Novel Small Molecule Inhibitors for the Human Kinesins

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

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

Table of content

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-2-nitro-1-tosylbenzene (57) ............................................................................ 76

4-Chloro-1-((4-methoxyphenyl)sulfonyl)-2-nitrobenzene (58) ...................................... 77

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

Introduction

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.

Introduction

2

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

Introduction

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

Introduction

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

Introduction

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

Introduction

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

Introduction

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.

Introduction

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

prognosis93-94

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

Introduction

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.

Introduction

10

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

Introduction

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.

Aim of the Work

12

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.

Aim of the work

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.

Results and Discussion Mklp2 and its inhibitors SH1 and Paprotrain

14

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 OCH2CH

2N

3 76%

42 F OCH2CH

2OCH

2CH

2N

3 59%

43 F O(CH2CH

2O)

3CH

2CH

2N

3 76%

44 Cl OCH2CH

2N

3 64%

45 Cl OCH2CH

2OCH

2CH

2N

3 64%

46 Cl O(CH2CH

2O)

3CH

2CH

2N

3 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 (Mklp2MD) was

used. As described in 1.4 the ECA is a well-established method to determine the

0%

20%

40%

60%

80%

100%

Mononuclear

Binuclear

Undefined


24

ATPase activity of kinesins and was used to investigate, if the synthesized small

molecules are able to inhibit the ATPase activity of Mklp2MD. First, the assay condition

were evaluated by titration of Mklp2MD, 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 Mklp2MD 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%

20%

40%

60%

80%

100%

120%

140%

ATP

ase

act

ivit

y re

l. t

o D

MSO

co

ntr

ol


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 Mklp2MD 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 Mklp2MD. In order to enhance the solubility of SH1 Triton X-100 was

0%

20%

40%

60%

80%

100%

120%

140%

160%

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

ATP

ase

act

ivit

y re

l. t

o D

MSO

co

ntr

ol

2 µM MT 400 nM MT


26

added in a final concentration of 0.1%. Mklp2MD 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 Mklp2MD 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: Mklp2MD 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

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

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

Mkl

p2

MD

acti

vity


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

Results and Discussion Kif18A and its inhibitor BTB-1

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).


32

Table 9: Synthetic route to the racemic sulfoxides. The yields and substitution patterns are given in the table.

Cpd. R1 R2 Yield

64 NO2 Ph 38%

65 Cl 2-naphthalene 66%

66 Cl 4-methoxybenzene 72%

67 H Ph 13%

68 H 4-methoxybenzene 2%

69 NO2 4-methylbenzene 20%

The NO2 substituent in ortho position to the linker was shifted to meta position.

Sulfone 70 was synthesized according to Moore et al. by an AlCl3 mediated reaction

of benzene and the corresponding sulfonyl chloride in 46% yield (Scheme 8).153

Scheme 8: AlCl3 mediated reaction of benzene and 4-chloro-3-nitrobenzenesulfonyl chloride to 70.

Further, the linker was changed from sulfone or sulfoxide to an ether by synthesizing

71 in 96% yield applying a SNAr of 1,4-dichloro-2-nitrobenzene with phenol

(Scheme 9).

Scheme 9: Synthesis of the biphenylether 71 by nucleophilic aromatic substitution under basic conditions.


33

Another effort to diversify the linker was to introduce a phosphinic acid residue. 4-

chloro-2-nitroaniline was converted into the iodide 72 using a diazetation-iodination

as described in literature (Scheme 10).154 The following copper mediated reaction of

the iodide with phenylphosphinic acid did not yield the desired small molecule 73

(Scheme 10).155 Only decomposition of the starting material was observed.

Scheme 10: Synthesis of iodide 72 in a one-pot procedure and the subsequent copper mediated reaction to 73.

In another attempt to synthesize 73 according to literature procedures 4-chloro-2-

nitroaniline was first converted into its diazonium salt and afterwards the copper

mediated reaction with dichloro(phenyl)phosphane was carried out

(Scheme 11).156-157 As well as the before mentioned approach this attempt did not

lead to the desired product but only to decomposition of the starting material. Since

at this time point in vitro studies revealed that the sulfone linker is essential for Kif18A

inhibition, as discussed later on (chapter 3.2.4 and 3.2.11), no further attempts to

synthesize 73 were carried out.

