Estrogen-like activity of glabrene and other constituents isolated from licorice root

Licorice root extract and its major isoflavan, glabridin, exhibited varying degrees of estrogen receptor (ER) agonism in different tissues in vitro and in vivo. Animals fed with licorice extract, compared with estradiol and glabridin, showed an increase in creatine kinase (CK) activity, a known marker for estrogen responsive genes, which was higher than expected from the levels of glabridin in the extract. This led us to test for other components that may contribute to this strong estrogen agonist activity. Results indicated that glabrene and isoliquiritigenin, (2',4',4-three hydroxy chalcone) (ILC) in the licorice extract can bind to the human ER with higher affinity (IC50, 1 and 0.5 microM) than glabridin (IC50, 5 microM). The stimulatory effects of glabrene in vivo were tissue specific and similar to those of estradiol. The effect of increasing concentrations of glabrene and ILC on the growth of breast tumor cell were biphasic. Both showed an ER-dependent growth-promoting effect at low concentrations (10 nM-10 microM), and ER-independent antiproliferative activity at concentrations >15 microM. This is the first study to indicate that glabrene, an isoflavene exerted varying degrees of ER agonism in different tissues.     

Flavonoids and isoflavonoids of chemotaxonomic significance from Glycyrrhiza pallidiflora (Leguminosae)

Chemical studies were carried out on the root of Glycyrrhiza pallidiflora (Leguminosae), a licorice of no medicinal or commercial value. Two isoflavone glycosides, wistin and ononin, were isolated as major constituents from the methanol extract. A series of chromatographic separations of the acetone extract yielded isoflavone aglycones (afromosin, 2′,7-dihydroxy-4′-methoxyisoflavone and formononetin), flavanones (liquiritigenin and 4′,7-dihydroxy-6,8-diprenylflavanone), an isoflavan [(-)-vestitol], a pterocarpan [(-)-medicarpin], chalcones (echinatin and isoliquiritigenin), dibenzoylmethanes (licodione and 2′-O-methyl-licodione), a flavone (4′,7-dihydroxyflavone), a 3-arylcoumarin (2′-7-dihydroxy-4′-methoxy-3-arylcoumarin), and a new isoflav-3-ene (2′-7-dihydroxy-4′-methoxyisoflav-3-ene). The co-occurrence of the retrochalcone echinatin and the biogenetically related licodione and 2′-O-methyl-licodione is of particular interest, and suggests that this species is closely related to G. echinata and G. inflata. The biogenesis of the retrochalcone is also discussed in relation to its significance in the chemotaxonomy of sects Echinatae and Bucharicae of the genus Glycyrrhiza.     

Estrogenic activities of extracts of Chinese licorice (Glycyrrhiza uralensis) root in MCF-7 breast cancer cells

Despite the wide use of Chinese licorice root (Glycyrrhiza uralensis) for the treatment of menopausal complaints, little is known on its potential estrogenic properties, and available information relative to its effects on cell proliferation is contradictory. In this study, the estrogenic properties of licorice root were evaluated in vitro by use of several assays. The effects of increasing concentrations of a DMSO extract of licorice root on the growth of MCF-7 breast cancer cells were biphasic. The extract showed an ER-dependent growth-promoting effect at low concentrations and an ER-independent anti-proliferative activity at high concentrations. In further experiments, licorice root was sequentially extracted to yield four fractions: hexane, EtOAc, methanol and H₂O. Only the EtOAc extract had effects on cell proliferation similar to the DMSO extract. The hexane extract had no effect on cell growth. In contrast, the methanol and water extracts showed an ER-independent, growth-promoting effect. Similar to its effects on cell proliferation, the EtOAc extract had a biphasic effect on S phase cell cycle distribution and the level of PCNA protein. This extract-induced transactivation of endogenous ERα in MCF-7 cells, supported by inducing down-regulation of ERα protein and mRNA levels, and up-regulation of ERα target genes pS2 and GREB1. These results suggest that the activity of licorice root and the balance between increased risk for cancer and prevention of estrogen-dependent breast cancer may depend on the amount of dietary intake.     

