Biotransformations can be diverse and not limited to simple oxidation or dealkylation. An oncologic agent, The BMS-690514, is an example of unexpected metabolism. This compound undergoes multiple biotransformations, among them are P450 mediated oxidations of its heterocycle pyrrolotriazine group. Two major metabolites A and B are resulting from + O biotransformation.  However if the metabolite A shows a hydroxylation of the heterocycle, the metabolite B seems to undergone a unusual rearrangement. The isolation of that metabolite demonstrated the formation a hydroxypyridotriazine group.

This structure was confirmed using NMR spectrometry. The metabolism study of deuterium and tritium isotope label at the oxidized position showed that the label was retain during the formation of the metabolite A and lost during the formation of the metabolite B. The authors concluded that this metabolite was formed via epoxidation.

Haizheng Hong et al. Chem. Res. Toxicol. 2011, 24, 125-134.

Several recent papers have highlighted the importance of identifying mitochondrial liabilities to completely assess the toxicity potential of a drug (1-3).  Mechanisms by which drugs induce organ toxicity include the production of reactive metabolites (4).  Examples such as high-dose acetaminophen, which produces metabolites toxic to the mitochondria are found in the literature (5-8). 

Metabolites or cocktails of metabolites obtained through the Hepatochem technologies may act on one or several mitochondrial targets to induce mitochondrial impairment.  ROS production or reduced anti-oxidant defenses, perturbation of the bioenergetic balance, induction of permeability transition, depletion of mtDNA or reduced mitochondrial mass are some of the various targets that may induce mitochondrial dysfunction, loss of susceptible cell integrity & ultimately organ malfunctions and/or failure (7,9-12).  Using the MitoSafe line of functional bioassays developed by ICDD will demonstrate whether metabolites of your drugs induce mitochondrial liabilities in live-cell models.  Mitochondria toxicity is most readily expressed clinically by hepatic injury & cardio-toxicity, which can both be flagged through the study of your compounds and their metabolites with the MitoSafe bioassays.

Don’t hesitate to ask questions to our mitochondria experts:Contact@icdd-sas.com

1- Marroquin LD, Hynes J, Dykens JA, Jamieson JD, Will Y. Toxicol Sci. 2007 ;97(2):539-47.

2- Dykens JA, Will Y. Drug Discov Today. 2007 ;12(17-18):777-85.

3- Begriche K, Massart J, Robin MA, Borgne-Sanchez A, Fromenty B.  J Hepatol. 2011 ;54(4):773-94.

4- Liebler DC, Guengerich FP.  Nat Rev Drug Discov. 2005 ;4(5):410-20.

5- Kostrubsky SE, Strom SC, Ellis E, Nelson SD, Mutlib AE. Chem Res Toxicol. 2007 ;20(10):1503-12.

6- Jaeschke H, McGill MR, Williams CD, Ramachandran A.  Life Sci. 2011 25;88(17-18):737-45.

7- Song Y, Shi Y, Yu H, Hu Y, Wang Y, Yang K. Toxicol Lett. 2011 ;202(1):55-60.

8- Chaudhuri L, Sarsour EH, Goswami PC. Environ Int. 2010 ;36(8):924-30.

9- Siu WP, Pun PB, Latchoumycandane C, Boelsterli UA.  Toxicol Appl Pharmacol. 2008 ;227(3):451-61.

10- Bai J, Nakamura H, Ueda S, Kwon YW, Tanaka T, Ban S, Yodoi J. J Biol Chem. 2004 ;279(37):38710-4.

11- Ramachandran A, Lebofsky M, Weinman SA, Jaeschke H.Toxicol Appl Pharmacol. 2011 ;251(3):226-33.

12- Zou W, Roth RA, Younis HS, Burgoon LD, Ganey PE. Toxicology. 2010 ;272(1-3):32-8. 

Metalloporphyrins are powerful catalysts capable of a wide variety of chemical transformations. Simple modifications to the catalyst system allow for tuning a catalyst for relatively mild oxidations or more difficult to oxidize substrates. Recently, Zhdankin and coworkers have demonstrated a co-catalyst system with an iron porphyrin with a mixture of iodobenzene and oxone allowing for the quantitative conversion of anthracene to anthroquine (1). This system has also shown promise for the oxidation of alkanes and alkenes such as tetrahydronaphthalene, dihydroanthrane and styrene in moderate yields.

