Pathway 8-hydroxycoumarin

Further transformation included additional hydroxylation steps leading to 2,6-dihydroxyquinoline and a trihydroxyquinoline (probably 2,5,6-trihydroxyquinoline). Shukla [322], working with Pseudomonas sp. identified an alternate pathway, involving additional metabolites, besides the 2-hydroxyquinoline and 8-hydroxycoumarin. These were 2,8-dihydroxyquinoline and 2,3-dihydroxyphenylpropionic acid. Quinoline-adapted cells were also able to transform 2-hydroxyquinoline and 8-hydroxycoumarin without a lag phase, providing additional support for their intermediate role as intermediates in the metabolism of quinoline. 

Schwarz et al. in agreement with Shukla observed the formation of 2-Oxo-l, 2-dihydroquinoline, 8-hydroxy-2-oxo-l, 2-dihydroquinoline, 8-hydroxycoumarin, and 2,3-dihydroxy-phenylpropionic acid were found as intermediates of quinoline transformation by P. fluorescens 3 and P. putida 86 [325], They compared that metabolic pathway with the one obtained for Rhodococcus strain B1 (Fig. 22). This bacterium was unable to yield denitrogenated metabolites (i.e., 2-oxo-l, 2-dihydroquinoline, 6-hydroxy-2-oxo-l, 2-dihydroquinoline, and 5-hydroxy-6-(3-carboxy-3-oxopropenyl)-lH-2-pyridone). 

Figure 23 sketches these four pathways, namely the 5,6-dihydroxy-2(lH)quinolinone pathway, the 7,8-dihy-droxy-2(lH)quinolinone pathway, the anthranilate pathway, and the 8-hydroxycoumarin pathway. 

C. tetosteroni 63 [320,349] Susanne Fetzner Quinoline 2-oxidoreductase (360 kDa) Nitrogen removal (hydroxycoumarin pathway). Reactivity demonstrated with heterocyclic-N... 

QN 2-oxidoreductase (OR), lH-2-Oxol, 2-dihydroQN-8monooxygenase. 35% OR yield Nitrogen removal (hydroxycoumarin pathway). OR Activity 19 units (33 times the extract activity)... 

Unlike in humans, the major metabolic pathway of coumarin in rats is the 3,4-epoxidation pathway. After a 100-mg/kg bw oral dose of [3- C]coumarin, urinary 3-hydroxycoumarin, 7-hydroxycoumarin, ort/zo-hydroxyphenyllactic acid and ortho-hydroxyphenylacetic acid accounted for 1.8, 0.4, 0.8 and 20% of the dose, respectively. Various metabolites including ort/zo-hydroxyphenylacetic acid were detected in the faeces (Kaighen Williams, 1961). Other studies in vivo have confirmed that rats are poor 7-hydroxylators of coumarin, with urinary 7-hydroxycoumarin accounting for < 1% of the dose (van Sumere Teuchy, 1971 Lake et al, 1989a). 

Because of the relative ease of measurement of 7-hydroxycoumarin, many studies have examined this pathway of coumarin metabolism after oral administration. Overall, several species including rats, most mouse strains, Syrian hamsters, guinea-pigs, dogs, marmosets and squirrel monkeys are poor 7-hydroxylators, excreting <5% of the administered dose as urinary 7-hydroxycoumarin (Cohen, 1979 Lake, 1999). 

Apart from glucuronic acid and sulfate conjugation of hydroxycoumarins, other phase II pathways of coumarin metabolism have been identified. For example, ortho-coumaric acid may be conjugated with glycine (Lake, 1999), and a coumarin mercapturic acid conjugate has also been reported (Huwer et al., 1991). Coumarin may also be metabolized by the gastrointestinal microflora to 3,4-dihydrocoumarin and ort/io-hydroxyphenylpropionic acid under anaerobic conditions (Scheline, 1968). 

