Identification of amino acid and glutathione N-conjugates of Toosendanin: Bioactivation of the furan ring mediated by CYP3A4

Article abstract of DOI:10.1021/tx5002145

Toosendanin (TSN) is a hepatotoxic triterpenoid extracted from Melia toosendan Sieb et Zucc. Considering that TSN contains the structural alert of the furan ring, it is believed that bioactivation of TSN may be responsible for its toxicity. Herein, the bioactivation potential and metabolism profiles of TSN were investigated. After an oral administration of 10 mg/kg TSN to rats, esterolysis and conjugation with amino acids were identified as the main metabolic pathways. The same types of conjugates were detected in liver microsomes in an NADPH-dependent manner. According to the remaining amount of the parent drug, the reactivity of trapping reagents with TSN reactive metabolites was sorted in a decreasing order of N^α-(tert-butoxycarbonyl)-L-lysine (Boc-Lys) > alanine, lysine, taurine, phenylalanine, serine, glutamic acid, glycine, and glutathione (GSH) > cysteine. No conjugates were observed in NADPH and N-acetyl cysteine (NAC)-supplemented human liver microsomal incubations. Further phenotyping studies and the chemical synthesis of the major conjugated standards proved that TSN was bioactivated by CYP3A4 and yielded a cis-butene-1,4-dial intermediate, which was prone to undergo 1,2-addition with the amino group of amino acids and GSH to form 3-pyrroline-2-one adducts. The sulfydryl group of GSH also attacked the intermediate and yielded S-conjugates by 1,4- or 1,2-addition, which would form pyrrole conjugates by further reacting with the amino group. Compared to the well-recognized Sconjugation of the furan ring, N-conjugation with multiple amino acids and GSH played a more important part in the elimination of reactive metabolites of TSN. The significance of these conjugates requires further investigation. (Chemical Equation Presented).

Full text of DOI:10.1021/tx5002145

Article
pubs.acs.org/crt
Identification of Amino Acid and Glutathione N‑Conjugates of
Toosendanin: Bioactivation of the Furan Ring Mediated by CYP3A4
Jinghua Yu, Pan Deng, Dafang Zhong, and Xiaoyan Chen*
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, P. R. China
ABSTRACT: Toosendanin (TSN) is a hepatotoxic triterpenoid extracted from
Melia toosendan Sieb et Zucc. Considering that TSN contains the structural alert of
the furan ring, it is believed that bioactivation of TSN may be responsible for its
toxicity. Herein, the bioactivation potential and metabolism profiles of TSN were
investigated. After an oral administration of 10 mg/kg TSN to rats, esterolysis and
conjugation with amino acids were identified as the main metabolic pathways. The
same types of conjugates were detected in liver microsomes in an NADPH-
dependent manner. According to the remaining amount of the parent drug, the
reactivity of trapping reagents with TSN reactive metabolites was sorted in a
α
decreasing order of N -(tert-butoxycarbonyl)-L-lysine (Boc-Lys) > alanine, lysine,
taurine, phenylalanine, serine, glutamic acid, glycine, and glutathione (GSH) >
cysteine. No conjugates were observed in NADPH and N-acetyl cysteine (NAC)-
supplemented human liver microsomal incubations. Further phenotyping studies and
the chemical synthesis of the major conjugated standards proved that TSN was bioactivated by CYP3A4 and yielded a cis-butene-
1,4-dial intermediate, which was prone to undergo 1,2-addition with the amino group of amino acids and GSH to form 3-
pyrroline-2-one adducts. The sulfydryl group of GSH also attacked the intermediate and yielded S-conjugates by 1,4- or 1,2-
addition, which would form pyrrole conjugates by further reacting with the amino group. Compared to the well-recognized S-
conjugation of the furan ring, N-conjugation with multiple amino acids and GSH played a more important part in the elimination
of reactive metabolites of TSN. The significance of these conjugates requires further investigation.
INTRODUCTION
Enedial intermediates are probably formed, as indicated by
adducts with trapping agents, such as GSH and amino acids in
in vitro experiments. The taurine adduct and GSH adducts
■
Toosendanin (TSN; Figure 1) is a triterpenoid derivative
extracted from the bark of Melia toosendan Sieb et Zucc and has
been used to treat parasitic diseases for many years. TSN also
exhibits antibotulism effects and anticancer properties.
18
1
,2
associated with the enedial intermediates of menthofuran and
19
3
,4
GSH conjugates of furan were also found in rat urine.
This study aimed to investigate the metabolic fate of TSN in
vivo, the bioactivation mechanism of TSN, and the enzyme
responsible for this bioactivation. Our results provided a
foundation to further investigate the hepatotoxicity of TSN.
However, liver injury has been observed in animals or humans
treated with excess TSN or herbal medicines containing
5
−7
TSN.
As such, the mechanism by which TSN elicits
hepatotoxic effects should be elucidated to promote the safe use
of TSN in clinical applications and further development in new
indications.
MATERIALS AND METHODS
■
The bioactivation of drugs can induce the formation of
reactive metabolites, which can interact with cellular macro-
molecules, such as proteins and nucleic acids; this interaction
causes protein dysfunction and DNA damage, resulting in liver
Materials. TSN was purchased from Xi’an Yinshate Biotech Co.,
Ltd. (Xi’an, Shaanxi, China). Boc-Lys, alanine, glycine, lysine, serine,
glutamic acid, cysteine, and phenylalanine were purchased from
Shanghai GL Biochem Ltd. (Shanghai, China). The following
chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA):
leucine enkephalin; taurine; NAC; GSH; NADPH; α-naphthoflavone;
sulfaphenazole; ticlopidine; quinidine; ketoconazole; clomethiazole;
and 1-aminobenzotriazole (1-ABT). Pooled human liver microsomes,
rat liver microsomes (RLM), and recombinant cytochrome P450
(CYP) enzymes CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4,
and 3A5 were supplied by BD Gentest (Woburn, MA, USA).
