PHOSPHOCHOLINE CONJUGATION: AN UNEXPECTED IN VIVO...

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PHOSPHOCHOLINE CONJUGATION: AN UNEXPECTED IN VIVO

CONJUGATION PATHWAY ASSOCIATED WITH HEPATITIS C NS5B

INHIBITORS FEATURING A BICYCLO[1.1.1]PENTANE

XIAOLIANG ZHUO, JOSEPH L. CANTONE, YINGZI WANG, JOHN E. LEET,

DIETER M. DREXLER, KAP-SUN YEUNG, XIAOHUA STELLA HUANG, KYLE J.

EASTMAN, KYLE E. PARCELLA, KATHLEEN W. MOSURE, MATTHEW G.

SOARS, JOHN F. KADOW, BENJAMIN M. JOHNSON

Departments of Pharmaceutical Candidate Optimization (X.Z., J.L.C., Y.W., D.M.D.,

X.S.H., K.W.M., M.G.S., B.M.J.), Synthesis and Analysis Technology Team (J.E.L.), and

Virology Chemistry (K-S.Y., K.J.E., K.E.P., J.F.K.), Bristol-Myers Squibb Co., 5

Research Parkway, Wallingford, CT 06492

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 9, 2016 as DOI: 10.1124/dmd.115.069062

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Running Title: Phosphocholine conjugation of HCV NS5B inhibitors

Correspondence should be addressed to:

Xiaoliang Zhuo, Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Co., 5

Research Parkway, Wallingford, CT 06492

Tel: (203) 677 5938; Fax: (203) 677 6193; e-mail: [email protected]

Number of Text Pages: 29

Number of Tables: 2

Number of Figures: 7

Number of References: 24

Number of Supplemental Figure: 1

Number of words: 245 (Abstract)

483 (Introduction)

1491 (Discussion)

Abbreviations used are: BDC, bile-duct-cannulated; COSY, 1H-1H homonuclear

correlation spectroscopy; CDP-choline, cytidine-diphosphocholine; CPT, CDP-choline:

1,2-diacylglycerol cholinephosphotransferase; Da, Dalton; DAG, diacylglycerol; DMSO,

dimethyl sulfoxide; ER, endoplasmic reticulum; HCV, hepatitis C virus; HMQC,

heteronuclear multiple quantum coherence; HMBC, heteronuclear multiple bond

correlation; HPLC, high pressure liquid chromatography; min, minute; MS, mass

spectrometry; NADPH, β-nicotinamide adenine dinucleotide phosphate tetrasodium salt;


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NS5B, nonstructural protein 5B; P450, cytochromes P450; UV, ultraviolet; POPC,

phosphocholine; 1, 5-(3-(bicyclo[1.1.1]pentan-1-ylcarbamoyl)-4-fluorophenyl)-2-(4-

fluorophenyl)-N-methyl-6-(3,3,3-trifluoropropyl)furo[2,3-b]pyridine-3-carboxamide; 2,

5-(3-(bicyclo[1.1.1]pentan-1-ylcarbamoyl)phenyl)-2-(4-fluorophenyl)-N-methyl-6-(3,3,3-

trifluoropropyl)furo[2,3-b]pyridine-3-carboxamide.


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Abstract

During a medicinal chemistry campaign to identify inhibitors of the hepatitis C

virus nonstructural protein 5B (RNA-dependent RNA polymerase), a

bicyclo[1.1.1]pentane was introduced into the chemical scaffold to improve metabolic

stability. The inhibitors bearing this feature, 5-(3-(bicyclo[1.1.1]pentan-1-ylcarbamoyl)-

4-fluorophenyl)-2-(4-fluorophenyl)-N-methyl-6-(3,3,3-trifluoropropyl)furo[2,3-

b]pyridine-3-carboxamide (1) and 5-(3-(bicyclo[1.1.1]pentan-1-ylcarbamoyl)phenyl)-2-

(4-fluorophenyl)-N-methyl-6-(3,3,3-trifluoropropyl)furo[2,3-b]pyridine-3-carboxamide

(2), exhibited low turnover in incubations with liver S9 or hepatocytes (rat, human), with

hydroxylation of the bicyclic moiety being the only metabolic pathway observed. In

subsequent disposition studies using bile-duct-cannulated rats, the metabolite profiles of

bile samples revealed, in addition to multiple products of bicyclopentane-oxidation,

unexpected metabolites characterized by molecular masses that were 181 Da greater than

those of 1 or 2. Further LC/MSn and NMR analysis of the isolated metabolite of 1

demonstrated the presence of a phosphocholine (POPC) moiety bound to the methine

carbon of the bicyclic moiety through an ester bond. The POPC conjugate of the NS5B

inhibitors was assumed to result from two sequential reactions: hydroxylation of the

bicyclic methine to a tertiary alcohol and addition of POPC by CDP-choline: 1,2-

diacylglycerol cholinephosphotransferase, an enzyme responsible for the final step in the

biosynthesis of phosphatidylcholine. However, this pathway could not be recapitulated

using CDP-choline-supplemented liver S9 or hepatocytes due to inadequate formation of

the hydroxylation product in vitro. The observation of this unexpected pathway prompted

concerns about the possibility that 1 and 2 might interfere with routine phospholipid


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synthesis. These results demonstrate the participation in xenobiotic metabolism of a

process whose function is ordinarily limited to the synthesis of endogenous compounds.


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Introduction

After a decade of research and clinical trials, the treatment of hepatitis C virus

(HCV) infection, which plagues nearly 3% of the world’s population and leads

potentially to progressive chronic liver diseases, has shifted away from the use of

pegylated interferon and ribavirin—medicines that offer only suboptimal sustained

virological responses and produce severe side effects (Eltahla et al., 2015). Instead,

direct-acting antiviral agents (DAAs) have emerged as the current standard of care to

treat HCV infection. These drugs are directed against multiple nonstructural (NS)

proteins that are essential to the virus life cycle, including the NS5B RNA-dependent

RNA polymerase, the NS5A replication complex, and the NS3/4A protease (Lindenbach

and Rice, 2005). Structural and functional characterization of NS5B revealed a right-

hand-like motif comprised of one active site and multiple allosteric sites, including 2

palm sites, 2 finger sites and 1 thumb site (Ago et al., 1999; Bressanelli et al., 1999;

Lesburg et al., 1999). Identification of these structural features laid a foundation for the

design of potent antiviral agents that, by binding to these NS5B allosteric sites, could

obstruct conformational changes of the polymerase that are required for initiation and

elongation of RNA strands (Caillet-Saguy et al., 2011; Eltahla et al., 2014). So far, anti-

HCV drug discovery targeting NS5B has identified both non-nucleotide inhibitors (e.g.

