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DMD #18572
1
Identification of the In Vitro Metabolites of ARQ 501 (β-Lapachone) in Whole Blood
Xiu-Sheng Miao§, Pengfei Song§, Ronald E. Savage, Caiyun Zhong, Rui-Yang Yang, Darin
Kizer, Hui Wu, Erika Volckova, Mark A. Ashwell, Jeffrey G. Supko, Xiaoying He, Thomas C.
K. Chan*
Department of Preclinical Development and Clinical Pharmacology, ArQule Inc. Woburn,
Massachusetts (X-S.M, P.S., R.E.S., C,Z., T.C.K.C.)
Department of Chemistry, ArQule, Inc. Woburn, Massachusetts (R.Y., D.K., H.W., E.V.,
M.A.A)
Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts (J.G.S. and
X.H.).
DMD Fast Forward. Published on January 7, 2008 as doi:10.1124/dmd.107.018572
Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics.
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Running Title Page
a) Running Title: Identification of ARQ 501 (β-lapachone) metabolites in whole blood
b) Corresponding author:
Thomas Chan, PhD
ArQule, Inc.
19 Presidential Way
Woburn, MA 01801
Tel: (781) 994-0300 (x467)
Fax: (781) 287-2908
Email: [email protected]
c) Number of text pages: 17
Number of tables: 1
Number of figures: 9
Number of schemes: 1
Number of references: 18
Number of words in the Abstract: 257
Number of words in the Introduction: 295
Number of words in the Discussion: 684
d) Abbreviations:
ARC, accurate radioisotope counting; RBCs, red blood cells; CID, Collision-induced
dissociation; HPLC, high-performance liquid chromatography; MRM, multiple reaction
monitoring; MS, mass spectrometry; NMR, Nuclear magnetic resonance; PK,
pharmacokinetic; TOF, time-of-flight; UPLC, ultra-performance liquid chromatography, WB,
whole blood
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Abstract
ARQ 501 (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b] pyran-5,6-dione, β-lapachone)
showed promising anticancer activity in Phase I clinical trials as monotherapy and in
combination with cytotoxic drugs. ARQ 501 is currently in multiple Phase II clinical trials. In
vitro incubation in fresh whole blood at 37ºC revealed that ARQ 501 is stable in plasma, but
disappears rapidly in whole blood. Our data showed that extensive metabolism in red blood cells
(RBCs) was mainly responsible for the rapid disappearance of ARQ 501 in whole blood. By
comparison, covalent binding of ARQ 501 and/or its metabolites to whole blood components
was a minor contributor to the disappearance of this compound. Sequestration of intact ARQ 501
in RBCs was not observed. Cross-species metabolite profiles from incubating [14C]-ARQ 501 in
freshly drawn blood were characterized using liquid chromatography-mass spectrometry-
accurate radioactivity counter. The results show that ARQ 501 was metabolized more rapidly in
mouse and rat blood than in dog, monkey and human blood, with qualitatively similar metabolite
profiles. Six metabolites were identified in human blood using ultra-high performance liquid
chromatography/time-of-flight mass spectrometry, and the postulated structure of five
metabolites was confirmed using synthetic standards. We conclude that the primary metabolic
pathway of ARQ 501 in human blood involved oxidation of the two adjacent carbonyl groups to
produce dicarboxylic and monocarboxylic metabolites, elimination of a carbonyl group to form a
ring-contracted metabolite, and lactonization to produce two metabolites with a pyrone ring to
form a ring-contracted metabolite. Metabolism by RBCs may play a role in clearance of ARQ
501 from the blood compartment in cancer patients.
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Introduction
ß-Lapachone (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b] pyran-5,6-dione) is found in
Pau d’arco trees (Tabebuia impetiginosa) and possesses anti-tumor activity against S-180
leukemic cells, Yoshida sarcoma cells, sarcoma 180 cells and Walker 256 carcinoma cells in
early studies (Docampo et al., 1979; Schaffner-Sabba et al., 1984; Chau et al., 1998). ARQ 501
is a fully synthetic ß-lapachone and has been identified as a potent activator of E2F1. The
compound causes tumor cell apoptosis and death in vitro and in vivo (Wang et al., 2005).
Promising anticancer activity was observed in Phase I and Phase II clinical trials.
Blood is generally assumed to be an inert vehicle for the transportation of drugs to target
tissues. The primary physiological objective of red blood cells (RBCs) is gas transport and
exchange. Beyond this, they perform metabolic functions to produce the necessary cofactors
(ATP, NADPH and NADH) enzymatically for their own persistence (Bossi and Giardina, 1996;
Wiback and Palsson, 2002). Though various enzymes in RBCs can metabolize drugs (Carruthers
and Melchior 1988; Hooks 1994), systematic investigation of whole blood metabolism of
therapeutic drugs has been, according to some researchers, traditionally neglected in drug
discovery and development (Cossum 1988; Hinderling 1997). In particular, few in-depth studies
of the metabolism of drugs in whole blood exist.