Recombinant enzymes (50 pmol/mL) were diluted with 0.1 M
phosphate-buffered saline (PBS, pH 7.4). All of the other reagents and
solvents were of analytical or HPLC grade.
8
injury. Therefore, the metabolism of TSN should be
investigated. In a previous study, the metabolism of TSN in9
human liver microsomes (HLM) has been described.
Oxidative metabolites, dehydrogenated metabolites, and
oxidative and dehydrogenated metabolites were found and
identified by ultraperformance liquid chromatography−quadru-
pole-time-of-flight mass spectrometry (UPLC/Q-TOF MS).
TSN contains a furan ring that functions as a structural
1
0
alert. Several furan-containing compounds, such as teucrin A,
4
-ipomeanol, and aflatoxin B1, exhibit toxicity attributed to a
1
1−14
bioactivated furan ring.
Butene-1,4-dial has been proposed
as a reactive metabolite of furans, and an epoxide intermediate
may be involved in the formation of this metabolite.
Received: May 29, 2014
Published: August 8, 2014
1
1,15−17
©
2014 American Chemical Society
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Figure 1. Mass spectra under high-collision energy of standards. (A) TSN, (B) Boc-Lys conjugate 1, (C) taurine conjugates 2a/2b, (D) lysine
conjugates 3a/3b, and (E) lysine conjugates 3c/3d.
Instrumentation. Chromatographic separation was performed
with an Acquity UPLC system (Waters Corp., Milford, MA, USA).
Mass spectra were recorded on the Synapt Q-TOF high-resolution
mass spectrometer (Waters Corp.). NMR experiments were
performed using a Bruker Avance III 400 spectrometer with
CD OD and D O as solvents. Semipreparative HPLC was performed
using a Shimadzu LC-6AD pump equipped with a UV detector.
Animal Study. Wistar rats (300 ± 20 g, n = 6) were provided by
the Experimental Animal Center, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences (China). This study was
performed in accordance with the principles of the Animal Care and
Use Committee of Shanghai Institute of Materia Medica (Shanghai,
China). The rats were randomly divided into two groups. One group
was placed into the metabolism cages and fasted overnight with free
access to water during the experiment. Blank feces and urine were then
collected. A single oral dose of TSN (10 mg/kg, in saline containing
Blank bile was collected. Afterward, the rats were orally treated with 10
mg/kg TSN; the bile was collected up to 24 h postdose. During the
experiment, the rats were allowed free access to food and water.
Biosynthesis of Boc-Lys Conjugate (1). TSN (2.3 mg, 0.004
mmol) dissolved in DMSO was diluted with 25 mL of 0.1 M PBS (pH
7.4). Boc-Lys (123 mg, 0.5 mmol) and NADPH (83.5 mg) dissolved
in 0.1 M PBS (pH 7.4) were then added. The resulting mixture was
preincubated at 37 °C for 3 min; afterward, the self-prepared dog liver
microsomes were added. The final incubation volume was 100 mL.
Incubations were performed at 37 °C for 60 min, and reactions were
terminated with an equal volume of ice-cold acetonitrile. These
incubations were performed in 10 replicates. The samples were
processed by centrifugation at 11,000g. The supernatants were
condensed and eluted with 20% acetonitrile on a C18 ODS-SPE
column. Then, the eluant was separated on HPLC-UV using a Gemini
C18 column (Phenomenex Corp., CA, USA). The mobile phase was a
mixture of acetonitrile and water with gradient elution from 20% to
50% acetonitrile in 11 min.
3
2
1% dimethyl sulfoxide and 1% Tween 80) was administered to rats,
and the urine and feces were collected up to 24 h postdose.
The other group was fasted overnight but provided free access to
water during the experiment. The animals were anesthetized with 3%
pentobarbital, and their bile ducts were cannulated with PE-10 tubing.
General Procedure for the Chemical Synthesis of Amino
Acid Conjugates. Taurine conjugates and lysine conjugates formed
from the enedial intermediates of TSN were synthesized by addition
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Scheme 1. Synthesis of Taurine Conjugates and Lysine Conjugates of TSN
after oxidation of dimethyldioxirane (DMDO; Scheme 1). They were
synthesized using the following procedures.
Japan). The mobile phase was a mixture of methanol and water
(20:80, v/v).
DMDO. DMDO was synthesized according to previously described
Trapping Experiments. TSN was dissolved in 0.1 M PBS (pH
7.4) containing 0.1% dimethyl sulfoxide. Trapping reagent (5 mM;
Boc-Lys, GSH, NAC, taurine, alanine, glycine, lysine, serine,
phenylalanine, cysteine, glutamic acid, or GSH combined with Boc-
Lys) was incubated in the presence of 1.0 mM NADPH and 20 μM
TSN in 0.1 M PBS (pH 7.4). The mixture was preincubated at 37 °C
for 3 min, and then HLM or RLM (4.0 mg/mL) was added to initiate
the reaction. The reactions were terminated with an equal volume of
ice-cold acetonitrile after 60 min of incubation. Control samples
without NADPH, trapping reagent, and/or TSN were also prepared.
Each incubation was performed in duplicate.
20,21
methods.
