Structure elucidation of ten autotaxin inhibitors and their … · and their metabolites using...

Submitted: 19/06/2014

MSc Chemistry

Analytical Sciences

“Structure elucidation of ten autotaxin inhibitors

and their metabolites using Liquid

Chromatography tandem Mass Spectrometry”

Master thesis

Georgios Psarros

Project Supervisor: Dr. H. Lingeman (Vrije Universiteit Amsterdam)

2

MSc Chemistry

Analytical Sciences

Master Thesis

Structural elucidation of ten autotaxin inhibitors using

Liquid Chromatography tandem Mass Spectrometry

by

Georgios Psarros

June 2014

Supervisor:

Prof Dr H. Lingeman

Daily Supervisor:

M. Mladic

Vrije Universiteit van Amsterdam

3

Preface

This thesis was prepared during the internship as a master student of the

Master of Analytical Sciences. The internship took place at the Department of

Biomolecular Analysis and Spectroscopy at VU University Amsterdam in the

period from February 2013 to October 2013.

I would like to thank Marija Mladic for her daily supervision and her

contribution at the experimental work, Dr. Jeroen Kool and Dr. Henk Lingeman

for their guidance during this project and Dr. Harald Albers for providing the

compounds.

4

Contents

Abstract................................................................................................................... 7

Introduction ............................................................................................................ 8

Anticancer drugs .................................................................................................. 8

Anticancer drugs directed at a drug target ........................................................... 9

Enzymes (as anti-cancer drug target) ................................................................. 11

Autotaxin ........................................................................................................... 12

Autotaxin inhibitors ........................................................................................... 14

Boronic acid-based autotaxin inhibitors ............................................................. 14

Metabolization of drugs ..................................................................................... 16

Experimental ......................................................................................................... 19

Apparatus .......................................................................................................... 19

Liquid Chromatography ................................................................................... 19

Mass Spectrometry ......................................................................................... 20

Methods............................................................................................................. 23

Metabolization with microsomes .................................................................... 23

Simulating Metabolism with Hydrogen Peroxide ............................................ 23

Results and Discussion .......................................................................................... 24

Proposed structure of Metabolites .................................................................... 28

Structural elucidation of metabolites using Mass Spectrometry ........................ 32

Fragmentation ................................................................................................ 32

5

Conclusion ............................................................................................................. 52

References ............................................................................................................ 54

Attachments.......................................................................................................... 56

6

List of Abbreviations

ACN Acetonitrile

ATX Autotaxin

CYPs Cytochrome P450 monooxygenases

ESI Electrospray ionization

GCPR G-Coupled Protein Receptors

GST Glutathione S-transferase

IC50 Half maximal inhibitory concentration

LPA Lysophosphatidic acid

LPC Lysophospholipids

MI Metabolic Incubation

NADPH Nicotinamide adenine dinucleotide phosphate-oxidase

NAT N-acetyl transferase

OE Oxidation Experiment

PST Phenol sulfotransferase

ST Sulfotransferase

TOF Time of Flight

UGT Glucuronosyl transferase

7

Abstract

This thesis describes the detection and the structure elucidation of ten potential

autotaxin inhibitors and their metabolites by the use of high performance liquid

chromatography (HPLC) tandem mass spectrometry (MS/MS). These compounds

were metabolized by incubation with pig liver microsomes and by oxidation

reactions with hydrogen peroxide (H2O2). The incubation mixtures then were

analyzed with HPLC-MS/MS. The principal metabolization pathway observed was

oxidative deboronation following by hydroxylation and in total 18 different

metabolites were identified. Useful in the structural investigation of the product

ions of the compounds and their metabolites were the accurate mass

measurements using a quadrupole-time of flight mass spectrometer.

8

Introduction

Anticancer drugs

What is Cancer?

Cancer is a term used for diseases in which abnormal cells divide without control

and are able to invade other tissues. Cancer cells can spread to other parts of the

body through the blood and lymph systems [1]. More than 100 different types of

cancer are known and they are named after the organ in which they start.

Anticancer drug development

Since cancer is the second leading cause of death in Europe and North America,

enormous resources are being invested in treatment, prevention and diagnosis of

this disease. For many pharmaceutical companies the key focus are the exploration

and the development of anticancer agents. Typical anticancer drug discovery and

development have focused on the cytotoxic agents and that was triggered by the

discovery of the toxic action of nitrogen mustards on cells of the haematopoietic

system 50 years ago [2].

Cytotoxic drugs

Cytotoxic drugs include any drug that inhibits or prevents the function of cells.

Cytotoxic drugs not only prohibit the rapid growth and division of cancer cells but

also affect the growth of other quick dividing cells in the body such as hair follicles

9

and the lining of the digestive system. As a consequence of the anticancer

treatment, many normal cells are damaged with the cancer cells [3].

Anticancer drugs directed at a drug target

In the last few decades drug development has been directed to a target-based drug

design focusing at the major drug targets categories, such as:

G Coupled Protein Receptors (44%)

Enzymes (29%)

Transporter proteins (15%)

ligands, structural and adhesion proteins, enzyme-interacting proteins and

other (12%) [4].

Figure 1: Major categories of drug targets.

Despite the fact that the traditional approach has achieved significant progress in

anticancer therapy, recent developments in molecular biology and the ability of

44%

29%

15%

3% 3% 3% 3%GPCR

Enzymes

Transporter proteins

Ligands

Enzyme-interacrion proteins

Structural and adhesion proteins

Other

10

understanding cancer at a molecular level have helped researchers to come up with

target-based drugs. These are agents that are designed to inhibit and/or modify a

selected molecular marker deemed important in cancer prognosis, growth, and/or

metastasis.

Several target-based compounds are developed in past years. Some examples are:

Imatinib mesylate (Gleevec1, Novartis) is a small-molecule compound that

inhibits a specific tyrosine kinase enzyme, the Bcr–Abl fusion oncoprotein. It

is used for gastrointestinal stromal tumor and chronic myeloid leukemia.

Gefitinib (Iressa1, AstraZeneca & Teva) is a small-molecule inhibitor of the

epidermal growth factor receptor’s (EGFR, or erbB1) tyrosine kinase domain.

It is used for non-small-cell lung cancer.

Bortezomib (Velcade1, Millenniums Pharmaceuticals) is the first of a new

class of agents called proteasome inhibitors and the first treatment in more

than a decade to be approved for patients with multiple myeloma.

