Novel Small Molecule Inhibitors for the Human Kinesins Mklp2 and Kif18A

Novel Small Molecule Inhibitors for the Human Kinesins

Mklp2 and Kif18A

DISSERTATION

zur Erlangung des Akademischen Grades des Doktors der Naturwissenschaften

(Dr. rer. nat.)

an der Universität Konstanz

Mathematisch‐Naturwissenschaftliche Sektion Fachbereich Chemie

vorgelegt von

Joachim Braun

aus Bessenbach

2015

Tag der mündlichen Prüfung: 17. April 2015 1. Referent: Prof. Dr. Andreas Marx 2. Referent: Prof. Dr. Thomas U. Mayer

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-287464

„Viele Menschen treten in dein Leben ein, aber nur ein paar besondere Menschen hinterlassen auch Spuren in deinem Herzen.“

Autor unbekannt.

Gewidmet Alfred und Anni Prößler

Danksagung

An erster Stelle möchte ich dem kürzlich verstorbenen Prof Dr. Ulrich Groth für die Aufnahme in seine Arbeitsgruppe und die jahrelange Finanzierung danken.

Des Weiteren gilt mein besonderer Dank Prof. Dr. Thomas U. Mayer für die Überlassung des sehr interessanten und interdisziplinären Themas sowie die Aufnahme in seine Arbeitsgruppe, viele wissenschaftliche Diskussionen, die Übernahme des Zweitgutachtens und die Möglichkeit als Chemiker molekular- und zellbiologisches Arbeiten erlernen zu können.

Außerdem danke ich Prof. Dr. Andreas Marx für die Übernahme des Erstgutachtens und zahlreiche Diskussionen in meinem Thesis Committee. Prof Dr. Müller danke ich für die Übernahme des Prüfungsvorsitzes.

Besonders möchte ich mich bei Johanna Kastl und Martin Möckel bedanken für die fruchtbare Zusammenarbeit, viele Diskussionen und die Hilfe bei den Versuchsdurchführungen.

Meinen drei Bachelor möchte ich für die schöne Zeit und gute Zusammenarbeit danken, sowie den zahlreichen Mitarbeiterpraktikanten.

Allen ehemaligen sowie übrig gebliebenen Mitgliedern der AG Groth danke ich für zahlreiche wissenschaftliche Diskussionen, die gute Arbeitsatmosphäre und vieles mehr. Besonderen Dank gebührt dem „Wölfsche“ für das „Käffsche“ um 15 Uhr bei dem sowohl wissenschaftliches als auch anderes diskutiert und in humoristischer Art verarbeitet wurde. Auch meinen zwei rumänischen Labormitbewohnerinnen Dana und Carmen ein herzliches Dankeschön für die unvergessliche Zeit mit euch beiden!

Der ganzen AG Mayer danke ich für ihre Hilfsbereitschaft, viele Diskussionen und das Geraderücken, wenn der Chemiker mal wieder etwas unsicher war und eine super Arbeitsatmosphäre.

Malin Bein danke ich für die Durchführung der Zytotoxizitätsstudien.

Für das Korrekturlesen dieser Arbeit danke ich Hüsnü Topal, Tobias Strittmatter, Johannes Drexler, Juliane Leutzow, Martin Möckel und Holger Bußkamp.

Besonders danken möchte ich all meinen Freunden, die meine Zeit hier in Konstanz zu einer unvergesslichen, wunderbaren Erfahrung gemacht haben.

Zuletzt gilt mein größter Dank meinen Eltern und meinen Geschwistern, die in jeglicher Lage zu mir gestanden und mich bedingungslos während der gesamten Studienzeit unterstütz haben. Ein sicherer Hafen in dem man sich wohlfühlen und zurückziehen kann, während alles andere seine Bedeutung verliert, ist unbezahlbar.

Die vorliegende Arbeit entstand in der Zeit von März 2010 bis Dezember 2014 in den Arbeitsgruppen von Prof. Dr. Ulrich Groth am Lehrstuhl für Organische Chemie im Fachbereich Chemie und Prof. Dr. Thomas U. Mayer am Lehrstuhl für Molekulare Genetik im Fachbereich Biologie an der Universität Konstanz.

Publikationen

Teile dieser Arbeit sind veröffentlicht in:

J. Braun, M. M. Möckel, T. Strittmatter, A. Marx, U. Groth, and T. U. Mayer

“Synthesis and Biological Evaluation of Optimized Inhibitors of the Mitotic Kinesin Kif18A”

ACS Chem. Biol. 2015, 10, 554–560

Weitere Publikationen:

H. Strobelt, E. Bertini, J. Braun, O. Deussen, U. Groth, T.U. Mayer, D. Merhof

“HiTSEE KNIME: a visualization tool for hit selection and analysis in high-throughput screening experiments for the KNIME platform”

BMC Bioinformatics 2012, 13 (Suppl 8):S4

J. Kastl, J. Braun, A. Prestel, H. Möller, T. Huhn, and T. U. Mayer

“M2I-1: A Small protein-protein interaction inhibitor targeting the mitotic spindle assembly checkpoint”

ACS Chem. Biol. 2015, submitted

1. Introduction ... 1

1.1 Cell Cycle ... 1

1.2 Kinesins in Mitosis and Cytokinesis ... 2

1.2.1 Structure of Kinesins ... 2

1.2.2 Mitosis and Cytokinesis ... 3

1.3 Relevance of Kinesins in Cancer ... 7

1.4 Screening for Kinesin Inhibitors ... 8

1.5 Chemical Genetics ... 10

2. Aim of the Work ... 12

3. Results and Discussion ... 14

3.1 Mklp2 and its Inhibitors SH1 and Paprotrain ... 14

3.1.1 Synthesis of Substituted Quinoxalines ... 15

3.1.2 Design of SH1 Analogs ... 15

3.1.3 Synthesis of Substituted 2-Phenylquinoxalines ... 16

3.1.4 Synthesis of 2-Thiophenquinoxalines ... 19

3.1.5. Prodrug Approach ... 20

3.1.6 Effects on Cells ... 22

3.1.7 Enzyme Coupled Assay ... 23

3.1.8 Malachite Green Assay ... 25

3.1.9 Summary and Outlook ... 26

3.2 Kif18A and its Inhibitor BTB-1 ... 29

3.2.1 Synthesis of BTB-1 ... 29

3.2.2 Design of BTB-1 Analogs ... 30

3.2.3 Synthesis of BTB-1 Analogs ... 31

3.2.4 Screening for Kif18A Inhibition and IC50 Determination ... 34

3.2.5 Mode of Inhibition and Selectivity ... 36

3.2.6 Cellular Toxicity Studies ... 39

3.2.7 Live Cell Imaging ... 42

3.2.8 Cellular Thermal Shift Assay ... 43

3.2.9 Immunofluorescence Imaging for Kif18A Localization ... 45

3.2.10 Tubulin Polymerization Assay and IC50 Determination ... 48

3.2.11 Structure Activity Relationships and Outlook ... 50

4. Summary ... 54

5. Zusammenfassung ... 56

6. Materials and Methods ... 58

6.1 General ... 58

6.2 Synthesis of SH1 Analogs ... 59

General Methods for Glyoxal Synthesis (Method A) ... 59

General Method for Preparation of Substituted Quinoxalines (Method B) ... 59

General Method for Preparation of Amide and Ester Analogs of SH1 (Method C) ... 59