Structural Aspects of The Inhibitory Effect of Glabridin on LDL Oxidation

The inhibitory effects of glabridin, an isoflavan isolated from licorice (Glycyrrhiza glabra) root, and its derivatives on the oxidation of LDL induced by copper ions or mediated by macrophages were studied, in order to evaluate the contribution of the different parts of the isoflavan molecule to its antioxidant activity. The peak potential (E_1/2) of the isoflavan derivatives, their radical scavenging capacity toward 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical and their ability to chelate heavy metals were also analyzed and compared to their inhibitory activity on LDL oxidation. In copper ion-induced LDL oxidation, glabridin (1), 4′-O-methylglabridin (2), hispaglabridin A (3), and hispaglabridin B (4), which have two hydroxyl groups at positions 2′ and 4′ or one hydroxyl at position 2′ on ring B, successfully inhibited the formation of conjugated dienes, thiobarbituric acid reactive substances (TBARS) and lipid peroxides, and inhibited the electrophoretic mobility of LDL under oxidation. Compounds 1–3 exhibited similar activities, whereas compound 4 was less active. In macrophage-mediated LDL oxidation, the TBARS formation was also inhibited by these isoflavans (1–4) at a similar order of activity to that obtained in copper ion-induced LDL oxidation. On the other hand, 2′-O-methylglabridin (5), a synthesized compound, whose hydroxyl at 2′-position is protected and the hydroxyl at 4′-position is free, showed only minor inhibitory activity in both LDL oxidation systems. 2′,4′-O-Dimethylglabridin (6), whose hydroxyls at 2′- and 4′-positions are both protected, was inactive. Resorcinol (7), which is identical to the phenolic B ring in glabridin, presented low activity in these oxidation systems. The isoflavene glabrene (8), which contains an additional double bond in the heterocyclic C ring, was the most active compound of the flavonoid derivatives tested in both oxidation systems. The peak potential of compounds 1–5 (300 μM), tested at pH 7.4, was similar (425–530 mV), and that for compound 6 and 8 was 1078 and 80 mV, respectively. Within 30 min of incubation, compounds 1, 2, 3, 4, 8 scavenged 31%, 16%, 74%, 51%, 86%, respectively, of DPPH radical, whereas compounds 5 and 6, which almost did not inhibit LDL oxidation, also failed to scavenge DPPH. None of the isoflavan derivatives nor the isoflavene compound were able to chelate iron, or copper ions. These results suggest that the antioxidant effect of glabridin on LDL oxidation appears to reside mainly in the 2′ hydroxyl, and that the hydrophobic moiety of the isoflavan is essential to obtain this effect. It was also shown that the position of the hydroxyl group at B ring significantly affected the inhibitory efficiency of the isoflavan derivatives on LDL oxidation, but did not influence their ability to donate an electron to DPPH or their peak potential values.     

甘草对大鲵抗嗜水气单胞菌感染的作用

通过投喂甘草提取物, 研究了甘草对大鲵(Andrias davidianus)抗嗜水气单胞菌(Aeromonas hydrophila)感染的作用。连续投喂56d, 从28d开始, 药物组血清溶菌酶活性先升后降, 呈抛物线趋势, 低剂量组在42d出现最大值(158.4±34.7) U/mL, 高剂量组在35d出现最大值(178.3±28.8) U/mL, 两者都显著高于同期对照组(P<0.05)。药物组在最后两次采样期, 即42d和56d时, 肾脏巨噬细胞吞噬活性显著高于对照组(P<0.05)。低剂量组和高剂量组最大值均出现在42d, 分别为(59.4±8.5)%和(58.4±5.2)%。同期相比, 药物组白细胞比容值均高于对照组。其中, 高剂量组在28d时白细胞比容值为(5.8±1.7)%, 低剂量组在56d时为(5.5±0.8)%, 高剂量组在56d时为(5.9±1.7)%, 三者都显著高于同期对照组(P<0.05)。药物组和对照组的脾脏脏器系数之间没有显示出显著差异。最后一次采样(56d)后人工感染嗜水气单胞菌, 对照组死亡率为90%, 低剂量组和高剂量组都为60%, 均低于对照组, 而药物组免疫保护率为33.3%, 也都高于对照组。结果表明, 投喂甘草提取物可在一定程度上提高大鲵对嗜水气单胞菌的抗性。     