Rituximab (Rituxan1, Biogen Idec & Genentech) is a monoclonal antibody

used in the treatment of B-cell non-Hodgkin’s lymphoma and B-cell leukemia.

It binds the CD20 antigen on the CD20+ B-cells, causing their apoptosis.

Trastuzumab (Herceptin1, Genentech) is a monoclonal antibody that binds

the cell surface HER2/neu (erbB2) receptor and is used in the therapy of

erbB2+ breast cancer [2].

11

Enzymes (as anti-cancer drug target)

Enzymes catalyze multistep chemical reactions and achieve phenomenal rate

accelerations by matching protein and substrate chemical groups in the transition

state [5]. The compounds that prevent that kind of chemical interactions to happen

are called inhibitors and they are among the most dominant and effective drugs in

the market. The key to design potent inhibitors is to study the catalytic chemistry

of enzymes which makes them a special class of drug target.

All the drug targets except for nucleic acids, evoke biological functions through

ligand binding. However, enzymes are catalysts, which means forming and breaking

covalent chemical bonds since enzymes are more capable from their nature of

chemical transformations than ligand binding. This makes enzymes a different drug

target.

Enzyme inhibitors are about 30% of the drugs marketed, therefore it is important

to consider enzymes as a separate class of drug targets.

Figure 2: Classification of enzyme target class. *enzymes are further classified as soluble or

membrane-associated; the number in the brackets corresponds to the number of

membrane-associated enzymes in each class.

12

Autotaxin

Autotaxin, ATX, is a secreted ecto-enzyme responsible for lysophosphatidic acid

(LPA) production in blood. It is a member of the family of ecto-nucleotide

pyrophosphatase /phosphodiesterase (NPP1-7) and is also referred, as NPP2 [6]. It

is the only family member capable of producing LPA by hydrolysis of IC50 (figure 3).

Figure 3: Autotaxin (ATX) is responsible for hydrolyzing the lipid lysophosphatidylcholine (LPC)

into lysophosphatidic acid (LPA) and choline [7].

The active LPA stimulates migration, proliferation and survival of many cells by

acting on specific G protein-coupled receptors [7].

Autotaxin plays an important role in vascular development and is found

overexpressed in some human cancers. Studies have shown that the main product

of ATX, LPA, mediates chemotaxis and proliferation in melanoma cells [6].

13

Recent experiments suggest that ATX expression is one of the factors involved in

metastasis of melanoma cells (Figure 4).

Figure 4: Metastasis of melanoma cells. Autotoxin’s main product, LPA, mediates chemotaxis and

proliferation in melanoma cells.

Inhibition of ATX production blocks LPA-induced migration of melanoma cells. It has

been detected that melanoma metastatic specimens have increased ATX level, and

ATX expression in primary melanoma is higher than in melanoma in situ [8].

Therefore inhibition of LPA production by ATX is therapeutic interesting.

14

Autotaxin inhibitors

The approved melanoma therapy lacks significant efficiency, hence, new potent

ATX inhibitors are under investigation. LPA receptors are not attractive targets since

LPA acts on multiple receptors that show overlapping activities. The first published

ATX inhibitor is L-Histidine and since then two additional categories of inhibitors

have been described. The first category consists of analogs of bioactive lipids

including LPA, and the second category of non-lipid small molecule inhibitors [6].

Boronic acid-based autotaxin inhibitors

In the early 1970s, boronic acids were initially used as inhibitors of proteasome and

they were established as possible transition state analogs of serine proteases [9].

During the 1980s, peptides with boronic moiety attached to them showed efficient

inhibition to trypsin, chymotrypsin, α-lytic protease, pancreatic elastase, leukocyte

elastase, thrombin, and β-lactamases [10]. Since then, they have been studied for

potent use in various disease states and lately, peptidyl boronic acids were

indicated as potent proteasome inhibitors. Furthermore, high antitumor and anti-

inflammatory effectiveness in vitro and in animal models was observed [12,13]. In

2003, the drug VELCADE (bortezomib, figure 5) was used for the medication of

relapsed refractory multiple myeloma and became the first boronic acid available

in the market as a therapeutic agent [12].

15

Figure 5: Structure of Bortezomib.

Recently, Albers et al. described the discovery of a boronic acid-based ATX inhibitor

which helped to shorten the half-life of LPA (∼5 min) in vivo. Interestingly, the

introduction of a boronic acid group, designed that way to target the active site

threonine in autotoxin, showed a great increase of inhibitory efficacy with the most

drastic of the compound to inhibit ATX activity in a nanomolar range.

The idea of the boronic acid moiety introduction was encouraged by the already in

use therapeutic proteasome inhibitor bortezomib, which targets the N-terminal

threonine oxygen nucleophile in the proteasome through a boronic acid.

The finding that ATX can be targeted by boronic acids may aid the development of

ATX inhibitors for therapeutic use in ATX/LPA-dependent pathologies, including

chronic inflammation, tumor progression and fibrotic diseases.

16

Figure 6: Hypothesis on the binding of boronic acid with the T210 oxygen nucleophile in the ATX

active site [13].

Metabolization of drugs

Most medicines, except for some very polar substances that might be directly

excreted into the urine via the kidneys, are liposoluble and they are subjected to

metabolism. Subsequently, more polar species are generating, which can easily

avoid the reabsorption from the kidneys and excreted into the urine [14]. Drug

metabolism together with bile and renal excretion determine for how long the drug

will stay in patients’ body. Metabolism can lead to many unfortunate effects to the

parent compound – a toxic parent medicine can be detoxified or a nontoxic parent

compound can generate active metabolites by biotransformation [15]. Other

adverse consequences are:

change or loss of selectivity

deposition/accumulation of metabolites

bioactive metabolites with irreversible action

acquired ability to cross the blood-brain barrier

17

Drug Metabolism has various chemical pathways, but they are now categorized into

two phases.

Phase 1 oxidation. A polar group, usually hydroxyl moiety (-OH group), is added to

a non-polar molecule. This oxidation is mainly carried out by cytochrome P450

monooxygenases (CYPs).

Phase 2 conjugation. A very polar endogenous molecule such as glucose or sulfate

is added to the organic group which was possibly formed from phase 1 reaction,

usually at –OH site. Conjugation reactions may directly occur in drugs with the

proper functional groups [14]. Conjugation enzymes like glucuronosyl transferase

(UGT) and phenol sulfotransferase (PST) are usually present at phase 2 reactions.