3,4-Dichlorophenylglyoxal (1) ... 60

3,4-Difluorophenylglyoxal (2) ... 60

Phenylglyoxal (3) ... 61

4-Bromophenylglyoxal (4) ... 61

4-Nitrophenylglyoxal (5) ... 61

1-(5-Chlorothiophen-2-yl)-2,2-dihydroxyethan-1-one (6) ... 62

5-Chloro-2-phenyl-7-(trifluoromethyl)quinoxaline (13) ... 62

2-(3,4-Dichlorophenyl)quinoxaline-6-carboxylic acid (14) ... 62

2-(3,4-Difluorophenyl)quinoxaline-6-carboxylic acid (15) ... 63

2-Phenylquinoxaline (16) ... 63

2-(4-Bromophenyl)-5-chloro-7-(trifluoromethyl)quinoxaline (17) ... 64

2-(5-chlorothiophen-2-yl)quinoxaline (18) ... 64

5-chloro-2-(5-chlorothiophen-2-yl)-7-(trifluoromethyl)quinoxaline (19) ... 65

Ethyl 2-(3,4-dichlorophenyl)quinoxaline-6-carboxylate (20) ... 65

2-(3,4-Dichlorophenyl)-N,N-diethylquinoxaline-6-carboxamide (21)... 66

(2-(3,4-dichlorophenyl)quinoxalin-6-yl)(4-methylpiperazin-1-yl)methanone (22) ... 67

(2-(3,4-Dichlorophenyl)quinoxalin-6-yl)(morpholino)methanone (23) ... 67

2-(3,4-Difluorophenyl)-N,N-diethylquinoxaline-6-carboxamide (24) ... 68

(2-(3,4-difluorophenyl)quinoxalin-6-yl)(4-methylpiperazin-1-yl)methanone (25) ... 69

(2-(4-Fluoro-3-morpholinophenyl)quinoxalin-6-yl)(morpholino)methanone (26) ... 69

6.3 BTB-1 Analogs Synthesis ... 70

General Methods for the Preparation of Sulfones and Sulfoxides ... 70

1-Chloro-2-nitro-4-(phenylsulfonyl)benzene (70) ... 71

2,4-Dinitro-1-(phenylsulfonyl)benzene (52) ... 72

2,4-Dinitro-1-(phenylsulfinyl)benzene (64) ... 72

4-Fluoro-2-nitro-1-(phenylsulfonyl)benzene (53) ... 73

2-Nitro-1-(phenylsulfonyl)-4-(trifluoromethyl)benzene (54) ... 74

2-((4-Chloro-2-nitrophenyl)sulfonyl)thiophene (55) ... 74

2-((4-Chloro-2-nitrophenyl)sulfonyl)naphthalene (56) ... 75

2-((4-Chloro-2-nitrophenyl)sulfinyl)naphthalene (65) ... 76

4-Chloro-1-((4-methoxyphenyl)sulfinyl)-2-nitrobenzene (66) ... 78

1-Nitro-2-(phenylsulfonyl)benzene (59) ... 78

1-Nitro-2-(phenylsulfinyl)benzene (67) ... 79

1-((4-Methoxyphenyl)sulfonyl)-2-nitrobenzene (60) ... 79

1-((4-Methoxyphenyl)sulfinyl)-2-nitrobenzene (68) ... 80

2,4-Dinitro-1-tosylbenzene (61) ... 81

2,4-Dinitro-1-(p-tolylsulfinyl)benzene (69) ... 81

4-Chloro-1-((4-chlorophenyl)sulfonyl)-2-nitrobenzene (62) ... 82

1-((4-Chlorophenyl)sulfonyl)-2,4-dinitrobenzene (63) ... 83

4-Chloro-2-nitro-1-phenoxybenzene (71) ... 83

4-Chloro-1-iodo-2-nitrobenzene (72) ... 84

4-Chloro-2-nitrobenzenediazonium (74) ... 85

6.4 Biochemical and Cellular Assays ... 85

Protein Expression and Purification from Bacteria via Poly-histidine (His) Tag ... 85

Polymerization of Taxol Stabilized Microtubules ... 86

Malachite Green Assay ... 86

Enzyme Coupled Assay ... 87

Enzyme Coupled Assay SH1 Analogs ... 87

Enzyme Coupled Assay SH1 Titration with Triton X-100 ... 87

Enzyme Coupled Assay BTB-1 Analogs ... 88

IC50 (Kif18A) ... 88

Basal Kif18A ATPase Activity ... 88

Inhibition Mode of 59 ... 88

Cellular Thermal Shift Assay (CETSA) ... 88

Western Blot Analysis ... 89

Tubulin Polymerization Assay ... 89

IC50 (Tub.Polym.) ... 90

Cell Culture ... 90

Immunofluorescence ... 90

Live Cell Imaging ... 91

Alamar Blue Assay and EC50 Values ... 91

7. References ... 93

8. Appendix ... 107

8.1 Appreviations ... 107

1

1. Introduction

1.1 Cell Cycle

Cell reproduction is an essential process in all higher eukaryotes in order to develop a multicellular organism and replace cells that died due to natural causes or environmental damage.¹ The cell cycle can be divided into different distinct phases:

gap1 phase (G1), synthesis phase (S phase), gap2 phase (G2) and the mitotic phase (M phase) (Figure 1). In the G1 phase the cell grows and verifies, if the conditions are ideal for proliferation or if there are any inhibitory signals. As a result of this, the cell enters a quiescent state or progress in the cell cycle. In the S phase the deoxyribonucleic acid (DNA) is replicated and the chromosomes as well as the centrioles are duplicated. To ensure an error free duplication of the DNA, the G2 phase includes a DNA damage checkpoint. G1, S and G2 phase are also referred to as interphase.

Figure 1: Schematic representation of the cell cycle and the different phases.

In the M phase the duplicated chromosomes are separated by the mitotic spindle machinery and equally distributed into two daughter cells. To ensure the correct distribution of the genetic material to the daughter cells, the spindle assembly checkpoint (SAC) monitors the correct attachment of chromosomes to the mitotic spindle.² The final physical separation of the daughter cells is achieved through cytokinesis. All these events are orchestrated by different mechanisms in order to sustain genomic integrity. One important class of motor proteins involved in these processes is the kinesin superfamily.

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1.2 Kinesins in Mitosis and Cytokinesis

1.2.1 Structure of Kinesins

Kinesins have a modular composition and consist of a head domain, also referred to as motor domain, connected to a tail and typically form dimers.^3-4 The head domain is built on a structurally highly conserved β-sheet backbone flanked by α-helices and contains the catalytically active ATPase site as well as the microtubule binding site.^4-

5 The connection between head and tail is called neck linker, a short sequence that synchronizes the ATPase cycle of the dimers, and is present in nearly all kinesins. In contrast to the highly conserved head domain, the tail domain differs in length and sequence depending on the kinesin family (Figure 2).