Biological activities of a specific ecdysteroid dimer and of selected monomeric structural analogues in the B(II) bioassay.

The biological activities of selected specific ecdysteroids obtained by photochemical or chemical transformation are compared in the B(II) bioassay, in which the potency reflects the affinity of binding to the ligand-binding site of the Drosophila melanogaster ecdysteroid receptor. The compounds tested represent 14-deoxy, 14-dehydroxy, 14-hydroperoxy and 14-epi derivatives of 20-hydroxyecdysone and were selected on the basis of their close structural relationship to elucidate the contribution of the 14-hydroxy group and the stereochemical configuration at C-14 to ecdysteroid agonist activity. The structure-activity relationship shows that a 14-hydroxy group is not required for activity. However, the alpha-configuration of -H, -OH or -OOH at C-14, which determines the C/D rings trans-annelation, is very significant for activity; it is as important for activity as the well studied A/B rings cis-annelation. Compounds containing a double bond involving C-14 showed low activity with the exception of the specific, and so far unique, ecdysteroid dimer 7,7'-bis-[14-deoxy-8(14)-ene-20-hydroxyecdysone], which was obtained as the main product of the photochemical transformation of 20-hydroxyecdysone. The relatively high biological activity of this dimeric compound is discussed.     

In vitro antileishmanial and antitrypanosomal activities of flavanones from Baccharis retusa DC. (Asteraceae)

Leishmaniasis and Chagas' are parasitic protozoan diseases that affect the poorest population in the world, causing a high mortality and morbidity. As a result of highly toxic and long-term treatments, novel, safe and more efficacious drugs are essential. In this work, the CH(2)Cl(2) phase from MeOH extract from the leaves of Baccharis retusa DC. (Asteraceae) was fractioned to afford two flavonoids: naringenin (1) and sakuranetin (2). These compounds were in vitro tested against Leishmania spp. promastigotes and amastigotes and Trypanosoma cruzi trypomastigotes and amastigotes. Compound 2 presented activity against Leishmania (L.) amazonensis, Leishmania (V.) braziliensis, Leishmania (L.) major, and Leishmania (L.) chagasi with IC(50) values in the range between 43 and 52 μg/mL and against T. cruzi trypomastigotes (IC(50)=20.17 μg/mL). Despite of the chemical similarity, compound 1 did not show antiparasitic activity. Additionally, compound 2 was subjected to a methylation procedure to give sakuranetin-4'-methyl ether (3), which resulted in an inactive compound against both Leishmania spp. and T. cruzi. The obtained results indicated that the presence of one hydroxyl group at C-4' associated to one methoxyl group at C-7 is important to the antiparasitic activity. Further drug design studies aiming derivatives could be a promising tool for the development of new therapeutic agents for Leishmaniasis and Chagas' disease.     

