NMR: An indispensable ADME tool
We highlight two recent papers this month, both of which feature the role of NMR spectroscopy in drug discovery and development, including its application as an indispensable analytical tool in ADME investigations [1, 3].
ADME Applications
Unlike LC-MS/MS based approaches, NMR spectroscopy is able to define the exact position of biotransformation in a drug compound. Not only that, by defining the position of biotransformation, NMR can also provide mechanistic insights into the formation of major metabolites observed in MetID studies.
An example given in paper [1] discusses the insights gained from the NMR data acquired for BMS-690514 M1, a cytochrome P450-mediated metabolite arising from hydroxylation followed by rearrangement.
BMS-690514 was shown to suffer from lower bioavailability in monkey, compared with rat and other species. Human and animal hepatocyte incubations revealed susceptibility to both phase I and phase II metabolism with the formation of several oxidative metabolites and direct glucuronides. The 14C-radiolabelling study in humans confirmed this, with circulating metabolites comprising a hydroxylated rearrangement product (M1), a direct ether glucuronide (M6), and multiple secondary glucuronides [2].
The position of hydroxylation in M1 was established by examination of the HMBC and ROESY correlations. Further, deuterium labelling of the parent compound revealed the presence of the label at the site of hydroxylation in M1 but subsequently lost during formation of another metabolite M37. The mechanism for formation of M1 was thus proposed as follows:
- Oxidation via epoxidation or iron-oxo addition pathway
- Opening of the pyrrolotriazine ring to form an aldehyde intermediate
- Reaction of the aldehyde with the secondary amine of the methoxyaniline group
- Formation of the pyridotriazine ring of M1
Circulating metabolites of BMS-690514 (* = position of carbon 16)
Use of 19F-NMR and mass spec as an alternative to traditional ADME studies
At the 50th DMDG meeting in York, Steve Thomas at GSK presented on a project in which both mass spec and NMR were instrumental in characterising the metabolic profile of alpibectir, a drug for TB that potentiates the efficacy of the “old drug” ethionamide by circumventing a resistance mechanism. This work has also recently been published in DMD [3].
Alpibectir has a relatively simple structure. It is extensively metabolised with major hydrolysis of the amide forming an N-dealkylated amine (M1) and trifluorobutyric acid product (M19), which are further biotransformed to a carbamoyl glucuronide conjugate (M18) and trifluoroacetic acid (M20), respectively.
Metabolites of alpibectir arising from amide hydrolysis and subsequent biotransformations
Due to the multiple sites of fluorination in the molecule, it was possible to fractionate human urine and plasma to detect and quantify metabolite responses using 19F-NMR [3]. Using this data and that derived from 1H-NMR, and UHPLC-MS/MS, the qualitative metabolic profile and quantitative determination of drug-related material circulating in plasma and excreted in urine could be determined without the use of radiolabelled drug. During discussion at the DMDG meeting, the need to be sure everything has been measured was brought up.
Also highlighted and worth noting here is the usefulness of authentic metabolite standards rather than relying on potentially misleading mass spectrometric intensities. In this example, the ionization efficiency of M1 was seen to be 5.3-fold higher than an equimolar standard of alpibectir under the same analytical conditions. Metabolites can also have markedly different ionization efficiencies compared with parent drug, potentially resulting in incorrect assignment of major and minor metabolites.
Cryoprobe technology: a game changer
Coupled with the use of high field magnets to increase sensitivity and discriminate between signals, cryoprobe technology enables significant sensitivity enhancements such that full structural information can be acquired on only small amounts (tens of micrograms or less) of material. Scientists at Hypha use data acquired on a 700 MHz instruments fitted with a 1.7mm cryoprobe as a tool for definitive structure elucidation of metabolites and degradation products of small molecule drugs [4], including larger small molecules such as macrocycles, degraders and cyclic peptides.
Quantitative NMR
The use of NMR as a quantitative technique for determining purity and concentration is becoming increasingly prevalent in the field of drug metabolism. The applications of quantitative NMR (qNMR) range from the determination of the absolute purity of metabolite materials purified in milligram-gram quantities to be used as analytical reference standards to the assessment of concentrations of metabolites purified in sub-milligram substrate amounts to enable bioactivity testing and SAR exploration.