BMS-791325, dasabuvir) and nucleotide inhibitors (e.g. sofosbuvir).

Recently, potent non-nucleotide NS5B inhibitors targeting the palm site of the

HCV RNA-dependent RNA polymerase were evaluated alongside inhibitors of other key

HCV enzymes, including NS5A, NS3/4A and NS5B (the thumb site), for possible use in

combination therapy (Gao et al., 2010; Gentles et al., 2014; Scola et al., 2014). During


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optimization to improve the metabolic stability of these NS5B inhibitors featuring a 6-

substituted furo[2,3-b]pyridine core, multiple isosteric replacements of a t-butyl moiety

were explored including a bicyclo[1.1.1.]pentane (Figure 1). As a precedent for this

approach, a bicyclo[1.1.1]pentane moiety was used during the discovery of metabotropic

glutamate receptor-1 antagonists as a phenyl replacement that maintained flanking

pharmacophores in a coplanar orientation (Pellicciari et al., 1996; Costantino et al.,

2001). In another example involving the optimization of a group of γ–secretase inhibitors,

the use of bicyclo[1.1.1]pentane as a phenyl isostere imparted improvements in metabolic

stability, aqueous solubility and permeability that were attributed to changes in

physicochemical properties (Stepan et al., 2012).

Compared to NS5B inhibitors featuring t-butyl groups in the same position, the

bicyclo[1.1.1.]pentane analogs evaluated in the present study retained comparable

antiviral activity against genotype 1a, 1b and 2a proteins (< 10 nM) and also avoided

rapid hydroxylation by P450s. Accordingly they were advanced for further evaluation of

ADME properties including the characterization of metabolite profiles in liver S9,

hepatocytes and bile-duct-cannulated (BDC) rats. Following the detection of unexpected

metabolites in rat bile, multiple approaches including LC/MSn and NMR were employed

to elucidate the structures of these metabolites in hopes of fostering a better

understanding of their mechanism of formation.

Materials and Methods

Chemicals and Liver Subcellular Fractions. Reduced β-nicotinamide adenine

dinucleotide phosphate tetrasodium salt (NADPH), potassium phosphate (monobasic and

dibasic), cytidine diphosphocholine (CDP-choline), ammonium formate,


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ethylenediaminetetraacetic acid (EDTA), formic acid and deuterated methanol

(methanol-d4) were purchased from Sigma (St Louis, MO). Magnesium chloride (1 M)

solution was obtained from ThermoFisher Scientific (Grand Island, NY). HPLC-grade

water and acetonitrile were obtained from Mallinckrodt Baker (Phillipsburg, NJ). Human

liver S9 (pooled from 20 male and female donors) and Aroclor-induced rat liver S9

(pooled from male animals) were obtained from BD Biosciences (Bedford, MA) and

from Xenotech (Lenexa, KS), respectively. Cryopreserved human hepatocytes (single

male donor) and rat hepatocytes (from male animals) were provided by CellzDirect, Inc

(Tucson, AZ). Everolimus was purchased from Cell Signaling (Danvers, MA). 5-(3-

(Bicyclo[1.1.1]pentan-1-ylcarbamoyl)-4-fluorophenyl)-2-(4-fluorophenyl)-N-methyl-6-

(3,3,3-trifluoropropyl)furo[2,3-b]pyridine-3-carboxamide (1), and 5-(3-

(bicyclo[1.1.1]pentan-1-ylcarbamoyl)phenyl)-2-(4-fluorophenyl)-N-methyl-6-(3,3,3-

trifluoropropyl)furo[2,3-b]pyridine-3-carboxamide (2) were synthesized and

characterized by Bristol-Myers Squibb in Wallingford, CT (Figure 1) [refer to Fused

Furans for the Treatment of Hepatitis C (WO 2014/159559 A1, October 2, 2014) by

Yeung K-S, Eastman KJ, and Parcella KE].

In Vitro Metabolism. Compounds 1 and 2 (10 µM each) were studied in

incubations (0.5 mL) with either human liver S9 (2 mg/mL) or Aroclor-induced rat liver

S9 (2 mg/mL) in potassium phosphate buffer (100 mM, pH 7.4). The reactions (n = 2)

were initiated by addition of NADPH (1 mM) and carried out at 37°C. At 0 and 30 min,

aliquots (150 µL) of the reaction mixtures were collected, and reactions were terminated

by adding three volumes of acetonitrile. Proteins were then removed using a Phenomenex

(Torrance, CA) Strata filter plate by centrifugation (Eppendorf 5804 R, Hamburg,


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Germany) at 2000g for 3 min. The filtrate was collected in a 96-well plate and evaporated

to 25% of the original volume under N2 gas. The remaining aqueous phase was analyzed

using HPLC/UV/MS.

In order to verify the presence of active CDP-choline:1,2-diacylglycerol

cholinephosphotransferase (CPT) in vitro, everolimus (10 µM) was incubated with

human liver S9 or Aroclor-induced rat liver S9 supplemented with CDP-choline (1 mM),

magnesium chloride (5 mM), EDTA (10 mM) in a Tris-HCl buffer (100 mM, pH 8.5)

(Wilgram et al., 1960) (Arthur and Choy, 1984). The reactions were carried out at 37°C

for 120 min, and the samples were then processed as described above. In a separate

experiment, 1 (10 µM) was incubated under the same experimental conditions in vitro,

but with an additional cofactor, NADPH (1 mM), added to facilitate phase I reactions.

