Characterization of a new anticancer agent, EAPB0203, and its main metabolites: Nuclear magnetic resonance and liquid chromatography-mass spectrometry studies

Article abstract of DOI:10.1021/ac3021483

The present study was conducted to assess the structures of the main unknown oxygenated metabolites of EAPB0203. The first step was to assign all the ¹H and ¹³C NMR of both EAPB0203 and its demethylated metabolite (EAPB0202) to the corresponding atoms in their molecular structures and to elucidate the fragmentation pathways for the [M + H]⁺ ions of these compounds using high-resolution mass spectrometry (MS). MS/MS spectra showed that both protonated molecules possessing an even number of electrons were unexpectedly losing radicals such as H^?, CH ₃^?, or even C₇H₇ ^? giving stable radical cations. In vitro metabolism studies were investigated in rat and dog liver microsomes and in the filamentous fungus Cunninghamella elegans. Structural elucidation of six oxygenated metabolites was performed based on the following: (i) their fragmentation pathways in liquid chromatography-MS/MS (LC-MS/MS) analyses; (ii) comparison of their changes in their molecular masses and fragment ions with those of the parent drugs; and (iii) the results of online H/D exchange experiments that provided additional evidence in differentiating hydoxylated metabolites from N-oxides. Structures of the metabolites were elucidated by LC-MS/MS and comparison with synthetic standards; structures of these standards were confirmed using one- and two-dimensional ¹H NMR spectroscopies.

Full text of DOI:10.1021/ac3021483

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Article
Characterization of a New Anticancer Agent, EAPB0203
and its Main Metabolites: Nuclear Magnetic Resonance
and Liquid Chromatography-Mass Spectrometry studies
Florian Lafaille, Bernard Banaigs, Nicolas Inguimbert, Christine Enjalbal, Pierre-Emmanuel
Doulain, Pierre-Antoine Bonnet, carine Deleuze-Masquefa, and Francoise MM BRESSOLLE
Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 17 Oct 2012
Downloaded from http://pubs.acs.org on October 23, 2012
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Characterization of a New Anticancer Agent, EAPB0203 and its Main
Metabolites: Nuclear Magnetic Resonance and Liquid Chromatography-Mass
Spectrometry studies
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†
Florian Lafaille1, Bernard Banaigs3, Nicolas Inguimbert3 , Christine Enjalbal4†, Pierre-
Emmanuel Doulain2, Pierre-Antoine Bonnet2, Carine Masquefa2*, Françoise M.M.
Bressolle1
*
1Clinical Pharmacokinetic Laboratory and 2Pharmacochemistry and Biomolecule Laboratory, EA4215,
Montpellier I University, Faculty of Pharmacy, 15 Avenue Ch. Flahault, 34093 Montpellier Cedex 5,
3
France; Biochemistry and Environment Laboratory, EA 4215, Perpignan University, 52 Avenue P.
4
Alduy, 66860 Perpignan Cedex 9, France; Institut des Biomolécules Max Mousseron (IBMM), UMR
5247, Universités Montpellier 1 et 2, CNRS, Bâtiment Chimie (17), Université Montpellier 2, Place
Eugène Bataillon, 34095 Montpellier Cedex 5, France
†N I and C E contributed equally to this work.
* Corresponding author E-mail:
fbressolle@yahoo.fr (tel.) (33) 4 11 75 95 76; (fax) (33) 4 11 75 97 37.
carine.masquefa@univ-montp1.fr (tel) (33) 4 11 75 95 36; (fax) 33 4 11 75 95 53
Abstract: 194 words; text: 4949 words; number of references: 13; number of tables: 1;
number of figures: 2
Supplemental data: 9 figures; 2 tables; 1 text.
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Abstract
The present study was conducted to assess the structures of the main unknown oxygenated
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metabolites of EAPB0203. The first step was to assign all the H and C nuclear magnetic
resonances (NMR) of both EAPB0203 and its demethylated metabolite (EAPB0202) to the
corresponding atoms in their molecular structures and to elucidate the fragmentation
pathways for the [M+H]+ ions of these compounds using high-resolution mass spectrometry
(MS). MS-MS spectra showed that both protonated molecules possessing an even number of
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electrons were unexpectedly losing radicals such as H•, CH3 or even C7H7 giving stable
radical cations. In vitro metabolism studies were investigated in rat and dog liver
microsomes and in the filamentous fungus Cunninghamella elegans. Structural elucidation of
six oxygenated metabolites was performed based on (i) their fragmentation pathways in
liquid chromatography-MS/MS analyses; (ii) comparison of their changes in their molecular
masses and fragment ions with those of the parent drugs; and (iii) the results of on-line H/D
exchange experiments which provided additional evidence in differentiating hydoxylated
metabolites from N-oxides. Structures of the metabolites were elucidated by LC-MS/MS and
comparison with synthetic standards; structures of these standards were confirmed using 1-
and 2-D1 H NMR spectroscopy.