Up-regulation of soyasaponin biosynthesis by methyl jasmonate in cultured cells of Glycyrrhiza glabra

Exogenously applied methyl jasmonate (MeJA) stimulated soyasaponin biosynthesis in cultured cells of Glycyrrhiza glabra (common licorice). mRNA level and enzyme activity of beta-amyrin synthase (bAS), an oxidosqualene cyclase (OSC) situated at the branching point for oleanane-type triterpene saponin biosynthesis, were up-regulated by MeJA, whereas those of cycloartenol synthase, an OSC involved in sterol biosynthesis, were relatively constant. Two mRNAs of squalene synthase (SQS), an enzyme common to both triterpene and sterol biosyntheses, were also up-regulated by MeJA. In addition, enzyme activity of UDP-glucuronic acid: soyasapogenol B glucuronosyltransferase, an enzyme situated at a later step of soyasaponin biosynthesis, was also up-regulated by MeJA. Accumulations of bAS and two SQS mRNAs were not transient but lasted for 7 d after exposure to MeJA, resulting in the high-level accumulation (more than 2% of dry weight cells) of soyasaponins in cultured licorice cells. In contrast, bAS and SQS mRNAs were coordinately down-regulated by yeast extract, and mRNA accumulation of polyketide reductase, an enzyme involved in 5-deoxyflavonoid biosynthesis in cultured licorice cells, was induced transiently by yeast extract and MeJA, respectively.     

Control of downy mildew (Pseudoperonospora cubensis) of greenhouse grown cucumbers with alternative biological agents

In organic cucumber production infection with downy mildew (Pseudoperonospora cubensis) is a major problem. Plant extracts from Glycyrrhiza glabra L. (licorice), a plant belonging to the family Fabaceae, and Salvia officinalis (sage) as well as cultures of the bacterium Aneurinibacillus migulanus were investigated for efficacy of disease control under commercial growing conditions. Contrary to bioassays, where sage extract and the microorganism showed highest activity, in the trials of 2008 G. glabra extract was more effective than sage extract or A. migulanus against P. cubensis. Parameters such as concentrations of the preparations or application intervals could have been the reason for this. In the following year's trial (2009) the concentration of these agents was therefore increased somewhat and plants were either treated in seven day application intervals or in ten day application intervals. In the semi-commercial trials of 2009 all alternative biological agents showed good efficacies up to around 80% against infection with downy mildew. The application interval seemed to have a marginal effect only. Again, the licorice extract tended to be the best agent.     

甘草根茎乙醇提取物抗菌活性研究

本实验采用琼脂扩散法和微量肉汤稀释法,研究了甘草根茎乙醇提取物对5种细菌(表皮葡萄球菌、金黄色葡萄球菌、枯草芽孢杆菌、大肠杆菌和绿脓杆菌)和2种真菌(白色念珠菌和黑曲霉)的抗菌活性。结果表明,甘草根茎乙醇提取物对革兰氏阳性菌非常敏感,而对革兰氏阴性菌和真菌不敏感,80%乙醇提取物对革兰氏阳性菌的MIC范围为0.156～0.312 mg·mL^-1,而10%乙醇提取物对革兰氏阳性菌的MIC范围为0.625～1.250 mg·mL^-1,表明甘草根茎抗菌活性成分在高浓度乙醇中溶解度较大,为临床上应用甘草根茎醇提物作为抗菌制剂提供了科学依据。     

Telmisartan inhibits advanced glycation end products (AGEs)-elicited endothelial cell injury by suppressing AGE receptor (RAGE) expression via peroxisome proliferator-activated receptor-gammaactivation

Advanced glycation end products (AGEs)-their receptor (RAGE) axis plays a central role in the pathogenesis of diabetic microangiopathy. Since the pathophysiological crosstalk between the AGEs-RAGE system and angiotensin II has also been associated with diabetic microangiopathy, we examined here whether and how telmisartan, a unique angiotensin II type 1 receptor blocker (ARB) with peroxisome proliferator-activated receptor-gamma (PPAR-gamma)-modulating activity, could inhibit the AGEs-elicited endothelial cell injury by suppressing RAGE expression in vitro. Telmisartan suppressed RAGE expression at both mRNA and protein levels in human cultured microvascular endothelial cells (ECs), which were prevented by GW9662, an inhibitor of PPAR-gamma. Further, telmisartan was found to inhibit up-regulation of mRNA levels for monocyte chemoattractant protein-1, intercellular adhesion molecule-1 and vascular endothelial growth factor in AGEs-exposed ECs. These results suggest that telmisartan inhibits the AGEs-elicited EC injury by down-regulating RAGE expression via PPAR-gamma activation. Our present study provides a unique beneficial aspect of telmisartan. Specifically, it could work as an anti-inflammatory agent against AGEs via PPAR-gamma activation and may play a protective role against diabetic microangiopathy.     