Figure 2: Schematic representation of mitotic kinesins and their structures divided into their families. Adapted by permission from Nature Publishing Group.⁴

The tail is typically build up of α-helical coiled coils to achieve multimerization, interrupted by unstructured, natively unfolded sequences and bears recognition

3 sequences for co-proteins, regulatory kinases, cargo and in some cases another microtubule binding site.^{3-4, 6-7}

1.2.2 Mitosis and Cytokinesis

Mitosis is divided into different distinct phases: Prophase, prometaphase, metaphase, anaphase and telophase.

The main events during prophase are centrosome separation, which is a motor- dependent event, chromosome condensation and nuclear envelope break down (NEBD) (Figure 3, a). Kif11, also known as Eg5, is the only member of the human kinesin-5 family and essential for centrosome separation.^{4, 8-11} It consists of two heterodimers forming a tetramer with two heads at each end functioning as a microtubule cross-linker (Figure 2 and Figure 3, right panel 1).^{8, 12-13} Besides, Eg5 has also microtubule binding sites in the tail (Figure 2) and moves towards the plus end of microtubules, if attached to antiparallel microtubules.^{8, 14-16} Therefore, Eg5 enables the separation of the centrosomes by sliding antiparallel microtubules apart, which originate from different centrosomes (Figure 3, right panel 1).^{8, 14} The Eg5-driven microtubule sliding resists forces that tend to speed up sliding, like pulling forces of dynein on the centrosomal microtubules (Figure 3 right panel 2 and 3).¹⁷

The end of prophase in mammalian cells is reached with NEBD and followed by prometaphase, in which a bipolar mitotic spindle is formed and chromosome congression takes place (Figure 3, b). The mitotic spindle self-organizes through the dynamics of microtubules, which are influenced by motor- and microtubule binding- proteins in order to nucleate, capture, slide and reorient microtubules from both asters.¹⁸ Microtubules originating from opposite poles can either form antiparallel overlaps or capture kinetochores forming parallel bundles known as kinetochore microtubules (K-fibers). The major difference between these kinds of microtubules is their dynamic behavior: non kinetochore microtubules have a half-life of approximately 10 seconds compared to K-fibers with a half-life of several minutes.¹⁹ At the beginning of spindle assembly the activity of Eg5 as well as pulling from cortical myosin and pushing forces from kinetochores are required, and the continued complete separation of the centrosomes take place (Figure 3, right panel 3-5).^20-22 It was found that Kif15 (KLP2) overexpression compensates the loss of Eg5, suggesting

4

that both kinesins are able to slide away overlapping antiparallel microtubules (Figure 3, right panel 6).^23-24 Recent studies revealed that Kif15 preferably associates with parallel microtubules in K-fibers counteracting the forces generated by Eg5.^{23, 25} After assembly of a bipolar spindle its maintenance requires outward sliding forces generated by Eg5 and/or Kif15 and inward forces generated by KifC1, also known as HSET, a member of kinesin-14 family (Figure 3, right panel 4-6).^{24, 26-27} As shown in Figure 2 kinesin-14 family members have their motor domain at the C terminus and are microtubule minus end directed motors (Figure 3, right panel 5). The HSET analogs in Drosophila melanogaster (Ncd) and Schizosaccharomyces pombe (Klp2) are able to stabilize parallel and slide apart antiparallel microtubules, in the opposite direction compared to Eg5.^28-30 These findings highlight the antagonistic activity of Eg5 and HSET during bipolar spindle formation.^{26, 31-33} In parallel to bipolar spindle formation the chromosomes have to be attached to microtubules and aligned in the metaphase plate.³⁴ Multiple kinesins like CENP-E (Kif10), Kif18A, Kif2B and C (MCAK) are necessary for this process (Figure 3, right panel 7 and 8). CENP-E, for example, is a plus end-directed motor, which is able to transport kinetochores, and therefore, the whole chromosome, to the plus ends of microtubules (Figure 3, right panel 7).^35-39 When sister kinetochores are attached to microtubules originating from opposite poles they are able to oscillate between the poles by utilizing microtubule dynamics.

This is achieved by the regulation of microtubules growth and shrinkage and switching between them by kinetochores with the help of kinesins, like Kif18A.

Kif18A, member of the kinesin-8 family, is a plus end-directed motor involved in modulating microtubule plus end dynamics and is localized at kinetochores (Figure 2, Figure 3 right panel 8).^40-49 It is under debate, if Kif18Aposseses a microtubule depolymerization activity. One model suggests that newly arriving Kif18A molecules “bounce” into already plus-tip localized Kif18A ,which than falls off together with one tubulin dimer. The ability to depolymerize microtubules is consistent with the observed higher oscillation amplitude of kinetochores following Kif18A depletion and the resulting severe chromosome congression defect.42, 46, 50-51

The processivity of Kif18A is increased by a C-terminal microtubule binding site, which is also required for the mitotic function of the kinesin.^43-45 Kif2A, Kif2B and MCAK (Kif2C), like Kif18A, are important regulators of kinetochore oscillation

5 (Figure 3, right panel 8). These kinesins are located at both ends of microtubules and act as microtubule depolymerases (Figure 2, Figure 3, right panel 8 and 10).

Figure 3: The first row a-d illustrate the single steps from mitosis and their characteristics are mentioned. The second row 1-12 shows kinesins and dynein and their involvement in spindle assembly and chromosome interactions.

Reprinted by permission of Nature Publishing Group^.4

6

At the minus end of K-fibers, they generate a pulling force on kinetochores whereas at the plus end of microtubules, they seem to be involved in correcting erroneous kinetochore attachments and regulating the speed of kinetochore motility.^{50, 52-55} When the chromosomes are properly aligned metaphase is reached, which is followed by anaphase (Figure 3 c and d). In anaphase, the two sister chromatids are separated and pulled to opposite poles. The motor requirements in human cells for anaphase are still unknown.⁴ In telophase in mammalian cells, the chromosomes decondens and new nuclei are formed.