In brief, oxone (48 g, 0.156 mol) was added in portions
to a stirred mixture of water (100 mL), acetone (80 mL, 1.102 mol),
and sodium bicarbonate (24 g). This mixture was stirred for
approximately 20 min before a slight vacuum was applied to the
reaction assembly. Yellow distillate was collected in a receiving flask
and cooled in a dry ice/acetone bath. The liquid was stored over
MgSO at −20 °C before use. The concentration of DMDO was
4
determined by HPLC-UV, yielding DMDO with concentrations
ranging from 0.05 to 0.08 M.
Taurine Conjugates (2). TSN (430 mg, 0.75 mmol) was dissolved
in 10.0 mL of acetone, and the solution was cooled in an acetone/dry
ice bath. Cold DMDO (11 mL, 0.77 mmol) was added in nitrogen
atmosphere. The reaction mixture was warmed to room temperature,
and reaction was completed in 1 h. Nitrogen was then blown through
the solution to eliminate unreacted DMDO; taurine (18.8 mg, 0.15
mmol) dissolved in 5 mL of 0.1 M PBS (pH 7.4) was then added
dropwise. The reaction mixture was incubated at 37 °C overnight. The
formed products were observed using HPLC-UV at 214 nm. Products
Recombinant Human CYP Enzymes Phenotyping. The
incubating conditions were similar to those of the microsomal
incubations except that the microsomes were substituted with human
recombinant CYP enzymes, including CYP1A2, 2A6, 2B6, 2C8, 2C9,
2
C19, 2D6, 2E1, 3A4, and 3A5. Taurine was used as the capture
reagent in the incubations. The concentrations of the CYP enzymes
were 50 pmol/mL. Each incubation was performed in duplicate. A
total normalized rate method was applied. The rates of metabolite
formation in individual incubations with recombinant CYP enzymes
were multiplied by the mean specific content of the corresponding
CYP enzyme in HLM to obtain the “normalized” reaction rates of each
2
a and 2b were then isolated on a Sunfire C8 column (Waters Corp.).
The mobile phase was a mixture of acetonitrile and water with gradient
elution from 10% to 40% acetonitrile in 11 min.
2
2
enzyme.
Lysine Conjugates (3). TSN was oxidized with DMDO by
conducting the procedures similar to those of taurine conjugates
synthesis. After oxidation was complete, lysine (21.9 mg, 0.15 mmol)
dissolved in 5 mL of 0.1 M PBS (pH 7.4) was added to the reaction
liquid and incubated at 37 °C overnight. Four products (3a−3d) were
isolated by semipreparative liquid chromatography using the YMC
ODS-AQ C18 reversed-phase column (Shimadzu Corp., Kyoto,
Microsomal Incubations in the Presence of Inhibitors. The
function of specific CYP enzymes in the bioactivation of TSN was
determined by adding seven chemical inhibitors listed as follows
(concentration and enzyme in parentheses): α-naphthoflavone (2.0
μM, CYP1A2); sulfaphenazole (6.0 μM, CYP2C9); ticlopidine (24
μM, CYP2B6); quinidine (8.0 μM, CYP2D6); ketoconazole (2.0 μM,
CYP3A); clomethiazole (24 μM, CYP2E1); and 1-ABT (1.0 mM,
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unspecified CYP inhibitor). Incubation mixtures contained 1 mg/mL
pooled HLM, 20 μM TSN, 5.0 mM taurine, 1.0 mM NADPH, and
inhibitors at various concentrations in 0.1 M PBS (pH 7.4). The final
incubation volume was 250 μL. Incubations were performed at 37 °C
for 60 min, and reactions were terminated with an equal volume of ice-
cold acetonitrile. Controls without chemical inhibitors were also
prepared. Each incubation was performed in duplicate.
Sample Pretreatment. Protein was precipitated by adding twice
the volume of acetonitrile to incubation, urine, and bile samples. The
samples were vortexed and centrifuged at 11,000g for 5 min. The
supernatant was transferred into a plastic tube, evaporated to dryness
under a stream of nitrogen at 40 °C, and reconstituted in 100 μL of
acetonitrile and water (10:90, v/v). Afterward, 10 μL aliquot of the
reconstituted solution was injected into UPLC/Q-TOF MS for
analysis.
Feces samples were mashed and mixed with methanol (200 mg of
feces/1 mL of methanol). The resulting mixture was placed in an
ultrasonic machine for 10 min and then centrifuged at 3,500g for 5
min. Approximately 300 μL of the aliquot of the supernatant was
transferred into a plastic tube, evaporated to dryness under a stream of
nitrogen at 40 °C, and reconstituted in 100 μL of acetonitrile and
water (10:90, v/v). An aliquot (10 μL) of the reconstituted solution
was injected into UPLC/Q-TOF MS for analysis.
Considering that the sodium adduct ion provided limited
information in a high-collision energy mass spectrum of a
positive mode, the negative mode was applied in this study.
The precursor ion detected in the negative mode was a
deprotonated TSN [M − H] at m/z 573.2324. The main
product ions at m/z 531.2225, 489.2143, 453.1919, and
−
4
25.1967 were formed by the neutral loss of C H O,
2 2
CH
COOH, H O, and/or CO, as shown in the high-collision
2
3
energy mass spectrum (Figure 1A).