Peroxisome proliferator-activated receptor-gamma agonist 15-deoxy-Delta(12,14)-prostaglandin J(2) ameliorates experimental autoimmune encephalomyelitis

Peroxisome proliferator-activated receptors (PPAR) are members of a nuclear hormone receptor superfamily that includes receptors for steroids, retinoids, and thyroid hormone, all of which are known to affect the immune response. Previous studies dealing with PPAR-gamma expression in the immune system have been limited. Recently, PPAR-gamma was identified in monocyte/macrophage cells. In this study we examined the role of PPAR-gamma in experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease multiple sclerosis. The hypothesis we are testing is whether PPAR-gamma plays an important role in EAE pathogenesis and whether PPAR-gamma ligands can inhibit the clinical expression of EAE. Initial studies have shown that the presence of the PPAR-gamma ligand 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ2) inhibits the proliferation of Ag-specific T cells from the spleen of myelin basic protein Ac(1-11) TCR-transgenic mice. 15d-PGJ2 suppressed IFN-gamma, IL-10, and IL-4 production by both Con A- and myelin basic protein Ac(1-11) peptide-stimulated lymphocytes as determined by ELISA and ELISPOT assay. Culture of encephalitogenic T cells with 15d-PGJ2 in the presence of Ag reduced the ability of these cells to adoptively transfer EAE. Examination of the target organ, the CNS, during the course of EAE revealed expression of PPAR-gamma in the spinal cord inflammatory infiltrate. Administration of 15d-PGJ2 before and at the onset of clinical signs of EAE significantly reduced the severity of disease. These results suggest that PPAR-gamma ligands may be a novel therapeutic agent for diseases such as multiple sclerosis.     

Licorice extract does not impair the male reproductive function of rats.

The effect of water extract of licorice (Glycyrrhiza uralensis), one of the most widely used medicinal plants in Oriental nations and in Europe, on male reproductive function was investigated in rats. Licorice extract was prepared as in Oriental clinics and orally administered at doses of 500, 1,000 or 2,000 mg/kg, the upper-limit dose (2,000 mg/kg) recommended in the Toxicity Test guideline of the Korea Food and Drug Administration, to 6-week-old male rats for 9 weeks. Licorice extract neither induced clinical signs, nor affected the daily feed consumption and body weight gain. There were no significant changes in testicular weights, gross and microscopic findings, and daily sperm production between vehicle- and licorice-treated animals, in spite of slight decreases in prostate weight and daily sperm production at the high dose (2,000 mg/kg). In addition, licorice did not affect the motility and morphology of sperm, although the serum testosterone level tended to decrease without significant difference, showing a 28.6% reduction in the high-dose (2,000 mg/kg) group. The results suggest that the no observed adverse-effect level of licorice extract is higher than 2,000 mg/kg, the upper-limit dose, and that long-term exposure to licorice might not cause profound adverse effects.     