Afterwards, cytokinesis as the final physical division takes place. A main component of cytokinesis is the contractile ring, consisting of actin and myosin.^56-58 It assembles through actin filament polymerization by GTPase RhoA and myosin motor activity.^59-62 Another key component is the central spindle, which forms during anaphase progression. It consists of bundled antiparallel microtubules and serves as a signaling platform for the positioning of the cleavage furrow.4, 56, 63-64 For the assembly of the central spindle activity of different microtubule-associated proteins (MAPs) like protein regulator of cytokinesis (PRC1), the centralspindlin complex and the chromosomal passenger complex (CPC) are required. Additionally, members of the kinesin-6 family like mitotic kinesin-like protein 2 (Mklp2, Kif20A) and M-phase phosphoprotein 1 (MPP1, Kif20B) contribute to central spindle assembly (Figure 3, right panel 12). PRC1 is localized at the central spindle and contains a conserved central domain, which induces microtubule bundling.^65-66 Further, the N- terminus of PRC1 comprises an oligomerization and a Kif4A binding domain that enhances the localization to the central region.^{65, 67} Despite its interaction with PRC1, Kif4A has a key role in the regulation of microtubule dynamics and therefore controlling the size of the central spindle (Figure 3, right panel 12).^68-69 A dimer of Mklp1 (Kif23) together with a dimer of Cyk4 - a Rho family GTPase-activating protein (GAP) - forms the tetrameric centralspindlin complex and localizes to the center of the central spindle.^70-71 It supports microtubule bundling, RhoA regulation and recruits regulators of abscission.^{60, 72} The third important multi protein complex for central spindle assembly, the CPC, consists of Aurora B, survivin, borealin and inner centromere protein (INCENP) and is not only active at the central spindle during

7 anaphase, but throughout mitosis. Its activity is not only constricted by activation and localization of Aurora B and, related to that phosphorylation of central spindle components, it is also suggested to be involved in microtubule bundling.^73-76 The accumulation of Aurora B and polo-like kinase 1 (PLK1) is achieved by Mklp2.^77-78 MPP1 as well as Mklp2 are essential in the late steps of cytokinesis.^79-80

In summary, kinesins have many talents and functions ranging from their importance for the correct distribution of the genetic material during mitosis to the precise abscission of the daughter cells.

1.3 Relevance of Kinesins in Cancer

Due to the rapid development of resistance in cancer cells to antimitotic agents like docetaxol and other taxol derivatives, and severe side effects of those, there is an emergent need for new antimitotic drugs and new targets beside the mitotic spindle.

In that context and as a result of their functions during cell division, kinesins have emerged as targets for cancer therapy.^81-84 For example, several inhibitors for Eg5 and CENP-E have entered clinical trials.⁸¹ For other kinesins so far no inhibitors are available albeit they could be used as promising targets. In Table 1 some examples of kinesins, their connection to different cancer types and their published inhibitors are shown. Kinesins are not only used as drug targets, but also serve as cancer and prognostic markers. ^85-90 Furthermore, in some cases overexpression of kinesins are involved in the development of drug resistance.⁹¹ It is obvious that kinesins can serve as targets for cancer chemotherapy but therefore their cellular functions have to be fully understood and selective inhibitors have to be developed.

8

Table 1: In the first row kinesins listed followed by their connection to cancer.

The last row shows the published inhibitors if available. Adapted by permission from Nature Publishing Group.⁸¹

Kinesin Expression status Lead

compounds/Inhibitors Kif2A Overexpression promotes development of

squamous cell carcinoma of tongue⁹²

MCAK

Prognostic marker in colon cancer;⁸⁸ overexpressed during breast tumorigenesis and in gastric cancer associated with poor prognosis^93-94

Sulfoquinovosylacylglycer ol inhibitors⁹⁵

Kif4A Overexpressed in cervical cancer;⁹⁶ prognostic marker in lung cancer⁸⁹

CENP-E

Down regulated in hepatocellular carcinoma;⁹⁷ overexpression is associated with poor prognosis in breast cancer⁹⁸

GSK923295⁹⁹

Eg5

Highly expressed in blast crisis chronic myelogenous leukemia;¹⁰⁰ overexpressed in pancreatic cancer¹⁰¹

Monastrol,¹¹ Ispinesib,¹⁰² Tritylcysteine¹⁰³ and

analogs Kif14

Predictor of grade and outcome in breast and ovarian cancer;^85-86 prognostic marker in lung cancer⁸⁷

Kif18A

Overexpressed in colorectal cancer;¹⁰⁴ overexpressed and associated with metastasis and poor survival in breast cancer¹⁰⁵

BTB-1¹⁰⁶

Mklp1 Overexpressed in glioma¹⁰⁷

Mklp2 Overexpressed in pancreatic cancer¹⁰⁸ Paprotrain¹⁰⁹ HSET

Predictive of brain metastasis of lung cancer;⁹⁰ overexpressed in docetaxel resistant breast cancer cells⁹¹

AZ82¹¹⁰

KifC3 overexpressed in docetaxel resistant breast cancer cells⁹¹

1.4 Screening for Kinesin Inhibitors

In order to find inhibitors for kinesins, robust and cost effective screening assays are necessary that are suitable for high-throughput screening (HTS) or high-content screening (HCS). One common property of kinesins is their ATPase activity, which

9 could be used as readout. Therefore the released phosphate has to be detected in a quantitative manner.

In a malachite green assay (MGA) the released phosphate forms a complex with molybdate of the stoichiometry PMo12O403-(Scheme 1).^111-113 The molybdate anion forms another complex with malachite green exposing its typically green color (Scheme 1). The exact nature of the complex is not described so far. The color change can be detected by measuring the absorption at 650 nm.

Scheme 1: Formation of molybdate and phosphate complex and further reaction to green colored complex with malachite green.

Another spectroscopic approach is the enzyme-coupled assay (ECA) (Figure 4).¹¹⁴ This assay utilizes the regeneration of ATP by pyruvate kinase and phosphoenolpyruvate (PEP). The resulting pyruvate is reduced to lactate under consumption of NADH by lactate dehydrogenase (Figure 4). The decrease of NADH can be monitored using its absorption at 340 nm.

Figure 4: Schematic representation of the ECA. The kinesin-consumed ATP is regenerated by an enzyme cascade using NADH. Therefore, the decrease of absorbance at 340 nm can be measured to quantify NADH consumption.

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1.5 Chemical Genetics

In the 1990´s Timothy J. Mitchison and Stuart L. Schreiber shaped the interdisciplinary approach of chemical genetics.^115-116 Like in classical genetic approaches, the function of proteins in cells or even in whole organisms is evaluated using small molecules.^117-118 In classical genetics, protein functions are disturbed by manipulation of the genomic information, antibody injection or RNA interference (RNAi). However, the chemical genetic approach has several advantages compared to classical genetics and enables the investigation of highly dynamic processes like mitosis in a highly spatial and temporal manner. Small molecules are able to penetrate the cell membrane without additives, show a fast and strong effect and therefore can be applied at specific time points and their activity can be timely restricted by wash-out.

As in classical genetics, one can distinguish between forward and reverse chemical genetic approaches (Figure 5).

Figure 5: Schematic representation of forward and reverse chemical genetic screens. Adapted from HFSP Journal.¹¹⁸

In a forward chemical genetic screen, organisms or cells are treated with a diverse collection of small molecules and afterwards the desired phenotype has to be selected. Following that, the cellular target of the small molecule and its specificity towards its target has to be identified. On the contrary, the reversed chemical genetic screen starts with a target protein and the search for small molecules that inhibit its

11 activity. After elucidation of the specificity, cells or organisms are treated with the small molecule and the received phenotype is examined. Both methods include critical steps. For the forward approach the identification of the cellular target is often challenging, because the received phenotype could be a consequence of perturbing multiple protein activities rather than that of a single one.¹¹⁹ For the reverse approach the identification of the phenotype is the bottle neck. Since the identified compound is so far only tested for selectivity in vitro it could act with different targets in a cellular environment.Nevertheless, chemical genetics is a powerful tool to dissect highly dynamic cellular processes. As a result of the rapid development in organic and combinatorial chemistry, compound libraries consisting of natural and synthetic products are readily available.^120-121 Hence, the discovered molecular tools from chemical genetic screens are not only of great value for basic science but also may open up novel avenues for the treatment of diseases.