Scheme 11: Proposed synthetic rout to 73. The diazonium compound 74 was readily available through diazetation, but the copper mediated reaction to 73 lead only to decomposition of the starting material.


34

The main focus for the synthesis was not to optimize the synthetic procedures, but

to yield pure substances in a fast manner for evaluation of their biological activity.

Therefore, all compounds were purified by column chromatography and/ or

successive recrystallization resulting in lower yields.

3.2.4 Screening for Kif18A Inhibition and IC50 Determination

With the synthesized BTB-1 analogs 52-71 in hand the next step was to evaluate their

ability to inhibit the ATPase activity of Kif18A. The motor-domain of Kif18A fused to

a His-Tag (Kif18AMD) was used in an enzyme coupled assay to evaluate the potency of

the respective analogs. The ECA conditions were adjusted to obtain 50% inhibition at

5 µM BTB-1 and the ATPase activity in the presence of DMSO was normalized to 100%

(Figure 11). All compounds that showed higher inhibition than 25% were considered

as active. This selection criteria was chosen in order to identify inhibitors with

comparable properties like BTB-1.


35

Figure 11: Quantification of the screening results of BTB-1 and its derivatives tested at 5 µM. Green bars indicate small molecules that were considered as active, since they showed an inhibitory activity above 25%. Average of three independent experiments and standard deviations are shown. In the lower panel the structure of the identified hits are shown.

Five out of 20 compounds were considered as active (52, 53, 54, 55, and 59, Figure

11 green bars) and selected for further analysis. Next, the IC50 values of the five small

molecules against Kif18AMD were determined using the ECA. All compounds showed

a concentration dependent inhibition in the low micromolar range (Figure 12).

Unfortunately, none of them showed a higher potency than BTB-1 (1.7 ± 0.2 μM).

-5%

10%

25%

40%

55%

70%

Inh

ibit

iion

in %

Cpd tested at 5 µM


36

10 -7 10 -6 10 -5 10 -4

0

20

40

60

80

100BTB-1

52

53

54

55

59

M

% A

TP

as

e a

cti

vit

y

Figure 12: Dose-response curves of BTB-1, 52, 53, 54, 55 and 59. All molecules showed a dose dependent inhibition in the low micromolar range, with BTB-1 being the most potent. Average of three independent experiments and standard deviations are shown.

With IC50 values of 3.0 ± 0.2 μM and 4.8 ± 0.4 μM for 59 and 53 respectively, they

were the most potent inhibitors, indicating that the replacement of the halogen atom

at phenyl moiety I is tolerated. The introduction of more bulky substituents at this

position like NO2 (52) or CF3 (54) as well as the isosterical exchange of the phenyl

moiety R2 to a thiophene (55) resulted in reduced potency (IC50 values:

(52) 10.2 ± 2.0 μM, (54) 10.3 ± 1.9 μM, and (55) 6.4 ± 0.9 μM).

3.2.5 Mode of Inhibition and Selectivity

In order to clarify if the newly found inhibitors, like BTB-1, act in an ATP competitive

manner106, ECA analysis of Kif18AMD activity in the absence of microtubules was

performed. The inhibitors BTB-1, 59 or 53 were applied at 50 µM concentration.


37

Figure 13: In the upper panel the raw data of the ECA with Kif18AMD and 50 µM BTB-1, 53 and 59 in the absence of microtubules are shown. The lower panel shows the decrease of absorbance at 340 nm relative to DMSO control. Both graphs show no inhibition of the tested small molecules for the basal ATPase activity of Kif18AMD compared to the DMSO control. Average of two independent experiments and standard deviations are shown.

Comparable to BTB-1, both small molecules did not show any inhibitory effect on the

basal, microtubule-independent ATPase activity of Kif18A (Figure 13), suggesting that

the newly found inhibitors behave like BTB-1. Additionally, ATP titration experiments

indicate that 59 acts in an ATP competitive manner like BTB-1 (Figure 14). These data

suggest, that the identified small molecules are ATP-competitive inhibitors, which are

only able to inhibit Kif18A when it is bound to microtubules.