With tuning of the catalyst and reaction conditions, metalloporphyrins are also capable of mild oxidations such as sulfoxidations even in the presence of reactive C-H or alcohol functional groups. Huang and coworkers have used a manganese porphyrin-hypochlorite system for the selective oxidation of glycosyl sulfides to the sulfoxides with high diasteromeric excesses (2). Very little sulfone formation and no oxidation on the sugar occurred. These two recent examples show both the selectivity and powerful oxidation capabilities of metalloporphyrins.

1. Yoshimura, A.; Neu, H. M.; Nemykin, V. N.; Zhdankin, V. V., Metalloporphyrin/Iodine(III)-Cocatalyzed Oxygenation of Aromatic Hydrocarbons. Advanced Synthesis & Catalysis 2010, 352, (9), 1455-1460.
2. Huang, J. Y.; Li, S. J.; Wang, Y. G., Selective Oxidation of Glycosyl Sulfides to Sulfoxides with Sodium Hypochlorite and Catalyzed by Metalloporphyrins. Journal of Carbohydrate Chemistry 2010, 29, (3), 142-153.

Drug induced liver injury is a major cause for withdrawing a drug from development or more dramatically from the market. In a recent article, Dr. Dennis J. Pelletier et Al. performed a SAR study of hepatotoxicity. They used the data from literature and built a structure searchable database. The resulting database was analyzed to identify the chemical structures associated with liver toxicity. Data from over 1266 compounds were collected and a SAR of 38 chemical structures was developed. An interesting chemical structure highlighted as a potential liver toxin is the thiophene ring. Metabolic activation of thiophene leads to a reactive intermediate that can undergo Michael type addition with cellular nucleophiles. See figure below.

Interestingly, hepatotoxicity is often due to an activation of the drug resulting from Phase I metabolization. Moreover, this kind of reactive metabolite is present at low levels in the blood stream which makes them difficult to be detected.
Biomimetic technology can allow for the production of such metabolites for biological and toxicology studies which could reduce the drug development attrition due to liver toxicity.
Dennis J. Pelletier et Al.; Chem. Res. Toxicol., 2010, 23, 1215-1222

We would like to bring to your attention to a new metabolism tool available online. Lars Olsen et al. have developed a web based platform that predicts potential sites of metabolization. This free tool is the first web service for prediction of CYP-mediated metabolism. Based on a recent publication from Simon E. Ward et al. where they describe the metabolism pathway of a novel clinical AMPA receptor positive modulator, we tested the prediction tool and the result confirmed the potential utility of this application for the prediction of CYP-mediated metabolism.

Patrik Rydberg, David E. Gloriam and Lars Olsen. Bioinformatics 2010, 26, 2988-2989. www.farma.ku.dk/smartcyp

Simon E. Ward et al. J. Med. Chem. 2010, 53, 5801-5812

In a recent article, Dr Angela D. Kerekes et al. described the PK optimization of a potent Aurora inhibitor 1. This compound presents a good PK profile for intravenous delivery but poor oral bioavailability in rats due to rapid metabolism and poor oral absorption. Metabolism studies determine that the compound undergoes N-desethyl and oxidation to generate metabolites 2 and 3.

To improve the PK, the initial strategy was to block N-Alkylation by introduction of fluorine (see compound 4). This modification improved the oral PK in rat but oral PK in monkey was unchanged. In further metabolism studies the failure of the fluorine addition to improve oral PK was attributed to an additional hydroxylation of the arylic position. The introduction of deuterium to that position resulted in a improved oral PK in monkey (see compound 5). This example demontrates that introduction of fluorine and deuterium when possible can be an excellent strategy to improve oral PK.

Angela D. Kerekes et al. J. Med. Chem. 2011 asap

Qualitative Analysis of the Role of Metabolites in Inhibitory Drug-Drug Interactions

In a recent guidance, the Food and Drug Administration recommended safety studies of metabolites. This guidance applied primarily to the metabolites formed specifically in humans for which toxicity could not be predicted by animal testing. However, in addition to directly mediating toxicity metabolites may also be responsible for Cyp inhibition. Preclinical animal studies are not predictive of potential drug-drug interaction which can become problematic. In two recent articles, Dr. Nina Isoherranen et al. presented results of literature analysis using the Metabolism and Transport Drug Interaction Database. Specifically, they found that in DDI studies where ther is a 20% increase in the area under the plasma concentration-time curve (AUC) of marker substrates, that 106 out of 129 inhibitors were confirmed to have metabolites that circulate in plasma. These data support that circulating metabolites are often present with inhibitors of P450 enzymes, suggesting a need for increased efforts to characterize the inhibitory potency of metabolites of candidate drugs and for newer models for in vitro to in vivo extrapolations.