For published examples illustrating the application of biosynthesised metabolites and quantitation using qNMR, we recommend a read of the paper published by Pfizer scientists on this topic [5]. Coupling of the two methods enabled generation of solutions of metabolites of known structure and concentration for evaluation in pharmacologic assays, standards for in vitro work to help establish clearance pathways, or as analytical standards for bioanalytical work to ascertain exposure.
qNMR provision at Hypha
qNMR is provided as part of our suite of analyses for synthesised metabolites. For metabolites purified in sufficient quantity, purity can be assessed by qNMR measurements made with reference to an internal standard certified as a reference material, selected so that the reference signals to be used for quantitation are well-resolved from any sample signals [5]. Similar amounts (≥ 3-4 mg) of analyte and reference standard are accurately weighed into the same vial in duplicate (or triplicate) and the combined samples dissolved in an appropriate deuterated solvent. The analyte purity is then determined by comparing the integral of a selected signal in its 1H NMR spectrum with that of the reference standard signal and calculating the purity based on the known reference purity.
For metabolites purified in ≤ 1 mg quantities, where the physical weight of the isolated material may not be reliable, the sample is valuable and avoidance of contamination through using an internal standard is undesirable, metabolite concentration can be estimated by comparison of discrete, well-resolved peaks in its 1H NMR spectrum with an artificial signal added during sample spectral acquisition. The protocol used is ERETIC (Electronic REference To access In vivo Concentration), using an electronically generated signal based on a previously acquired 1H NMR spectrum of a certified standard.
Considering all the various applications, it will be interesting to see how far NMR spectroscopy will reach in helping understand and meet the challenges of drug discovery in the future, and not just as a tool for structure elucidation.
References
[1] Perspectives on Nuclear Magnetic Resonance Spectroscopy in Drug Discovery Research. Janet Caceres-Cortes, Bradley Falk, Luciano Mueller, and T G Murali Dhar. Journal of Medicinal Chemistry 2024 67 (3), 1701-1733 DOI: 10.1021/acs.jmedchem.3c02389
[2] Metabolism and disposition of [14C]BMS-690514, an ErbB/vascular endothelial growth factor receptor inhibitor, after oral administration to humans. Christopher LJ, Hong H, Vakkalagadda BJ, Clemens PL, Su H, Roongta V, Allentoff A, Sun H, Heller K, Harbison CT, Iyer RA, Humphreys WG, Wong T, Zhang S. Drug Metab Dispos. 2010 Nov;38(11):2049-59. doi: 10.1124/dmd.110.034850.
[3]Human Met-ID of Alpibectir; Combining 19F and 1H-NMR with HRMS. , , and
[4] Chapter 4 – Methods for metabolite generation and characterization by NMR. In: Identification and Quantification of Drugs, Metabolites, Drug Metabolizing Enzymes, and Transporters (Second Edition). Liam Evans, Richard Phipps, Julia Shanu-Wilson, Jonathan Steele, Stephen Wrigley. Editors: Shuguang Ma, Swapan K. Chowdhury. Elsevier, 2020, Pages 119-150, ISBN 9780128200186.
https://doi.org/10.1016/B978-0-12-820018-6.00004-1
[5] Biosynthesis of Metabolites for Use in Pharmacologic Studies. Gregory S. Walker, Jonathan N. Bauman, Tim F. Ryder, Evan B. Smith, Douglas K. Spracklin and R. Scott Obach. Drug Metabolism and Disposition October 1, 2014, 42 (10) 1627-1639; DOI: https://doi.org/10.1124/dmd.114.059204
[6] Importance of purity evaluation and the potential of quantitative 1H NMR as a purity assay. G.F. Pauli, S.-N. Chen, C. Simmler, D.C. lankin, T. Gödecke, B.U. Jaki, J.B. Friesen, J.B. McAlpine, J.G. Napolitano. J. Med. Chem. 57 (2014) 9220-9231. https://doi.org/10.1021/jm500734a
Recommended further reading on the application of NMR in biotransformation studies
The Role of NMR as a Qualitative and Quantitative Analytical Technique in Biotransformation Studies. Gregory S. Walker, Raman Sharma, Shuai Wang. Chapter 13. In: Handbook of Drug Metabolism, Third Edition. 2019. https://www.taylorfrancis.com/chapters/edit/10.1201/9780429190315-14/role-nmr-qualitative-quantitative-analytical-technique-biotransformation-studies-gregory-walker-raman-sharma-shuai-wang