The aim of this thesis was the development and biological evaluation of inhibitors targeting the mitotic kinesins Mklp2 and Kif18A.

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2. Aim of the Work

Kinesins are essential for the correct distribution of the genetic material into the daughter cells during mitosis and meiosis.⁴ Different kinesins can have multiple functions at different time points during the cell cycle. Specific small molecule inhibitors are therefore ideal tools to dissect the different functions of kinesins, because they act in a very rapid and often reversible manner. For some kinesins inhibitors are already available as described in 1.3.

For the kinesin Mklp2, Paprotrain was discovered in 2010 by Tcherniuk et al.¹⁰⁹, whereas a small molecule named SH1 was identified and characterized in the laboratory of Prof. Thomas U. Mayer in 2009 ¹²², which served as lead compound for the SH1 project. SH1 showed a low water solubility, which complicated its characterization. Thus, the aim of this part of the thesis was to synthesize polar SH1 derivatives in order to enhance water solubility and establish structure activity relationships. Therefore, the synthesized small molecules should be studied for their ability to inhibit Mklp2 in vitro as well as their effect on cells. To achieve this, different synthetic approaches, biochemical and cellular assays were performed.

For the kinesin Kif18A, a small molecule named BTB-1 was identified as potent inhibitor by Catarinella et al. in 2009 ¹⁰⁶. BTB-1 shows the desired inhibition of the ATPase activity of Kif18A in vitro and led to an increased percentage of cells in mitosis upon cellular treatment. Nevertheless, cells treated with high concentration of BTB-1 did not show elongated spindles that are characteristic for the depletion of Kif18A.

Here, the aim was to establish a synthetic pathway in order to synthesize a small molecule collection of BTB-1 analogs. The small compound collection should be investigated for their inhibitory activity and selectivity towards Kif18A. Further, structure activity relationships should be established for a better understanding of the mode of action of BTB-1. Next, the small compound library should be studied for their effect on the cellular level. Different biochemical and cellular assays were carried out to characterize the new small molecule collection.

13 In addition to their undeniable value for molecular biology, small molecules inhibitors of Kif18A and Mklp2 could serve as starting point for the development of novel drugs for mitosis-related diseases, such as tumors that overexpress these kinesins104-105, 108.

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3. Results and Discussion

3.1 Mklp2 and its Inhibitors SH1 and Paprotrain

As described in 1.2 Mklp2 is indispensable for cytokinesis. In the group of Prof.

Thomas U. Mayer an inhibitor for Mklp2, called SH1, was identified in a reversed chemical genetic screen (Scheme 2, a).¹²² The treatment of HeLa cells with SH1 caused a cytokinesis defect resulting in binucleated cells.¹²² SH1 was successfully resynthesized in my diploma thesis by condensation of ortho-phenylenediamines with glyoxales and the biological activity on HeLa cells were reproduced (Scheme 2, a).¹²³ The main problem of SH1 was its poor water solubility.

Scheme 2: The structure of SH1 and Paprotrain as well as their synthesis are shown. a) SH1 was synthesized using phenylglyoxal and phenylenediamines receiving the desired quinoxaline. b) Literature described synthesis of Paprotrain by aldol condensation of 3-pyridinecarboxaldeyde and 3-indolylacetonitrile.

In 2010 Tcherniuk et al. published Paprotrain as a selective Mklp2 inhibitor with a half-maximal inhibitory concentration (IC50) of 0.83±0.1 µM.¹⁰⁹ It was synthesized using an aldol reaction of 3-indolylacetonitrile with 3-pyridinecarboxaldehyde (Scheme 2, b). The treatment of cells with Paprotrain resulted in a binucleated cellular phenotype similar to that received from RNAi-mediated depletion of Mklp2.

Paprotrain compared to SH1 shows a higher solubility in aqueous media but is sensitive to light induced isomerization due to its double bond. In the following, the focus will be on SH1 and the synthesis of analogs while Paprotrain serves as point of comparison. In order to establish structure activity relationships (SARs) and enhance water solubility new compounds based on SH1 should be synthesized in fast and efficient manner.

15 3.1.1 Synthesis of Substituted Quinoxalines

Quinoxalines are readily available by the reaction of 1,2-arylenediamins and various two carbon unit donors like α-diketones, α-halocarbonyl compounds, oxalic acid derivatives, epoxides and dihalides to mention some.¹²⁴ All these reactions are restricted to symmetrical diamine building blocks, because otherwise regioisomeric mixtures of quinoxalines are obtained. To achieve regioselectivity addition of different catalysts is described in literature.^125-130 As described in my diploma thesis SH1 can be synthesized in a regioselective manner using glyoxal hydrate,¹²³ which served as the synthetic approach for further synthesis of SH1 analogs (Scheme 2, a).

3.1.2 Design of SH1 Analogs

In order to improve the water solubility of SH1 and establish SARs, different approaches were used (Scheme 3). One approach was to introduce different polar substituents at the phenyl or quinoxaline ring, which was started in my diploma thesis and followed up (Scheme 3, a).¹²³ The second approach was to isosterically exchange the phenyl moiety to a thiophene (Scheme 3, b). This exchange should improve the solubility remarkably since heterocycles, like quinoxalines and thiophenes, are readily water soluble compared to the aromatic phenyl moiety. The last approach was to enhance the solubility by conjugation to an enzymatic cleavable carbohydrate, so-called prodrug approach (Scheme 3, c). As linker between the carbohydrate and the bioactive quinoxaline ethylene glycol scaffolds of different length were chosen.

Despite their enhancement of water solubility, they also show a high flexible backbone, which could be beneficial for enzymatic cleavage of the ester bond or if not cleaved for binding of the quinoxaline to its target. A second effect is, that the compounds uptake is not restricted to passive diffusion, because carbohydrates are actively transported into cells. All approaches are based on the initial reaction of phenylenediamines with glyoxales. In the next section the approach a will be described.

16

Scheme 3: Different approaches for enhancement of water solubility. a) Introduction of polar groups at the phenyl or quinoxaline ring. b) Isosterical exchange of the phenyl moiety to more water soluble thiophene. c) Conjugation of SH1 analog to propargyl-2-acetamido-2-deoxy-α-D-glucoside.

3.1.3 Synthesis of Substituted 2-Phenylquinoxalines

First, analog to methods described in literature, differently substituted 2-phenyl- glyoxal hydrates (compound 1-5) were synthesized as well as one thiophene analog 6.123, 131-133 Therefore, the selective oxidation of activated methyl groups of acetophenones with selenium (IV) oxide (SeO2) was applied (Scheme 4, compounds 1-5). For the thiophene glyoxal hydrate 6 no yield is given, because the crude product was directly used for condensation. The next step consists of the condensation of phenylenediamines with the received glyoxal hydrates (Table 2). Yields marked with * correspond to small molecules synthesized during my diploma thesis using phenacylbromids instead of glyoxales and are listed for completeness (Table 2, SH1 and compounds 7-12).¹²³

17 Scheme 4: Synthesis of glyoxal hydrates by SeO2 mediated oxidation of activated methyl groups. The yields are shown except for the thiophene glyoxal, which was directly used for further synthesis due to instability.