The in vitro metabolism of 1 and 2 was also examined in incubations with

cryopreserved primary human or rat hepatocytes. The frozen cells (1 tube each) were

thawed in a 37°C water bath and then added to 3 mL of CHRM medium (Life

Technologies, Grand Island, NY) for human cells or to 3 mL of Williams Medium E

supplemented with hepatocyte maintenance supplement pack (Life Technologies) for rat

cells. Following a brief centrifugation at 100g for 10 min (for human hepatocytes) or at

55g for 3 min (for rat hepatocytes), the cell pellet was resuspended in 0.5 mL of pre-

warmed hepatocyte incubation medium (Life Technologies), and cell viability and yield

were determined using trypan blue dye exclusion staining. The hepatocytes (with at least

80% viability) in suspension (1×106 cells/mL) were incubated with a substrate (10 μM)

for 0 and 2 h at 37°C in a humidified CO2 (5%) incubator. The reaction was stopped by

addition of an equal volume of acetonitrile. After centrifugation at 1500g for 20 min, the


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supernatant was evaporated to 50% of the original volume using N2 gas, and the

remaining aqueous phase was analyzed using HPLC/UV/MS.

The activity of CPT was also assessed by incubating everolimus (10 µM) with rat

hepatocytes (1×106 cells/mL) under the same conditions described above and analyzing

for the presence of the phosphocholine conjugate reported previously (Zollinger et al.,

2008).

BDC Rat Study. All animal procedures were reviewed and approved by the

Animal Care and Use Committee at Bristol-Myers Squibb Co. Compounds 1 and 2 were

formulated (0.67 mg/mL) in a vehicle of dimethyl acetamide/PEG400 (10/90, v/v) and

administered intravenously (2 mg/kg of body weight; n = 3 for each compound) to BDC

male rats (Hilltop Lab Animals, Inc., Scottdale, PA), and bile, plasma, urine, and feces

were collected over 24 h. Serial blood samples were collected from the jugular vein into

K3EDTA-containing tubes at 0, 0.5, 1, 3, 6, and 24 h post-dose. Plasma samples were

obtained by centrifugation at 4°C (1500 to 2000 g) and stored at -80°C until analysis.

Aliquots (100 μL) of plasma samples were pooled across time points and mixed

with 3 volumes of acetonitrile by vortexing. Following precipitation of the protein pellet

by centrifugation at 15,000g for 10 min, the supernatant was evaporated to 25% of the

original volume under N2 gas. The remaining aqueous phase was analyzed by

HPLC/UV/MS.

Aliquots (250 µL) of pooled bile or urine samples were mixed with 3 volumes of

acetonitrile, centrifuged at 15,000g for 5 min, and the supernatants were evaporated to

25% of the original volume under N2 gas. The remaining aqueous phase was analyzed by

HPLC/UV/MS.


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Feces samples (2 g) were homogenized in 3 volumes of a potassium phosphate

buffer (50 mM, pH 7.4), and extracted twice with ethyl acetate. Following centrifugation

at 15,000g for 5 min, the supernatants were dried using N2 gas. The extracts were

resuspended in 300 µL of a solvent (water/acetonitrile/formic acid, 50/50/0.1, v/v/v) for

HPLC/UV/MS analysis.

Isolation of 1-3. Aliquots (70 mL) of bile from the BDC rats administered with 1

were extracted with ethyl acetate followed by n-butanol. The butanol extract was

evaporated to dryness, re-dissolved in methanol:water (65/35, v/v), and extracted with

chloroform that had been pre-equilibrated with methanol/water (65/35, v/v). The aqueous

methanol extract was further enriched by SPE using a Waters Oasis HLB cartridge (1 g,

20 cc). After sample application (10 mL in water), samples were eluted using a step-

gradient of water:acetonitrile (9:1 v/v), followed by 3:1, 1:1, 1:3 water:acetonitrile, and

100% acetonitrile (10 mL each). 1-3 was detected primarily in the 1:1 water:acetonitrile

eluent and was purified further on a Waters Sunfire C18 column (5 µm, 4.6 × 150 mm)

using an Agilent 1100 series HPLC system (Agilent Technologies, Santa Clara, CA). The

mobile phase consisted of solvent A (10 mM ammonium acetate in 5% acetonitrile) and

solvent B (acetonitrile) with a linear gradient from 15% to 100% B over 25 min at a flow

rate of 1.2 mL/min. The fractions were collected into Beckman 96-deep-well

polypropylene blocks (Beckman Coulter, Inc., Brea, CA) using an Agilent G1364C

fraction collector. Fractions containing 1-3 were pooled and repurified to remove UV-

transparent bile-related impurities using a shallow linear gradient on the same C18

column: 15% to 75% solvent B over 25 min. The fractions containing 1-3 (~ 25 µg) were

evaporated to dryness under nitrogen at room temperature.


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HPLC/UV/MS Analysis. The HPLC/UV/MS system consisted of a Waters

Acquity binary solvent manager, a Waters Acquity sample manager, a Waters Acquity

photodiode array detector, and a Waters Xevo Q-TOF mass spectrometer (Waters

Corporation, Milford, MA). Chromatographic separations were carried out on a Waters

BEH C18 column (1.7 μm, 2.1 × 100 mm). The mobile phase consisted of

water/acetonitrile/formic acid (95/5/0.1, v/v/v) (solvent A) and acetonitrile with 0.1%

formic acid (solvent B) at a flow rate of 0.5 mL/min with a linear gradient as follows: 5%

B isocratic for 0.5 min, 2% to 35% B over 5.5 min, and finally 35% to 100% B over 3.5

min. The gradient was maintained for 1 min and then returned to initial conditions for a

1.5-min equilibration period. The eluent from the column was analyzed by the

photodiode array detector (scanning 200 – 400 nm at 5 Hz) followed by the mass

spectrometer equipped with an electrospray ionization source and operated in a positive-

ion mode. To obtain maximum sensitivity based on the ionization of the parent

molecules, the source temperature was set to 125°C, the desolvation temperature was

250°C, the capillary voltage was 3 kV, the sampling cone was 40 (arbitrary units) and the

extraction cone was 1.7 (arbitrary units). All mass spectrometry data was acquired using

a low collision voltage (6 V) and a high-voltage ramp (25 – 50 V), and data was

processed using Metabolynx software (Waters Corp). Metabolite structures were assigned

based on their LC/MS/MS product-ion mass spectra.