In vitro incubation in whole blood and in plasma revealed that ARQ 501 was stable in
plasma but disappeared rapidly from whole blood. Potentially, sequestration in RBCs, covalent
binding to blood components, metabolism by RBCs, or a combination of the above processes
could be responsible. The objectives of this study were to explore the underlying mechanism for
the rapid disappearance of ARQ 501 from whole blood and to identify its probable in vitro
metabolites.
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Materials and Methods
Materials. [14C] ARQ 501 was synthesized by ABC Laboratories Inc. (Columbia, MO) with a
radiochemical purity of 99.6%. The specific activity was 5.22 µCi/mg (10mCi/mmol). Non-
labeled ARQ 501 and deuterated ARQ 501 (D6-ARQ 501) were synthesized by ArQule, Inc.
Scintillation cocktail ULTIMA-FLO was purchased from PerkinElmer Life and Analytical
Sciences Inc. (Waltham, MA). Metabolite standards were synthesized by ArQule, Inc (Woburn,
MA) (Kizer et al., 2007) and their synthetic routes will be published separately (Yang et al.,
2007). High purity acetonitrile and water were purchased from EMD Chemicals (Gilbbstown,
NJ). All other chemicals used were of reagent grade or better.
Sample collection. Fresh mouse and rat whole blood was collected within ArQule, Inc. Fresh
dog and monkey whole blood was supplied by Charles River Laboratories, Inc. (Wilmington,
MA). Fresh human whole blood was obtained from healthy volunteers. Blood samples were
collected into K3-EDTA Vacutainer (BD, Franklin Lakes, NJ) over ice and were used within 24
hrs. Plasma was prepared by centrifuging fresh whole blood at 3000 g at 25°C for 5 min. Non-
labeled ARQ 501 was used to determine drug stability and partitioning in whole blood, while
[14C] ARQ 501 was used to investigate covalent binding, metabolite profiling and structural
elucidation. Since ARQ 501 is light sensitive, photoprotection was achieved by using amber
vials or aluminum foil as a seal for sample plates.
Drug stability and partitioning studies were conducted using fresh whole blood and
plasma from four species: mouse, rat, dog and human. ARQ 501 was spiked into whole blood
and plasma, respectively, to prepare a 10 µM solution, which was incubated at 37ºC in a
thermomixer. An aliquot of 150 µL of whole blood was collected at 0, 30, 60 and 120 minutes
post incubation, then immediately centrifuged at 6,000 g for 1 min to separate plasma from
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RBCs. The resulting RBCs were mixed with a two-fold volume of distilled water and incubated
at 37ºC to generate RBC lysate. The concentrations of ARQ 501 in plasma and RBC lysate were
determined with LC-MS-MS after protein precipitation by a three-fold volume of acetonitrile
containing D6-ARQ 501 as internal standard (300 ng/mL).
Covalent binding and metabolism studies were conducted by incubating [14C] ARQ 501
at 10 µM and 100 µM with human whole blood at 37ºC. An aliquot of 150 µL of whole blood
was collected at 0, 30, 60, 120 and 180 min post incubation, and mixed with a two-fold volume
of distilled water to generate whole blood lysate. Aliquots (20 µL) of the whole blood lysate at
each time point were used for total radioactivity counting. To eliminate the color quenching
effect of whole blood lysate, four volumes of 30% H2O2 were added into each aliquot for
decolorization. The sample was mixed with scintillation cocktail for radioactivity measurement
on a Wallac 1450 MicroBeta liquid scintillation counter (PerkinElmer Life and Analytical
Sciences Inc., Waltham, MA). Aliquots (20 µL) of the whole blood lysate at each time point
were mixed with three volumes of acetonitrile and centrifuged to separate the protein pellet from
supernatant. The resulting protein pellets were used for covalent binding determination and the
supernatant was used for metabolite characterization by LC-MS-ARC. Protein pellets were
extensively washed with 80% aqueous ethanol until the radioactivity in the washings was
reduced to less than twice background radioactivity (∼150 cpm). The protein pellets were then
dissolved in 100 µL of 0.1 N NaOH solution and neutralized with an equal volume of 0.1 N HCl
solution prior to the decolorization and radioactivity measurement. Covalent binding (%) was
calculated as percentage of the total radioactivity remaining in protein pellets after extensive
washing.