•
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A new series of compounds has been synthesized by our group. These compounds are
analogs of imiquimod, the first member of the imidazoquinoline family, efficacious as a
topical therapy for certain types of skin cancers.1,2 The cytotoxic activity of these new
compounds has been evaluated, in vitro, against human cancer cell lines. Among them,
EAPB0203 and EAPB0503, belonging to the imidazo[1,2-a]quinoxaline series, have emerged
as the most promising drugs with interesting antitumoral activities against melanoma, T-
lymphoma, colon and breast cancer cell lines.3-8 In a mice melanoma model, EAPB0203 was
more potent than fotemustine.3,7 Some data are now available on the pharmacokinetics of
these compounds.7,8
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The present paper deals with the metabolism of EAPB0203. In vitro metabolism studies
carried out using hepatic microsomes from four mammalian species have previously shown
that EAPB0203 was metabolized via two main metabolic pathways (supplemental Figures 1
and 2): first, N-demethylation leading to the formation of EAPB0202 exhibiting similar in
vitro cytotoxicity against A375 melanoma cancer cell line as the parent compound;3 second,
arousing through a series of hydroxylation and/or N-oxidation leading to the formation of
three major compounds named M2, M6 and M7 and their corresponding demethylated
products, M1, M3 and M5.8 A minor metabolite (M4) was only detected in mouse. The
percentage of each metabolite according to the species is provided in supplemental data
Figure 2. Unfortunately, in this previous paper,8 the precise position of the oxygen atom has
not been elucidated. Thus, the objective of the present paper was to characterize the major
oxygenated metabolites of EAPB0203.
In the present study, several spectroscopic approaches such as nuclear magnetic
resonance (NMR) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) with
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triple quadrupole (QqQ) and QqTof mass analyzer configurations allowing on-line mass
measurement of precursor and product ions, were used to identify the main unknown
oxygenated metabolites of EAPB0203 (i.e., M1, M2, M3, M5, M6 and M7). To facilitate
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elucidation of metabolite structures, the first step of this work was to assign all the H and
13C NMR resonances of EAPB0203 and EAPB0202 to the corresponding atoms in their
molecular structures, and to characterize fragmentation pathways and to identify product
ions of both compounds in MS/MS. Moreover, hydrogen/deuterium (H/D) exchange
experiments were performed in MS/MS to aid in identification of the metabolites.9,10 To
generate these metabolites, in vitro metabolism studies were first investigated in rat and
dog hepatic microsomes, the two species metabolizing EAPB0203 at higher rates.8 Then,
EAPB0203 and EAPB0202 were incubated with Cunnighamella elegans, a relevant in vitro
model for drug metabolism studies allowing the formation of large amounts of
metabolites.11
ꢀ EXPERIMENTAL SECTION
Solvents, Chemicals and Reagents.
EAPB0203
(N-methyl-1-(2-phenethyl)imidazo[1,2-a]quinoxalin-4-amine; molecular weight, 302) and its
demethylated metabolite, EAPB0202, (1-(2-phenethyl)imidazo[1,2-a]quinoxalin-4-amine;
molecular weight, 288) were synthesized by the Pharmacochemistry and Biomolecule
Laboratory (Montpellier I University, France).12 Their structures are presented in
supplemental Figure 1; each compound may exist in tautomeric forms. EAPB0203 and
EAPB0202 were stored at 20 °C protected from light. Acetonitrile and methanol (Carlo Erba,
Val de Reuil, France), ammonium formate (Fluka, Vandoeuvre, France), and
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dimethylsulfoxide (DMSO) and formic acid (Prolabo, Paris, France) were all of analytical
grade. Methanol-D4 and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (St.
Louis, MO, USA), and deuterium oxide (100.0 atom % D) and formic acid-D2 (99+ atom %,
95% solution in D2O) from Acros Organics (Geel, Belgium). Water used throughout the
experiments was generated by a Milli-Q water purification system (Milford, MA, USA).
Ammonium formate buffer was prepared by mixing ammonium formate and water to a
concentration of 2 mM; the pH was adjusted to pH 3 ± 0.05 with formic acid.
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Synthesis of metabolites M1, M2 and M6. Compounds M1, M2 and M6 were prepared as
previously described.13 All compounds were obtained as solids and the structures were
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confirmed by LC-MS/MS, H, C-spectra and 2D NMR experiments. Yields for the synthesis
were not optimized. Methodological details are given in the data supplement.
NMR Spectroscopy. The NMR spectra of EAPB203 and EAPB202, and that of the
metabolites were obtained with a NM-40TH5 dual 1H, 13C probe in a JEOL EX400 operating at
400 MHz for proton and 100.53 MHz for carbon-13 at 298 K. 1H and 13C NMR chemical shifts
are referenced to solvent peaks: δH 2.49 (residual DMSO-d5H) and δC 39.5 for DMSO-d6.
1
The assignment of all H resonances was based on connectivity information transmitted
via DQF 1H-1H COSY spectra. Assignment of the 13C NMR resonances was performed by using
HSQC (direct 1JH-C connectivities) and HMBC (long range nJH-C couplings) experiments.
Two-dimensional (2D) homonuclear correlated experiments DQF-COSY and NOESY were
all acquired using standard procedures with a spectral width of ca. 4000 Hz in both columns
F1 and F2. The time domain matrix consisted of 256 points in t1 and 1024-2048 points in t2
with 64-128 acquisitions for 256 experiments in t1. Data sets were zero-filled to 512 points in
t1 prior to Fourier transformation to obtain a frequency domain matrix of 512 x 1024-2048
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real data points. Squared sine bell apodization functions were used. Heteronuclear
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correlated experiments were performed in H-detected mode using the standard pulse
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programs HSQC and HMBC with a spectral width of ca. 20000 Hz in F1 and 4000 Hz in F2. The
time domain matrix consisted of 256 points in t1 and 2048 points in t2 with 128 acquisitions
for 256 experiments in t1. Data sets were zero-filled to 512 points in t1 prior to Fourier
transformation to obtain a frequency domain matrix of 512 x 2048 real data points. The
evolution delay was set to optimize 140 Hz couplings for HSQC and 8 Hz couplings for HMBC.