Compound 7 and 8 were regioisomeric mixtures. Compared to the previously used phenacylbromid approach the yields were improved. As mentioned above, the thiophene glyoxal was used directly without further purification and converted to the corresponding quinoxalines. Two examples 18 and 19 are given in Table 3 and this compound class is discussed in chapter 3.1.5 in further detail.

Table 2: Reaction scheme for the synthesis of substituted 2-phenylquinoxalines.

The substitution pattern and yields are given in the table. Yields marked with * correspond to small molecules synthesized during my diploma thesis.

Cpd. R¹ R² R³ R⁴ R⁵ Yield

SH1 Cl Cl Cl H CF₃ 83%*

7 Cl Cl Cl H CF₃ 33%

regioisomers*

8 F F Cl H CF₃ 28%

regioisomers*

9 Cl Cl H H H 47%*

10 F F H H H 35%*

11 F F H COOEt H 14%*

12 OMe OMe H H H 71%*

13 H H Cl H CF₃ 65%

14 Cl Cl H COOH H 94%

15 F F H COOH H 75%

16 H H H H H 97%

17 H Br Cl H CF₃ 72%

18

Table 3: The reaction scheme depicts the synthesis of 2-thiophenequinoxalines and the corresponding compounds and yields are given in the table.

Cpd. R³ R⁵ Yield

18 H H 51%

19 Cl CF3 50%

To enhance cell membrane permeability the free acids were converted into amides or ethyl esters. N-Methylpiperazine and morpholine were selected as suitable amines, because they bear another hetero atom, which enhances the solubility for polar solvents, and diethylamine as a less bulky substituent. Further, the sterical demand of this position for biological activity was addressed. Therefore, the carbonic acid was transformed into an acid chloride to enhance reactivity and reacted with the corresponding nucleophile. In a first attempt to synthesize the morpholide 26, the base was used as solvent and the reaction was heated, which resulted in a nucleophilic aromatic substitution (SNAr) where morpholine replaced one fluoride of the phenyl ring (Table 4, 27). To circumvent this problem the corresponding amine was used in a small excess (1.1 equiv.) and triethylamine (NEt3) was added as acid scavenger (Table 4). Interestingly, all difluoro substituted compounds 24-26 were received in lower yields compared to their dichloro analogs 21-23. This could be explained by the altered electronegativity through the two fluoride substituents.

19 Table 4: Reaction pathway to amides and ethyl ester are shown. The corresponding acid is first converted into its acid chloride followed by conversion to the desired amid/ester. The yields and different products are listed in the table.

Cpd. R¹ R² R⁴ Yield

20 Cl Cl COOEt 59%

21 Cl Cl diethylamide 74%

22 Cl Cl N‐methylpiperazide 53%

23 Cl Cl morpholide 68%

24 F F diethylamide 53%

25 F F N‐methylpiperazide 37%

26 morpholine F morpholide 24%

3.1.4 Synthesis of 2-Thiophenquinoxalines¹³⁴

The synthesis of 27-40 described in this part was carried out by Kim M. Leitner during her bachelor thesis. First, differently chlorinated 2-acetylthiophenes were synthesized according to literature procedures applying the swamping catalyst effect (Scheme 5).^135-137 The swamping catalyst effect, which is an excess of AlCl3, leads to the complexation of the carbonyl group and prevents α-halogenation of it.

Scheme 5: Synthesis of differentially chlorinated 2-acetothiophenes by temperature control and excess of 3 equiv. AlCl3.

The regioselectivity is achieved by temperature control. This was done to receive electron poor thiophenes comparable to dichloroacetophenones used for the

20

synthesis of SH1 analogs. For the formation of 2-thiophenequinoxalines the synthetic procedures described in 3.1.4 were used. The acetylthiophenes were oxidized to glyoxales followed by condensation to phenylenediamines. The amides and esters were synthesized using the corresponding acid chloride as described in 3.1.4. Table 5 shows the received compounds and achieved yields. For further details and exact synthetic procedures see bachelor thesis of Kim M. Leitner.¹³⁴

Table 5: Synthesized 2-thiophenequinoxalines and yields. For the amides/esters the yields correspond to the amidation/esterification via the acid chloride pathway.

Cpd. R¹ R² R³ R⁴ R⁵ Overall yield

27 H H H H H 51%

28 Cl Cl Cl H CF₃ 50%

29 Cl H Cl H CF₃ 13%

30 H Cl Cl H CF₃ 11%

31 Cl H H H H 19%

32 H Cl H H H 16%

33 Cl H H COOH H 11%

34 Cl Cl H COOH H 54%

35 H Cl H COOH H 30%

36 Cl H H diethylamide H 30% (in respect to the acid)

37 Cl H H morpholide H 18% (in respect to the

acid)

38 Cl H H COOMe H 32% (in respect to the

acid)

39 H Cl H COOMe H 33% (in respect to the

acid)

40 Cl H H COOEt H 22% (in respect to the

acid)

3.1.5. Prodrug Approach¹³⁸

The work presented in this section was carried out by Sabrina Batke during her bachelor thesis. The main reaction used herein was the Huisgen 1,3 dipolar

21 cycloaddition of alkines and azides.¹³⁹ Beforehand, the quinoxaline structures were synthesized as described in 3.1.4. The acid chlorides were esterified with ethylene glycol linkers of different lengths bearing a terminal azide group (Table 6). With the azide in hand the next step was the copper (I) catalyzed 1,3 dipolar cycloaddition.

Table 6: Schematic representation of the synthesis of azide bearing ethylene glycol linker and the corresponding yields are given in the table.

Cpd. R¹ R² Yield

41 F OCH₂CH₂N₃ 76%

42 F OCH₂CH₂OCH₂CH₂N₃ 59%

43 F O(CH₂CH₂O)₃CH₂CH₂N₃ 76%

44 Cl OCH₂CH₂N₃ 64%

45 Cl OCH₂CH₂OCH₂CH₂N₃ 64%

46 Cl O(CH₂CH₂O)₃CH₂CH₂N₃ 65%

Table 7: Scheme of the copper (I) mediated 1,3 dipolar cycloaddition of azides and alkine modified carbohydrates. The yields of the synthesized compounds are shown in the table.

Cpd. R n Yield

47 Cl 1 35%

48 F 1 31%

49 Cl 2 60%

50 F 2 34%

51 F 4 36%

22

Therefore, the reaction was carried out with propargyl-2-acetamido-2-deoxy-α-D- glucoside under microwave irradiation and copper catalysis.^140-142 The results are summarized in Table 7. For further details of synthetic procedures see bachelor thesis of Sabrina Batke.¹³⁸

3.1.6 Effects on Cells

First, the cellular effect of selected compounds was investigated and the results received during my diploma thesis were completed using the same experimental setup. As described in the PhD thesis of Stefan Hümmer, the inhibition of Mklp2 should result in a failure of cytokinesis and therefore lead to binucleated cells.^122-123 S-phase synchronized HeLa cells were treated with 100 µM compound or 1% DMSO as solvent control and imaged for 18 h using live cell imaging (Figure 6). It was distinguished between mononucleated, binucleated and undefined cells. All compounds marked with “*” showed crystallization during the experiments.