LC-MS-MS. An API4000 mass spectrometer (Applied Biosystems, Foster City, CA) equipped
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with Agilent 1100 binary pump (Agilent Technologies, Palo Alto, CA) and an HTS PAL injector
(LEAP Technologies, Inc., Carrboro, NC.) was used for LC-MS-MS analyses in blood stability
and partitioning experiments. Chromatographic separations were performed on an Atlantis dC18
HPLC column (2.1 × 50 mm, 5µm) using a binary gradient. The mobile phase solvents (water
[A] and acetonitrile [B] ) were both modified with 0.1% formic acid. The gradient employed was
as follows: solvent B started at 40% and held for 0.3 min, then linearly increased to 95% from
time 0.3 to 2.75 min, held at 95% for 0.5 min, then decreased to 40% at 3.27 min. Effluent from
HPLC was introduced to the mass spectrometer via an electrospray ionization source in positive-
ion mode. Multiple reaction monitoring (MRM) was employed to monitor ARQ 501 and D6-
ARQ 501 using m/z 243.1>159.0 and 249.1>159.0, respectively. Good linearity was achieved
over the concentration range of 3-2000ng/mL with an R2 >0.99 when a weighting of
1/(concentration)2 was used.
LC-MS-ARC. Radioactivity profiles in whole blood samples were monitored using liquid
chromatography coupled with an accurate radioisotope counting (LC-ARC) system. HPLC was
performed on an Agilent 1100 Series Modules system coupled to a Packard Radiomatic 500TR
series flow scintillation analyzer (Perkin-Elmer, Meriden, CT) and an accurate radioisotope
counting XFlow controller (AIM Research Co. Newark, DE). Mass spectra were collected with a
Waters Quattro LC mass spectrometer (Waters Corp., Milford, MA) operated in positive-ion
electrospray mode. The capillary voltage and cone voltage were set to 3.5kV and 30V,
respectively. The source and desolvation temperatures were 100oC and 350oC, respectively.
Collision-induced dissociation (CID) experiments were conducted for the selected target masses
with argon as the collision gas. The LC-MS-ARC system was controlled by an ARC data system
(Version 2.6).
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The chromatographic separation was achieved using an Atlantis dC18 column (250mm ×
4.6mm, 5µm) or an Xterra MS column (250 × 4.6mm, 5µm) (Waters Corp., Milford, MA) at a
flow rate of 1.0 ml/min. The Xterra MS column was applied to run the orthogonal
chromatographic conditions at pH 3 and 10. The mobile phase effluent was split to allow 20% to
the mass spectrometer via the electrospray ionization source. The remaining 80% of the flow was
mixed with scintillation cocktail ULTIMA-FLO and analyzed with ARC using a 500 µL liquid
cell. The splitting was conducted through a T-piece with an on-line check valve that allowed the
flow to go only in one direction. The mobile phase solvents (water [A] and acetonitrile [B]) were
both modified with 0.1% formic acid (pH 3) or 10 mM ammonium hydroxide (pH 10). The
gradient employed was as follows: solvent B started at 15% and held for 1 min, then linearly
increased to 95% from time 1 to 30 min, held at 95% for 4 min, then decreased to 15% at 35 min.
UPLC-QTOF MS. High-resolution MS experiments were conducted on a Waters Q-TOF
Premier operated in positive-ion electrospray mode. The instrument was calibrated across the
mass range of 50-1000 Da using a solution of sodium formate (0.3 mM). Data were acquired
using a desolvation temperature of 350oC, source temperature of 100oC and cone voltage of 30V.
Acquisition and analysis of the data were performed using MassLynx software (Version 4.1).
Full scan TOF MS spectra were acquired for measurement of accurate masses of ARQ 501 and
its metabolites. Data were centroided and mass was corrected during acquisition using an
external reference (Lock-Spray) solution of 500 ng/ml warfarin infused at 5 µL/min which
provided a reference ion at m/z 309.1127 ([M+H]+). One scan of the LockSpray channel was
interleaved with 10 scans of the analyte channel. Scans were of 0.2 s duration with a 0.1 s
interscan delay.
A Waters Acquity UPLC using an HSS T3 column (2.1 × 100mm, 1.8µm) was
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employed for the separation of metabolites. The mobile phase consisted of a binary solvent
system using water (A) and acetonitrile (B), both acidified with 0.1% formic acid. The gradient
program began with 5% eluent B and maintained for 1 min, then ramped linearly to 95% from 1
to 15 min and held for 2 min. The flow rate was 200 µl/min and the injection volume was 5 µL.
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Results
In vitro studies of ARQ 501 in whole blood. When incubated with fresh human whole blood or
the RBC fraction at 37ºC, more than 90% of ARQ 501 rapidly disappeared within 30 min.