Structure Elucidation of Synthesized Standards 2a,
1
13
2
b, and 3a−3d. A comparison of the H and C NMR
spectra of 2a with those of TSN (Table 1) indicated that the
terminal furan ring (δH 6.14, s, H-21, 7.38, s, H-22, 7.17, s, H-
2
3; δC 124, s, C-20, 142, d, C-21, 113, d, C-22, 144, d, C-23)
was replaced with an N-(2-sulfethyl)-3-substituted-3-pyrroline-
-one moiety (δH 6.98, s, H-21, 3.86, t, H -22; δC 139, s, C-20,
2
2
1
13
141, d, C-21, 53.3, t, C-22, 173, s, C-23). The H and C NMR
spectra of 2a and 2b (Table 1) indicated that the two
metabolites shared the same framework. Compound 2b differed
from 2a only in the substitution position at the newly formed 3-
pyrroline-2-one moiety. On the basis of NMR data, 2a and 2b
were confirmed as N-(2-sulfethyl)-3-substituted-3-pyrroline-2-
one TSN and N-(2-sulfethyl)-4- substituted-3-pyrroline-2-one
TSN, respectively.
UPLC/Q-TOF MS Analyses. Metabolite profiling was performed
under chromatographic conditions using an Acquity UPLC HSS T3
column (1.8 μm, 2.1 mm × 100 mm; Waters Corp.) in an Acquity
UPLC system (Waters Corp.). The mobile phase was a mixture of 5.0
mM ammonium acetate (A) and acetonitrile (B). Gradient elution was
started from 5% B and maintained for 1 min; the gradient was
increased linearly to 40% B for 12 min and maintained for 2 min.
Afterward, the gradient was increased to 99% B for 1 min and
decreased to 5% B to equilibrate the column. The flow rate was 0.4
mL/min. The column temperature was maintained at 45 °C.
MS detection was performed using a Synapt Q-TOF high-resolution
mass spectrometer (Waters Corp.) operated in negative ion
electrospray ionization (ESI) mode. Source and desolvation temper-
atures were maintained at 100 and 350 °C, respectively, at a cone
voltage of 40 V. Data were centroid from 50 to 1800 Da during
acquisition by using an internal reference containing 400 ng/mL of
leucine enkephalin solution infused at 5 μL/min, generating a
The H and 13C NMR spectral data revealed a close
relationship between 3a/3c and 2a and between 3b/3d and 2b,
respectively. Thus, 3a/3c possibly contained a 3-substituted-3-
pyrroline-2-one moiety, and 3b/3d contained a 4-substituted-3-
pyrroline-2-one moiety. However, conjugation with α- or a
terminal amino group of lysine could not be decided only on
the basis of the NMR data. To confirm the structures of 3a−3d,
we isolated the Boc-Lys conjugate of TSN (1) from the
microsomal incubations. The chemical structure of 1 was
1
1
13
obtained using the detailed MS (Figure 1B) and NMR [ H, C,
heteronuclear single quantum coherence and heteronuclear
multiple-bond correlation] data. The similarities between the
chemical shifts of ε-carbon (C-31) in 3c/3d and 1 indicated
that the terminal amino group of lysine reacted with
unsaturated dialdehydes to form 3c/3d. Accordingly, the α-
amino group of lysine possibly participated in the formation of
substituted-3-pyrroline-2-one moiety in 3a/3b. These results
showed that 3a, 3b, 3c, and 3d were N-(5-amino-1-
carboxypentyl)-3-substituted-3-pyrroline-2-one TSN, N-(5-
amino-1-carboxypentyl)-4-substituted-3-pyrroline-2-one TSN,
N-((R)-5-amino-5-carboxypentyl)-3-substituted-3-pyrroline-2-
one TSN, and N-((R)-5-amino-5-carboxypentyl)-4-substituted-
E
reference ion in ESI (−) mode at m/z 554.2615. MS function was
applied in data acquisition with two separate scan functions that were
programmed with independent collision energies. Low collision energy
provided information regarding intact precursor ions; the following
scan at a higher range of collision energy revealed information related
23
to the corresponding fragment ions.
MS and UPLC were conducted using MassLynx 4.1 software. The
actual samples were compared with the control samples using
MetaboLynx, a subroutine of the MassLynx software. Mass defect
filtering (MDF) was applied to screen metabolites with a filter of 30
mDa between the filter template and target metabolites. Comparison
fragment ion spectra between parent molecule and metabolites further
aided in the identification of metabolite structures and site(s) of
modifications in the parent molecule.
3
-pyrroline-2-one TSN, respectively.
Metabolism of TSN in Rats. A total of 21, 14, and 30
metabolites of TSN were detected in rat feces, urine, and bile,
respectively (Figures 2, 3, and 4), compared with the blank
samples. These metabolites could be mainly classified as
esterolysis metabolites, oxygenated metabolites, dehydrogen-
ated metabolites, amino acid conjugates, and GSH conjugates.
The structures of these metabolites were characterized by
comparing the retention times and mass spectra of the
metabolites in rat excrement samples with those in samples
spiked with authentic standards. Phase I metabolites were
named in the order of molecular weight and retention time
(Rt). These metabolites were identified as follows.
RESULTS
■
Mass Spectral and Chromatographic Behaviors of
TSN. The fragmentation and chromatographic behaviors of
parent compounds should be completely understood to help
identify metabolites. In this study, the chromatogram of TSN
displayed two peaks at retention times of 12.1 and 12.8 min,
respectively, with an area ratio of 3 because of the presence of a
hemiacetal group. The two LC peaks were named M0-1 and
M0-2 for convenience. If the hemiacetal group was reserved,
the metabolite likely exhibited two chromatographic peaks
similar to those of the parent drug. In a positive scan mode, the
Metabolite M1. The two isomers M1-1 and M1-2 detected
in urine, feces, and bile were eluted at 8.3 and 8.7 min,
respectively. These isomers showed a deprotonated molecule at
m/z 489.2169. The molecular formula of C H O was
+
sodium adduct ion of TSN [M + Na] at m/z 597.2310 was
detected instead of a protonated molecule [M + H] .