Everolimus and its conjugate were analyzed using the same LC/UV/MS system,

except that the mobile phase was composed of solvent A (5 mM ammonium formate with

0.1% formic acid) and solvent B (methanol with 0.1% formic acid) (Bruns et al., 2015)

and delivered at 0.3 mL/min using a linear gradient as follows: 30% B for 0.5 min, and


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then 30 to 100% B over 5.5 min. The column was then reequilibrated to the initial HPLC

conditions for 2 min.

The isolated 1-3 was also analyzed using an LTQ Orbitrap mass spectrometer

(Thermo Fisher, Waltham, MA) equipped with a nano-electrospray source and operated

in a positive-ion mode. The source voltage, capillary voltage, and tube lens voltage were

set to 1.4 kV, 25 V, and 170 V, respectively, while the capillary temperature was set to

150°C. The scan ranges were m/z 100 – 1000, 50 – 200 and 50 – 150 for MS/MS, MS3,

and MS4 spectral data acquisition, respectively, with a normalized collision energy of

35% and an isolation width of m/z 2.0. Chromatographic separation was achieved using a

Waters Nanoacquity system with a Waters Symmetry C18 column (5 µm, 20 × 180 mm)

as a trap column connected to a Michrom Magic C18AQ (5 µm, 0.1 × 150 mm) column

(Bruker-Michrom Inc., Auburn, CA) as an analytical column. Following sample injection

(3 µL) to the trap column and a 1-min wash using solvent A (water/acetonitrile/formic

acid, 95/5/0.1, v/v/v) at a flow rate of 10 µL/min, the trapped analyte was back-eluted

with solvent B (acetonitrile) and further separated on the analytical column by applying a

10-min linear gradient from 5% to 95% solvent B at a flow rate of 500 nL/min.

Relative abundances of drug-related components detected in the incubations were

estimated according to their UV peak areas at the maximum absorption wavelength (λ =

303 nm) of the corresponding parent compounds. Molar extinction coefficients of

compounds and their metabolites were assumed to be similar.

NMR. The isolated 1-3 was reconstituted in 50 μL of methanol-d4 and transferred

to a 1.7 mm NMR tube that was placed in a 5 mm NMR carrier tube. All 1H spectra were

acquired on a Bruker Avance 500 MHz spectrometer (Bruker BioSpin Corporation,


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Billerica, MA) equipped with a 5-mm TCI cryo probe, and 13C spectra were acquired at

125 MHz on the same instrument. The chemical shifts were referenced to the residual

solvent (methanol-d4) signals of 4.78 and 49.3 ppm for 1H and 13C, respectively. The two

dimensional spectra, including heteronuclear multiple quantum coherence (HMQC),

heteronuclear multiple bond correlation (HMBC), and homonuclear correlation

spectroscopy (COSY) experiments, were acquired according to standard protocols

provided by the instrument manufacturer.

Results

In vitro metabolism. The biotransformation of compounds 1 and 2 was studied

using liver S9, primary hepatocytes and BDC rats, and the results of these experiments

are summarized in Table 1. The relative abundances of metabolites in vitro were

expressed as percentages of the initial amount of the corresponding parent compound,

based on UV peak areas. Relative amounts of metabolites in vivo were expressed as

percentages of the total drug-related material detected in the BDC rat bile based on UV

peak areas.

In incubations (30 min) of 1 with NADPH-supplemented liver S9 (human,

Aroclor-induced rat) or in incubations (2 h) with hepatocytes (human, rat), the only

metabolites detected, 1-1 and 1-2, were identified as hydroxylation products based on

their observed molecular weight shifts (16 Da) relative to the parent mass. Metabolite 1-1

was detected in both rat and human liver homogenates, whereas 1-2 was detected only in

incubations with human liver S9. Both metabolites were minor components relative to the

parent compound. Additionally, when compound 1 was incubated with Aroclor-induced


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rat or human liver S9 supplemented with NADPH and CDP-choline, the metabolite

profile was identical with that observed from the incubation with NADPH as the sole

cofactor.

The in vitro metabolism of 2 was also investigated in incubations with liver S9

and primary hepatocytes. One minor hydroxylation product, 2-1, was observed across the

in vitro samples.

In vitro formation of an everolimus conjugate. When everolimus was

incubated with Aroclor-induced rat or human liver S9, a conjugate (P + 165 Da), was

detected by LC/UV/MS. The same product was also observed in incubations with the rat

hepatocytes. The structural assignment of this product is described in the Mass

Spectrometry section.

In vivo metabolism. The metabolite profile of 1 was then characterized

following intravenous administration (2 mg/kg) to BDC rats. In the bile samples collected

between 0 and 24 h, 1 represented only about 1% of the total drug-related material

recovered from these matrices. The most abundant metabolites of 1 in bile were 1-1

(27%), the hydroxylation product also detected in the liver S9 and hepatocyte

incubations, and 1-3 (52%), a metabolite that exhibited a net molecular weight addition

of 181 Da relative to 1. Multiple minor metabolites were also formed along with

pathways involving either mono-hydroxylation (1%, 1-2), bis-hydroxylation (1%, 1

product), or hydroxylation (mono- or bis-) accompanied by dehydrogenation (13% total,

4 products). In plasma, 1 represented the most abundant drug-related material (> 76%)

with presence of 1-1 and 1-2.


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Separately, only 5% of the total drug-related material was attributed to unchanged

2 in bile samples (0 � 24h) collected from BDC rats receiving an intravenous

administration of 2 mg/kg. Compound 2 was converted to 2-1 as a major hydroxylation

product (55%), along with multiple minor metabolites that were formed by either mono-

hydroxylation (2-2, 6%), bis-hydroxylation (4%, 1 product) or hydroxylation and

dehydrogenation (18% total, 4 products). Another product, 2-3, exhibiting a mass shift of

181 Da, represented a minor metabolite (6%) in bile. The conjugates (P + 181 Da) of 1

and 2 were only detected in BDC rat bile and not in plasma, urine or feces. No metabolite

was detected in plasma.