However, ARQ 501 was shown to be stable in plasma for 2h (Fig. 1). Similar results were
observed for other species, including mouse, rat, dog and monkey. Rational explanations for the
rapid disappearance of ARQ 501 in whole blood could include sequestration of ARQ 501 in
RBCs, covalent binding of ARQ501 (and/or its metabolites) to blood components, metabolism
by enzymes in blood, or a combination of the above processes. In light of this hypothesis, in vitro
studies were conducted. ARQ 501 was distributed between human RBCs and plasma with a
partition coefficient of 1 (range: 0.82-1.09), which suggested that the sequestration of ARQ 501
into RBCs was not significant. After 60 min incubation of [14C] ARQ 501 in human whole blood
at 37ºC (Figure 2), covalent binding reached a plateau of approximately 23% of the total
radioactivity of [14C] ARQ 501 at 10µM and 100µM, which represents 11.5 and 112.3 pmole/mg
protein at 10 and 100 µM, respectively. Given that ARQ 501 was stable in plasma, these data
clearly suggest that metabolism by an enzyme or enzymes located in RBCs was responsible for
the rapid disappearance of the ARQ 501 in whole blood.
Identification of metabolites. Radioactivity detection facilitated metabolite profiling, and
accurate mass measurement provided structural identification of the metabolites. This study used
[14C] ARQ 501 for metabolite identification as well as covalent binding studies. Figure 3 depicts
the results from in vitro time-course studies on the metabolism of [14C] ARQ 501 in human
whole blood. After 60 min incubation at 37ºC, ARQ 501 was not detectable while four major
metabolites (M1, M2, M3 and M5) and two minor metabolite peaks (M4 and M6) were
observed. At 120 min, the radioactivity (based on ARC peak area) of M1, M2, M3, M4, M5 and
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M6 was approximately 13%, 35%, 14%, 1%, 20% and 5%, respectively, of the total radioactivity
from [14C] ARQ 501. These six major radioactive peaks (corresponding to M1 through M6) were
neither found in the control samples (collected at time point 0) nor in incubated plasma samples.
Figure 4 illustrates comparative radiochromatograms derived from LC-ARC analysis of samples
from an interspecies metabolic study. The metabolite profiles show qualitative but not
quantitative similarities among the different studied species. For example, M5 was not a major
metabolite in mouse or rat whole blood, but was a major one in dog, monkey and human blood.
In addition, an unidentified peak at 11.75 min (labeled with an asterisk) was only observed in
dog blood.
Detection and identification of the metabolites was based on radioactive peak monitoring
and molecular ion detection. The same protocol was conducted with non-labeled ARQ 501, and
the samples were analyzed using UPLC-TOF MS in order to obtain accurate mass measurement
and high-resolution product ion mass spectra for each of the selected metabolite peaks. Table 1
summarizes the accurate masses, elemental compositions and the postulated molecular structures
of the metabolites.
Interpretation of product ion mass spectra of metabolites frequently hinges upon the
similarity of product ions from the metabolite to those of the parent compound. Figure 5
illustrates the product ion mass spectra of ARQ 501 in positive-ion mode. The characteristic
product ion at m/z 187 in the CID mass spectrum of m/z 243 (Fig. 5a) indicated a neutral loss of
56Da by a cross-ring cleavage at the C(2)-O(1) and C(3)-C(4) bonds on the ether ring C, along
with charge retention on the rings A and B moiety (Scheme 1). Further fragmentation of m/z 187
led to prominent loss of a carbonyl group, giving rise to another characteristic ion at m/z 159,
which in turn eliminated the second carbonyl group to form m/z 131 (Fig. 5b). The product ion at
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m/z 105 from m/z 159 indicates whether or not phenyl ring A is modified through metabolism
(Fig. 5c). These characteristic fragmentation patterns were applied toward the structural
identification of metabolites. In addition, the synthesized standard of ARQ 501 was characterized
as: 1H NMR (400 MHz, CDCl3), δ: 8.06 (dd, J = 7.6 Hz, J’ = 1.2 Hz, 1H), 7.81 (d, J = 8.0 Hz,
1H), 7.64 (td, J = 8.0 Hz, J’ = 1.2 Hz, 1H), 7.50 (td, J = 8.0 Hz, J’ = 1.2 Hz, 1H), 2.57 (t, J = 6.4
Hz, 2H), 1.85 (t, J = 6.8 Hz, 2H), 1.47 (s, 6H).
To identify potential pH-sensitive groups (e.g. –OH and -COOH) within the metabolites,
the pH of the mobile phase was run at pH 3 and 10, respectively, and retention times were
monitored to detect shift. Significant shift in retention time was observed for M1-M3 but not for
M4-M6 between pH 3 and 10, indicating that pH-sensitive groups were present in M1-M3 but
not in M4-M6. The chromatographic behavior of the metabolites was integrated with their mass
spectral data for structural characterization.