+
2
6
34
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Figure 4. MDF metabolic profiles after a single oral dose of 10 mg/kg
TSN was administered to Wistar rats in pooled bile (0 to 8 h) (A) and
MDF total ion chromatograms of pooled bile (0 to 8 h) (B) and rat
blank bile (C).
Figure 2. MDF metabolic profiles after a single oral dose of 10 mg/kg
TSN was administered to Wistar rats in pooled urine (0 to 12 h) (A)
and MDF total ion chromatograms of pooled urine (0 to 12 h) (B)
and rat blank urine (C).
suggesting that hydrolysis occurred at position C-3. Therefore,
M2 was confirmed as the 3-esterolysis product of TSN. M2-1
and M2-2 represented either the LC peak of the 3-esterolysis
product of TSN at retention times of 10.7 and 11.2 min,
respectively, based on their LC area ratios.
Metabolite M3. M3, eluting at 13.8 min, was found only in
bile. The deprotonated molecule of M3 was m/z 571.2186,
providing the molecular formula of C H O . Thus, M3 was
30
36 11
inferred as the dehydrogenation metabolite of TSN. The main
fragment ions at m/z 527.2335 (−CO ), m/z 451.1742
2
(
−C H O , −CO , −H O), m/z 425.2032 (−CH COOH,
2
2
2
2
2
3
−
C H O, −CO ), and m/z 407.1847 (−2CH COOH, −CO )
2
2
2
3
2
were detected in the high-energy mass spectrum. The neutral
loss of CO indicated the presence of a carboxylic group or
2
lactone. Moreover, the chromatogram of M3 did not display
double peaks, indicating that the hemiacetal group was
changed. Therefore, dehydrogenation tentatively occurred at
C-28 to form lactone. The chemical structure of M3 is shown in
Scheme 2.
Metabolite M4. M4-1 (Rt = 10.3 min), M4-2 (Rt = 11.0
min), and M4-3 (Rt = 11.4 min) contained a deprotonated
molecule at m/z 589.2318 with an elemental composition of
C H O ; this result indicated that one oxygen atom was
Figure 3. MDF metabolic profiles after a single oral dose of 10 mg/kg
TSN was administered to Wistar rat in pooled feces (0 to 12 h) (A)
and MDF total ion chromatograms of pooled feces (0 to 12 h) (B)
and rat blank feces (C).
30
38 12
added to the parent drug. M4-1 and M4-2 were detected in
feces, and M4-1 and M4-3 were found in bile. The high-energy
mass spectra of M4-1 to M4-3 showed fragment ions at m/z
547.2266 and m/z 505.2174 from the neutral loss of C H O;
obtained on the basis of its accurate mass, indicating that two
acetyl groups were removed from the parent drug. M1 was
deduced as a diesterolysis product of TSN. M1-1 and M1-2
were two isomers resulting from a hemiacetal group based on
their LC area ratios.
2
2
this result resembled the mass behavior of the parent drug.
However, the position where oxygenation occurred was not
confirmed without the authentic standards.
Metabolite M5. M5 (Rt = 5.2 min) with a deprotonated
molecule at m/z 603.2110 was found in bile. The molecular
formula of C H O indicated that TSN was dioxygenated
Metabolite M2. M2 with a deprotonated molecule at m/z
31.2223 was observed in urine, feces, and bile. The elemental
5
30
36 13
composition was C H O , revealing that one acetyl group
and dehydrogenated. The fragment ions of M5 at m/z
541.2734 (−CO , −H O), m/z 523.2588 (−CO , −2H O),
2
8
36 10
was removed from the parent drug. The authentic standard was
2
2
2
2
1
synthesized by hydrolyzing TSN with 2 M K CO . The H
and m/z 445.1929 (−CH COOH, −CO , −3H O) were
2
3
3
2
2
NMR spectrum of M2 was similar to that of TSN; however, the
yielded as a consequence of the neutral loss of CO ,
2
proton signal at δ 5.17 (1H, d, J = 3.9 Hz, H-3) disappeared,
CH COOH, and H O. The neutral loss of CO was similar
3
2
2
1
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a
Scheme 2. Proposed Metabolic Pathways of TSN in Rats
a
*, the chemical structures of the compounds were confirmed by authentic standards.
to that of M3, suggesting that dehydrogenation possibly
occurred at C-28; oxygenation sites were not assigned on the
basis of the limited MS information.
The identification of the conjugate metabolites was described in
the following section.
Taurine Conjugates. The metabolites T1-1 (Rt = 4.6 min),
T1-2 (Rt = 5.1 min), and T1-3 (Rt = 5.2 min), which were
detected in urine, feces, and bile contained a deprotonated
molecule at m/z 696.2324. Accurate mass measurement
provided the chemical formula of C H NO S, suggesting
Phase I metabolites were characterized, and the results
showed that TSN underwent esterolysis, oxygenation, and/or
dehydrogenation after this triterpenoid entered the body.
Esterolysis was the primary phase I metabolic pathway.
Identification of Conjugate Metabolites in Rats. The
high-energy mass spectral and chromatographic behaviors of
metabolites were compared with those of the parent drug. The
amino acid conjugates were named after the first letter of the
conjugated amino acid; the conjugates with the same molecular
weight were named in the sequential order of retention time.