The metabolization enzymes are in principal in the liver, however metabolism may

occur in different sites such as in the brain, lungs or intestines. The Cytochromes

P450 (CYPs) are the most intensively studied enzymes due to the complicated

processes that they produce. The past years, scientists using the knowledge from

the study of CYPs were enabled to prognosticate the effects of drug metabolism in

vivo. A few in vitro liver models have been established during the past decades

which are: perfused liver, liver slices, primary hepatocytes, cytosol, S9 fractions,

supersomes, cell lines, transgenic cell lines and microsomes [16].

The most efficient in vitro model to study drug metabolism seems to be with the

use of microsomes which are subcellular fractions consisting of fragmented

endoplasmic reticulum to which ribosomes are attached [17]. The absence of

enzymes like N-acetyl transferase (NAT), sulfotransferase (ST), glutathione S-

18

transferase (GST) and cytosolic cofactors (Phase 2 enzymes) leads to an incomplete

production of metabolites, however due to the simplicity in use and the fact that

they are one the best characterized in vitro models for drug metabolism research

they are still extremely helpful [16].

In this project pig liver microsomes were used to metabolize the lead compounds

and mixtures of metabolites were produced therefore an analytical separation was

necessary for their study.

19

Experimental

Apparatus

Liquid Chromatography

An autosampler Shimadzu SIL-30AC was used. For the gradient LC separations (with

two Shimadzu LC-30AD parallel pumps), a Waters XBridge C18 column (4.6

x100mm, 5μm particles) with a guard column was used. A column oven Shimadzu

CTD-30A was used with a temperature of 40oC. The injection volume was 50 μl.

Mobile phase A consisted of H2O-ACN-formic acid (98%-2%-0.1%) and mobile phase

B of H2O-ACN-formic acid (2%-98%-0.1%). The following gradient (0.6mL/min flow)

was used: 3 min at 5% B → 10 min linear increase to 95% B →5 min at 95% B → 1

min linear decrease to 5% B→10 min at 5% B (figure). On-line UV measurements

were performed with a Shimadzu SPD-M20A prominence diode array detector. The

flow from the UV detector was directed into the mass spectrometer with a minimal

amount of PEEK tubing. Data analysis was performed with Postrun Analysis

software.

Figure 7: Graphic display of HPLC gradient program.

20

Mass Spectrometry

ULTIMA MICROMASS

In the early phase of the experiments a less accurate Mass Spectrometer was used.

The MS was a MICROMASS Q-TOF Ultima equipped with an electrospray ionization

source. Positive ion electrospray was performed with a capillary voltage of 4 kV.

Spectrum acquisition was performed between m/z 50 and 700 with 1 sec Scan time

and 0.1 sec Inter-Scan delay in MS mode and 0.5 sec Scan time and 0.1 sec Inter-

Scan delay in MS2 mode. The Source and Desolvation Temperature was 120 and

300 respectively. With the help of a divert valve, a solvent delay of 4 min in the

beginning and 4 min in the end of the run was used in order to prevent

contamination of the ESI source. Data analysis was performed with Mass Lynx

software.

BRUKER

A triple Quadrupole Time of Flight (Q-TOF) mass spectrometer from BRUKER

(Bremen-Germany) was used for the mass spectrometric detection. All experiments

were performed with electrospray ionization (ESI) in the positive ion mode. The

capillary voltage was 4.5 kV, the dry gas Temperature was set at 200oC and the Gas

flow at 10 l/min. Spectrum acquisition was performed with a scan range of 50-3000

m/z. Data depended MS2 analysis was performed with collision energy of 5eV. The

instrument was calibrated with recommended solutions (Sodium Formate). In order

to achieve higher mass accuracy, in the beginning of every measurement 1 min of

calibrant was measured giving the option to “correct” the masses afterwards if that

was necessary. To manage that an extra pump was introduced into the system and

21

a loop with the calibrant (figure). Data analysis was performed with Bruker

DataAnalysis software.

0-1 minute of the run

1-29 minute of the run

Figure 8: System scheme after calibration loop was installed.

22

Figure 9: Schematic representation of BRUKER micrOTOF compass 1.3.

23

Methods

Metabolization with microsomes

Pig liver microsomes were diluted 10 times in a reaction buffer consisted of 50 mM

KH2PO4- , 5mM MgCl2and 5 mM glucose-6-phosphate at pH 7,4 (adjusted with

NaOH). At 4 oC the solution was divided into Eppendorf tubes and compounds of

final concentration 100μM were added with 5 activity units/mL of glucose-6-

phosphate-dehydrogenase. The incubation was initiated at room temperature by

the addition of 10% (v/v) 30mM NADPH (which was used as a regenerator) and then

the tubes were transferred in a water bath at 37oC. At 30 and 60 minutes same

volume of 10mM NADPH was added and after 90 minutes the incubation was

stopped with 200% (v/v) ice-cold acetonitrile. The tubes were centrifuged at 10.000

rpm for 5 min at 4 oC and the supernatant was transferred to new tubes and

thereafter evaporated in a LABCONCO SpeedVac. The residues were re-dissolved in

mobile phase A and stored at -20oC [18].

Simulating Metabolism with Hydrogen Peroxide

Boiling compounds in hydrogen peroxide (H2O2) is a way to simulate metabolism

and since it is cheap and fast procedure high quantities of metabolites can be easily

produced. This method was tested for all the compounds, and in some cases

different metabolites were produced. Two series of samples were obtained, the

first one after 1 hour of boiling the parent compounds in H2O2 and the second after

7 hours. In that way, the comparison of the metabolite formation rate was enabled,

which is a crucial step in the study of metabolites.

(The experiments were performed at the Netherlands Cancer Institute (NKI) by

Harald Albers)

24

Results and Discussion

Aim of the thesis

The aim of this project was the study of 10 potential autotaxin inhibitors regarding

their metabolization pattern and the structural elucidation of their metabolites.

Also the metabolites produced by metabolic incubations with pig liver microsomes

in vitro were qualitatively compared with the ones produced with the oxidation of

the parent compounds with hydrogen peroxide.

Structure of compounds to be metabolized

We were provided with 10 different parent compounds which we can divide in 5

different groups regarding to their structure. Groups A-D contain a boron moiety in

their structure whereas group E does not.