Squared sine bell apodization functions were used.
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QqQ MS/MS Fragmentation of EAPB0203 and EAPB0202 using the Syringe-Pump
Method. MS/MS experiments were performed on an AB Sciex API 3000 triple quadrupole
mass spectrometer (Courtaboeuf, France) with electrospray ionization (ESI) source. The
ionisation mode was positive.
The stock solutions of EAPB0203 and EAPB0202 (1 g/L) were prepared in acetonitrile.
They were extemporaneously diluted in acetonitrile-purified water-formic acid (50:49:1,
v/v/v) to prepare working solutions at concentrations of 0.01 g/L. Working solutions were
analyzed by direct continuous infusion (200 µL) at a flow rate of 10 µL/min using a syringe
pump. Operating conditions were as follows: curtain gas, 10 psig; ion spray voltage, +6000V;
entrance potential, 11 V; desolvatation temperature, 450 °C; declustering potential, 70 V;
and focusing potential, 400 V. Nitrogen gas was used as the collision gas. Collision energy
varied from 30 to 80 eV. The dwell time for each ion was set at 0.2 second.
We also performed H/D exchange studies that provide a convenient mean for elucidation
of fragmentation mechanisms in MS/MS that is complementary to accurate mass
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measurement. For which, samples were dissolved in methanol-D4 at concentration of 0.01
g/L. After a 2-hour incubation period at 20 °C in the dark, they were infused into the source.
High resolution MS and MS2 Analyses of Directly Infused EAPB0203 and EAPB0202. For
the determination of exact masses, a Q-Tof I mass spectrometer (Waters, Milford, CA) fitted
with an ESI ion source was used. The mass spectrometer was calibrated in the positive ion
mode using 1% phosphoric acid in H2O/CH3CN solution (50:50, v/v). Data were acquired by
the Tof analyzer at 1 acquisition/s from m/z 50 to m/z 3000 with a resolution of 7500.
Depending on the sample, three to eight acquisitions were summed to produce the final
spectrum. Samples were dissolved as previously described and infused into the ESI source at
a flow rate of 5 μL/min. The following MS conditions were used: voltages were set at +3.0 kV
for the capillary and adjusted for the sampling cone from 10 to 60 V according to the sample.
The source was heated at 100 °C. Nitrogen constituted both nebulizing and desolvatation
gas. The latter was heated at 120 °C. In MS/MS experiments, the sampling cone was set at
50 V. The second quadrupole analyzer was set in the rf mode using argon as collision gas and
the collision energy varied from 10 to 30 eV depending on the samples to give optimal
fragmentation. MassLynx v. 4.1 (Waters) software was used for data acquisition and
processing.
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LC-MS/MS (QqQ) Method for the Separation and Identification of Metabolites of
EAPB0203. The LC system (Agilent 1200 LC, Agilent Technologies, Les Ulis, France) was
connected to the API 3000 tandem triple quadrupole mass spectrometer. MS conditions
were similar to those described above. LC separations were performed using a method
similar to that previously published.7 The column used was a C8 Zorbax eclipse XDB (5 µm,
4.6 mm × 150 mm) preceded by a guard column of the same nature (3.9 mm × 20 mm)
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(Agilent Technologies), both maintained at 40 °C. Chromatography was carried out via a
gradient system; the flow rate was set at 800 μL/min. The eluent was post-column split and
approximately 0.5 mL/min was directed to the LC-MS interface. Mobile phase A was
acetonitrile and mobile phase B contained 2 mmol/L ammonium formate buffer (pH 3.0 ±
0.05). The gradient elution was employed according to the following linear programme: time
zero, 30% solvent A; 9 min, 100% solvent A; 10 min, 100% solvent A; 11 min, 30% solvent A;
and 14 min, 30% solvent A. The autosampler was maintained at 4 °C.
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Incubation of EAPB0203 and EAPB0202 with Rat and Dog liver microsomes. EAPB0203
and EAPB0202 (20 µM) were incubated with 1 g/L of pooled rat and dog liver microsomal
proteins in 0.1 M phosphate buffer solution (pH 6.3) as previously described.8 Reactions
were initiated by addition of NADH (10 mM). The incubations were carried out at 37 °C in a
shaking incubator. The total incubation mixture volume was 250 µL. After incubation for 4 h,
0.25 mL of purified water and 0.5 mL of water containing 10 mL/L TFA were added. After
centrifugation (4 °C) for 10 min at 4000g, the sample pretreatment procedure was the same
as that published previously.7 Briefly, the supernatants were transferred onto
preconditioned Oasis HLB cartridges (30 mg). The cartridges were then washed with 1 mL of
water and the analytes were eluted with 2x1 mL of dichloromethane. The eluates were
evaporated to dryness under nitrogen at 40 °C. The residues were reconstituted with 100 µL
of a mixture containing acetonitrile-water-formic acid (50:49:1, v/v/v) then 10 μL were
injected into the LC-MS/MS system. All determinations were performed in triplicate on
three different days.