Based on the total UV peak areas of the drug-related materials in bile, urine and

feces as well as the volumes of these three matrices, the disposition of 1 and 2 was

accomplished mainly via metabolism followed by excretion in bile (data not shown).

Mass spectrometric analysis and structural assignments of these products are described in

the next section.

Mass Spectrometry. The MSe product-ion spectrum of the protonated 1 (m/z

570) showed that fragmentation occurred on the bicyclo[1.1.1]pentan-1-ylcarbamoyl

moiety resulting in neutral losses of the bicyclic group (66 Da) and the amino-bicyclic

group (83 Da) to yield the two most abundant product ions of m/z 504 and 487,

respectively (Figure 2A). The ion of m/z 504 further shed a CH2 group (14 Da) to give

rise to the product ion of m/z 473. The product ion of m/z 427 was derived from a loss of

the bicyclo[1.1.1]pentan-1-ylcarbamoyl moiety along with loss of CH2 (14 Da) and HF

(20 Da) groups.


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During MS/MS, the protonated 1-1 (m/z 586) experienced a neutral loss of one

molecule of H2O (18 Da) to form a product ion of m/z 568 in the MSe spectrum (Figure

2B), confirming it as a hydroxylation product. The diagnostic base peak of m/z 487 and

an ion of m/z 504 together indicated hydroxylation of the bicyclic moiety. Two minor

product ions of m/z 459 and 430 were proposed to result from a neutral loss of the

hydroxyl-bicyclo[1.1.1]pentan-1-ylcarbamoyl moiety and subsequent loss of NHCH2 (29

Da), respectively. A second hydroxylation product (1-2) exhibited a fragmentation

pattern similar to 1-1 (data not shown), suggesting that it was also a bicyclo-

hydroxylation product.

Similarly, CID of 2 and its hydroxylation product (2-1) occurred on the

bicyclo[1.1.1]pentan-1-ylcarbamoyl moiety, giving rise to m/z 469 as the most abundant

product ion in the MSe spectra of both compounds (Figure 3A and 3B). Thus, 2-1 was

interpreted to have undergone hydroxylation on the bicyclic structure. In addition, 2-2, a

hydroxylation product, showed a similar product ion spectrum to 2-1 and was proposed to

be converted by bicyclo-hydroxylation (data not shown). Compounds 2 and 2-1 also gave

rise to similar fragmentation patterns as 1 and its hydroxylation products (Figure 2A and

2B).

Compound 1-3 (m/z 751, P + 181 Da), the unexpected metabolite of 1 in vivo,

exhibited a base peak of m/z 184 in its MS/MS product ion spectrum (Figure 2C). The

presence of a fragment ion of m/z 487, which was also observed in the MS/MS spectrum

of 1, suggested that the entire metabolic addition was associated with the amino-bicyclic

group. Likewise, the MS/MS analysis of 2-3 revealed fragment ions of m/z 184 and m/z

469, with the latter product ion indicating metabolic modification of the bicyclic portion


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(Figure 3C). In order to elucidate the structural features of the added group, multi-stage

mass spectrometry experiments were carried out using an LTQ-Orbitrap mass

spectrometer, and MS3 and MS4 product ion spectra of the ion of m/z 184 were collected.

The exact mass of the added moiety (184.0736 Da) and observed mass defect suggested

an elemental composition of C5H15NO4P (Sleno, 2012). The MS3 ion spectrum exhibited

two ions of m/z 60.0808 and 86.0965, corresponding to elemental compositions of

C3H10N+ and C5H12N

+, suggesting alkylamines characterized by different carbon chain

lengths. The mass of another fragment ion of m/z 125.0000 was consistent with the

elemental composition of an ethylphosphoric acid (C2H6O4P+). The MS4 spectrum of the

m/z 125 ion (from the MS3 spectrum) gave rise to another ion of m/z 98.9842, consistent

with the molecular weight of a phosphoric acid (H4O4P+).

About 95% and 83% of biliary metabolites of 1 and 2, respectively, were formed

via metabolism of the bicyclopental moiety, as evidenced by the observation of the

diagnostic fragment ions of m/z 487 or m/z 469 in their MS/MS product ion spectra (data

not shown). On the other hand, the remaining in vivo metabolites of 1 and 2 were

proposed to form by metabolism on the remaining portions of the molecules based on

their product ion spectra (data not shown) and are beyond the scope of the current paper.

However, the relative abundances of these products are summarized in Table 1.

The MS/MS product ion spectrum of the everolimus conjugate (m/z 1123, P + 165

Da) exhibited product ions resulting from the fragmentation of both everolimus and the

conjugate moiety (Supplemental Figure 1). The ions of m/z 1091 and 1073 were formed

by a neutral loss of methanol and further loss of water, respectively. The ions of m/z 829

and 532 were formed by fragmentation of the everolimus moiety as illustrated in Figure


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S1. In addition, multiple products ions could be ascribed to fragmentation of the

conjugate moiety. The ions of m/z 184 and 86 were identical to those observed in the

product ion spectra of 1-3 and 2-2 mentioned previously. The product ion spectrum was

consistent with that of the everolimus phosphocholine conjugate detected in human and

multiple preclinical species (Zollinger et al., 2008).

NMR Spectroscopy The 1H and 13C chemical shifts of 1 and its respective

conjugate (1-3), isolated from the BDC rat bile, were obtained during the acquisition of

1H, HMQC and HMBC spectra and were assigned as summarized in Table 2. The 8

protons of the aromatic rings, including those of the two fluorophenyl groups (H11/15,

H12/14, H23, H27, and H26) and the furo[2,3-b]pyridine (H9), in 1 and 1-3 were

characterized by no, or minor, changes in chemical shifts. No extra aromatic proton(s)

was observed. In addition, the aliphatic protons of the N-methyl group (H30) and

trifluoropropyl group (H18 and H19) also remained intact. On the bicyclic moiety, 1H

chemical shifts of the 6 identical methylene protons (H38/40/41) and the single methine

proton (H39) of 1 were 2.20 and 2.49 ppm, respectively. The peak area ratio between

these signals was approximately 6:1 (data not shown). However, only one group of the

bicyclic protons in 1-3 (2.47 ppm) were observed, and their NMR peak area was

approximately 3-fold greater than that of the H18 (3.05 ppm) or H19 (2.66 ppm)

methylene protons (data not shown). Furthermore, a long-range C-H correlation study

(HMBC) of 1-3 revealed 3 distinct correlations between the bicyclic protons (2.47 ppm)

and the bicyclic carbons of 42.9 ppm, 56.7 ppm and 63.5 ppm (Figure 5 and Table 2).

