“Structure elucidation of ten autotaxin inhibitors and their metabolites using Liquid Chromatography tandem Mass Spectrometry”

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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

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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

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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.

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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

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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.

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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

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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

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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].

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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.

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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].

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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.

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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].

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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.

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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

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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

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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.

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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 40o_{C. 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.

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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 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 200o_C_{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

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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

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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 o_{C 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 37o_{C. 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 o_{C 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 -20o_{C [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)

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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)

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Group C (compounds HA281/HA288)

Group D (compounds HA295/HA296)

Group E (compounds HA286/HA287)

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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.

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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.

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Proposed structure of Metabolites

Compound HA278

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Compound HA281

Compound HA285

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Compound HA287

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Compound HA289

Compound HA295

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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 %)

10_B _10.0129 _19.9

11_B _11.0093 _80.1

19_F _18.9984 ₁₀₀

35_Cl _34.9688 _75.78

37_Cl _36.9659 _24.22

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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 195.0911 218.9578 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 4 x10 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 4 x10 Intens. 100 150 200 250 300 350 400 450 500 m/z Full MS MS2 ₅₀₅

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Metabolite M1

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

On the right: proposed structure and fragmentation of M1.

Fragment Formula Exact mass Measured mass Mass error (mDa) 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 223.0455256.2490 296.2500 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

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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 255.2323 281.1755 352.2451 368.2408 398.2118425.2170 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

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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 4 x10 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 500m/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

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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

Table 6: Mass accuracy measurements of HA289 parent fragment ions. 520.1570 186.2221 520.6913 103.9553 +MS, 9.4-9.5min #(555-563) 0 1 2 3 4 4 x10 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 5 x10 Intens. 50 100 150 200 250 300 350 400 450 500 m/z Full MS MS2 ₅₂₀

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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

Table 7: Mass accuracy measurements of HA289 M1 fragment ions. 290.1858 492.1452 493.1481 334.1750 159.9789 +MS, 9.6-9.6min #(567-571) 0 1 2 3 4 x10 Intens. 100 150 200 250 300 350 400 450 500m/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 4 x10 Intens. 100 150 200 250 300 350 400 450 500 m/z

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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

Table 8: Mass accuracy measurements of HA289 M2 fragment ions. 136.0587 185.9413 218.9547 508.1394 509.1428 255.9204 105.9553 +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 4 x10 Intens. 100 150 200 250 300 350 400 450 500 m/z Full MS MS2 ₅₀₈

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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 509.1422 229.1431 +MS, 9.4min #(560) 0 250 500 750 1000 1250 Intens. 50 100 150 200 250 300 350 400 450 500m/z 137.0241 220.1022 372.1289 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

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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

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Group C HA288

Parent

F2 C9H5F6N + 227.0290 227.0291 -0.1

F3 C12H10F6N + 282.0712 282.0718 -0.6

F4 C14H15F6N2+ 325.1134 325.1130 0.4

Table 11: Mass accuracy measurements of HA288 parent fragment ions. 369.1031 +MS2(533.1688), 17.3255eV, 10.5min #622 0 1 2 3 4 5 6 5 x10 Intens. 100 150 200 250 300 350 400 450 500 m/z 369.1026 533.1677 149.9523 103.9549 +MS, 10.4-10.5min #(617-625) 0.0 0.2 0.4 0.6 0.8 1.0 5 x10 Intens. 50 100 150 200 250 300 350 400 450 500 m/z

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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.

Table 12: Mass accuracy measurements of HA288 M1 fragment ions. 103.9543 185.9438218.9577 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 4 x10 Intens. 100 150 200 250 300 350 400 450 500 m/z

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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 4 x10 Intens. 100 150 200 250 300 350 m/z 113.0710 227.0279 313.1126 357.1023 +MS2(357.1023), 15eV, 9.9min #589 0 2 4 6 4 x10 Intens. 100 150 200 250 300 350 m/z

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Metabolite M3

F2 C5H10NO2+ 116.0706 116.0710 -0.4

F3 C6H10NO2+ 128.0706 128.0713 -0.7

F4 C16H17F6N2O2+ 383.1189 383.1178 1.1

Table 14: Mass accuracy measurements of HA288 M2 fragment ions. 89.0677 103.9541 149.9512 185.9415 218.9559 314.0584 445.1199 419.0582 386.0484 +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 4 x10 Intens. 100 150 200 250 300 350 400 450 m/z

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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

Table 15: Mass accuracy measurements of HA295 parent fragment ions. 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 4 x10 Intens. 100 150 200 250 300 350 400 450 m/z 161.0401 275.0023 319.0076 449.0813 +MS2(450.0873), 15eV, 11.8min #699 0 1 2 3 4 4 x10 Intens. 150 200 250 300 350 400 450 m/z Full MS MS2 ₄₄₉

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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

Table 16: Mass accuracy measurements of HA295 M1 fragment ions. 112.0288 151.0381 195.0912 228.1963 250.1784 343.2947 421.0746 439.0839 313.2395 295.2252 440.0888 +MS, 11.0-11.2min #(655-667) 0.00 0.25 0.50 0.75 1.00 1.25 4 x10 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 4 x10 Intens. 100 150 200 250 300 350 400 m/z Full MS MS2 ₄₃₉