Incubation of EAPB0203 and EAPB0202 with Cunnighamella Elegans. Stock culture of C.
elegans ATCC 36112 (LGC Standards, Molsheim, France) was maintained on Sabouraud
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dextrose agar plates at 4 °C. Spores and/or mycelia were used to inoculate 125-mL
Erlemmeyer flasks containing 30 mL of Sabouraud dextrose medium. After 48-h incubation
at 25 °C on a rotary shaker (150 rpm), the mycelia were removed and transferred to 125-mL
Erlemmeyer flasks containing 30 mL of Sabouraud. 0.5 milligram of EAPB0203 or EAPB0202
dissolved in 100 µL DMSO (i.e., 1.65 and 1.73 µmol, respectively) was added to each flask (4
flasks per compound). Control included an incubation of the fungus without the drugs (2
flasks). All of the flasks were incubated at 25 °C for 168 h. Thereafter mycelium was
separated from the culture broth by filtration. Under these conditions, EAPB0203 and
EAPB0202 were found to be stable. After lyophilisation of the media, the pellets were
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extracted with dichloromethane (2 mL) then with
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dichloromethane/methanol (50:50, v/v). The organic phases were evaporated to dryness
under a stream of nitrogen (40 °C). Each residue was dissolved in 0.3 mL DMSO, and then 2.7
mL of water containing 5 mL/L TFA were added. Purification was accomplished by means of
solid-phase extraction. Thus, each solution was loaded onto Oasis HLB 60 mg cartridges
previously conditioned with 2 mL of methanol and equilibrated with 2 mL of water. The
cartridges were washed with 2x1.5 mL of purified water. The analytes were eluted with 3x2
mL of dichloromethane. Eluates were combined then evaporated to dryness under nitrogen
at 40 °C. The residues were finally redissolved in 5 mL of a mixture containing acetonitrile-
water-formic acid (50:49:1, v/v/v) then 5 μL were injected into the LC-MS/MS system. These
determinations were replicated on two different days.
ꢀ RESULTS AND DISCUSSION
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Nuclear magnetic resonance (NMR) spectroscopy. Structure and NMR assignments of
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EAPB0203 and EAPB0202 are presented in supplemental data Figure 3A,B. The H NMR
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spectrum (400 MHz, DMSO-d6) of EAPB0202 showed eleven signals ascribable to ten
aromatic protons, four aliphatic ones and two exchangeable protons. Interpretation of 1H-1H
DQF-COSY spectrum suggested that these eleven signals could be assigned to the three
isolated spin systems (H6-H7-H8-H9, H1'-H2', and H4'-H5'-H6'-H7'-H8'), the isolated proton
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(H2) and to the two amine protons. The C NMR (100 MHz, DMSO-d6) spectrum showed 6
quaternary carbons, eight sp2 carbons, and two sp3 carbons. HMQC and HMBC connectivities
allowed the unambiguous assignments of all proton and carbon resonances to the structure
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of EAPB0202. In the H NMR spectrum, the multiplicity of the two methylene groups, H1'
and H2', two triplets instead of two AB systems, clearly indicated the presence of two pairs
of equivalent protons and consequently a free rotation around the bonds C1-C1', C1'-C2' and
C2'-C3'. But, analysis of through-space interactions observed in NOESY experiment
(supplemental Figure 3A) showed two nOe cross-peaks H2/H2’ and H1’/H9 (orange arrays in
3D EAPB202 structure). This seems to indicate that a preferential conformation occurs, in
DMSO, with the three bonds C1-C1', C1'-C2' and C2'-C3' in the plane of the tricyclic aromatic
ring system. As expected, EAPB0203 showed remarkable NMR spectral similarities to
EAPB0202, the most significant differences being the presence of an additional signal
corresponding to the N-methyl group. COSY, HSQC and HMBC NMR spectra are presented in
supplemental data Figure 4A,B.
MS/MS fragmentations of EAPB0203 and EAPB0202. Results of the ESI-MS/MS analyses
carried out on the QqTof mass spectrometer are tabulated in supplemental Table 1. High
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resolution experiments were performed for both EAPB0203 and EAPB0202 compounds on
the protonated molecular ion and some of the observed fragment ions. The elemental
compositions of the selected precursor ions and of fragment ions were deduced from the
measured exact masses. The mass errors between the theoretical calculated and measured
masses ranged from 2 to 11 ppm, which indicated relatively good mass accuracies. QqTof-
MS/MS spectra of the [MH+H]+ precursor ions at m/z 303 (EAPB0203) and m/z 289
(EAPB0202) are shown in supplemental data Figures 5 and 6 that accompany this paper,
respectively. Very surprisingly, MS/MS spectra showed that both protonated molecules
possessing an even number of electrons were unexpectedly losing radicals such as H• (loss of
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•
•
1 Da), CH3 (loss of 15 Da) or even C7H7 (loss of 91 Da) giving stable radical cations. Such
assignments were unambiguously confirmed by exact mass measurements of the observed
fragment ions as shown in supplemental Table 1. The loss of H• was very weak and was
clearly detected under QqTof MS/MS experiments. Besides, the 91 mass loss was tricky to be
•
identified as the tropyl radical (C7H7 ) being isobaric with the neutral molecule C6H5N (91 Da)
that can be formally lost from the fused benzene ring side. Following elucidation of the
fragmentation mechanisms using ESI-QqTof-MS2 experiments supported by exact mass
measurements, we can propose, for EAPB0203 and EAPB0202, the fragmentation schemes
illustrated in Figure 1. Stabilization of distonic radical cations is allowed by delocalisation of
the radical as shown by the variety of isomeric forms that can be suggested, which explained
the propensity of such heterocyclic compounds to spontaneously fragment upon low energy
collision-induced dissociations to form radicals.