Compounds marked with ⁺ were only tested once. Compounds 12, 15 and 20-26 did not show any significant effect on cells. The resynthesized and commercial SH1 showed similar activity in contrast to the regioisomeric mixture of SH1 7, which was less active. Small molecules 9 and 11 showed an activity comparable to SH1.

23 Figure 6: Schematic representation of assay setup. S-phase synchronized HeLa cells were treated with 100 µM compound or 1% DMSO as solvent control and observed for 18 h. It was distinguished between mononucleated and binucleated cells. Compound marked with * showed crystallization during the experiment, whereas compounds marked with ⁺ were only tested once. For all other small molecules the mean of three independent experiments and standard deviations are shown.

These results suggest that the substitution pattern of the phenyl moiety is essential for activity and that small changes at the quinoxaline are tolerated (9, 11), but bulkier substituents diminish activity (21-26). It is noticeable that all active small molecules showed crystallization during the assay and thus, the exact active concentration of the corresponding small molecule is unknown. With the first hints for bioactivity, the next step was to confirm Mklp2 as the target in vitro.

3.1.7 Enzyme Coupled Assay

For the in vitro assays His-tagged recombinant Mklp2 motor domain (Mklp2^MD) was used. As described in 1.4 the ECA is a well-established method to determine the

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Mononuclear Binuclear Undefined

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ATPase activity of kinesins and was used to investigate, if the synthesized small molecules are able to inhibit the ATPase activity of Mklp2^MD. First, the assay condition were evaluated by titration of Mklp2^MD, taxol stabilized microtubules and ATP. After that a compound collection was applied at a concentration of 50 µM. DMSO was used as solvent control and the ATPase activity was set to 100%, whereas 50 µM Paprotrain was used as a positive control.

Figure 7: The ATPase activity of Mklp2^MD is shown relative to the DMSO control.

Small molecules were used in a concentration of 50 µM. All compounds were tested with two different end concentrations of DMSO. The first bar shows the activity with a final concentration of 5% and the second bar of 10% DMSO. Bars colored in red mark compounds, which showed no linear decrease of absorption whereas yellow bars indicate that linear ranges were observable. Green bars point to linear absorbance decrease during the whole assay. Average of three independent experiments and standard deviations are shown.

Due to the solubility problems experienced in the cellular assays two different DMSO concentrations (5% and 10%) were used, in order to enhance the solubility of the small molecules in polar media. The screening results are depicted in Figure 7.

Despite the increased concentration of DMSO, crystallization of some compounds was observed. Owing to the high absorbance of SH1 at 341 nm the crystallization resulted in a nonlinear absorption course resulting in a more difficult determination of the ATPase activity. All compounds showing that behavior are colored in red in

1. Bar ATPase activity

5% DMSO 2. Bar ATPase activity 10% DMSO 0%

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ATPase activity rel. to DMSO control

25 Figure 7 (SH1, 8, 13, 17, 19, 21 and 7). Yellow bars indicate that a linear range is observable but not for the whole assay and green bars show a linear behavior for the whole time. For 19 and 20 the solubility was enhanced as a result of the higher DMSO concentration, leading to better assay results. Paprotrain, for example, showed a linear decrease of absorption at 340 nm independent of the DMSO concentration and showed the highest inhibitory activity. In a second approach Triton X-100 was added additionally to higher DMSO concentration as detergent in a final concentration of 0.1%, which resulted in crystallization inhibition but also in a complete loss of inhibitory activity of SH1 (Figure 8). Furthermore, two different microtubule (MT) concentrations were used, but they did not show any influence on the ATPase activity. As a consequence of the readout problem caused by the absorption of SH1, the malachite green assay was used as an alternative approach.

Figure 8: The ATPase activity of Mklp2^MD in presence of 0.1% Triton X-100 is shown relative to the DMSO control. SH1 was applied in different concentrations ranging from 100-25 µM. Blue bars indicate the ATPase activity at 2 µM microtubules (MT) and orange at 400 nm MT´s. No inhibition of SH1 and no significant difference between the applied MT concentrations was observed.

Average of three independent experiments and standard deviations are shown.

3.1.8 Malachite Green Assay

Since the readout of the MGA is absorbance measurements at 650 nm SH1 should not interfere with the assay. In the beginning, the assay conditions were determined by titration of Mklp2^MD. In order to enhance the solubility of SH1 Triton X-100 was

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25 µM SH1 50 µM SH1 75 µM SH1 100 µM SH1

ATPase activity rel. to DMSO control

2 µM MT 400 nM MT

26

added in a final concentration of 0.1%. Mklp2^MD showed no sensitivity towards Triton X-100 in the assay, excluding interactions of the additive with the kinesin or other assay components. First, different concentrations of SH1 were used to elucidate if SH1 inhibits Mklp2^MD in a concentration dependent manner. As described above, DMSO was used as solvent control and the activity was set to 100%. Unexpectedly, no inhibition was observed at the different concentrations of SH1 (Figure 9).

Figure 9: Mklp2^MD activity is shown at different SH1 concentrations. No concentration dependent inhibition was observed. Average of three independent experiments and standard deviations are shown.

3.1.9 Summary and Outlook

In summary, the established synthesis route using glyoxales as starting material is a valuable tool to build up a library of varied substituted quinoxalines starting from commercial available building blocks. With the help of this approach compounds 13- 26 were synthesized in good and the thiophene analogs 27-40 in medium yields. The difference in the yields of the 2-thiophenequinoxalines is due to the lower stability of thiopheneglyoxales compared to phenylglyoxales.¹³⁴ All in all this approach is suitable for multistep synthesis as described for 2-acetamido-2-deoxy-α-D-glucosides in 3.1.6 Prodrug approach.¹³⁸ Further, the solubility of the compounds in aqueous media was raised by the introduction of polar groups like methoxy, carboxylic acid or amid groups. The biochemical evaluation for SH1, 7-11, 13, 17-19 and 21 failed caused by their low water solubility and crystallization during the ECA. Even the

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SH1 100 µM SH1 50 µM SH1 25 µM SH1 10 µM Mklp2MDactivity