32
43
14
that taurine was added to TSN. T1-1, T1-2, and T1-3 shared
the same fragment ions of m/z 654.2220 (−C H O), m/z
2
2
636.2113 (−CH COOH), m/z 608.1911 (−CH COOH−,
3
3
CO), m/z 548.1977 (−2CH COOH, −CO), and m/z
3
106.9771. The neutral loss of C H O, CH COOH, and/or
2
2
3
CO was similar to the fragmentation behavior of TSN. The
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Figure 5. Mass spectra under high-collision energy of (A) GSH conjugates GS1-1/GS1-2 and (B) dehydrated GSH conjugates GS2-1/GS2-2 in rat
bile.
Figure 6. Metabolic profiles of TSN in liver microsomes. (A) HLM supplemented with taurine; (B) RLM supplemented with taurine; (C) HLM
supplemented with GSH; (D) RLM supplemented with GSH; (E) HLM supplemented with GSH and Boc-Lys; (F) HLM supplemented with Boc-
Lys; and (G) HLM supplemented with NAC.
fragment ion of m/z 106.9771 in low m/z field was probably
generated from the cleavage of the C−N bond of taurine; this
result indicated that taurine was most likely bound to TSN via
an amino group. The authentic standards 2a and 2b were
synthesized to further elucidate their structures. Compound
2a/2b showed identical MS fragmentation as that of T1 (Figure
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C). Similar to the chromatographic behavior of the parent
Article
1
as those detected in rats; more taurine conjugates were found in
HLM incubations (Figure 6A and B). The formation of the
taurine conjugates was NADPH dependent, indicating that
CYP enzyme(s) would be involved in the bioactivation of TSN.
As a commonly used trapping agent, GSH was used to trap
the reactive metabolites of TSN in HLM and RLM. The
amount of GSH conjugates was significantly less than that of
taurine conjugates under the same incubation conditions. The
parent and dehydrated GSH conjugates were also observed in
liver microsomal incubations as those detected in rats. The
metabolic profiles of TSN in GSH and NADPH-supplemented
HLM and RLM are illustrated in Figure 6C and D.
In HLM incubation supplemented with both GSH and Boc-
Lys as trapping agent, GSH and Boc-Lys conjugates (GB1-1,
GB1-2, m/z 1106.4498) were observed. GB1-1 and GB1-2 were
speculated as S-substituted pyrrole conjugates base on GSH
characteristic fragment ion at m/z 272.0935 and 210.1265 in
their high-energy mass spectra. The isomers probably resulted
from different substituted positions. The metabolic profile of
TSN in Boc-Lys and GSH-supplemented HLM are illustrated
in Figures 6E.
compound, 2a displayed two chromatograph peaks at retention
times 4.6 and 5.1 min, which were the same as those of T1-1
and T1-2, respectively. Therefore, T1-1 and T1-2 were both
inferred as N-(2-sulfethyl)-3-substituted-3-pyrroline-2-one
TSN. The same situation also occurred in 2b. T1-3 was
assigned as N-(2-sulfethyl)-4-substituted-3-pyrroline-2-one
TSN. However, the corresponding isomeric peak of T1-3 was
not detected in vivo possibly because of the low response of this
compound.
The metabolites T2-1 and T2-2 found in urine, feces, and
bile displayed a deprotonated molecule at m/z 654.2213. The
elemental composition of T2-1 and T2-2 was C H NO S;
3
0
41
13
this result indicated that taurine was added to deacetylated
TSN. The retention times of T2-1 and T2-2 were 3.6 and 3.8
min, respectively. The fragment ions of T2 at m/z 106.9802,
189.0086, and 216.0376 were also presented in the high-energy
mass spectrum of T1; this result suggested that T2 contained a
structure similar to that of T1. With a chromatographic area
ratio of approximately 3, T2-1 and T2-2 were considered as two
isomers resulting from the hemiacetal group, and both were
deduced as the 3-esterolysis product of 2a.
The metabolic profile of TSN in HLM supplemented with
Boc-Lys showed 13 conjugates, which were produced in an
NADPH-dependent manner (Figure 6F). These conjugates
could be classified into four types according to their accurate
masses, i.e., the parent (BL1-1 to BL1-4, m/z 817.3756),
dehydrogenated (BL2-1 to BL2-4, m/z 815.3637), esterolytic
Lysine Conjugates. The metabolites L1-1 (Rt = 5.1 min),
L1-2 (Rt = 5.7 min), and L1-3 (Rt = 6.3 min) with a
deprotonated molecule at m/z 717.3235 were detected in rat
urine, feces, and bile, respectively. Accurate mass measurement
provided the chemical formula of C H N O ; this result
3
6
50
2
13
indicated that lysine was added to TSN. Four authentic
standards of lysine conjugates (3a−3d) were obtained in a
manner similar to the synthesis of taurine conjugates. By
comparison of chromatographic retention time and MS
behaviors of the conjugates (Figure 1D and E), L1-1 and L1-
(
BL3-1 to BL3-3, m/z 775.3683), and dehydrogenated and
esterolytic (BL4-1 and BL4-2, m/z 773.3528) Boc-Lys
conjugates. BL1-1 (1) was isolated from the liver microsomal
incubations, and its chemical structure was confirmed as N-
(
(R)-5-((tert-butoxycarbonyl)amino)-5-carboxypentyl)-3-sub-
3
were identified as N-((R)-5-amino-5-carboxypentyl)-3-sub-
stituted-3-pyrroline-2-one TSN based on the NMR and MS
data. BL1-3 was proposed as the isomer of BL1-1 formed by a
hemiacetal group based on their LC area ratios. Accordingly,
BL1-2 and BL1-4 were a pair of isomers, and their structures
were both inferred as the 4-substituted isomer of 1. As such,
BL2, BL3, and BL4 were, respectively, assigned as dehydro-
genated, 3-esterolytic, and dehydrogenated and 3-esterolytic
products of BL1.