Additionally, the bicyclic protons (2.47 ppm) exhibited a correlation with the bicyclic

carbons (56.7 ppm) in the HMQC spectrum (Figure 6), indicating direct connectivity.


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The 1H spectrum of 1-3 also showed 3 additional and distinct groups of aliphatic

signals at 3.24, 3.65 and 4.28 ppm (Table 2). The peak area ratio among these signals was

approximately 9:2:2 (data not shown). The HMQC spectrum demonstrated direct

connectivity between these protons (3.24, 3.65 and 4.28 ppm) and carbons at 54.5, 67.3

and 60.3 ppm, respectively (Figure 6), and COSY was used to show a correlation

between the protons of 3.65 ppm and 4.28 ppm (Figure 7). Moreover, the HMBC

experiment established three long-range C-H correlations: the 1H of 3.24 ppm and 13C of

54.5 ppm, the 1H of 3.24 ppm and 13C of 67.3 ppm, and the 1H of 3.65 ppm and 13C of

54.5 ppm (Figure 5).

Discussion

The metabolite profiles of 1 and 2 in the BDC rat studies demonstrated extensive

metabolism of both molecules in liver and pointed to the bicyclopentane moiety as the

main site of metabolism. In addition to hydroxylation, with or without dehydrogenation,

metabolism of the bicyclic group also yielded unexpected products characterized by a

mass addition of 181 Da, which did not match any of the metabolic mass shifts monitored

routinely in our laboratory.

The MS/MS product ion spectra of the unknown products, 1-3 and 2-3, included a

base peak of m/z 184, whose multi-stage MSn fragmentation spectra assisted in the

characterization of multiple structural features of the added moiety (Figure 4). The m/z

60.0808 and m/z 86.0965 ions were in agreement with elemental compositions of alkyl

amines, C3H10N and C5H12N, respectively, whereas the ions of m/z 98.9842 and m/z

125.0000 matched the masses of phosphoric acid (H4O4P+) and ethylphosphoric acid


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(C2H6O4P+), respectively. Therefore, the added moiety appeared to consist of an ethyl-

substituted phosphoric acid connected to an alkyl amine, with the degree of substitution

at the nitrogen atom still undetermined. All elemental compositions listed above

represented the theoretical formulas that were closest to the observed masses, providing

additional support for the structure assignments.

In order to elucidate the structure of the added moiety, 1-3 was isolated from rat

bile and analyzed by NMR. The 1H spectra indicated that the metabolic change did not

occur on any of the aromatic carbons, N-methyl group or trifluoropropyl group,

consistent with the MS/MS analysis implicating the bicyclic group as the likely soft spot.

Comparison of the 1H spectra describing the bicyclic portions of 1 and 1-3 led to one of

the most important findings: only one group of 6 equivalent protons (2.47 ppm) in 1-3

was observed, in contrast to the two groups of protons (2.20 and 2.49 ppm) that were

apparent in 1. This observation excluded the possibility of modification of one of the

three equivalent methylene carbons (C38/C40/C41) which would have otherwise resulted

in three different groups of protons—one H39 proton, 4 equivalent methylene protons,

and the one proton corresponding to the site of metabolism. Therefore, metabolism was

shown to occur at the bridgehead carbon (C39), causing the 6 equivalent protons

H38/H40/H41 to shift downfield (2.47 ppm) and leaving no proton connected to C39.

Moreover, the chemical shift of C39 of 1-3, which was assigned as 63.5 ppm, exhibited a

large downfield shift from 26.1 ppm, providing clear evidence that C39 was connected to

the added moiety through an oxygen.

The conjugated group also left a diagnostic footprint in the aliphatic region of the

NMR spectra. First, the characteristic NMR peak area ratio of the three distinct protons, 9


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(3.24 ppm), 2 (3.65 ppm), and 2 (4.28 ppm), suggested the presence of three equivalent

methyls and two different methlyene groups. Second, the HMQC and COSY spectra

supported the notion that the protons of 3.65 ppm and 4.28 ppm were attached to

neighboring carbons (67.3 and 60.3 ppm, respectively)—likely an ethylene as suggested

by the MSn results. Furthermore, the HMQC and HMBC spectra provided evidence of a

direct connection and through-bond proximity between the carbon of 54.5 ppm and the 9

equivalent protons of 3.24 ppm, suggesting the presence of three methyl groups likely

attached to a nitrogen. Collectively, the LC/MS/MS and NMR data supported a structure

assignment of 1-3 where a phosphocholine group [H2PO4-CH2CH2N(CH3)3, POPC] was

connected to the methine carbon (C39) of the bicyclic moiety through an ester bond.

It is well known that CPT catalyzes the final step of the Kennedy reaction by

adding POPC from CDP-choline to a diacylglycerol (DAG), yielding

phosphatidylcholine, a major component of biological membranes including microsomal

membranes of all eukaryotes (McMaster and Bell, 1997) (Blank et al., 1979; Demopoulos

et al., 1979). The membrane-bound CPT resides in the endoplasmic reticulum (ER), a

primary location for the biosynthesis of nearly all kinds of phospholipids from fatty acyl

CoA and glycerol 3-phosphoate (Vance and Vance, 2008) (Bishop and Bell, 1988)

(Daleke, 2003) (Lagace and Ridgway, 2013). The substrate, DAG, docks in the active site

of CPT with its hydroxyl group exposed to the cytosol, where the cofactor, CDP-choline,

is present (McMaster and Bell, 1997) (Alberts et al., 2008). In the current study, the

POPC conjugation of 1 and 2 was presumably catalyzed by CPT following hydroxylation

of the bicyclic methine, a prerequisite reaction that was catalyzed by P450. It is

reasonable that, after being released by P450, the tertiary alcohol product of 1 or 2 binds


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to the CPT active site in the ER membrane, with the newly added hydroxyl group facing

the cytosol, and is then conjugated by addition of a POPC to the hydroxyl group.