M1. Accurate mass measurement indicated that M1 was produced by oxidation of ARQ 501
(Table 1). The characteristic ion at m/z 203 suggests an intact ether ring C, and the product ions
at m/z 175 and 147 from m/z 203 (produced by the elimination of one and two carbonyl groups,
respectively) indicate an intact dicarbonyl ring B (Fig. 6). In addition, the product ion at m/z 121
clearly localized the oxidation to the phenyl ring A. However, none of the synthesized oxidative
metabolite standards, 7-, 8-, 9- and 10-hydroxy-ARQ 501, matched the chromatographic
retention time of M1, which eluted much earlier than any of the above standards, indicating that
M1 was very polar. Therefore, the structure of M1 was not postulated here.
M2. M2 represents most of the radioactivity among the six metabolites detected in the human
whole blood sample after 2 hours of incubation. Accurate mass measurement indicated that M2
was formed by the introduction of two oxygen and two hydrogen atoms (34Da) to the ARQ 501
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molecule (Table 1). Figure 7 demonstrates the product ion mass spectra of M2 in positive- and
negative-ion modes. In positive-ion mode, the relatively unstable parent ion at m/z 277
undergoes facile elimination of H2O to produce a major product ion at m/z 259. Ion at m/z 159
derived from the characteristic ion of m/z 203 by losing a CO2 (44Da) indicated a carboxylic
structure.
The product ion mass spectrum in negative-ion mode clearly revealed carboxylic
structural information consistent with that of a carboxylic acid (Fig. 7b), because deprotonation
in negative-ion mode initially occurred on the carboxylic group and decarboxylation could
readily take place on the negatively charged group upon collisional activation (Miao and
Metcalfe, 2007). This is further supported by the fact that sp2 carbons can facilitate the loss of
CO2 by stabilizing negative charges via the increases s character of the carbon (Bandu, et al.,
2004), which indicates a carboxylic group is located in the phenyl ring. The ions at m/z 231 and
187 were generated by subsequent neutral losses of CO2 from the precursor ion m/z 275.
Therefore, interpretation of mass spectra led to a structure corresponding to oxidative opening of
the carbonyl ring B and the forming of a dicarboxylic acid structure (Table 1). Ultimately, the
structure of M2 was confirmed by comparing its retention time and product ion spectra to the
synthesized standard (Table 1). The standard compound was characterized as: mp. 185-187oC,
1H NMR (400 MHz, DMSO-d6) δ: 12.00 (br, 2H), 7.79 (d, J = 7.2 Hz, 1H), 7.49 (t, J = 7.6 Hz,
1H), 7.40 (t, J = 7.6 Hz, 1H), 7.19 (d, J = 8.0 Hz, 1H), 2.33 (t, J = 6.4 Hz, 2H), 1.69 (t, J = 6.4
Hz, 2H), 1.26 (s, 6H). The IUPAC name of M2 is 6-(2-carboxyphenyl)-2,2-dimethyl-3,4-
dihydro-2 H-pyran-5-carboxylic acid.
M3. Accurate mass measurement indicated that M3 was formed by the addition of one O and
two H atoms (18Da) to ARQ 501 (Table 1). Figure 8 shows the product ion mass spectra of M3
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in positive- and negative-ion modes. Compared to positive-ion mode (Fig. 8a), more structural
information was revealed in negative-ion mode (Fig. 8b). One of the major product ions in
negative-ion mode was observed at m/z 215 and appeared to arise from a neutral loss of 44Da,
which suggested that a carboxylic group was within the molecule. Interpretation of the product
ion mass spectra suggested that the cleavage between C(5)-C(6) of the two carbonyl groups was
initiated by an oxidative process that generated a carboxylic as well as an aldehyde group.
Further investigation with 1H NMR on synthesized standards confirmed the proposed structure
and provided the positional information of the carboxylic group, which was located at position 6
(Scheme 1 and Table 1). The standard compound was characterized as: mp. 146-147oC, 1H NMR
(400 MHz, CDCl3) δ: 9.20 (s, 1H), 8.10 (dd, J = 8.0, 1.2 Hz, 1H), 7.61 (td, J = 7.6, 1.2 Hz, 1H),
7.56 (td, J = 7.6, 1.6 Hz, 1H), 7.40 (dd, J = 7.2, 1.2 Hz, 1H), 2.43 (t, J = 6.8 Hz, 2H), 1.79 (t, J =
6.4 Hz, 2H), 1.37 (s, 6H). The IUPAC name of M3 is 2-(5-formyl-2,2-dimethyl-3,4-dihydro-2 H-
pyran-6-yl)benzoic acid.