H/D exchange experiments on EAPB0203 and EAPB0202. H/D exchange experiments
were conducted on the QqQ. When EAPB0203 was dissolved in methanol-D4, it generated
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an [MD+D]+ ion at m/z 305. The 2 Da increase is in agreement with the presence of one
exchangeable hydrogen atom in its structure and the supplemental 1 Da being contributed
by the ionizing agent D+. The MS/MS spectrum of the m/z 305 ion afforded predominant
fragment ions at m/z 214, m/z 199/198, m/z 172/171 and m/z 129/128 thus confirming the
fragmentation pathway deduced from the high resolution MS experiments. Concerning the
metabolite EAPB0202, the [MD+D]+ ion was at m/z 292; the 3Da increase is in agreement
with the exchange of two hydrogen atoms by deuterium and the presence of one deuterium
affording the ionized species. The MS/MS spectrum of the m/z 292 ion gave the following
predominant fragment ions: m/z 201/200 and m/z 174/173/172 indicative of the presence
of two to three exchangeable hydrogen atoms in these fragments; other predominant
fragment ions were m/z 129/128 and m/z 118/117. Results of H/D exchange experiments
are reported in Table 1. These results were in agreement with the fragmentation schemes
shown in Figure 1.
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LC-MS/MS Experiments. After incubation of EAPB0203 with dog and rat liver
microsomes, 71.3±2.9 and 57.0±1.7% (n=9) of the drug were metabolized, respectively. The
LC-MS analysis of the extracts carried out on the ESI-QqQ mass spectrometer allowed
identification of four major metabolites (EAPB0202, M7, M2 and M1, supplemental Figure
7A) in dog and six metabolites (EAPB0202, M7, M6, M3, M2 and M1, supplemental Figure
7B) in rat. After incubation of EAPB0202, two metabolite peaks were generated both in dog
(M5 and M1, 75±3.0% of EAPB0202 metabolized, supplemental Figure 7C) and in rat (M3
and M1, 55±2.5% of EAPB0202 metabolized, supplemental Figure 7D). The retention times
of EAPB0203, EAPB0202, M7, M6, M5, M3, M2 and M1 were 6.56, 5.62, 4.8, 4.34, 4.28, 4.11,
4.08, and 3.57 min, respectively (supplemental Figure 7). They were detected as their
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[MH+H]+ ions at m/z 303 (EAPB0203), 289 (EAPB0202), 319 (M7, M6 and M2; 16 Da higher
than the protonated molecular ion of EAPB0203) and 305 (M5, M3, M1; 16 Da higher than
the protonated molecular ion of EAPB0202). Thus, M7, M6 and M2 are oxygenated
derivatives of EAPB0203, and M5, M3 and M1 that of EAPB0202. LC-MS/MS analysis of the
extracts from cultures of C. elegans grown with EAPB0203 and EAPB0202 allowed
identification of two peaks from EAPB0203 which eluted at 4.08 min (M2, m/z 319) and 3.57
min (M1, m/z 305), and one peak from EAPB0202 (M1, m/z 305) which eluted at 3.57 min
(supplemental Figure 8). On-line H/D exchange LC-MS/MS was also performed using D2O and
formic acid-D2 in the mobile phase as well as for the reconstitution of the extracts to aid in
identification of the metabolite structures and to discriminate between hydroxylation and N-
oxidation (Table 1). The structures of metabolites were elucidated based on the combination
of different techniques: (i) their fragmentation pathways in LC-MS/MS; (ii) the comparison of
their changes in their molecular masses and fragment ions with those of the parent drugs;
and (iii) the results of H/D exchange experiments. Although the retention time was slightly
shifted with deuterated solvents, metabolites were traced by mass measurements. For M6,
M2 and M1 comparisons with authentic standards in LC-MS/MS were performed to confirm
the structures.
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Identification of metabolites M2 and M1. M2 was produced by dog and rat hepatic
microsomal preparations containing EAPB0203 (supplemental Figure 7A,B) and M1 was
formed in microsomes from rat and dog containing EAPB0203 and EAPB0202 (supplemental
Figure 7). When H2O was used as the aqueous component in the mobile phase, M2 and M1
gave [MH+H]+ molecular ions at m/z 319 and 305, respectively. When molecular ions of M2
and M1 were fragmented upon collision induced dissociations (30-80 eV), they not only
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showed the same fragmentation ions as EAPB0203 and EAPB0202, but also displayed ion
intensities similar to those of EAPB0203 and EAPB0202 in the MS/MS spectra, respectively
(supplemental Table 2). Ions at m/z 212 and 198 were produced through loss of the radical
C7H7O• from the molecular ions, respectively. These results suggested that the mass
increment of 16 Da occurred on the phenethyl moiety of the parent compounds. The
retention times and fragmentation patterns of M2 and M1 found in the extracts from
cultures of C. elegans were well in agreement with those of M2 and M1 found after
incubation of EAPB0203 and EAPB0202 with hepatic microsomes. Noninoculated controls
without the drugs had no peaks corresponding to any of the two metabolites. The on-line
H/D exchange LC-MS/MS experiments using D2O and formic acid-D2 afforded [MD+D]+ ions
at m/z 322 for M2 (predominant fragment ions at m/z 214, 199 and 172) and m/z 309 for M1
(predominant fragment ions at m/z 201, 173/172 and 129/128) (Table 1). This revealed that
M2 had three exchangeable hydrogen atoms and M1 four exchangeable hydrogen atoms in
their structures. Thus, M2 and M1 could be formed by aromatic hydroxylation of the phenyl
ring D (supplemental Figure 1). Based on LC-MS/MS spectra and coelution with available
standards, M2 and M1 were hydroxylated at the C-6’ position of the ring D. M1 is the
demethylated compound of M2.
