. The occupancy affinity shift was consistent with in vitro binding affinity in autoradiography. Our studies established the optimal conditions for [(3)H]AZ11637326 in vivo specific binding in the rat brain and support the use of [(3)H]AZ11637326 as a pre-clinical tool for assessment of novel alpha-7 compounds in drug discovery.

Phosphorus-nitrogen compounds. 21. Syntheses, structural investigations, biological activities, and DNA interactions of new N/O spirocyclic phosphazene derivatives. The NMR behaviors of chiral phosphazenes with stereogenic centers upon the addition of chiral solvating agents.

The reactions of hexachlorocyclotriphosphazatriene, N(3)P(3)Cl(6), with N/O-donor-type N-alkyl (or aryl)-o-hydroxybenzylamines (1a-1e) produce mono- (2a-2e), di- (3a-3d), and tri- (4a and 4b) spirocyclic phosphazenes. The tetrapyrrolidino monospirocyclic phosphazenes (2f-2i) are prepared from the reactions of partly substituted compounds (2a-2d) with excess pyrrolidine. The dispirodipyrrolidinophosphazenes (3e-3h) and trispirophosphazenes (3i-3k) are obtained from the reactions of trans-dispirophosphazenes with excess pyrrolidine and sodium (3-amino-1-propanoxide), respectively. Compounds 3a-3d have cis and trans geometric isomers. Only the trans isomers of these compounds are isolated. Compounds 3a-3h have two stereogenic P atoms. They are expected to be in cis (meso) and trans (racemic) geometric isomers. In the trans trispiro compounds (3i-3k), there are three stereogenic P atoms. They are expected to be in racemic mixtures. The stereogenic properties of 3a-3k are confirmed by (31)P NMR spectroscopy upon the addition of the chiral solvating agent; (S)-(+)-2,2,2-trifluoro-1-(9'-anthryl)ethanol. The molecular structures of 3i-3k, 4a, and 4b look similar to a propeller, where the chemical environment of one P atom is different from that of others. Additionally, 4a and 4b are also expected to exist as cis-trans-trans and cis-cis-cis geometric isomers, but both of them are found to be in cis-trans-trans geometries. The solid-state structures of 2a, 2e, 2f, 3e, and 3f are determined by X-ray crystallography. The compounds 2f-2i, 3e-3i, and 3k are screened for antibacterial activity against gram-positive and gram-negative bacteria and for antifungal activity against yeast strains. These compounds (except 3f) have shown a strong affinity against most of the bacteria. Minimum inhibitory concentrations (MIC) are determined for 2f-2i, 3e-3i, and 3k. DNA binding and the nature of interaction with pUC18 plasmid DNA are studied. The compounds 2f-2i, 3e-3i, and 3k induce changes on the DNA mobility. The prevention of BamHI and HindIII digestion (except 2g) with compounds indicates that the compounds bind with nucleotides in DNA.

Next-generation spirobenzazepines: identification of RWJ-676070 as a balanced vasopressin V1a/V2 receptor antagonist for human clinical studies.

We have continued to explore spirobenzazepines as vasopressin receptor antagonists to follow up on RWJ-339489 (2), which had advanced into preclinical development. Further structural modifications were pursued to find a suitable backup compound for human clinical studies. Thus, we identified carboxylic acid derivative 3 (RWJ-676070; JNJ-17158063) as a potent, balanced vasopressin V(1a)/V(2) receptor antagonist with favorable properties for clinical development. Compound 3 is currently undergoing human clinical investigation.

Pyrrolo- and pyridoazepine alkaloids as chemical markers in Stemona species.

Broad-based phytochemical investigations on 31 Stemona species and geographical provenances led to an overview concerning characteristic accumulation trends and the distribution of different Stemona alkaloids. Two major metabolic differences suggested a taxonomic segregation of the complex Stemona tuberosa group from the other species, and was supported by morphological characters. Whereas most of the Stemona species were characterised by protostemonine type alkaloids, the S. tuberosa group clearly deviated by accumulation trends towards tuberostemonine or croomine derived alkaloids belonging to two different skeletal types. Also of chemotaxonomic relevance was the structural divergence of protostemonine type alkaloids into pyrrolo- or pyridoazepine derivatives represented by stemofoline or oxystemokerrine, respectively, as major constituents. Their common occurrence in different provenances of S. curtisii, also deviating from the other species by various chromosome numbers, deserves special taxonomic attention. Species specific chemical markers were given by the unique accumulation of didehydrostemofoline (=asparagamine A) in S. collinsae and stemokerrine in S. kerrii. In contrast to previous reports, no bisdehydro derivatives with an aromatic pyrrole ring were detected supporting the hypothesis that these alkaloids are artifacts. A new stereoisomer of tuberostemonine was isolated and identified by spectroscopic methods.