Design and synthesis of (Z)-1,2-diphenyl-1-(4-methanesulfonamidophenyl)alk-1-enes and (Z)-1-(4-azidophenyl)-1,2-diphenylalk-1-enes: novel inhibitors of cyclooxygenase-2 (COX-2) with anti-inflammatory and analgesic activity

A group of novel (Z)-1,2-diphenyl-1-(4-methanesulfonamidophenyl)alk-1-enes was designed for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. In vitro COX-1/COX-2 enzyme inhibition studies identified (Z)-1,2-diphenyl-1-(4-methanesulfonamidophenyl)oct-1-ene (8d) as a highly potent (IC50=0.03 microM), and an extremely selective [COX-2 SI (selectivity index)>3,333], COX-2 inhibitor that showed good anti-inflammatory (AI) activity (ID50=2.8 mg/kg). A molecular modeling (docking) study showed that the p-MeSO2NH group present in (Z)-8d inserts deep inside the 2 degrees-pocket of the COX-2 binding site, it undergoes a hydrophobic interaction with Ala516 and Gly519, and one of the O-atoms of the MeSO2 group participates in a weak hydrogen bonding interaction with the NH2 of Arg513 (distance= 3.85 angstroms). Similar in vitro COX-1/COX-2 enzyme inhibition studies showed that the azido compound 1-(4-azidophenyl)-1,2-diphenyloct-1-ene (9c) is also a potent and selective COX-2 inhibitor (COX-2 IC50=0.11 microM: SI>909) that exhibits good AI activity (ID50=5.0 mg/kg). A docking experiment to determine the orientation of (Z)-9c within the COX-2 binding site showed that the linear p-N3 group inserts into the COX-2 2 degrees-pocket, where it undergoes an ion-ion (electrostatic) interaction with Arg513. Structure-activity data acquired indicate that an olefin having either a C-1 p-MeSO2NH-phenyl, or a p-N3-phenyl, substituent, that is, cis to a C-2 unsubstituted phenyl substituent, in conjunction with C-1 unsubstituted phenyl and C-2 alkyl substituents, provides a novel template to design acyclic olefinic COX-2 inhibitors.     

Design and synthesis of acyclic triaryl (Z)-olefins: a novel class of cyclooxygenase-2 (COX-2) inhibitors

A group of acyclic 2-alkyl-1,1-diphenyl-2-(4-methylsulfonylphenyl)ethenes was designed for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. In vitro COX-1 and COX-2 isozyme inhibition structure-activity studies identified 1,1-diphenyl-2-(4-methylsulfonylphenyl)hex-1-ene as a highly potent (IC(50) = 0.014 microM), and an extremely selective [COX-2 selectivity index (SI) > 7142], COX-2 inhibitor that showed superior anti-inflammatory (AI) activity (ID(50) = 2.5 mg/kg) relative to celecoxib (ID(50) = 10.8 mg/kg). This initial study was extended to include the design of a structurally related group of acyclic triaryl (Z)-olefins possessing an acetoxy (OAc) substituent at the para-position of the C-1 phenyl ring that is cis to a C-2 4-methylsulfonylphenyl substituent. COX-1 and COX-2 inhibition studies showed that (Z)-1-(4-acetoxyphenyl)-1-phenyl-2-(4-methylsulfonylphenyl)but-1-ene [(Z)-13b] is a potent (COX-1 IC(50) = 2.4 microM; COX-2 IC(50) = 0.03 microM), and selective (COX-2 SI = 81), COX-2 inhibitor which is a potent AI agent (ID(50) = 4.1mg/kg) with equipotent analgesic activity to celecoxib. A molecular modeling (docking) study showed that the SO(2)Me substituent of (Z)-13b inserts deep inside the 2 degrees -pocket of the COX-2 active site, where one of the O-atoms of SO(2) group undergoes a H-bonding interaction with Phe(518). The p-OAc substituent on the C-1 phenyl ring is oriented in a hydrophobic pocket comprised of Met(522), Gly(526), Trp(387), Tyr(348), and Tyr(385), and the C-2 ethyl substituent is oriented towards the mouth of the COX-2 channel in the vicinity of amino acid residues Arg(120), Leu(531), and Val(349). Structure-activity data acquired indicate that a (Z)-olefin having cis C-1 4-acetoxyphenyl (phenyl) and C-2 4-methylsulfonylphenyl substituents, and a C-1 phenyl substituent in conjunction with either a C-2 hydrogen or short alkyl substituent provides a novel template to design acyclic olefinic COX-2 inhibitors that, like aspirin, have the potential to acetylate COX-2.     