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Identification of metabolites M6 and M3. When H2O was used in the mobile phase, M6
(found in the rat microsomes incubated with EAPB0203, supplemental Figure 7B) gave rise
to a pseudo-molecular ion at m/z 319. At collision energy of 30 eV, one product ion was
• +•
obtained at m/z 228 [MH+H-C7H7 ] . At 50 eV, the following product ions were observed:
m/z 228 and the further loss of H• (m/z 227), m/z 213 and 201 by loss of CH3• and HCN from
m/z 228, respectively. At 80 eV, the most abundant fragment ions were detected at m/z 213,
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186/185, 145/144 and 133 (supplemental Table 2). M3 (found in the rat microsomes
incubated with EAPB0203 and EAPB0202, supplemental figure 7B,D) gave protonated
molecule at m/z 305. For the MS/MS experiment, the molecular ion was fragmented at
collision energy of 30 eV, which formed daughter ion fragment at m/z 214 corresponding to
the loss of the tropyl radical. At collision energy of 50 eV, other fragment ions were
generated from m/z 214: m/z 213, 197 and 187 by the loss of H•, NH3 and HCN, respectively;
the ion at m/z 213 also loosing HCN to give m/z 186. At higher collision energy (such as 80
eV), product ions were similar to those already observed for M6 but with slightly different
relative abundances (supplemental Table 2). For both M6 and M3, all detected fragment
ions were exhibiting a mass increment of 16 Da compared to those formed from EAPB0203
and EAPB0202, respectively and were detected with similar percent relative abundances.
Thus, the incorporation of an oxygen atom had occurred on the phenyl ring A of the
quinoxaline ring (supplemental Figure 1). M3 is the demethylated compound of M6. The
MS/MS spectrum of M6 in D2O gave the [MD+D]+ ion at m/z 322 indicating the presence of
three exchangeable hydrogen atoms in the structure. At 30-80 eV, the main product ions
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• +•
were: m/z 231 [MD+D-C7H7 ] , m/z 216 (loss of CH3• from m/z 231) and m/z 188 (loss of DCN
from m/z 216) (Table 1). In D2O, M3 was detected at m/z 309 suggesting four exchangeable
hydrogen atoms in its structure; the most abundant product ions were m/z 218 ([MD+D-
• •+
C7H7 ] ), 217 by loss of H•, 190 by loss of DCN, 189 (losses of H• and DCN) and 146/145
(Table 1). Based on these results, M6 and M3 can be rationalized as hydroxylated
metabolites. M6 was identified by LC-MS/MS co-analysis with synthetic standard. NMR
experiments, 1H NMR and NOE DIFF, confirmed the proposed structure for M6, with the site
of hydroxylation clearly located on position C8 (supplemental Figure 9).
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Identification of metabolites M7 and M5. M7 (found in the dog microsomes incubated
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with EAPB0203, supplemental Figure 7A) gave a pseudo-molecular ion [MH+H]+ at m/z 319
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• +•
which fragmented to m/z 228 [MH+H-C7H7 ] , m/z 227 by further loss of H•, and m/z 213 by
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loss of CH3• using a collision energy of 50 eV. These product ions have a mass difference of
16 Da relative to the fragment ions of m/z 212, 211 and 197 formed from EAPB0203
(supplemental Table 2). Thus, the oxygen atom should be positioned on the
imidazoquinoxaline ring. This collision energy gave informative structural data since other
fragments detected at m/z 199 and 185 were generated from m/z 227 and 213 by the loss of
CO, respectively. In contrast to metabolite M6 where the incorporation of the oxygen atom
had occurred on the phenyl ring A of the quinoxaline ring leading to the loss of 27 Da (HCN)
from the same fragment ions, such neutral loss of 28 Da (CO) clearly indicated that the
hydroxyl group was precisely located onto the imidazole ring as seen in Figure 2. At 80 eV,
predominant product ions were: m/z 227, 213/212, 199/198, 185/184, 171/170, 144/143,
and 131/130/129/128 (supplemental Table 2). The demethylated product of M7, M5, found
after incubation of EAPB0202 with the dog hepatic microsomes (supplemental Figure 7C)
gave protonated molecule at m/z 305. MS/MS spectra (collision energy, 30 eV) revealed
• +•
daughter ion fragment at m/z 214 [MH+H-C7H7 ] . At 50 eV, the loss of H• and NH3 from the
ion at m/z 214 leads to fragment ions at m/z 213 and 197, respectively. As previously
reported for M7, at 80 eV, the abundant fragment with m/z 185 resulted from the loss of CO
from m/z 213. At this high collision energy, most of the obtained fragment ions were similar
to those observed for M7. Results are presented in supplemental Table 2. The proposed
fragmentation pathway is depicted in Figure 2. These data indicated that, for both M7 and
M5, the oxygen atom was positioned at the C-2 position on the imidazole C ring. Compared
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to EAPB0203 that yielded an abundant signal at m/z 212 by loss of the tropyl radical
(intensity, 98%) while that at m/z 211 was not very pronounced (<5%), the presence of the
hydroxyl group on the imidazole ring decreased the stability of the radical cation at m/z 228
(20%) and high abundant fragment ion at m/z 227 (100%) was observed (supplemental Table
2). The MS/MS spectrum of M7 in D2O afforded the m/z 322 ion (corresponding to the m/z
319 in H2O) indicating that there were three exchangeable hydrogen atoms on this structure.