M4. Accurate mass measurement confirmed the elemental composition of M4 as C14H15O3, only
one carbon atom different from that of ARQ 501 (Table 1). Figure 9a shows the product ion
mass spectrum of M4 and the major fragmentation pathways. The characteristic neutral loss of
56Da occurred in M4 and generated one major ion, m/z 175, through cross-ring cleavage of the
C(2)-O(1) and C(3)-C(4) bonds. The proposed structure of M4 was confirmed by a synthesized
standard characterized as: mp. 115-117oC, 1H NMR (400 MHz, DMSO-d6) δ: 7.74 (d, J = 7.2
Hz, 1 H), 7.61 (t, J = 8.0 Hz, 1 H), 7.39-7.33 (m, 2 H), 2.45 (t, J = 6.4 Hz, 2 H), 1.87 (t, J = 6.4
Hz, 2 H), 1.41 (s, 6 H). The IUPAC name of M4 is 2,2-dimethyl-3,4-dihydro-2H,5H-pyrano[3,2-
c]chromen-5-one.
M5. M5 represents the largest peak observed in human blood after 60 min incubation (Fig. 3).
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Accurate mass measurement provided the elemental composition of C14H15O2. The difference of
28Da between M5 and ARQ 501 implies the loss of CO group. Figure 9b illustrates the product
ion mass spectrum of M5 and the major fragmentation pathways. The product ion spectrum of
[M+H]+ (m/z 215) was dominated by an intense signal at m/z 159, which was produced from the
characteristic cross-ring cleavage of the C(2)-O(1) and C(3)-C(4) bonds. This ion (m/z 159)
underwent further fragmentation to yield the product ion m/z 131 by a neutral loss of CO. In
addition, the product ion at m/z 105 suggests no metabolic modification to phenyl ring A. Hence,
the structure of M5 was postulated to occur by the elimination of a carbonyl group through
inductive cleavage followed by subsequent formation of a five-membered ring. The structure of
M5 was confirmed with a synthesized standard characterized as: mp. 53-55oC, 1H NMR (400
MHz, DMSO-d6) δ: 7.41-7.29 (m, 3H), 7.12 (d, J = 6.8 Hz, 1H), 2.20 (t, J = 6.4 Hz, 2H), 1.78 (t,
J = 6.4 Hz, 2H), 1.41 (s, 6H). The IUPAC name of M5 is 2,2-dimethyl-3,4-dihydroindeno[1,2-
b]pyran-5(2H)-one.
M6. Accurate mass measurement shows that this metabolite has the same elemental composition
as M4, C14H15O3 (Table 1). Figure 9c illustrates the product ion mass spectrum of M6 and the
major fragmentation pathways. Apparently, the only major ion at m/z 175 was generated by the
characteristic cross-ring cleavage with a neutral loss of 56Da. The ion at m/z 105 confirms that
phenyl ring A remained intact and was not the site of metabolism. Upon locating the
biotransformation that occurred on ring B, an ester structure was assigned to M6 and was
confirmed by a synthetic standard. The standard compound was characterized as: 1H NMR (400
MHz, CDCl3) δ: 8.24 (d, J = 7.8 Hz, 1H), 7.74-7.72 (m, 2H), 7.49-7.47 (m, 1H), 2.65 (t, J = 6.6
Hz, 2H), 1.90 (t, J = 6.6 Hz, 2H), 1.39 (s, 6H). The IUPAC name of M6 is 2,2-dimethyl-3,4-
dihydropyrano[3,2-c]isochromen-6(2H)-one.
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Discussion
Potential reasons for the rapid disappearance of ARQ 501 in blood were investigated by
incubating ARQ 501 in freshly drawn blood at 37oC. Metabolic profiling demonstrated that ARQ
501 was extensively metabolized in whole blood under in vitro conditions, and that the
enzymatic activity was located in the RBC fraction. These results further showed that
metabolism by RBCs was a major contributor, while covalent protein binding of ARQ 501
and/or its metabolites was a minor contributor (23%) to the loss of ARQ 501 in whole blood.
Interspecies metabolic profiling implied that the enzymes responsible for ARQ 501
metabolism exist in the RBCs of the studied species, but for each species, they exhibit notably
different enzymatic activity. ARQ 501 showed concentration-dependant metabolism in human
whole blood; the metabolic rate of ARQ 501 decreased with increasing concentrations over a
range of 10-500 µM. In addition, ARQ 501 was completely metabolized after 60 min incubation
in human whole blood (Fig. 3), which coincides with the plateau at 60 min in the time-course
covalent binding study (Fig. 1).