Although mono- and bis-hydroxylation of the bicyclic moiety of 1 or 2 led to the

formation of multiple metabolites, only one mono-hydroxylation product appeared to be a

suitable substrate of CPT, indicating a degree of selectivity on the part of the enzyme. In

addition, replacement of the fluorophenyl ring with an unsubstituted phenyl as in 2, a

subtle structural change relative to 1, resulted in a markedly diminished extent of POPC

conjugation, also reflecting the substrate specificity of the enzyme. On the other hand, the

hydroxylation products of 1 or 2 do not appear to be closely related to the structures of

the endogenous substrates of CPT, which include DAGs possessing varied lengths of

saturated or unsaturated fatty acid chains (usually 16 to 22 carbons) and a primary

alcohol group, demonstrating that CPT can recognize molecules whose chemical scaffold

differs from those of its endogenous substrates. Previously, Zollinger et al reported that

everolimus, a potent immunosuppressant, formed a POPC ester via direct conjugation of

a hydroxyl group as a prominent metabolite in rats, mice, cynomolgus monkeys and

human (Zollinger et al., 2008). Formation of POPC conjugates of everolimus was also

unexpected in light of the structural differences between DAGS and everolimus.

In the present study, the formation of the everolimus POPC conjugate was

confirmed in incubations with liver S9 fortified with CDP-choline and with rat

hepatocytes, demonstrating the activity of CPT in these in vitro systems. However, no

POPC product was observed following an attempt to recapitulate formation of the POPC

conjugate of 1 in Aroclor-induced rat liver S9 supplemented with NADPH and CDP-

choline, nor were such conjugates formed in incubations with hepatocytes despite the


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presence of all enzymes and cofactors required to carry out this two-step reaction. The

apparent discrepancy between the in vitro and in vivo results is explained by inadequate

turnover to the appropriate tertiary alcohol product during the first of these two steps in

vitro (Table 1). On the other hand, this was not an issue in the everolimus example, since

it features a primary alcohol and acts as a direct CPT substrate without the need for

phase-I biotransformation. Whereas the synthesis of the methine-hydroxylation products

of 1 or 2 and the subsequent incubation of these metabolites in vitro might have

facilitated the detection of the phosphocholine conjugates and allowed this disconnect to

be resolved fully, the deprioritization of the bicyclic series in the medicinal chemistry

program precluded the availability of the requisite resources to address this question.

Whether the hydroxylation products of 1 and 2 would be converted to the POPC

conjugates in human subjects remains unknown. The observation of the POPC conjugates

only in bile from the BDC rats, as well as the structural dissimilarity between these

antiviral compounds and the endogenous substrates of CPT, make it difficult to predict

whether and to what extent the analogs of 1 and 2 would undergo POPC conjugation in

vivo without conducting additional experiments.

From the standpoint of program decision-making, the observation of this unusual

reaction in BDC rats was accompanied by uncertainty over the possibility that the POPC-

xenobiotic conjugate formation might interfere with the de novo synthesis and function of

phospholipids. However, the results of these studies did not allow the magnitude of this

effect to be estimated, since the BDC rat study was not designed to measure mass-

balance or determine the flux through any specific metabolic pathway in vivo. Although

substitution of the methine of the bicyclic group was considered as a means of blocking


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POPC conjugation, this idea was not pursued given the difficulty in predicting whether

hydroxylation and phosphocholine conjugation might still occur on another site within

the bicyclic group.

In summary, we observed an uncommon POPC conjugation pathway associated

with HCV NS5B inhibitors featuring a bicyclic moiety. The LC/MSn and NMR analysis

was used to identify the bicyclopental methine carbon as the site that underwent

sequential hydroxylation and esterification with POPC. The observation of this reaction

in vivo, but not in vitro, complicated our routine efforts to establish relationships across

species and anticipate what might happen in a clinical setting. These findings stand as

another example of how enzymes with seemingly exclusive functions in the

transformation of endogenous substrates may, in certain circumstances, get recruited to

participate in the metabolism of xenobiotics.


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Acknowledgments

The authors thank Nicholas Meanwell for manuscript review and valued

discussion, and Dawn D. Parker, Jennifer G. Pizzano and Jean Simmermacher-Mayer for

technical assistance.


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Authorship Contribution

Participated in research design: Zhuo, Cantone, Wang, Drexler, Mosure

Conducted experiments: Zhuo, Cantone, Wang, Leet

Contributed new agents or analytical tools: Yueng, Eastman, Parcella, Kadow, Zhuo,

Cantone, Wang, Drexler

Analyzed and interpreted data: Zhuo, Cantone, Wang, Drexler, Leet, Huang, Johnson

Wrote or contributed to the writing of the manuscript: Zhuo, Johnson, Wang, Leet,

Drexler, Soars


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Figure Legends

Figure 1 Structures of two selected HCV NS5B inhibitors featuring a

bicyclo[1.1.1]pentane.

Figure 2 High energy LC/MSe product ion spectra of 1 (panel A) and its two

metabolites, 1-1 (panel B) and 1-3 (P + 181 Da) (panel C), acquired using a Waters Xevo

QTOF mass spectrometer. The proposed fragment ions and corresponding theoretical

masses are shown.

Figure 3 High energy LC/MSe product ion spectra of 2 (panel A) and its two

metabolites, 2-1 (panel B) and 2-3 (P + 181 Da) (panel C), acquired using a Waters Xevo

QTOF mass spectrometer. The proposed fragment ions and their theoretical masses are

shown.

Figure 4 The product ion spectra of the fragment ion of m/z 181.0022 (MS3, panel A)

and the fragment ion of m/z 125.0000 (MS4, panel B) selected from the product ion

spectrum (LC/MS/MS) of 1-3. The spectra were acquired using a Thermo LTQ-Orbitrap

mass spectrometer. The proposed fragment ions and theoretical masses are shown.