No conjugates were detected in HLM when NAC was used
as a trapping agent. Figure 6G shows the metabolic profile of
TSN in HLM supplemented with NAC and NADPH.
Using other amino acids, including alanine, lysine, phenyl-
alanine, serine, glutamic acid, glycine, and cysteine, as trapping
reagents, the corresponding conjugates could be found in
NADPH-supplemented liver microsomal incubations. Accord-
ing to the remaining amount of the parent drug, the reactivity
of trapping reagents with TSN reactive metabolites was sorted
in a decreasing order of Boc-Lys > alanine, lysine, taurine,
phenylalanine, serine, glutamic acid, glycine, and GSH >
cysteine.
CYP Enzymes Involved in the Bioactivation of TSN.
CYP enzymes involved in the bioactivation of TSN were
investigated by recombinant human P450 screening, including
CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5,
mixed with taurine as a trapping reagent. The metabolite T1-1
was mainly detected in taurine-fortified incubated samples with
CYP3A4 and CYP3A5. These products were not detected
when the same samples were incubated with the other isoforms.
CYP3A4 was identified as the major enzyme involved in the
bioactivation of TSN after the relative content of the
stituted-3-pyrroline-2-one TSN and N-((R)-5-amino-5-carbox-
ypentyl)-4-substituted-3-pyrroline-2-one TSN, respectively.
The minor chromatographic peak of 3c and the major
chromatographic peak of 3d exhibited the same retention
time at 5.7 min. Thus, L1-2 was inferred as the mixture of 3c
and 3d.
GSH Conjugates. The GSH conjugates (GS1-1 to GS1-2,
m/z 878.3052) and further dehydrated GSH conjugates (GS2-1
to GS2-2, m/z 860.2977) were detected in rat bile. As shown in
Figure 5A, a fragment ion resulting from the neutral loss of H2S
(
m/z 844.3244) and a GSH characteristic fragment ion
containing sulfydryl group (m/z 160.0108) were found in the
high-energy mass spectra of GS1-1 and GS1-2, which provided
the evidence that GSH bound to TSN via an amino group, not
a sulfydryl group. Their conjugation site was reasonably
identified in the furan ring of TSN based on the structures of
taurine conjugates and MS behaviors. For GS2-1 and GS2-2, no
such characteristic ions were observed (Figure 5B), and their
structures were proposed based on the accurate masses. The
proposed chemical structures of GS1 and GS2 are shown in
Scheme 2.
Other Amino Acid Conjugates. Glycine conjugates and
glutamic acid conjugates of TSN were detected in rat excreta.
The 3-esterolysis products of the amino acid conjugates were
also found in vivo.
Formation of Conjugated Metabolites in HLM or RLM.
Taurine conjugates were the most abundant conjugated
metabolites in rats. The mechanism of taurine conjugate
formation was investigated in RLM and HLM. The same
taurine conjugates were yielded in liver microsomal incubations
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Figure 7. Formation of T1-1 in incubations with recombinant human CYP enzymes. (A) The rates of T1-1 formation obtained from individual
incubations with recombinant CYP enzymes were multiplied by the mean specific content of the corresponding CYP enzyme in HLM. (B) Effect of
selective CYP inhibitors on the formation of T1-1 in incubations of TSN with HLM and taurine. Data are reported as the mean of two separated
determinations.
corresponding CYP enzyme in HLM was normalized (Figure
A).
To further determine the primary enzyme(s), specific
enzyme inhibitors, including α-naphthoflavone (CYP1A2),
sulfaphenazole (CYP2C9), ticlopidine (CYP2B6), quinidine
response factor of metabolites, the major drug-related
compounds were identified as the esterolysis metabolite (M2-
1 and M2-2), lysine conjugates (L1-1 and L1-2), and taurine
conjugates (T1-1 and T1-2) in feces; esterolysis products (M2-
1 and M2-2) in urine; and esterolysis product (M2-1), taurine
conjugates (T1-1 and T1-2), and glycine conjugates (G1-1 and
G1-2) in rat bile. These results suggested that TSN was mainly
metabolized by esterolysis and/or amino acid conjugation in
rats. GSH conjugates as minor metabolites were observed in rat
bile. The metabolic pathways of TSN in rats are proposed in
Scheme 2.
7
(
(
CYP2D6), clomethiazole (CYP2E1), or ketoconazole
CYP3A), were used in the incubations in the presence of
taurine. The formation of the major taurine conjugate T1-1 was
monitored. Among the inhibitors used, only ketoconazole
elicited a significant inhibitory effect on the bioactivation of
TSN (Figure 7B), which was similar to the inhibition effect of
1
-ABT. These results confirmed that CYP3A4 was the primary
The formation of these conjugated metabolites indicated that
TSN may be bioactivated in vivo. The furan ring, hemiacetal
group, and epoxy group present in the chemical structure of
enzyme responsible for the bioactivation of TSN.