Group A (compounds HA278/HA285)

Group B (compounds HA280/HA289)

25

Group C (compounds HA281/HA288)

Group D (compounds HA295/HA296)

Group E (compounds HA286/HA287)

26

Metabolization results of compounds HA278-HA296

After the metabolization of the parent compounds the usage of a chromatographic

separation technique was necessary since mixtures of parent compounds and

metabolites were formed. For the structural elucidation of the metabolites a Q-TOF

mass spectrometer was used coupled to a HPLC system. The experimental setup is

outlined in figure 10. Online UV measurements were performed with the flow

directed after the separation column into the UV detector and after that into the

MS detector with minimal amount of PEEK tubing.

Figure 10: Experimental setup

27

Using a high resolution Bruker Mass Spectrometer we could confirm that masses

within a range of +/- 5ppm (or +/-3 mDa) from the calculated mass correspond to

the right fragments.

After a series of analyses we manage to identify in total 18 different metabolites

from 10 parent compounds with 2 different metabolization techniques (metabolic

incubation and oxidation with H2O2). The proposed structures of the metabolites

for all ten compounds and the table with the experimental conditions are given

below.

Table 1: Experimental conditions during metabolite identification.

28

Proposed structure of Metabolites

Compound HA278

Compound HA280

29

Compound HA281

Compound HA285

Compound HA286

30

Compound HA287

Compound HA288

31

Compound HA289

Compound HA295

Compound HA296

32

Structural elucidation of metabolites using Mass Spectrometry

The molecular structure of the metabolization products originating from 10

different autotaxin inhibitors was elucidated using a High resolution Q-TOF

instrument.

Fragmentation

For practical reasons in the next paragraphs we will show in detail the

fragmentation of one only compound per group. All the needed information can be

found in the end of this chapter in table 20.

It would be useful, before starting with fragmentation, to give some information

about the isotopic abundances of our compounds’ characteristic elements.

Isotope Atomic Mass (ma/u) Natural abundance (atom %)

10B 10.0129 19.9

11B 11.0093 80.1

19F 18.9984 100

35Cl 34.9688 75.78

37Cl 36.9659 24.22

Table 2: Isotopes of Boron, Fluorine and Chlorine.

33

Group A

HA278

Parent

Figure 11: MS and MS/MS spectra of parent compound.

On the right: proposed fragmentation of HA278.

Fragment Formula Exact mass Measured mass Mass error (mDa)

F1 C7H6BO3+ 149.0405 149.0407 -0.2

F2 C6H8BO3+ 139.0561 139.053 3.1

F3 C6H6BO2+ 121.0455 121.0458 -0.3

F4 C9H5F6+ 227.0290 227.0272 1.8

F5 C20H19F6N2O+ 417.1396 417.1349 4.7

Table 3: Mass accuracy measurements of HA278 parent fragment ions.

79.0188

103.9558

195.0911218.9578

264.9584

318.0691 350.0621 425.2246

505.1369

146.0061

385.0986

+MS, 11.9min #704

0.0

0.2

0.4

0.6

0.8

4x10

Intens.

100 150 200 250 300 350 400 450 500 m/z

149.0407

227.0272

505.1366

+MS2(505.1366), 15.364eV, 11.8min #701

0.0

0.5

1.0

1.5

4x10

Intens.

100 150 200 250 300 350 400 450 500 m/z

Full MS

MS2 505

34

Metabolite M1

Figure 12: MS and MS/MS spectra of M1.

On the right: proposed structure and fragmentation of M1.


F1 C14H15F6N2O2+ 357.1032 357.1025 0.7

F2 C9H5F6+ 227.0290 227.0272 1.8

F3 C7H5BO2+ 121.0284 121.0284 0

F4 C6H9N2O3+ 157.0608 157.0603 0.5

Table 4: Mass accuracy measurements of HA278 M1 fragment ions

144.9806 194.1162

223.0455 256.2490296.2500 352.2461

477.1214

103.9551

141.9238

+MS, 12.1-12.2min #(718-722)

0

250

500

750

1000

1250

1500

Intens.

100 150 200 250 300 350 400 450 m/z

121.0281

357.1022

477.1266

+MS2(477.1266), 15eV, 12.1min #719

0

1000

2000

3000

4000

5000

6000

Intens.

100 150 200 250 300 350 400 450 m/z

Full MS

MS2 477

35

Metabolite M2




F1 C7H5F6+ 105.0355 105.0344 1.1

F2 C9H5F6+ 227.0290 227.0312 -2.2

Table 5: Mass accuracy measurements of HA278 M2 fragment ions.

The fragments that indicate a change in the structure of the parent compound are

found at the A part of the molecule. Specifically we can see that the main fragment

(m/z 149.0407) of the parent compound corresponds to the part A. At MS/MS

spectra of the metabolites the m/z difference compared to the parent compound

is Δ -28.0126 and -44.0063 for each metabolite respectively. These differences

indicate loss of boron and one hydroxyl group (BOH) for the M1 and loss of boron

and both hydroxyl groups [B(OH)2] for the M2. Taking the above into consideration

the proposed structures of M1 and M2 are verified.

105.0344

227.0312

461.1307

+MS2(461.1307), 15eV, 13.2min #782

0

200

400

600

800

1000

Intens.

100 150 200 250 300 350 400 450 500 m/z

103.9555

144.9824

195.0891

210.1069 255.2323281.1755

352.2451

368.2408

398.2118 425.2170

461.1313

483.1106

104.0389

+MS, 13.2-13.2min #(781-785)

0

500

1000

1500

2000

2500

Intens.

100 150 200 250 300 350 400 450 m/z

Full MS

MS2 461

36

Figure 14: MS/MS spectra of parent compound, M1 and M2.

On the right: structure of autotaxin inhibitor HA278.

149.0407

227.0272

505.1366

+MS2(505.1366), 15.364eV, 11.8min #701

0.0

0.5

1.0

1.5

4x10

Intens.

100 150 200 250 300 350 400 450 500 m/z

105.0344

227.0312

461.1307

+MS2(461.1307), 15eV, 13.2min #782

0

200

400

600

800

1000

Intens.

100 150 200 250 300 350 400 450 500 m/z

MS2 461

121.0281

357.1022

477.1266

+MS2(477.1266), 15eV, 12.1min #719

0

1000

2000

3000

4000

5000

6000

Intens.

