Cyanide Trapping of Iminium Ion Reactive Intermediates Followed by Detection and Structure Identific

1
Cyanide Trapping of Iminium Ion Reactive
Intermediates Followed by Detection and Structure
Identification Using Liquid Chromatography-Mass
Spectrometry Dayana Argoti1, Li Liang2, Abdul
Conteh2, Liangfu Chen2 , Dave Bershas2 ,
Chung-Ping Yu2 , Paul Vouros1, Eric
Yang2 1Barnett Institute and Dept. of Chemistry,
Northeastern University, Boston, MA 2Drug
Metabolism and Pharmacokinetics, GlaxoSmithKline
Pharmaceuticals, King of Prussia, PA

• Overview
• The goal of this research was to develop a
  relatively high throughput LC/MS method for the
  detection of iminium ions as reactive
  intermediates in rat and human microsomal
  incubations
• Iminium ions were detected via a constant neutral
  loss (CNL) scan and further characterized via
  MS/MS
• A total of fourteen alicyclic amine compounds
  were investigated with parallel trapping
  experiments of cyanide and glutathione (GSH)
• Detailed results of the cyanide trapping method
  for nefazodone and prochloroperazine are
  discussed, while overall results for assays are
  listed in Table 1.
• This work may provide an effective LC/MS method
  for the detection of iminium ion intermediates
  from alyciclic amine compounds

Results Summary for Compounds Screened
Results and Conclusions

• Cyanide efficiently and consistently traps
  reactive iminium ion intermediates
• In addition to detection of parent drug iminium
  ions as a cyano adduct, several metabolite based
  iminium ions were detected that are consistent
  with reported metabolism for the compound
• Bioactivation of alicyclic amines does not always
  necessarily involve an iminium ion intermediate
• Generally, overall results for both cyanide and
  GSH trapping were complementary for alicyclic
  amine compounds
• A high-throughput LC-MS screening method has
  been developed for detection of reactive
  intermediates of alicyclic amine drugs

Liquid Chromatography/Mass Spectrometry
Adduct Detection in Constant Neutral Loss and
Proposed Metabolite Structures

• Introduction
• Cytochrome P450 mediated bioactivation of a drug
  to its reactive metabolite has been reported as
  the first step in many adverse drug reactions1
• A commonly used technique for reactive metabolite
  detection is the GSH trapping assay, however,
  this method cannot detect some specific reactive
  metabolites, such as iminium ions, acyl
  glucuronides and aldehydes2-4
• Metabolic activation of alicyclic amines
  generates a number of oxidative products that can
  be formed though an iminium ion intermediate5
• Iminium ions have the potential to bind to
  cellular macromolecules and cause
  agranulocytosis1,5

Nefazodone
Prochlorperazine
A
A
References 1 Hess, D. A. and Rieder, M. J.
(1997) The role of reactive drug metabolites in
immune-mediated adverse drug reactions. Ann
Pharmacother. 31, 1378-87. 2 Chen, W. G., Zhang,
C., Avery, M. J. and Fouda, H. G. (2001) Reactive
metabolite screen for reducing candidate
attrition in drug discovery. Adv Exp Med Biol.
500, 521-4. 3 Castro-Perez, J., Plumb, R., Liang,
L. and Yang, E. (2005) A high-throughput liquid
chromatography/tandem mass spectrometry method
for screening glutathione conjugates using exact
mass neutral loss acquisition. Rapid Commun Mass
Spectrom. 19, 798-804. 4Evans, D. C., Watt, A.
P., Nicoll-Griffith, D. A. and Baillie, T. A.
(2004) Drug-protein adducts an industry
perspective on minimizing the potential for drug
bioactivation in drug discovery and development.
Chem Res Toxicol. 17, 3-16. 5 Gorrod, J. W. and
Aislaitner, G. (1994) The metabolism of alicyclic
amines to reactive iminium ion intermediates. Eur
J Drug Metab Pharmacokinet. 19, 209-17.
Mass Spectrometric Structural Analysis

• Methods
• Microsomal Incubations
• Each test compound was incubated with rat or
  human liver microsomes in an NADPH regenerating
  system and trapping species, either cyanide or
  GSH.
• Two separate cyanide incubations were performed,
  one with cyanide and the other with stable
  isotope labeled cyanide.
• Nicotine incubations were initially used to
  establish and optimize method conditions.
  Subsequently incubations of nicotine were used as
  a positive procedure control in addition to
  negative controls.
• Two negative controls were used with each
  incubation set. The first one without test
  compound, and the second without NADPH.
• After 90 minutes, incubations were terminated and
  protein was removed by centrifugation at 4C for
  20 minutes
• Liquid Chromatography/Mass Spectrometry
• Incubate supernatant was used for LC/MS analysis
• Chromatographic separations were performed on a
  Waters YMC ODS-AQ, C18 column, 2.1 mm ID, 50mm
  length, 5 mm particle size
• Solvents A 10mM Ammonium Acetate pH4 in water
  B Methanol
• LC Method Isocratic at 4 B for 0.5 minutes, B
  increased linearly to 90 over 2.5 minutes,
  isocratic at 90 B for 2 minutes, imediately
  returned to 4
• Mass spectral analyses were performed using an
  MDS Sciex API-4000 triple quadrupole mass
  spectrometer equipped with a TurboIonSpray
  interface

Table 1. Summary of cyanide and GSH trapping
results for fourteen alicyclic amine compounds.
Iminium ion intermediates from the parent drug
were detected for triprolidine, mianserin,
indinavir, ketoconazole and clozapine. Metabolite
based iminium ions consistent with reported
metabolisms for these compounds include,
N-dealkylation (thioridazine and clozapine),
possible a-carbonyl fromation (triprolidine),
oxygenation (mianserin, indinavir, ketoconazole,
ciprofloxacin, thioridazine, and clozapine), and
ring opening (indinavir). Iminium ions were not
detected for four of the fourteen compounds
listed (phencyclidine, clemastine, rifampin, and
ticlopidine). However, several electrophilic
metabolites for rifampin and ticlopidine were
detected with GSH. These results can be due to a
number of reasons, not all alicyclic amines form
iminium ion intermediates or iminium ions formed
were below the detection limit of the LC-MS
system employed. Results from rat and human
microsomal incubations were found to be similar.
Acknowledgements

• Elaine Ricicki
• Susan Schiavo
• NIH grant no. IROICA69390

Figure 2. A. Total Ion Current for CNL 27 of
prochlorperazine incubate and proposed metabolite
structures B. MS/MS spectrum of
prochloroperazine m/z 374 C. MS/MS of
cyano-adduct PM5 m/z 399 D. MS/MS of 37Cl
isotope of PM5 m/z 401 formed in KCN E. MS/MS
spectrum of cyano-adduct PM5 m/z 401 formed in
K13C15N.
Figure 1. A. Total Ion Current for CNL 27 of
nefazodone incubate and proposed metabolite
structures B. MS/MS of nefazodone m/z 470 C.
MS/MS of cyano-adduct NM5 m/z 511 formed in KCN
D. MS/MS of NM5 m/z 513 formed in K13C15N E.
MS/MS of 37Cl isotope of NM5 m/z 513 formed in
KCN F. MS/MS of cyano-adduct NM4 mono-oxygented
isomer m/z 511 G. MS/MS di-oxygenated cyano
adduct NM3 m/z 527.