Protein tyrosine phosphatase 1B inhibitory activity of triterpenes isolated from Astilbe koreana

Bioassay-guided fractionation of a MeOH extract of the rhizomes of Astilbe koreana (Saxifragaceae), using an in vitro protein tyrosine phosphatase 1B (PTP1B) inhibitory assay, resulted in the isolation of a new triterpene, 3alpha,24-dihydroxyolean-12-en-27-oic acid (4), along with four triterpenes, 3-oxoolean-12-en-27-oic acid (1), 3beta-hydroxyolean-12-en-27-oic acid (beta-peltoboykinolic acid; 2), 3beta-hydroxyurs-12-en-27-oic acid (3), and 3beta,6beta-dihydroxyolean-12-en-27-oic acid (astilbic acid; 5). Compounds 1-5 inhibited PTP1B with IC50 values of 6.8+/-0.5, 5.2+/-0.5, 4.9+/-0.4, 11.7+/-0.9, and 12.8+/-1.1 microM, respectively. Our results indicate that 3-hydroxyl group and a carboxyl group in this type of triterpenes may be required for the activity, while addition of one more hydroxyl group at C-6 or C-24 may be responsible for a loss of activity. Thus, compounds 2 and 3 which possess only one hydroxyl group at C-3 and a carboxyl group at C-27 could be potential PTP1B inhibitors.     

Synthesis and antimalarial activity of new haemanthamine-type derivatives

Thirty one derivatives were prepared from the natural alkaloids haemanthamine (1), haemanthidine (2) and 11-hydroxyvittatine (3). They were evaluated for their in vitro antimalarial activity against chloroquine-sensitive strains of Plasmodium falciparum and some structure-activity relationships were outlined. For haemanthamine derivatives having a methoxy group at C-3, the presence of a free hydroxyl group at C-11 is important for the activity. The double bond at C-1-C-2 plays also an important role to achieve good inhibitory activity. Compound 35 with two nicotinate groups at C-3 and at C-11 was the most active compound with a IC(50)=0.8±0.06μM.     

Biosynthesis of the 2-(aminomethyl)-4-(hydroxymethyl)furan subunit of methanofuran

2H- and 13C-labeled precursors were used to establish the pathway for the biosynthesis of the 2-(aminomethyl)-4-(hydroxymethyl)furan (F1) component of methanofuran in methanogenic archaebacteria. The extent and position of the label incorporated into F1 were measured from the mass spectrum of the diacetyl derivative of F1. [1,2-13C2]Acetate was found to be incorporated into two separate positions of the F1 molecule as a unit. The extent of incorporation of 13C2 into each of these positions was the same as that observed for the incorporation of acetate into the alanine and proline produced by the cells. From [2,2,2-2H3]acetate, deuterium was incorporated into two separate sites of the F1 molecule, one containing up to two deuteriums and the other only one. On the basis of the fragmentation pattern of the F1 diacetyl derivative, it was determined that two deuteriums were incorporated into the hydroxymethyl group at C-4 and one was incorporated at C-3 of the furan ring. The extent and distribution of the incorporated deuterium at the C-4 methylene were the same as that observed for C-6 of the glucose produced by the cells. On the basis of this and additional information presented in this paper, it is concluded that F1 is generated by the condensation of dihydroxyacetone phosphate with pyruvate. The resulting dihydroxy-substituted tetrahydrofuran after elimination of 2 mol of water would produce the phosphate ester of 2-carboxy-4-(hydroxymethyl)furan. Reduction of the carboxylic acid to an aldehyde and subsequent transamination would produce the phosphate ester of F1.