Predominant fragment ions were: m/z 231/230/229, 216/215/214, 201/200 and
188/187/186 (Table 1). Concerning M5, on-line H/D exchange LC-MS/MS experiment
produced [MD+D]+ ion at m/z 309, suggesting four exchangeable hydrogen atoms.
Predominant fragment ions were: m/z 218/217/216 and 188/187/186 (Table 1). These data
confirmed that M7 and M5 were obtained by hydroxylation and were in agreement with the
fragmentation schemes shown in Figure 2. The MS/MS spectra combined with accurate mass
measurements provided rapid and reliable information to identify the C-2 location of the
hydroxyl group on the C ring of the parent molecules.
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ꢀ CONCLUSION
In the present study, LC-MS/MS with QqTof mass analyzer configuration was used to
identify all fragment ions of EAPB0203 and EAPB0202. Most measured mass errors were less
than 10 ppm using external mass calibration, indicating high mass accuracy. The combination
of different analytical techniques was used to elucidate the structures of oxygenated
metabolites of the two parent compounds. All of them were hydroxylated compounds. This
was confirmed by on-line H/D exchange LC-MS/MS experiments which clearly facilitate the
differentiation of hydroxylated metabolites from N-oxydes. Structures of M1, M2 and M6
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have been assigned based on LC-MS/MS techniques together with comparison with
synthetic standards. The chemical structures of these standards were confirmed by LC-
MS/MS and NMR analyses. Structures of M5 and M7 were confirmed by LC-MS/MS and
accurate mass measurement. Using cryopreserved rat, dog and human hepatocytes, we
have found that hydroxylated metabolites were partially further conjugated with glucuronic
acid (data not shown).
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ꢀ ACKNOWLEDGEMENTS
The authors would like to thank Bénédicte Marion and Jacques Bompart for their assistance.
This work was partially supported by the Gefluc Languedoc-Roussillon, Montpellier, France.
ꢀ ASSOCIATED CONTENT
Supporting Information.
This material is available free of charge via the Internet at http://pubs.acs.org.
ꢀ AUTHOR INFORMATION
Corresponding Author
*E-mail: FBressolle@aol.com. Tel: (33) 4 11 75 95 76. Fax: (33) 4 11 75 97 37
*
carine.masquefa@univ-montp1.fr (tel) (33) 4 11 75 95 36; (fax) 33 4 11 75 95 53
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ꢀ REFERENCES
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(1) Steinmann, A.; Funk, J.O.; Schuler, G.; Von den Driech, P. J. Am. Acad. Dermatol. 2000,
43, 555-556.
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(2) van Egmond, S.; Hoedemaker, C.; Sinclair, R. Int. J. Dermatol. 2007, 46, 318–319.
(3) Moarbess, G.; Deleuze-Masquéfa, C.; Bonnard, V.; Paniagua, S.; Vidal, J.R.; Bressolle, F.;
Pinguet, F.; Bonnet, P.A. Bioorg. Med. Chem. 2008, 16, 6601-6610.
(4) Moarbess, G.; El-Hajj, H.; Kfoury, Y.; El-Sabban, M.E.; Lepelletier, Y.; Hermine, O.;
Deleuze-Masquéfa, C.; Bonnet, P.A.; Bazarbachi, A. Blood 2008, 111, 3770-3777.
(5) Deleuze-Masquéfa, C.; Moarbess, G.; Khier, S.; David, N.; Gayraud-Paniagua, S.;
Bressolle, F.; Pinguet, F.; Bonnet, P;A. Eur. J. Med. Chem. 2009, 44,3406–3411.
(6) Khier, S.; Moarbess, G.; Deleuze-Masquéfa, C.; Margout, D.; Solassol, I.; Pinguet, F.;
Bonnet, P.A.; Bressolle, F. J. Sep. Sci. 2009, 32:1363-1373.
(7) Khier, S.; Deleuze-Masquéfa, C.; Moarbess, G.; Gattacceca, F.; Margout, D.; Solassol, I.;
Cooper, J.F.; Pinguet, F.; Bonnet, P.A.; Bressolle, F.M. Eur. J. Pharm. Sci. 2010, 39:23-29.
(8) Khier, S.; Gattacceca, F.; El Messaoudi, S.; Lafaille, F.; Deleuze-Masquéfa, C.; Bompart,
J.; Cooper, J.F.; Solassol, I.; Pinguet, F.; Bonnet, P.A.; Bressolle, F.M.M. Drug Metab.
Dispos. 2010, 38, 1836-1847.
(9) Ohashi, N.; Furuuchi, S.; Yoshikawa, M. J. Pharm. Biomed. Anal. 1998, 18, 325-334.