Mass spectra of hydroxy- and alkoxy-coumarins have been very intensively studied. The decomposition sequence of 3-hydroxycoumarin is initiated by carbon monoxide loss from the molecular ion giving a 2-hydroxybenzofuran ion. Subsequent fragmentation occurs by two major pathways, involving a further loss of CO and expulsion of a formyl radical. The former leads to the base peak, and thence by another loss of CO to give the abundant benzene radical cation at m/e 78. The other main pathway gives a benzoyl cation which leads to the phenonium ion at m/e 77 (77IJC(B)816). 

Extensive studies involving 57 aryl-substituted 4-hydroxycoumarins and using electrospray ionization (ESI) mass spectrometry have allowed the effect of the substituents in the aromatic ring on the fragmentation patterns to be determined. Deuterated compounds were used to prove some of the proposed fragmentation pathways, and the effect of tautomerism on the formation of quasimolecular ions and subsequent fragmentation was explained <2004EJS523>. 

Dermal exposure by-passes the first-pass effect of initial metabolism by the liver. Coumarin in the blood first passes through the lung, where significant amounts can be exhaled, prior to being metabolically processed by the liver. Metabolic pathways are highly species- and, sometimes, strain-specific. DBA/2J mice have been reported to have a high level of coumarin hydroxylase activity, resulting in metabolism mainly to 7-hydroxycoumarin. CH3/HeJ mice, on the other hand, have been reported to have very little hydroxylase activity. 

In rats and many strains of mice other than the DBA/2J, oral coumarin exposure results in hepatic metabolism of coumarin, with the formation of the coumarin 3,4-epoxide (CE), which spontaneously rearranges extremely rapidly to the o-hydroxyacet-aldehyde (o-HPA), the toxic metabolite. The o-HPA is then further metabolized to the nontoxic o-hydro-xyacetic acid (o-HPAA) and o-hydroxyethanol (o-HPE). Rodents also metabolize coumarin by several lesser pathways, to the nontoxic 3-hydroxycoumarin and several other more minor metabolites. It is the balance between the formation of the toxic o-HPA and the nontoxic o-HPAA and o-HPE that is critical to the determination of hepatotoxicity at high exposure levels of coumarin. Mice form more o-HPA than do rats, but detoxify it much more rapidly and efficiently than do rats. The result is that hepatotoxicity at doses 150 mg coumarin kg body weight is observed in rats, but not mice. Similarly, when high doses of coumarin result in high plasma levels, mice demonstrate pulmonary toxicity whereas rats do not. This is the result of the formation of higher levels of CE and o-EIPA in the Clara cells in the lungs of mice, which is not observed in rats. 

The bicoumarin 229 was obtained using a double Fries rearrangement of the diacetate 226 promoted by TiCU as a Lewis acid, and a subsequent cyclization of the dicarbonate 228 derived from the diketone 227 (equation 103) . The Fries rearrangement of hydroxycoumarin chloroacetates 230 provides a new short pathway to furocoumarins 231 (equation 104) °°. 

Seven hydroxycoumarins were further tested at a single concentration (5.0x1 O 4 M) for their ability to influence Cl and C3 functional activities after preincubation with undiluted NHS. 7-Methylesculin (11) had a good effect on reducing total, Cl, and C3 hemolysis via both pathways. Scoparone (6) strongly inhibited C3 alternative activity but in the case of the classical pathway only the total hemolysis was diminished without influence on Cl and C3. Esculin (1) slightly increased C3 classical activity but caused exhaustion of alternative C3 activity. 

Biosynthesis 7-Hydroxycoumarin ( umbelliferone) is formed on the shikimic acid pathway and geraniol is the precursor of the side chain. 

Isotopic enrichment and Vcc couplings have been a great help in tracing the biosynthetic route of the 3,4-dihydrobenzoate moieties of the siderophore petrobactin, produced by Bacollus anthracis str. Sterne and in investigation of biosynthetic pathways to hydroxycoumarins during postharvest physiological deterioration in cassava roots. 

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