100 150 200 250 300 350 400 450 m/z

MS2 477

MS2 505

37

Group B

HA289

Parent




F1 C12H15BNO3+ 232.1140 232.1142 -0.2

F2 C7H6BO3+ 149.0405 149.0407 -0.2

F3 C6H6BO2+ 121.0455 121.0458 -0.3

F4 C8H13N2O2+ 169.0972 169.0971 0.1

F5 C7H5Cl2+ 158.9763 158.9757 0.6


520.1570

186.2221 520.6913103.9553

+MS, 9.4-9.5min #(555-563)

0

1

2

3

4

4x10

Intens.

50 100 150 200 250 300 350 400 450 500 m/z

149.0407

169.0971

232.1142

370.1097

520.1565

+MS2(520.9081), 16.4575eV, 9.4min #560

0.0

0.2

0.4

0.6

0.8

5x10

Intens.

50 100 150 200 250 300 350 400 450 500 m/z

Full MS

MS2 520

38

Metabolite M1




F1 C12H14NO2+ 204.1019 204.1012 0.7

F2 C7H5O2+ 121.0284 121.0277 0.7

F3 C8H13N2O2+ 169.0972 169.0964 0.8

F4 C17H25Cl2N3O2+ 372.1240 372.1224 1.6

F5 C7H5Cl2+ 158.9763 158.9739 2.4


290.1858

492.1452

493.1481

334.1750159.9789

+MS, 9.6-9.6min #(567-571)

0

1

2

3

4x10

Intens.

100 150 200 250 300 350 400 450 500 m/z

Full MS

MS2 492

121.0277

204.1012

372.1224

492.1449

+MS2(492.8918), 15eV, 9.6min #568

0

2

4

6

8

4x10

Intens.

100 150 200 250 300 350 400 450 500 m/z

39

Metabolite M2

Oxidation Experiment




F1 C7H5O2+ 121.0284 121.0275 0.9

F2 C12H14NO2+ 204.1019 204.1013 0.6

F3 C7H5Cl2+ 158.9763 158.9758 0.5


136.0587 185.9413 218.9547

508.1394

509.1428

255.9204105.9553

+MS, 9.8min #(579)

0

2000

4000

6000

8000

Intens.

100 150 200 250 300 350 400 450 500 m/z

121.0275

204.1013

508.1438

+MS2(509.1415), 15.6277eV, 9.7min #576

0

2

4

6

4x10

Intens.

100 150 200 250 300 350 400 450 500 m/z

Full MS

MS2 508

40

Metabolic Incubation



Metabolite M2 with m/z 508 is present with two different oxidation positions. With

metabolic incubation, the oxidation took place in A-ring whereas in oxidation

experiment in C-ring. The fragments that indicate this difference are 121 and 204 in

figure 17 while in figure 18 these fragments are found with a difference of +16

which indicates the addition of a hydroxyl group. However, the exact position in the

ring is unclear.


F1 C7H5O3+ 137.0233 137.0241 -0.8

F2 C12H14NO3+ 220.0968 220.0971 -0.3

F3 C17H24Cl2N3O2+ 372.1240 372.1289 -4.9*


*even though the error is higher than expected, this is the proposed structure.

123.0549

155.1103

195.0901

252.1601

327.0742

508.1403

212.1650

509.1422229.1431

+MS, 9.4min #(560)

0

250

500

750

1000

1250

Intens.

50 100 150 200 250 300 350 400 450 500 m/z

137.0241

220.1022372.1289

508.1398

+MS2(509.1534), 15.6288eV, 9.5min #562

0

200

400

600

800

1000

Intens.

100 150 200 250 300 350 400 450 500 m/z

Full MS

MS2 508

41

Metabolite M3




F1 C7H5O+ 105.0335 105.0330 0.5

F2 C12H14NO+ 188.1070 188.1078 -0.8


112.0276

155.1083

170.1148

195.0918

226.1804

246.0187

266.1753

295.2291

335.2249

353.2338

392.1742

414.3021

476.1544

432.3088

477.1551

450.3237

+MS, 10.1min #600

0

500

1000

1500

Intens.

100 150 200 250 300 350 400 450 m/z

105.0330

188.1078

476.1519

+MS2(477.1539), 15eV, 10.1min #602

0

100

200

300

400

500

Intens.

100 150 200 250 300 350 400 450 m/z

Full MS

MS2 476

42

Group C

HA288

Parent




F1 C15H15F6N2O2+ 369.1032 369.1031 0.1

F2 C9H5F6N + 227.0290 227.0291 -0.1

F3 C12H10F6N + 282.0712 282.0718 -0.6

F4 C14H15F6N2+ 325.1134 325.1130 0.4


369.1031

+MS2(533.1688), 17.3255eV, 10.5min #622

0

1

2

3

4

5

65x10

Intens.

100 150 200 250 300 350 400 450 500 m/z

369.1026

533.1677

149.9523103.9549

+MS, 10.4-10.5min #(617-625)

0.0

0.2

0.4

0.6

0.8

1.0

5x10

Intens.

50 100 150 200 250 300 350 400 450 500 m/z

43

Metabolite M1



The fragmentation of the metabolite M1 was not god enough so fragments of the

part of the molecule that changed were not observed. However, comparing the

mass difference Δm/z= - 28 with previous compounds (metabolite M1 of HA278 and

HA289) we concluded that this loss derives from the deboronation of the parent

compound following with hydroxylation in the same ring.


F1 C15H15F6N2O2+ 369.1032 369.1017 1.4


103.9543

185.9438 218.9577 253.9233 369.1043 403.0732

505.1552

144.9798154.0024

+MS, 10.6-10.7min #(628-636)

0

1000

2000

3000

4000

5000

6000

Intens.

100 150 200 250 300 350 400 450 500 m/z

369.1017

505.1551

+MS2(505.1551), 15.3807eV, 10.7min #633

0.0

0.5

1.0

1.5

2.0

2.5

4x10

Intens.

100 150 200 250 300 350 400 450 500 m/z

44

Metabolite M2




F1 C13H15F6N2+ 313.1134 313.1126 0.8

F2 C9H5F6+ 227.0290 227.0279 1.1

F3 C4H9N2+ 85.0760 85.0733 2.7

F4 C5H9N2O+ 113.0709 113.0710 -0.1

F5 C11H10F6N+ 270.0712 270.0688 2.4

F6 C5H9N2O2+ 129.0659 129.0659 0


357.1025

103.9541 185.9488

+MS, 9.8-9.9min #(584-588)

0

2

4

6

4x10

Intens.