(10) Liu, D.Q.; Hop, C.E.; Beconi, M.G.; Mao, A.; Chiu, S.H. Rapid Commun. Mass Spectrom.
2001, 15, 1832-1839. Erratum in: Rapid Commun. Mass Spectrom. 2003, 17, 264.
(11) Asha, S.; Vidyavathi, M. Biotechnol. Adv., 2009, 27, 16-29
(12) Masquéfa, C.; Moarbess, G.; Bonnet, P.A.; Pinguet, F.; Bazarbachi, A.; Bressolle, F.
Patent Corporation Treaty; European patent, 2008/063290.
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(13) Deleuze-Masquefa. C.; Gerebtzoff, G.; Subra, G.; Fabreguettes, J.R.; Ovens, A.; Carraz,
M.; Strub, M.P.; Bompart, J.; George, P.; Bonnet, P.A. Bioorg. Med. Chem. 2004,12,1129-
1139.
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Legends of Figures
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Figure 1. Proposed fragmentation pathways of EAPB0203 and EAPB0202.
Figure 2. Proposed fragmentation pathways of M7 and M5.
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Table 1 LC-MS/MS behavior of EAPB0203, EAPB0202 and their metabolites after H/D
exchanges in triple quadrupole in the positive ionization mode.
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Collision Precursor
Ion fragmentsa,b
energy
(eV)
Ion
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EAPB0203
30
50
305 (100%)
None
214 (60%).
214
(80%)/213
(20%),
199
m/z 214
(100%)/198 (37%), 187 (10%).
ND
m/z 172
NDCH
N
80
None
213 (15%), 199 (75%)/198 (44%), 172
(100%)/171 (83%)/170 (20%), 154
(40%), 144/143 (40%), 129/128
(50%), 118/117 (25%), 102 (15%).
N
3
m/z 199
rt=6.82 min
EAPB0202
30
50
292 (100%)
None
201 (50%).
201 (100%)/200 (65%)/199 (10%),
181 (10%), 174 (10%)/173 (10%).
m/z 201
ND
N
80
None
200 (63%)/199 (25%), 173 (75%)/172
(100%)/171 (24%), 154 (30%), 145
(40%)/144 (27%), 129/128 (65%),
118/117 (55%), 103/102 (50%).
m/z 173
ND
N
2
rt=5.71 min
M1
30
50
309 (100%)
None
201 (74%).
DO
201 (100%)/200 (30%), 181 (10%),
174 (10%)/173 (10%).
m/z 201
ND
N
80
None
200 (43%)/199 (10%), 173 (65%)/172
(100%)/171 (20%), 129/128 (60%),
118/117 (45%), 102/103 (30%).
m/z 173
ND2
N
rt = 3.74 min
M2
30
50
322 (87%)
None
214 (100%).
DO
m/z 214
214
(100%)/213
(20%),
199
(63%)/198 (15%), 187 (5%).
ND
N
80
None
213 (5%), 199 (60%)/198 (5%), 172
(100%)/171 (42%), 129/128 (30%),
118/117 (15%).
m/z 172
NDCH3
N
m/z 199
rt = 4.28 min
M3
30
50
309 (100%)
None
218 (60%).
218 (100%)/217 (70%), 198 (10%),
190 (10%).
m/z 218
ND
80
None
217 (50%), 190/189 (100%), 161/160
(40%), 146/145 (53%), 135/134
(30%)
DO
N
N
m/z 190
ND2
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rt = 4.34 min
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M5
30
309 (88%)
None
218 (100%)
m/z 218
50
80
218 (57%)/217 (100%), 198 (22%),
188 (50%)
OD
m/z 188
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ND
None
217 (100%)/216 (40%), 188/187/186
N
(30%), 133/132/131/130 (30%).
ND2
N
rt = 4.36 min
M6
30
50
322 (100%)
None
231 (50%).
231
(100%)/230
(10%),
216
m/z 231
(90%)/215 (18%), 204 (10%) / 203
(10%).
ND
DO
N
m/z 188
80
None
216 (60%), 188 (100%)/187 (40%),
161/160 (28%), 146/145 (45%),
134/133 (35%).
NDCH3
N
m/z 216
rt = 4.48 min
M7
30
50
322 (75%)
None
231 (100%)
m/z 231
OD
231 (20%)/230 (100%), 216/215
(20%), 201/200 (40%), 188/187/186
(50%).
m/z 201
ND
N
N
80
None
230/229 (100%), 216/215/214 (50%),
201/200 (40%), 188/187/186 (40%),
172 (30%), 160/159/158 (25%),
145/144 (45%), 133/132/131/130
(30%).
NDCH3
m/z 216
rt = 5.05 min
Similar fragmentation patterns were obtained in rat and dog.
aBoldface indicates the most prominent product ion(s); Values in parentheses are relative
peak intensities. rt, retention time. Retention times were slightly shifted with deuterated
solvents
b
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OH
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OH
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N-demethylation
NH
N
N
OH
NH
N
N
hydroxylation
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NHCH3
NH2
Phase 1 metabolites of a new anticancer agent: EAPB0203.
NMR and LC-MS/MS analyses
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Proposed fragmentation pathways of EAPB0203 and EAPB0202.
396x529mm (96 x 96 DPI)
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Proposed fragmentation pathways of M7 and M5.
396x529mm (96 x 96 DPI)
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