DMD Fast Forward. Published on January 7, 2008 as...

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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.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on January 7, 2008 as DOI: 10.1124/dmd.107.018572

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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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References

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Helander A, Tottmar O (1987) Metabolism of biogenic aldehydes in isolated human blood cells, platelets

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Hinderling PH (1997) Red blood cells: a neglected compartment in pharmacokinetics and

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Hooks MA (1994) Tacrolimus, a new immunosuppressant - a review of the literature. Abb Pharmacother

28: 501-511.

Kizer D, Miao X, Yang R-Y, Wu H, Volckova E, Nguyen K, Ali S, Tandon M, Savage R, Ashwell MA,

Chan T (2007) Synthesis and characterization of the metabolites of ARQ 501 in whole blood. The 10th

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lapachone: synthesis of derivatives and activities in tumor models. J Med Chem 27: 990-994.

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USA.

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blood metabolites. Submitted to Bioorg Med Chem.


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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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