Figure 5 HMBC (Heteronuclear Multiple Bond Correlation) spectrum of 1-3, illustrating

long-range correlation of 1H and 13C nuclei.


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Figure 6 HMQC (Heteronuclear Multiple-Quantum Correlation) spectrum of 1-3,

showing 2D heteronuclear chemical shift correlations between directly-bonded 1H and

13C nuclei.

Figure 7 COSY (homonuclear correlation spectroscopy) spectrum of 1-3, depicting

through-bond 1H-1H correlations.


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TABLE 1 Summary of the metabolite profiles of 1 and 2 obtained from incubations with

liver S9 (human, rat), hepatocytes (human, rat), and bile (BDC rats). The relative

abundances (%) of the drug-related materials in incubations or matrices were estimated

based on the UV peak areas (λ = 303 nm) assuming absorption coefficients of the parent

compounds and their metabolites were similar. The metabolite names are in bold letters.

Metabolism on the bicyclo[1.1.1]pentane moiety Metabolism on

other moieties

Parent Mono-oxidation P + 181 Da Bis-oxidation Mono-/bis-oxidation +

dehydrogenation

Mono-oxidation,

etc.

1 AIRLS9 1 (30 min) 98 2 (1-1) ND 6 ND ND ND

HLS9 2 (30 min) 99 < 1 (1-1, 1-2) ND ND ND ND

RHep 3 (2h) 99 < 1 (1-1) ND ND ND ND

HHep 4 (2h) 99 < 1 (1-1) ND ND ND ND

Rat bile 5 (0 – 24h) 1 27 (1-1), 1 (1-2) 52 (1-3) 3 (2 products) 13 (4 products) 4

Rat plasma (0.5, 1, 3, 6, 24h) 76�95 4-16 (1-1), 2-11 (1-2) ND ND ND ND

2 AIRLS9 (30 min) 95 5 (2-1) ND ND ND ND

HLS9 (30 min) 99 < 1 (2-1) ND ND ND ND

RHep (2h) 99 < 1 (2-1) ND ND ND ND

HHep (2h) 99 < 1 (2-1) ND ND ND ND

Rat bile (0 - 24h) 4 55 (2-1), 6 (2-2) 6 (2-3) 4 (1 product) 18 (4 products) 13

Rat plasma (0.5, 1, 3, 6, 24h) 100 ND ND ND ND ND

1 Aroclor-induced rat liver S9

2 Human liver S9

3 Rat hepatocytes

4 Human hepatocytes

5 Bile duct-cannulated rats

6 ND: not detected


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TABLE 2 Proton (1H NMR) and carbon chemical shifts (13C NMR for parent, and

HMQC and HMBC for conjugates) of 1 and 1-3 (P + 181 Da). The proposed structure of

the conjugate is shown below. The numbering system is for illustrative purposes only and

does not correspond to IUPAC nomenclature.

δH ppm δC ppm

Position 1 1-3 1 1-3

9 7.95 7.95 132.9 132.5

11,15 8.02 8.02 131.4 130.7

12,14 7.29 7.29 117.2 116.8

18 3.05 3.05 28.9 28.6

19 2.67 2.66 33.5 33.1

23 7.66 7.66 132.5 132.1

26 7.35 7.35 117.8 117.9

27 7.57 7.57 135.1 135.5

30 2.94 2.95 26.9 26.5

37 NA1 NA 50.1 42.9

38, 40, 41 2.20 2.47 53.9 56.7

39 2.49 ND 26.1 63.5

45 NA 4.28 NA 60.3

46 NA 3.65 NA 67.3

48, 51, 52 NA 3.24 NA 54.5

1 NA: not applicable


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F

N O

NHO

OHN

X

F3C

Compound 1: X = FCompound 2: X = H

Figure 1

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

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d.115.069062 at ASPET Journals on February 22, 2020 dmd.aspetjournals.org Downloaded from


100 200 300 400 500 600 700

Inte

nsity

(%)

0

100184.0734

86.0971124.9999

751.2325

487.1047

50

m/z

1-3 (P + 181 Da)

C

m/z100 180 260 340 420 500 580

Inte

nsity

(%)

0

100570.1825

487.1095

484.1292427.1071

320.0882136.0567123.0244 318.0740346.0681 473.0937

504.1361

50

Compound 1

A

m/z100 180 260 340 420 500 580

Inte

nsity

(%)

0

100 487.1071

430.0912

366.0791123.024481.0564

346.0610319.0819137.0587 392.0563459.1253

586.1763

568.1666

50

504.1342

1-1 (P + 16 Da)

B

Figure 2




DM

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m/z100 180 260 340 420 500

Rela

tive

Inte

nsity

(%)

0

100 552.1902

469.1164

302.0977123.0248

466.1366412.0943

486.1437

50

Compound 2

A

m/z100 180 260 340 420 500 580

Rela

tive

Inte

nsity

(%)

0

100 469.1162

301.0909

568.1838

50

412.0926

2-1 (P + 16 Da)

550.1834

B

m/z200 300 400 500 600 700 800 900 1000

Rela

tive

Inte

nsity

(%)

0

100 184.0716

125.0000

733.2360

469.1077

50

2-3 (P + 181 Da)

C

Figure 3




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50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000

20

40

60

80

100

Rela

tive

Abun

danc

e (%

)

86.0965

125.0000

184.0736

166.063060.0808

50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150m/z

0

20

40

60

80

100

Rela

tive

Abun

danc

e (%

) 98.9842

MS3

MS4

Figure 4




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H38/40/41- C37

H38/40/41- C38/40/41

H38/40/41- C39

H38/40/41H48/51/52

H48/51/52 – C48/51/52

H48/51/52 – C46

Figure 5

H46 – C48/51/52




DM

D Fast Forw


OI: 10.1124/dm



Figure 6

H45 H46

H38/40/41- C38/40/41

H38/40/41H48/51/52

H48/51/52 – C48/51/52

H45 – C45

H46 – C46




DM

D Fast Forw


OI: 10.1124/dm



H45 H46H38/40/41H48/51/52

H45 – H46

H45 – H46

Figure 7




DM

D Fast Forw


OI: 10.1124/dm



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