1. Nina Isoherranen, Houda Hachad, Catherine K. Yeung, and Rene H. Levy. Chem. Res. Toxicol. 2009, 22, 294-298 and C.K. Yeung, Y. Fujioka, H. Hachad, R.H. Levy and N. Isoherranen. Clinical pharmacology & Therapeutics 2011, 89, 105-113

By Prof. John T Groves

For many years it has been thought that chlorination reactions involving C-H bonds were intrinsically unselective, especially for complex molecules. However, highly selective chlorinations are now recognized in natural product biosynthesis that are mediated by metalloenzymes such as chloroperoxidases and the non-heme enzyme Syr3. The development of a practical catalyst for such halogenations would offer numerous possibilities for late stage drug diversification or selective fluorination. A recent publication from the Groves lab at Princeton University has made an important advance in biomimetic halogenation. Aspects of this work were supported by the National Institutes of Health, the National Science Foundation and the Department of Energy.

Graduate student Wei Liu has developed a manganese catalyst that shows great promise for the C-H chlorination of complex, drug-like molecules. In one interesting example, the terpenoid natural product sclareolide was selectively chlorinated at a single methylene position in 42% yield, despite the large number of other sites available.¹

1. Wei Liu and John T. Groves, Manganese Porphyrins Catalyze Selective C-H Bond Halogenations, J. Am. Chem. Soc., 2010, 132, 12847-12849.

Surprising biotransformation leading to active metabolite

The structure elucidation and biological testing of metabolites can allow for discovery of new chemical entities.

In their CB1 therapeutic program, Dr Chien-Huang Wu et al. discovered the formation of a unique metabolite resulting from a biotransformation of their lead compound 1. Surprisingly, the imide moity of that molecule undergoes metabolic N-demethylation to form the intermediate 2. This compound rearranges by cyclization to generate the imidazole-4-one 3. More interestingly, the resulting compound showed potency and was used as a starting point to develop a new chemical series. The metabolite isolation allowed for the discovery of a new chemical series.

J. Med. Chem. 2009, 52, 4496-4510

By Prof. John T Groves

For many years the ‘Holy Grail’ in oxidative catalysis has been the selective functionalization of unactivated C-H bonds in complex molecules. In the case of hydroxylation, such processes could provide access to metabolites and analogs of drug molecules much the way cytochrome P450 enzymes do. As for halogenation, highly selective chlorinations are now recognized in natural product biosynthesis that are mediated by metalloenzymes such as chloroperoxidases and the non-heme enzyme Syr3. Two recent publications from the Groves group at Princeton University have made advances in biomimetic catalysis on both fronts. Aspects of this work were supported by the National Institutes of Health, the National Science Foundation and the Department of Energy.

Work by graduate student Seth Bell has led to an extraordinarily efficient and fast catalysis of C-H hydroxylation.(1) The catalyst is a water-soluble iron porphyrin that is simple to prepare and to use (A). Xanthene hydroxylationoccurred with a very large rate constant, greater than 106 M-1s-1. The highly sensitive hydroxylation product, 9-xanthydrol, could be obtained in 90% yield at 48% conversion (B). Kinetic and mechanistic analysis revealed that strong C-H bonds, up to 100 kcal/mol, could be hydroxylated with this catalyst. Notably, over oxidation, which is a normal course of events with other oxidants, was very limited in this case.

In another recent development in the Groves lab, graduate student Wei Liu has developed a manganese catalyst that shows great promise for the C-H chlorination of complex, drug-like molecules. In one interesting example (C), the terpenoid natural product sclareolide was selectively chlorinated at a single methylene position in 42% yield, despite the large number of other sites available.(2)

1. Seth R. Bell and John T. Groves, A Highly Reactive P450 Model Compound I, J. Am. Chem. Soc., 2009, 131, 9640-9641.
2. Wei Liu and John T. Groves, Manganese Porphyrins Catalyze Selective C-H Bond Halogenations J. Am. Chem. Soc., 2010, 132, ASAP.