Drug evaluation: ranirestat--an aldose reductase inhibitor for the potential treatment of diabetic complications.

Ranirestat is an orally available aldose reductase inhibitor under development by Dainippon Sumitomo Pharma Co Ltd (formerly Dainippon Pharmaceutical Co Ltd), Kyorin Pharmaceutical Co Ltd and Eisai Co Ltd for the potential treatment of diabetic complications, such as neuropathy, cataracts, retinopathy and nephropathy. Phase III trials were ongoing in February 2006.

A new phenanthrene with a spirolactone from Dendrobium chrysanthum and its anti-inflammatory activities.

Investigation of phenolic patterns from the stems of Dendrobium chrysanthum by HPLC-PDA-MS has led to the isolation of a new phenanthrene derivative with a spirolactone ring, dendrochrysanene (1), that proved to suppress the mRNA level of TNF-alpha, IL8, IL10, and iNOS in murine peritoneal macrophages. The structure of 1 was characterized on the basis of various NMR (1H, 13C, 1H-1H COSY, HMQC, and HMBC), mass spectrometry, and X-ray crystal diffraction data.

Discovery and preclinical characterization of (+)-3-[4-(1- piperidinoethoxy)phenyl]spiro[indene- 1,1'-indane]-5,5'-diol hydrochloride: a promising nonsteroidal estrogen receptor agonist for hot flush.

In our studies of the development of a novel class of selective estrogen receptor modulators, (+)-3-[4-(1-piperidinoethoxy)phenyl]spiro[indene-1,1'-indane]-5,5'-diol hydrochloride (1) was found to be an estrogen receptor ligand with beneficial effects in rat models for human hot flush. Moreover, 1 was found to have beneficial effects on lipid and bone metabolism while maintaining marginal effects on the uterus and breasts. These findings suggest that 1 would provide a new treatment for hot flush.

Chlorinated alkaloids in Menispermum dauricum DC: root culture.

Feeding experiments using (36)Cl showed that Menispermum dauricum root culture produces four alkaloids containing chlorine. They included the novel alkaloids dauricumine and dauricumidine as well as the known alkaloids acutumine and acutumidine. The structures of novel alkaloids were established by spectroscopic, crystallographic, and chemical methods. These four alkaloids were labeled with (36)Cl, isolated, and fed independently to root cultures. Mutual conversion between acutumine and acutumidine, and between dauricumine and dauricumidine by N-methylation and N-demethylation, was demonstrated. Moreover, dauricumine was converted to acutumine and acutumidine. Epimerization of acutumidine to dauricumidine or vice versa was not observed. These results suggest that dauricumine is the first chlorinated alkaloid formed in cultured M. dauricum roots. Skewed distribution of radioactivity derived from labeled dauricumine is proof that epimerization at C-1 proceeds at a lower rate than N-demethylation.

Binding of 125I-labeled ghrelin to membranes from human hypothalamus and pituitary gland.

Ghrelin has been proposed as a natural ligand of the GH secretagogue receptor(s) (GHS-R), which was an orphan receptor activated by synthetic peptidyl (hexarelin) and non-peptidyl (MK-0677) GHS to strongly release GH in animals and humans. Herein we studied: 1) the binding of 125I-labeled human ghrelin to membranes from human hypothalamus and pituitary gland; 2) the ability of human ghrelin (either octanoylated or desoctanoylated), as well as of some GHS and neuropeptides to compete with the radioligand. The saturation binding analysis showed, in both tissues, the existence of a single class of high-affinity binding sites with limited binding capacity. The Bmax (maximal number of binding sites) values of ghrelin receptors in the hypothalamus were significantly greater (p<0.001) than those detected in the pituitary, whereas the Kd (dissociation constant) values in the two tissues were similar. 125I-ghrelin bound to hypothalamic membranes was displaced by ghrelin, hexarelin, MK-0677, various GHS antagonists (EP-80317, [D-Arg1-D-Phe5-D-Trp7,9-Leu11]-substance P) and some natural (cortistatin-14) and synthetic (vapreotide) SRIH-14 agonists. In contrast, no competition was seen in the presence of GHRH-44, SRIH-14 or desoctanoylated ghrelin, a ghrelin precursor that is devoid of GH-releasing properties. In conclusion, this preliminary study firstly demonstrates that ghrelin needs octanoylation to bind its hypothalamo-pituitary receptors. These receptors are the specific binding sites for GHS and their antagonists, as well as for SRIH analogs (vapreotide and cortistatin- 14), but not for native SRIH.