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Group E HA286

Parent

F1 C7H5Cl2+ 158.9763 158.9739 2.4

F2 C13H15Cl2N2O2+ 301.0505 301.0491 1.4

Table 17: Mass accuracy measurements of HA286 parent fragment ions. 103.9545 158.9752 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 4 x10 Intens. 150 200 250 300 350 400 450 m/z Full MS MS2 ₄₇₈

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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

Table 18: Mass accuracy measurements of HA286 M1 fragment ions. 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 4 x10 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 5 x10 Intens. 100 120 140 160 180 200 220 240 260 280 m/z Full MS MS2 ₂₈₉

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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

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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)

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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

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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.

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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 4 x10 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 4 x10 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 5 x10 Intens. 100 200 300 400 500 600 m/z

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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 5 x10 Intens. 100 200 300 400 500 m/z 227.0286 576.1931 576.7081 112.0268 288.6017 +MS, 10.3min #(612) 0 1 2 3 4 5 6 4 x10 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 5 x10 Intens. 100 150 200 250 300 350 400 450 500 550 m/z

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58 HA281-parent MS and MS/MS HA281-metabolite1 MS and MS/MS 158.9763 301.0489 +MS2(466.1152), 15eV, 10.0min #591 0 2 4 6 4 x10 Intens. 150 200 250 300 350 400 450 m/z 103.9555 149.9518 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 4 x10 Intens. 100 150 200 250 300 350 400 450 m/z 103.9552 144.9813 195.0930 226.1800 301.0512 437.1019 438.1059 302.0495 112.0275 +MS, 10.1-10.1min #(598-602) 0 2000 4000 6000 Intens. 100 150 200 250 300 350 400 m/z

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59 HA281-metabolite2 MS and MS/MS 158.9761 301.0508 +MS2(437.8954), 10.1min #(599) 0 1 2 3 4 5 4 x10 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 4 x10 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 4 x10 Intens. 100 125 150 175 200 225 250 275 m/z

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60 HA281-metabolite3 MS and MS/MS HA285-parent MS and MS/MS 123.0551 149.9516 195.0877 218.9570 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 158.9775 _200.0811 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 218.9561 267.1228 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

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61 HA285-metabolite1 MS and MS/MS 149.0398 262.9995 439.0849 +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 409.8189 121.0948 +MS, 11.8min #(701) 0 1 2 3 4 4 x10 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 4 x10 Intens. 100 150 200 250 300 350 400 m/z

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62 HA287-parent MS and MS/MS HA287-metabolite1 MS and MS/MS 103.9559 185.9404 218.9568 369.1056 546.1455 253.9241 149.9525 +MS, 10.6min #(627) 0.0 0.2 0.4 0.6 0.8 1.0 4 x10 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 4 x10 Intens. 50 100 150 200 250 300 350 400 450 500 m/z 112.0285 195.0919 357.1045 236.1576 259.1005 +MS, 9.8-9.9min #(582-590) 0 1 2 3 4 4 x10 Intens. 50 100 150 200 250 300 350 m/z

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63 HA296-parent MS and MS/MS 113.0708 227.0297 313.1135 357.1043 +MS2(357.1043), 15eV, 9.9min #587 0 2 4 6 4 x10 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 4 x10 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 4 x10 Intens. 100 150 200 250 300 350 400 450 500 550 m/z

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64 HA296-metabolite1 MS and MS/MS 112.0283 151.0378 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 4 x10 Intens. 150 200 250 300 350 400 450 500 m/z 133.0281 151.0385 247.1080 _357.1046 489.1258_507.1361 +MS2(507.1361), 15.5161eV, 11.5min #686 0 1 2 3 4 x10 Intens. 150 200 250 300 350 400 450 500 m/z

“Structure elucidation of ten autotaxin inhibitors and their metabolites using Liquid Chromatography tandem Mass Spectrometry”

MSc Chemistry

Analytical Sciences

“Structure elucidation of ten autotaxin inhibitors

and their metabolites using Liquid

Chromatography tandem Mass Spectrometry”

Master thesis

Georgios Psarros

MSc Chemistry

Analytical Sciences

Master Thesis

Structural elucidation of ten autotaxin inhibitors using

Liquid Chromatography tandem Mass Spectrometry

Georgios Psarros

Preface

Contents

List of Abbreviations

Abstract

Introduction

Anticancer drugs

What is Cancer?

Anticancer drug development

Anticancer drugs directed at a drug target



Enzymes (as anti-cancer drug target)

Autotaxin

Autotaxin inhibitors

Boronic acid-based autotaxin inhibitors

Metabolization of drugs

Experimental

Apparatus

Liquid Chromatography

Mass Spectrometry

Methods

Metabolization with microsomes

Simulating Metabolism with Hydrogen Peroxide

Results and Discussion

Aim of the thesis

Structure of compounds to be metabolized

Metabolization results of compounds HA278-HA296

Proposed structure of Metabolites

Structural elucidation of metabolites using Mass Spectrometry

Fragmentation

Conclusion

References

Attachments