100 150 200 250 300 350 m/z

113.0710

227.0279

313.1126357.1023

+MS2(357.1023), 15eV, 9.9min #589

0

2

4

6

4x10

Intens.

100 150 200 250 300 350 m/z

45

Metabolite M3




F1 C15H15F6N2O2+ 369.1032 369.1014 1.8

F2 C5H10NO2+ 116.0706 116.0710 -0.4

F3 C6H10NO2+ 128.0706 128.0713 -0.7

F4 C16H17F6N2O2+ 383.1189 383.1178 1.1


89.0677

103.9541149.9512 185.9415 218.9559

314.0584

445.1199

419.0582386.0484

+MS, 10.3-10.4min #(613-617)

0

1000

2000

3000

4000

Intens.

100 150 200 250 300 350 400 450 m/z

116.0679

369.1014

445.1174

+MS2(445.1174), 15eV, 10.4min #618

0.0

0.5

1.0

1.5

4x10

Intens.

100 150 200 250 300 350 400 450 m/z

46

Group D

HA295

Parent




F1 C8H6BO3+ 161.0405 161.0401 0.4

F2 C7H8BO3+ 151.0561 115.0546 1.5

F3 C7H6BO2+ 133.0455 133.0451 0.4

F4 C5H9N2O+ 113.0709 113.0716 -0.7


103.9553

144.9815

449.0843

450.0859

244.2614

+MS, 11.7-11.8min #(694-702)

0.0

0.5

1.0

1.5

4x10

Intens.

100 150 200 250 300 350 400 450 m/z

161.0401

275.0023 319.0076449.0813

+MS2(450.0873), 15eV, 11.8min #699

0

1

2

3

4

4x10

Intens.

150 200 250 300 350 400 450 m/z

Full MS

MS2 449

47

Metabolite M1




F1 C8H7O3+ 151.0390 151.0389 0.1

F2 C8H5O2+ 133.0284 133.0285 -0.1

F3 C6H9N2O3+ 157.0608 157.0594 1.4

F4 C7H5Cl2+ 158.9763 158.9747 1.6


112.0288151.0381

195.0912228.1963 250.1784

343.2947421.0746

439.0839

313.2395295.2252

440.0888

+MS, 11.0-11.2min #(655-667)

0.00

0.25

0.50

0.75

1.00

1.25

4x10

Intens.

100 150 200 250 300 350 400 450 m/z

113.0708

133.0285

151.0389

219.1140 291.0034

+MS2(440.0807), 15eV, 11.1min #660

0

1

2

3

4

5

6

4x10

Intens.

100 150 200 250 300 350 400 m/z

Full MS

MS2 439

48

Group E

HA286

Parent




F1 C7H5Cl2+ 158.9763 158.9739 2.4

F2 C13H15Cl2N2O2+ 301.0505 301.0491 1.4


103.9545158.9752 266.1717

478.0913

479.0952

226.1793

301.0505

+MS, 9.9-10.0min #(590-594)

0

2000

4000

6000

8000

Intens.

100 150 200 250 300 350 400 450 m/z

158.9739

301.0491

+MS2(479.1041), 15eV, 10.0min #595

0

1

2

3

4x10

Intens.

150 200 250 300 350 400 450 m/z

Full MS

MS2 478

49

Metabolite M1




F1 C5H9N2O+ 113.0709 113.0721 -1.2 F2 C9H10Cl2N+ 202.0185 202.0184 0.1 F3 C5H9N2O2

+ 129.0659 129.0652 0.7 F4 C11H15Cl2N2

+ 245.0607 245.0600 0.7 F5 C7H5Cl2+ 158.9763 158.9763 0 F6 C7H4Cl+ 122.9996 123.0013 -1.7 F7 C4H9N2

+ 85.0760 85.0743 1.7


129.0659

158.9765 245.0610

289.0509

195.0902

290.0548

112.0292

+MS, 9.1-9.3min #(543-551)

0

1

2

3

4

4x10

Intens.

100 120 140 160 180 200 220 240 260 280 m/z

158.9763

245.0600

+MS2(289.0495), 15eV, 9.2min #548

0.00

0.25

0.50

0.75

1.00

1.25

1.50

5x10

Intens.

100 120 140 160 180 200 220 240 260 280 m/z

Full MS

MS2 289

50

Table 19: Structural elucidation of the compounds HA285, HA280, HA281, HA296, HA287 and their metabolites.

Compound m/z (mass error) Fragments (mass error) Proposed Structure

HA285 437.0837 (0.7mDa) 158.9763 (0.7mDa)

149.0405 (0.7mDa)

-

M1 409.0716 (0.8mDa) 158.9763 (-0.5mDa),

121.0284 (0.1mDa),

93.0335 (-1.3mDa)

289.0505 (-2.1mDa)

Parent - BOH

HA280 588.2099 (1.7mDa)

149.0405 (0.4mDa)

232.1140 (0.2mDa)

438.1611 (-2.8mDa)

M1 560.1979 (1.4mDa) 121.0284 (-0.1mDa)

204.1019 (-0.7mDa)

357.1032 (-1mDa)

440.1767 (-0.8mDa)

227.0290 (1.1mDa)

Parent - BOH

M2 576.1928 (0.8mDa) 121.0284 (1.1mDa)

204.1019 (0 mDa)

227.0290(0.4mDa)

Parent – BOH,

+OH

HA281 465.1150 (1.4mDa) 301.0505 (1.6mDa)

158.9763 (0mDa)

245.0607 (0.9mDa)

M1 437.1029 (0.2mDa) 301.0505(-0.1mDa) 158.9761 (0.2mDa)

Parent - BOH

M2 289.0505 (-0.1mDa) 245.0607 (0.4mDa) 158.9765 (-0.2mDa)

N- dealkylation of

parent compound

(-C9H9BO3)

M3* 377.0666 (0mDa) 301.0477 (2.8mDa) 158.9763 (-1.2mDa) 128.0706 (0.8mDa) 116.0706 (0.2mDa)

M2 + C3H4O3

51

HA296 517.1364 (-1.5mDa) 161.0405 (0.5mDa)

277.0290 (0.1mDa)

133.0455 (3.1mDa)

M1 507.1349 (-0.4mDa) 151.0390 (1.7mDA)

227.0290 (2.2mDa)

357.1032 (1.7mDa)

157.0608 (0.9mDa)

133.0284 (2.3mDa)

Parent – BOH,

+OH

HA287 546.1458 (0mDa) 369.1032 (0.3mDa)

227.0290 (-2.9mDa)

M1 357.1032 (-0.9mDa) 227.0290 (-0.9mDa)

313.1134 (-0.6mDa)

129.0659 (0.9mDa)

85.0760 (0.4mDa)

113.0709 (-0.3mDa)

270.0712 (-2.9mDa)

N- dealkylation of

parent compound

(-C10H7NO3)

*M3 is visible only at oxidation experiment

52

Conclusion

Metabolic stability of ten ATX inhibitors was successfully assessed using HPLC-

MS/MS analysis of metabolic mixtures generated from the parent compounds. Two

different approaches were used to generate metabolic mixtures of ATX inhibitors.