Human microsomal epoxide hydrolase is the target of germander-induced autoantibodies on the surface of human hepatocytes.

Germander, a plant used in folk medicine, caused an epidemic of cytolytic hepatitis in France. In about half of these patients, a rechallenge caused early recurrence, suggesting an immunoallergic type of hepatitis. Teucrin A (TA) was found responsible for the hepatotoxicity via metabolic activation by CYP3A. In this study, we describe the presence of anti-microsomal epoxide hydrolase (EH) autoantibodies in the sera of patients who drank germander teas for a long period of time. By Western blotting and immunocytochemistry, human microsomal EH was shown to be present in purified plasma membranes of both human hepatocytes and transformed spheroplasts and to be exposed on the cell surface where affinity-purified germander autoantibodies recognized it as their autoantigen. Immunoprecipitation of EH activity by germander-induced autoantibodies confirmed this finding. These autoantibodies were not immunoinhibitory. The plasma membrane-located EH was catalytically competent and may act as target for reactive metabolites from TA. To test this hypothesis CYP3A4 and EH were expressed with human cytochrome P450 reductase and cytochrome b(5) in a "humanized" yeast strain. In the absence of EH only one metabolite was formed. In the presence of EH, two additional metabolites were formed, and a time-dependent inactivation of EH was detected, suggesting that a reactive oxide derived from TA could alkylate the enzyme and trigger an immune response. Antibodies were found to recognize TA-alkylated EH. Recognition of EH present at the surface of human hepatocytes could suggest an (auto)antibody participation in an immune cell destruction.

Biotransformation of hinesol isolated from the crude drug Atractylodes lancea by Aspergillus niger and Aspergillus cellulosae.

Biotransformation of the sesquiterpene alcohol hinesol (1) with spasmolytic activity, which was prepared from the rhizome of Atractylodes lancea, was carried out by Aspergillus niger and Aspergillus cellulosae IFO 4040. Compound 1 was easily converted to compounds 2-9 by A. niger, and compounds 10 and 11 by A. cellulosae, respectively. Their stereostructures were established by a combination of high-resolution NMR spectral analysis, X-ray crystallographic analysis, and chemical reactions such as epoxydation.

Specific receptors for synthetic GH secretagogues in the human brain and pituitary gland.

In vitro studies have been performed to demonstrate and characterize specific binding sites for synthetic GH secretagogues (sGHS) on membranes from pituitary gland and different human brain regions. A binding assay for sGHS was established using a peptidyl sGHS (Tyr-Ala-hexarelin) which had been radioiodinated to high specific activity at the Tyr residue. Specific binding sites for 125I-labelled Tyr-Ala-hexarelin were detected mainly in membranes isolated from pituitary gland and hypothalamus, but they were also present in other brain areas such as choroid plexus, cerebral cortex, hippocampus and medulla oblongata with no sex-related differences. In contrast, negligible binding was found in the thalamus, striatum, substantia nigra, cerebellum and corpus callosum. The binding of 125I-labelled Tyr-Ala-hexarelin to membrane-binding sites is a saturable and reversible process, depending on incubation time and pH of the buffer. Scatchard analysis of the binding revealed a finite number of binding sites in the hypothalamus and pituitary gland with a dissociation constant (Kd) of (1.5 +/- 0.3) x 10(-9) and (2.1 +/- 0.4) x 10(-9) mol/l respectively. Receptor activity is sensitive to trypsin and phospholipase C digestion, suggesting that protein and phospholipids are essential for the binding of 125I-labelled Tyr-Ala-hexarelin. The binding of 125I-labelled Tyr-Ala-hexarelin to pituitary and hypothalamic membranes was displaced in a dose-dependent manner by different unlabelled synthetic peptidyl (Tyr-Ala-hexarelin, GHRP2, hexarelin, GHRP6) and non-peptidyl (MK 0677) sGHS. An inhibition of the specific binding was also observed when binding was performed in the presence of [D-Arg1-D-Phe5-D-Trp7,9-Leu11]-substance P, a substance P antagonist that has been found to inhibit GH release in response to sGHS. In contrast, no competition was observed in the presence of other neuropeptides (GHRH, somatostatin, galanin or Met-enkephalin) which have a known influence on GH release. In conclusion, the present data demonstrate that sGHS have specific receptors in human brain and pituitary gland and reinforce the hypothesis that these compounds could be the synthetic counterpart of an endogenous GH secretagogue involved in the neuroendocrine control of GH secretion and possibly in other central activities.