RBCs contain moderate cytochrome P450-like activity due to the existence of various
enzymes, such as aldehyde dehydrogenase, esterase, hemoglobin, methyl transferases, N-acetyl
transferases, glutathione transferases, etc. (Helander and Tottmar, 1987; Ericsson et al., 1999;
Owen and Natatsu, 1983; Mieyal, 1985; Awasthi and Singh, 1984). Atul et al. (2002) reported
that the same metabolite of centpropazine was formed in rat liver S9, intestine and RBCs. It
provided support that the RBCs may share many metabolic enzymes with liver and intestine, and
can act as an extra-hepatic system for drug clearance. In the present study, the metabolites of
ARQ 501 observed in blood were not detected in in vitro incubations of ARQ 501 with liver
microsomes or hepatocytes in any species. In liver microsomes, the major metabolites were
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formed through monohydroxylation on the phenyl ring A or the ether ring C. In hepatocytes,
most of the metabolites were produced through phase II metabolic pathways, such as
glucuronidation, sulfation and glucosidation (Miao et al., 2007). This suggested a unique
biotransformation pathway in blood compared to the hepatic system. Consistent with the
previous studies that few Phase II metabolites are generated by RBCs (Cossum, 1988), no phase
II metabolites were detected in the current study.
Work is underway to identify the specific enzymes responsible for the biotransformation
of ARQ 501 in whole blood and to evaluate the metabolic pathways for other ARQ 501 related
drug candidates. It is conceivable that formation of M2 occurs via initial oxidative
biotransformation of quinone, insertion of molecular oxygen by monooxygenase, followed by
hydrolysis would lead to dicarboxylic acid M2. Similar biotransformations were observed for the
metabolism of acenaphthene (Vila et al., 2001). Reduction is an important reaction in the
metabolism and activation of quinonic drugs and xenobiotics (Testa 1995). Alternatively, initial
reduction of the quinone ring by carbonyl reductase or quinone reductase through a two-electron
reduction would lead to unstable hydroxyquinone (Gaikwad et al., 2007). Oxidative cleavage of
the hydroxyquinone would be expected to provide the dicarboxylic acid M2, as reported for the
metabolism of pyrene (Vila et al., 2001). We have observed that the reduction of ARQ 501 is
involved in its metabolism in hepatocytes (Miao et al., 2007). The conjugation metabolites (e.g.
glucuronides, sulfates, etc.) of ARQ 501 arise from a two-step process, which requires reduction
of the quinone ring B to a hydroquinone, followed by conjugation at the reduced site(s).
Following the formation of M2, the carboxylic group on the pyran ring C of M2 can be reduced
by carboxylic acid reductase, which yields the aldehyde metabolite M3. The selective generation
of M3 indicates regioselectivity of the carboxylic acid reductase since the regioisomer of M3
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with the carboxylic group at position 5 was not observed. As observed for M2 and M3 of ARQ
501, etoposide, an anticancer agent, was also metabolized to an open ring metabolite when
incubated with intact human RBCs (Loo et al., 1986).
The two conjugated lactone regioisomers, M4 and M6, might be generated from ARQ
501 through similar multiple metabolic steps. The formation of M5 could be initiated from M2,
followed by spontaneous enzymatic dehydration and ring cyclization. Funabiki et al (1986)
demonstrated several oxygenase model reactions using 3,5-di-tert-butylcatechol as a probe
compound. The studied compound was oxygenated with insertion of molecular oxygen to give
intra- and extradiol oxygenation intermediates, which further produced pyrone products through
eliminating a carbonyl group. Similarly, ARQ 501 can form unstable intermediates containing
seven-membered ring formed through insertion of oxygen extra-diketone, and the intermediates
yield M4 and M6, based on the position of the inserted oxygen, through eliminating a carbonyl
group. Likewise, M5 can be produced directly from ARQ 501 through the carbonyl elimination
pathway. To our knowledge, this novel ring-contraction biotransformation has not been reported
previously.
In conclusion, five in vitro metabolites of ARQ 501 were identified in whole blood. The
rapid disappearance of ARQ 501 in blood clearly emphasized the importance of separating
plasma from RBCs immediately upon collection of clinical blood samples. Further studies have
shown that the identified metabolites here were also detected in clinical samples from cancer
patients treated with ARQ 501, the next logical step is to ask how important is the blood
metabolism for the systemic clearance of ARQ 501. Investigations are ongoing to search for
potential Phase II metabolites derived from the blood metabolites and to evaluate the
contribution of blood metabolism to the in vivo clearance in patients.