10
DISCUSSION
TSN are all reported as structural alerts. The functional
group(s) involved in the bioactivation of TSN was unclear
because of the lack of diagnostic fragment ions. In order to
confirm the activated group(s), we scaled in vitro incubation
and isolated a Boc-Lys conjugate (BL1-1). The NMR data
showed that the BL1-1 was 3-substituted 3-pyrroline-2-one
adduct, indicating that the furan ring other than epoxy and
hemiacetal participated in the bioactivation of TSN. To
■
This study is the first to describe the metabolism of TSN in
vivo. A total of 21, 14, and 30 metabolites of TSN were detected
in rat feces, urine, and bile, respectively, after the Wistar rats
received 10 mg/kg TSN. Considering that MS responses differ
among the parent drug and metabolites, it is necessary to apply
a correction factor to the measured areas in UPLC/Q-TOF MS
chromatograms. Multiplying the peak area by the mole
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investigate the mechanism, three kinds of trapping agents,
namely, GSH with both nucleophilic sulfydryl group and amino
group, Boc-Lys with only one nucleophilic amino group, and
NAC with only one nucleophilic sulfydryl group were used to
trap the reactive metabolites. No conjugates were found in
NAC-supplemented incubations, and Boc-Lys conjugates were
the most abundant among all of the conjugates based on the
amount of the remaining parent. In addition, the neutral loss of
protected by the acetyl group, it could not form pyrrole
adducts. Besides, the electrophilicity of the R group may also
affect the nucleophilic attack of NAC. These may contribute to
the absence of NAC S-conjugates in the present study.
According to the remaining amount of the parent drug in the
trapping experiments, the cis-butene-1,4-dial intermediate of
TSN greatly preferred the 1,2-addition of the amino group.
In summary, esterolysis and amino acid conjugation were the
major metabolic pathways of TSN in Wistar rats. In vitro studies
demonstrated that the furan ring of TSN was bioactivated by
CYP3A4 to form the cis-butene-1,4-dial intermediate. It
underwent 1,2-addition with the amino group and yielded a
3-pyrroline-2-one adduct. The sulfydryl group yielded con-
jugates by 1,4- or 1,2-addition, which could generate pyrrole
adducts by further reacting with the amino group. The 1,2-
addition of the amino group was the predominate pathway.
Compared to the well-recognized S-conjugation of the furan
ring, N-conjugation from multiple amino acids and GSH played
more a important role in the elimination of reactive metabolites
of TSN. In addition, taurine conjugates and unusual lysine
conjugates, derived from the cis-butene-1,4-dial intermediate,
were first reported in vivo as the major metabolites. The cis-
butene-1,4-dial intermediate of TSN could likely react with
peptides or proteins, which might be responsible for the
hepatotoxicity induced by TSN.
H S and the GSH characteristic fragment ion containing the
2
sulfydryl group was found in the high-energy mass spectra of
GSH conjugates. These findings seemed to support that the
reactive metabolites of TSN possibly reacted with an amino
group rather than a sulfydryl group. However, the trapping
experiment with both Boc-Lys and GSH generated S-
substituted pyrrole adducts. This result indicated that the
sulfydryl group could also attack the intermediates of TSN. The
detection of dehydrated GSH conjugates provided additional
evidence.
The bioactivation of the furan ring has been extensively
investigated. The cis-butene-1,4-dial intermediate of furan has
been verified in furan and furan-containing com-
1
5,16,24,25
pounds,
and an epoxide intermediate probably
produced cis-butene-1,4-dial. According to the chemical
structures of the conjugates of TSN, the mechanism of
bioactivation of TSN is depicted in Scheme 3. The furan ring
AUTHOR INFORMATION
Corresponding Author
Tel/Fax: +86-21-50800738. E-mail: xychen@simm.ac.cn.
Scheme 3. Proposed Mechanism of TSN Bioactivation
■
*
Funding
This work was supported by the National Natural Science
Foundation of China (Nos. 81202579 and 81173115).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Liang Li for kindly providing assistance in NMR
analysis and Peng Lei for facilitating the synthesis of taurine
conjugates and lysine conjugates.
ABBREVIATIONS
TSN, toosendanin; Boc-Lys, N -(tert-butoxycarbonyl)-L-lysine;
■
α
of TSN was mainly bioactivated by CYP3A4 to form the cis-
butene-1,4-dial intermediate. It underwent 1,2-addition with
the amino group of amino acids (taurine, lysine, Boc-Lys, and
GSH) to form 3-pyrroline-2-one N-conjugates after ring closure
and dehydration. The amino group preferred to attack the 4-
aldehyde group with less steric hindrance to form major 3-
substituted pyrroline conjugates. The 1-aldehyde group formed
a minor 4-substituted conjugate via a similar mechanism. In
addition, the electrophilicity of the substituted group of cis-
butene-1,4-dial may affect the reaction. In the meantime, the
cis-butene-1,4-dial intermediate also underwent 1,4- or 1,2-
addition with the sulfydryl group and gave rise to the S-
conjugates, which were still unstable and would further react
with the amino group from the intra- or intermolecular amino
group to form pyrrole derivatives (see Scheme 3). When cis-
butene-1,4-dial reacted with the sulfydryl group by 1,4-addition
only, there should be just one kind of dehydrated GSH
conjugate. Since two kinds of dehydrated GSH conjugates were
detected in in vivo and in vitro studies, 1,2-addition probably
involved in the reaction. As the amino group of NAC is
NAC, N-acetyl cysteine; GSH, glutathione; CYP, cytochrome
P450; HLM, human liver microsomes; RLM, rat liver
microsomes; 1-ABT, 1-aminobenzotriazole; Rt, retention
time; DMDO, dimethyldioxirane; ESI, electrospray ionization;
UPLC/Q-TOF MS, ultraperformance liquid chromatography−
quadrupole-time-of-flight mass spectrometry
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