The incubation with pig liver microsomes was first used to simulate the in vivo

metabolism of the compounds and estimate the relative stability of the compounds.

Next, oxidation experiment using hydrogen-peroxide was used to test the ability of

alternative way of producing the metabolites of investigated compounds.

Metabolic mixtures generated in both experiments were then subjected to the

HPLC-MS/MS analysis. The mixtures were easily separated on a C18 column and

accurate MS and MS/MS measurements allowed full or partial structure elucidation

of the formed metabolites.

For the compounds with the boron moiety, the primary route of metabolism

observed in metabolic incubation was the deboronation of the parent compound

followed by hydroxylation in the same position (M1). Metabolites with double

hydroxylation after the deboronation were also formed for two of the compounds

and simple deboronation was also observed in some cases but with low

concentration. N-dealkylation of the parent compounds was also present.

For the two compounds without the boron moiety in their structure only one

metabolite was observed, which was formed after N-dealkylation of the parent

compound. Therefore, these structures were marked to be relatively stable.

The same main metabolites were produced in the oxidation experiment with the

hydrogen-peroxide, which demonstrated that this technique could be used for the

53

study of metabolic stability of these and similar compounds. Furthermore, it could

be used to generate large amounts of the metabolites for eventual pharmacological

characterization, and toxicology and pharmacokinetic studies.

54

References

[1] “What Is Cancer? - National Cancer Institute.” [Online]. Available: http://www.cancer.gov/cancertopics/cancerlibrary/what-is-cancer. [Accessed: 03-Mar-2014].

[2] A. S. Narang and D. S. Desai, Pharmaceutical Perspectives of Cancer Therapeutics. New York, NY: Springer US, 2009.

[3] “Cytotoxic Drugs < Health and safety | CUPE.” [Online]. Available: http://cupe.ca/health-and-safety/cytotoxic-drugs. [Accessed: 03-Mar-2014].

[4] M. Rask-Andersen, M. S. Almén, and H. B. Schiöth, “Trends in the exploitation of novel drug targets.,” Nat. Rev. Drug Discov., vol. 10, no. 8, pp. 579–90, Aug. 2011.

[5] J. G. Robertson, “Enzymes as a special class of therapeutic target: clinical drugs and modes of action.,” Curr. Opin. Struct. Biol., vol. 17, no. 6, pp. 674–9, Dec. 2007.

[6] A. B. Hoeglund, A. L. Howard, I. W. Wanjala, T. C. T. Pham, A. L. Parrill, and D. L. Baker, “Characterization of non-lipid autotaxin inhibitors.,” Bioorg. Med. Chem., vol. 18, no. 2, pp. 769–76, Jan. 2010.

[7] H. M. H. G. Albers, L. J. D. Hendrickx, R. J. P. van Tol, J. Hausmann, A. Perrakis, and H. Ovaa, “Structure-based design of novel boronic acid-based inhibitors of autotaxin.,” J. Med. Chem., vol. 54, no. 13, pp. 4619–26, Jul. 2011.

[8] M. Jankowski, “Autotaxin: its role in biology of melanoma cells and as a pharmacological target.,” Enzyme Res., vol. 2011, p. 194857, Jan. 2011.

[9] G. E. Lienhard and K. A. Koehler, “2-Phenylethaneboronic acid, a possible transition-state analog for chymotrypsin,” Biochemistry, vol. 10, no. 13, pp. 2477–2483, Jun. 1971.

[10] C. A. Kettner and A. B. Shenvi, “Inhibition of the serine proteases leukocyte elastase, pancreatic elastase, cathepsin G, and chymotrypsin by peptide boronic acids.,” J. Biol. Chem., vol. 259, no. 24, pp. 15106–15114, Dec. 1984.

55

[11] J. Adams, M. Behnke, S. Chen, A. A. Cruickshank, L. R. Dick, L. Grenier, J. M. Klunder, Y.-T. Ma, L. Plamondon, and R. L. Stein, “Potent and selective inhibitors of the proteasome: Dipeptidyl boronic acids,” Bioorg. Med. Chem. Lett., vol. 8, no. 4, pp. 333–338, Feb. 1998.

[12] V. Uttamsingh, C. Lu, G. Miwa, and L.-S. Gan, “Relative contributions of the five major human cytochromes P450, 1A2, 2C9, 2C19, 2D6, and 3A4, to the hepatic metabolism of the proteasome inhibitor bortezomib.,” Drug Metab. Dispos., vol. 33, no. 11, pp. 1723–8, Nov. 2005.

[13] H. M. H. G. Albers, L. a van Meeteren, D. a Egan, E. W. van Tilburg, W. H. Moolenaar, and H. Ovaa, “Discovery and optimization of boronic acid based inhibitors of autotaxin.,” J. Med. Chem., vol. 53, no. 13, pp. 4958–67, Jul. 2010.

[14] M. S. Lennard and A. V. Stachulski, “Drug Metabolism: The Body’s Defense against Chemical Attack,” J. Chem. Educ., vol. 77, no. 3, p. 349, Mar. 2000.

[15] A. P. Li, “Preclinical in vitro screening assays for drug-like properties,” Drug Discov. Today Technol., vol. 2, no. 2, pp. 179–185, Jun. 2005.

[16] S. Asha and M. Vidyavathi, “Role of human liver microsomes in in vitro metabolism of drugs-a review.,” Appl. Biochem. Biotechnol., vol. 160, no. 6, pp. 1699–722, Mar. 2010.

[17] J. Kool, A. F. Rudebeck, F. Fleurbaaij, S. Nijmeijer, D. Falck, R. A. Smits, H. F. Vischer, R. Leurs, and W. M. A. Niessen, “High-resolution metabolic profiling towards G protein-coupled receptors: rapid and comprehensive screening of histamine H₄ receptor ligands.,” J. Chromatogr. A, vol. 1259, pp. 213–20, Oct. 2012.