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Footnote page
* Corresponding author
§. The authors contributed equally
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Figure Legends
FIG. 1. The stability of ARQ 501 (10µM) in human plasma (�) and whole blood (� for plasma and �
for RBCs) at 37oC
FIG. 2. The covalent binding of [14C] ARQ 501 to blood components after incubation with human
whole blood at 37oC
FIG. 3. In vitro time-course studies of human whole blood metabolism with [14C] ARQ 501
FIG. 4. Comparative radiochromatograms of [14C] ARQ 501 incubated with whole blood of different
species for 2hrs (1,2,3…p = M1, M2, M3…parent)
FIG. 5. Mass spectra of ARQ 501. Product ion mass spectra of m/z 243 (a), m/z 187 (b) and m/z 159
(c)
FIG. 6. Product ion mass spectrum of M1 in positive-ion mode
FIG. 7. Product ion mass spectra of M2 in positive-ion mode (a) and negative-ion mode (b)
FIG. 8. Product ion mass spectra of M3 in positive-ion mode (a) and negative-ion mode (b)
FIG. 9. Product ion mass spectra of M4 (a) M5 (b) and M6 (c) in positive-ion mode. The insets show
the CID mass spectra of m/z 175 (a) and m/z 159 (b), respectively
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TABLE 1. Accurate mass measurement and the postulated structures of the ARQ 501metabolites in
whole blood
Compound [M+H]+ ∆M Calculated
(Da)
Measured
mass (Da)
Difference
mDa
Elemental
composition
Structure
ARQ 501 243 - 243.1021 243.1018 -0.3 C15H15O3
O
O
O
M1 259 +16 259.0970 259.0965 -0.5 C15H15O4
O
O
O
O
M2 277 +34 277.1076 277.1080 0.4 C15H17O5
O
COOHCOOH
M3 261 +18 261.1127 261.1136 0.9 C15H17O4
O
COOHCHO
M4 231 -12 231.1021 231.1025 0.4 C14H15O3 O O
O
M5 215 -28 215.1072 215.1080 0.8 C14H15O2
O
O
M6 231 -12 231.1021 231.1024 0.3 C14H15O3
O
O
O
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O
O
O
1
2
3
4
56
7
8
9
10 *
A B
C
Scheme 1. Structure of [14C] ARQ 501 (the 14C-label is indicated with an asterisk)
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FIG. 1.
0 30 60 120
0
20
40
60
80
100
120
% A
RQ
501
rem
aini
ng
Time (min)
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FIG. 2.
0 30 60 120 1800
20
40
60
80
100
Cov
alen
t bin
ding
(%
)
Time (min)
10uM 100uM
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FIG. 3
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
0
5000
10000ARC(CPM)
Min
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
0
5000
10000ARC(CPM)
Min
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
01000200030004000ARC(CPM)
Min
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
0
2000
4000
6000ARC(CPM)
Min
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
0
2000
4000
6000ARC(CPM)
Min
0 min
30 min
60 min
120 min
180 min
M1
M2
M3 M4M5
M6
ARQ501
ARQ501
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FIG. 4
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
05000
1000015000ARC(CPM)
Min
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
0
5000
10000ARC(CPM)
Min
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
010002000
ARC(CPM)
Min
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
0
5000
10000ARC(CPM)
Min
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
0200040006000ARC(CPM)
Min
Mouse
Rat
Dog
Monkey
Human1
2
3 4 56p
*
1
1
1
1
2
2
2
2
3
3
3
3
5
5
5
5
6p
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FIG. 5.
m/z80 100 120 140 160 180 200 220 240 260
%
0
100
%
0
100
%
0
100 187
159
105103155
131 133
183 243225201
159103
7795
105
131
113 187
103
77
95
159105
131
[M+H]+
a.
b.
c.
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FIG. 6.
m/z80 100 120 140 160 180 200 220 240 260
%
0
100203
175
121
10791
147
131
173
191
217
259
[M+H]+
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FIG. 7.
m/z80 100 120 140 160 180 200 220 240 260 280 300
%
0
100
%
0
100 x10173
149
111
95
159
191 259
203 241
213 223277
129
101
231
187
275
a.
b.
[M-H]-
[M+H]+
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FIG. 8.
m/z80 100 120 140 160 180 200 220 240 260 280
%
0
100
%
0
100 149
95
133113175 225
187 243 261
197
158
157111
129 182
215
259
a.
b.
[M+H]+
[M-H]-
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FIG. 9.
m/z80 100 120 140 160 180 200 220 240
%
0
100 x2175
107
79 121 231
m/z80 100 120 140 160 180 200 220 240
%
0
100 x4159
131105 215
m/z80 100 120 140 160 180 200 220 240
%
0
100 x2 175
147
133
129
10591157 231
m/z60 80 100 120 140 160 180
%
0
100 79
77
65
95
91
107121
119 175133
m/z60 80 100 120 140 160 180
%
0
100103
9577105
131 159
a
b
c
[M+H]+
[M+H]+
[M+H]+
CID of m/z 159
CID of m/z 175
O
O
159131-CO
105
O
O
O
175147-CO
-H2O
157
105
-H2O
129
O O
O
175
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