Structures of metabolites

Figure 5.44 Structures of metabolites of Indinavir proposed on the basis of the product-ion scans of the (M + H)+ ions at m/z 483, 523 and 539 obtained during the LC-MS analysis of an incubation of Indinavir with rat liver S9. Reprinted by permission of Elsevier Science from Identification of in vitro metabolites of Indinavir by Intelligent Automated LC-MS/MS (INTAMS) utilizing triple-quadrupole tandem mass spectrometry , by Yu, X., Cui, D. and Davis, M. R., Journal of the American Society for Mass Spectrometry, Vol. 10, pp. 175-183, Copyright 1999 by the American Society for Mass Spectrometry.

H-labeled substrates have been used to determine the dissipation and degradation of aromatic hydrocarbons in a contaminated aquifer plume (Thierrin et al. 1995). Its application was particularly appropriate since the site was already contaminated with the substrates. With suitable precautions, this procedure seems capable of extension to determining the presence—though not the complete structure—of metabolites, provided that the possibility of exchange reactions were taken into account. 

Chapters 5 and 6 attempt to provide a brief introduction to experimental procedures with emphasis on procedures for establishing the structure of metabolites using isotopes and physical methods. 

The electrospray mass spectrum of metabolite 2 indicates it has a molecular weight of 522 Da, while the MS-MS spectrum of the (M + H)+ ion contains an intense ion at m/z 422, 1 Da greater than the base peak of the MS-MS spectrum of the protonated molecular ion of the parent drug. If we assume a similar relationship between these ions as assumed for m/z 465 and m/z 466 above, it is not unreasonable to postulate the structure of metabolite 2 to be that shown in Figure 5.44. 

Tab. 1.1 Chemical structures of metabolites involved in selective isotope labeling strategies (from Ref. [14] with kind permission)...

In the present paper, emphasis will be placed on findings obtained in the last three years. Attention will focus on aromatic hydrocarbon accumulations and on the formation, retention, and structure of metabolites. Material appearing in recent reviews will not be emphasized (2,3,4). 

During the course of clinical development, it is often important to identify the structures of metabolites. This information provides an opportunity to better understand interpatient variability in pharmacokinetics and toxicity. Clinical studies performed by Lokiec and coworkers, 1996 on a semisynthetic derivative of 20(S)-camptothecin, CPT-11, demonstrate the use of LC/MS to investigate the in vivo metabolic pathways. CPT-11 is a potent inhibitor of topoisomerase II, which is an enzyme involved in DNA duplication, and exhibits significant activity against various types of tumors in clinical studies. The understanding and control of the main biotransformation pathways are particularly important for anticancer drugs because therapeutic doses are often close to the maximum tolerated dose. 

Structure of metabolites not identified Pathways similar to 2,3,7,8-TCDD ... 

Figure 5.4. Alphabetical list of biochemical compounds at Klotho. The 1D (Smile strings), 2D, and 3D structures of metabolites can be viewed/retrieved from Klotho server.

Nevertheless, certain features of terran metabolism might benefit from a biosolvent whose properties differ from water s. For example, the instability of C=N in water constrains the structure of metabolites in water. The compound HN=C=NH, an analog of 0=C=0 (carbon dioxide), immediately hydrolyzes in water to give urea (H2NCO-NH2), whose thermodynamic instability with respect to hydrolysis in water yields carbon dioxide and ammonia. Thus, water as a solvent requires that the dominant form of carbon at the +4 oxidation state be carbon dioxide. 

Table 1. Mathematical structure of metabolite and isotope balances...

In most instances classical approaches in isolation of metabolites followed by MS and NMR analyses are still necessary to obtain the definitive structural information. This is due for complex structures of metabolites as they are drugs in development in most instances with the need of multiple NMR experiments in addition to an initial one dimensional1H NMR experiment. 

The thiophene and the 3-substituted thiophenes 233 have been found to undergo ring dihydroxylation yielding the as fraar-dihydrodiol metabolites 234. Evidence is provided for a dehydrogenase-catalyzed desaturation of a heterocyclic dihydrodiol 234-< /234-rraas (R= Me) to yield the corresponding 2,3-dihydroxythiophene 235 as its preferred thiolac-tone tautomer 236. A simple model to allow prediction of the structure of metabolites, formed from Toluene dioxygenase (TDO)-catalyzed bacterial oxidation of thiophene substrates, has been presented (Scheme 17) <20030BC984>. 

Liquid chromatographic methods for determining ezetimibe and its metabolites are summarized in Table 3.10 and described in subsequent sections. The chemical structure of metabolites of ezetimibe is illustrated in Fig. 3.4. 

Similar research has attempted to gain insight into protein function prediction based on information hidden in the molecular structure of metabolites (35). Such work may eventually identify the relationship between metabolite structure and protein function, thus possibly improving techniques in the prediction of enzyme function and novel metabolic pathways (36). 

Positive ion thermospray LC/MS and LC/MS/MS methods carried out on a triple quadrupole mass spectrometer have been successfully utilized in our laboratory to elucidate the structures of metabolites of several compounds currently undergoing development as drug candidates. Samples were obtained from both in vivo sources (urine, bile, or plasma) and analyzed directly from the biological fluid or from in vitro enzymatic/chemical methods. In all cases, buffer ionization mode using ammonium acetate in the HPLC mobile phase was employed for ionization of the metabolites of interest. 

It must be stressed that it is useless to perform any chemical modification it is thus possible to study the structures of metabolites produced in vivo directly, with all associated advantages. 

Drug metabolism studies in animal models provides important information concerning the structures of metabolites before embarking on costly human studies. MS is used to provide confirmatory information if synthetic metabolites are available and to help in the structural characterization of novel metabolites. Many... 

Porphyrin synthesis. The synthesis of porphyrins is shown, with the structures of metabolites up to porphobilinogen (a pyrrole) presented. After that point, only an... 

This technique may be used in two distinct ways. The drug may be labeled at a specific site(s) and the mass spectra of metabolites derived from the labeled compound compared with those obtained from the unlabeled drug. Recent examples of this application include reports on the metabolism of oxybutynin,tocainide, and ketamine. Alternatively, a heavy isotope (usually H) is introduced into the metabolite of interest by reaction with a labeled derivatizing reagent. For example, perdeutero-methylation results in a mass difference of 3 daltons between labeled and unlabeled derivatives per methyl group. Structures of metabolites obtained from caffeine and phenytoin have been confirmed by this technique. Tri(deuteromethyl)silylation has been employed in the identification of metabolites of carbamazepine and afloqualone. ... 

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