[18] J. Reinen, J. Kool, and N. P. E. Vermeulen, “Reversed-phase liquid chromatography coupled on-line to estrogen receptor bioaffinity detection based on fluorescence polarization.,” Anal. Bioanal. Chem., vol. 390, no. 8, pp. 1987–98, Apr. 2008.

56

Attachments

HA280-parent MS and MS/MS

HA280-Metabolite1 MS and MS/MS

588.2072

227.0284

+MS, 10.1min #597

0

2

4

6

4x10

Intens.

100 200 300 400 500 600 m/z

149.0401

232.1138

438.1583

588.2086

+MS2(588.2086), 21.1795eV, 10.0min #594

0

2

4

6

8

4x10

Intens.

150 200 250 300 350 400 450 500 550 m/z

560.1960

227.0279 334.1734

+MS, 10.2min #604

0.0

0.5

1.0

1.5

5x10

Intens.

100 200 300 400 500 600 m/z

57

HA280-metabolite2 MS and MS/MS

121.0273

204.1008

440.1758

560.1964

+MS2(560.5099), 19.2314eV, 10.2min #605

0.0

0.2

0.4

0.6

0.8

1.0

5x10

Intens.

100 200 300 400 500 m/z

227.0286

576.1931

576.7081112.0268 288.6017

+MS, 10.3min #(612)

0

1

2

3

4

5

6

4x10

Intens.

100 200 300 400 500 m/z

121.0273

204.1009

576.1920

+MS2(576.1920), 20.3517eV, 10.3min #609

0.0

0.5

1.0

1.5

5x10

Intens.

100 150 200 250 300 350 400 450 500 550 m/z

58



158.9763

301.0489

+MS2(466.1152), 15eV, 10.0min #591

0

2

4

6

4x10

Intens.

150 200 250 300 350 400 450 m/z

103.9555149.9518

465.1132

466.1145

302.0518

226.1799

+MS, 9.9-10.0min #(590-594)

0.0

0.2

0.4

0.6

0.8

1.0

4x10

Intens.

100 150 200 250 300 350 400 450 m/z

103.9552144.9813 195.0930

226.1800301.0512

437.1019

438.1059

302.0495112.0275

+MS, 10.1-10.1min #(598-602)

0

2000

4000

6000

Intens.

100 150 200 250 300 350 400 m/z

59


158.9761

301.0508

+MS2(437.8954), 10.1min #(599)

0

1

2

3

4

5

4x10

Intens.

50 100 150 200 250 300 350 400 m/z

158.9750

245.0604

+MS2(289.8899), 15eV, 9.2min #547

0.0

0.5

1.0

1.5

2.0

4x10

Intens.

75 100 125 150 175 200 225 250 275 m/z

129.0668 195.0904

289.0499

290.0534

246.0648

158.9744

+MS, 9.2min #(545)

0.00

0.25

0.50

0.75

1.00

1.25

4x10

Intens.

100 125 150 175 200 225 250 275 m/z

60



123.0551 149.9516 195.0877218.9570

377.0675

378.0711

141.9586

103.9549

+MS, 9.7-9.8min #(574-582)

0

500

1000

1500

2000

Intens.

100 150 200 250 300 350 m/z

116.0704

128.0698 158.9775200.0811

301.0477

361.0519

377.0671

+MS2(378.0724), 15eV, 9.8min #579

0

1000

2000

3000

Intens.

100 150 200 250 300 350 m/z

121.0655144.9805 173.1542

218.9561267.1228

369.1667

387.1805

404.2057

437.0822

249.2058

105.0699

+MS, 11.4-11.6min #(677-689)

0

2000

4000

6000

8000

Intens.

100 150 200 250 300 350 400 m/z

61


149.0398

262.9995439.0849

+MS2(437.0772), 15eV, 11.4min #678

0

1000

2000

3000

Intens.

150 200 250 300 350 400 450 m/z

103.9568 195.0891

409.0731

409.8189121.0948

+MS, 11.8min #(701)

0

1

2

3

4

4x10

Intens.

100 150 200 250 300 350 400 m/z

121.0285

158.9760

289.0541

+MS2(409.0749), 15eV, 11.8min #698

0.0

0.5

1.0

1.5

2.0

2.5

3.0

4x10

Intens.

100 150 200 250 300 350 400 m/z

62



103.9559185.9404 218.9568 369.1056

546.1455

253.9241149.9525

+MS, 10.6min #(627)

0.0

0.2

0.4

0.6

0.8

1.0

4x10

Intens.

50 100 150 200 250 300 350 400 450 500 550 m/z

369.1029

+MS2(546.1448), 18.2488eV, 10.6min #628

0.0

0.5

1.0

1.5

2.0

2.5

4x10

Intens.

50 100 150 200 250 300 350 400 450 500 m/z

112.0285195.0919

357.1045

236.1576 259.1005

+MS, 9.8-9.9min #(582-590)

0

1

2

3

4

4x10

Intens.

50 100 150 200 250 300 350 m/z

63


113.0708

227.0297

313.1135 357.1043

+MS2(357.1043), 15eV, 9.9min #587

0

2

4

6

4x10

Intens.

100 150 200 250 300 350 m/z

103.9560

144.9823

517.1379

245.1058

+MS, 12.1-12.2min #(718-722)

0.0

0.5

1.0

1.5

2.0

4x10

Intens.

100 150 200 250 300 350 400 450 500 m/z

161.0400

227.0289

517.1384

+MS2(517.1384), 16.2022eV, 12.1min #719

0

1

2

3

4

5

4x10

Intens.

100 150 200 250 300 350 400 450 500 550 m/z

64


112.0283151.0378 195.0907 249.2062

507.1368

387.1806

409.1631

425.1381

+MS, 11.5-11.7min #(681-693)

0.0

0.5

1.0

1.5

2.0

4x10

Intens.

150 200 250 300 350 400 450 500 m/z

133.0281

151.0385

247.1080 357.1046

489.1258507.1361

+MS2(507.1361), 15.5161eV, 11.5min #686

0

1

2

3

4x10

Intens.

150 200 250 300 350 400 450 500 m/z

Structure elucidation of ten autotaxin inhibitors and their … · and their metabolites using...

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