27 attempt to enhance solubility with a higher DMSO concentration was not sufficient to prevent crystallization. Hence, SH1 serves as point of reference it was titrated using Triton X-100 as additive for solubility enhancement. As a result the crystallization of SH1 was avoided but no inhibitory activity was detectable. To avoid the interference of SH1 absorption at 341 nm with the assay readout the MGA was applied. As described before Triton X-100 was added as detergent and SH1 was used in different concentrations. In accordance with the ECA results addition of Triton X-100 resulted in a complete loss of inhibitory activity of SH1. It seems that the low solubility of SH1 is necessary for its activity. In the cellular assay it was also proven, that only small molecules that crystallized during the assay showed bioactivity. Therefore, the idea is that the low solubility of SH1 is necessary in order to develop its bioactivity and that SH1 acts as an aggregation based inhibitor. In literature different approaches are described to detect aggregation based inhibitors and β-lactamase was used as model system.^143-147 The general approach was to diminish inhibition by addition of Triton X-100. As described above SH1 shows this effect and could be termed as detergent dependent inhibitor. To identify SH1 as an aggregation based inhibitor the proof of the inhibitory activity without detergence has to be shown. The ECA could not be used for this, but the MGA as well as other assays suitable for detection of ATPase activity like using radioactive labeled ATP or Förster resonance energy transfer (FRET) based ATP probes can be applied.^148-149 Another possibility would be to alter the molecular nature of SH1 in a way that the inhibition of Mklp2 remains and the water solubility is enhanced. Looking at the structure of Paprotrain the pyridine moiety, as a nitrogen containing aromatic heterocycle, could be replaced by the substituted quinoxaline scaffold of SH1, resulting in a fusion of SH1 and Paprotrain (Scheme 6). Further, the 3,4-dichlorophenyl residue could be replaced by methylindol as described by Finlay et al. (Scheme 6).¹⁵⁰ In their work they described the exchange of a dichlorophenyl by a methylindol moiety resulting in a higher efficacy of the small molecule as IKur

inhibitor. For the synthesis of these indole substituted quinoxalines the glyoxal approach can be used. The synthesis of 5-chloro-2-(1-methyl-1H-indol-3-yl)-7- (trifluoromethyl)quinoxaline is under current investigation.

28

Scheme 6: Synthetic approaches to enhance solubility in polar media by combination of SH1 and Paprotrain (upper panel) or exchange of dichlorophenyl by methylindole (lower panel).

The last mentioned approach to modify the structure of SH1 has the benefit that a soluble inhibitor is in general a better analyzable small molecule concerning its bioactivity and could be used as a tool in molecular biology. Furthermore, the newly developed inhibitor could serve as starting point for additional synthesis of selective Mklp2 inhibitors and could open up new avenues for treatment of cytokinesis related diseases like Mklp2 overexpressing tumors.¹⁰⁸

29

3.2 Kif18A and its Inhibitor BTB-1

The unique mitotic kinesin Kif18A integrates plus-end directed motility with the ability to affect microtubule dynamics. It is essential for the accurate architecture of the mitotic spindle and proper alignment of chromosomes in mammalian cells.^42-47 Cells depleted of Kif18A by RNAi display elongated spindles with hyperstable microtubules and the majority of chromosomes is scattered throughout the spindle.^42-46 Since multiple cancer types show an elevated level of Kif18A, its function might be beneficial for the survival and development of these cells.^104-105 So far BTB-1 is the only known inhibitor of Kif18A. As shown by Catarinella et al. in 2009¹⁰⁶, BTB-1 inhibited the microtubule-stimulated ATPase activity of Kif18A with an IC50 value of 1.7 µM in a reversible manner. Treatment of HeLa cells with high concentrations of BTB-1 did not result in a phenotype with elongated spindles reminiscent of Kif18A depletion, but resulted in an increase of the mitotic index. This result showed that BTB-1 is bioactive in tissue culture cells. Based on the different phenotypes, it was speculated that Kif18A might not be the only or relevant binding partner of BTB-1 in cells. Therefore, a small molecule collection of structurally near analogs of BTB-1 should be synthesized in order to establish SARs and to get a better understanding of the inhibition mode of BTB-1. Further, their biological activity on cultured tissue cells and in vitro should be evaluated.

3.2.1 Synthesis of BTB-1

BTB-1 was synthesized according to known literature procedures (Scheme 7).106, 151-152 The first step consists of a nucleophilic aromatic substitution (SNAr) under basic conditions to displace the activated chloride at the aromatic core by a thiophenolate. Subsequent oxidation of the resulting sulfide by H2O2 yields BTB-1. This general approach was used to build up the small library of BTB-1 analogs as described in 3.2.3. The synthetic pathway starts from commercially available building blocks and represents a robust and cost-efficient method to assemble a small scale compound collection in a fast manner.

30

Scheme 7: BTB-1 was synthesized using a nucleophilic aromatic substitution under basic conditions followed by oxidation to the corresponding sulfone with H2O2 in an overall yield of 74%.

3.2.2 Design of BTB-1 Analogs

A subset of closely related analogs of BTB-1 were synthesized to get a better understanding, which structural motifs of BTB-1 are necessary for its inhibitory activity against its target Kif18A, and to potentially find more effective inhibitors. To this end, the unsymmetrically substituted diphenylsulphone BTB-1 was divided into three scaffolds: the two phenyl moieties I, II and a linker between them (Figure 10).

To enhance lipophilicity and cell membrane permeability of BTB-1, lipophilic groups like fluorine and trifluoromethyl were introduced at phenyl moiety I in para position to the linker. Additionally, the electron withdrawing group NO2 was introduced to elucidate how altered electronegativity and sterical demand at this position affect the inhibitory activity towards Kif18A. In order to investigate the importance of the NO2 group in ortho position at phenyl moiety I for Kif18A inhibition, it was shifted to the meta position to the linker.The phenyl moiety II was isosterically exchanged to thiophene and the aromatic system was expanded to naphthalene. Various substituents (Cl, OMe, Me) were introduced in para position to the linker (phenyl moiety II). Finally, the sulfone linker was changed to an ether or sulfoxide to analyze the effect of the geometry of the linker on the inhibition of Kif18A.

Figure 10: Structure of BTB-1 and its division into scaffolds I, II and linker.

31 3.2.3 Synthesis of BTB-1 Analogs

The synthetic approach for BTB-1, as described in 3.2.1, was used in order to synthesize differently substituted sulfones. Starting with a nucleophilic aromatic substitution under basic conditions followed by H2O2 oxidation the sulfones 52-63 were received in low to good yields (Table 8).

Table 8: Reaction scheme for the synthesis of substituted diphenylsulfones. The substitution pattern and yields over two steps are given in the table. For BTB-1, the literature yield is given for comparison. Compounds marked with * were synthesized using sodium thiophenolate and 0.3 equiv. NaOH.

Cpd. R¹ R² Overall yield

BTB-1 Cl Ph 74%¹⁰⁶

52* NO2 Ph 25%

53* F Ph 12%

54* CF3 Ph 26%

55 Cl 2-thiophene 68%

56 Cl 2-naphthalene 6%

57 Cl 4-methylbenzene 7%

58 Cl 4-methoxybenzene 12%

59 H Ph 3%

60 H 4-methoxybenzene 5%

61 NO2 4-methylbenzene 18%

62 Cl 4-chlorobenzene 55%

63 NO2 4-chlorobenzene 77%

The racemic sulfoxides 64-69 were synthesized by changing the oxidant to meta-chloroperoxybenzoic acid (m-CPBA) and using it in stoichiometric quantities to receive a single oxidation process to the sulfoxides (Table 9).