0,5

0,55

0,6

0,65

0,7

0,75

0,8

0,85

0,9

0,95

1

0 100 200 300 400 500 600

Ab

sorb

ance

at

34

0 n

m

Time in sec

BTB-1 50 µM 53 50 µM 59 50 µM DMSO 0.5% Background

0%

20%

40%

60%

80%

100%

BTB-1 53 59

ΔA

34

0n

m/m

in r

el t

o D

MSO

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ol


38

0 2 0 0 4 0 0 6 0 0 8 0 0

0

1

2

3

4

5

6

7

8

9

1 0

M ic h a e lis -M e n te n

A TP in µM

AT

P/s

0

20 µM

10 µM

5 µM

2 .5 µM

1 µM

0 .5 µM

0 .2 5 µM

0 .1 µM

Figure 14: The ATPase activity of Kif18AMD was determined by increasing concentrations of ATP and different concentrations of 59. The received data were fitted to competitive inhibition mode using GraphPad Prism. Average of three independent experiments and standard deviations are shown.

In order to analyze compound selectivity towards Kif18A, the two most potent

compounds 53 and 59 were tested in vitro for their effects on the ATPase activity of

other kinesins related to Kif18A (Figure 15). Therefore, ECA was performed in the

presence of 100 μM BTB-1, 53 or 59 and His-tagged motor domains of the mitotic

kinesins Kif3A, Kif4A, Kif5A, Mklp1, Eg5, MPP1 or MCAK. 53 and 59 showed reduced

inhibitory activity towards Kif3A and Kif5A compared to BTB-1 and comparable

inhibitory activity towards the other tested kinesins (Figure 15). These findings

suggest, that the substitution of chloride with hydrogen and fluoride atoms has a

selectivity increasing effect.


39

Kif18A Kif5A Kif4A Kif3A Mklp1 Eg5 MPP1 MCAK

0

50

100

% In

hib

itio

n

BTB-1

53

59

Figure 15: The inhibitory effect of BTB-1, 53 and 59 at 100 µM on different mitotic kinesins are shown. Compared to BTB-1 53 and 59 show reduced inhibition of Kif5A and Kif3A and comparable inhibitory activity towards the other tested kinesins. Average of three independent experiments and standard deviations are shown.

3.2.6 Cellular Toxicity Studies

With the first in vitro results in hand, the next step was to analyze, if the identified

novel Kif18A inhibitors are active in cells. Therefore, the whole small molecule

collection was tested for their cytotoxicity on HeLa cells using an alamar blue assay

(Figure 16 upper panel).158 In this assay the percentage of viable cells are detected

through the conversion of Resazurin in living cells into the fluorescent dye Resorufin.

The fluorescence serves as readout and is directly linked to the number of viable cells.

Interestingly, determination of the half maximal effective concentration (EC50 values)

showed that all synthesized sulfoxides 64-69 had EC50 values in the low micromolar

range (Table 10). The dose response curves of the five most potent compounds for

Kif18A inhibition 52, 53, 54, 55, and 59 as well as BTB-1 are shown in Figure 16. Out

of this set of inhibitors (Figure 16 lower panel) compound 52 showed the highest

cytotoxicity with 1.1 ± 0.3 μM followed by 54 with 2.6 ± 0.4 μM. BTB-1 and 55 were

less toxic with EC50 values of 35.8 ± 9.0 μM and 23.1 ± 7.3 μM, respectively. For 53


40

the lowest cytotoxicity was observed with EC50 values above 50 μM and 59 did not

show a significant cytotoxicity at all.

10 -8 10 -6 10 -4 10 -2

0

50

100

BTB-1

52

53

54

55

59

M

% C

ell

via

bil

ity

Figure 16: The upper panel shows a schematic representation of the alamar blue assay. Viable cells reduce the nonfluorescent dye Resazurin into the fluorescent Resorufin. The fluorescence serves as readout. The lower panel shows the dose response curves of BTB-1, 52-55 and 59 received from HeLa cells treated with different concentrations of inhibitors. Average of three independent experiments and standard deviations are shown. The structures of the tested compounds are shown in the lower panel.


41

Table 10: EC50 values in µM of the BTB-1 analogs received from the alamar blue assay. No cytotoxicity or values above 60 µM are described with “ “.

Cpd. EC50 (µM)

BTB-1 35.8 ± 9

52 1.1 ± 0.3

53 54.7 ± 7.8

54 2.6 ± 0.4

55 23.1 ± 7.3

56 13.8 ± 2.9

57 -

58 36.2 ± 9.3

59 -

60 -

61 2.6 ± 0.4

62 20.8 ± 6.3

63 -

64 1.9 ± 0.2

65 5.5 ± 1.2

66 9.3 ± 2.1

67 20.8 ± 3.0

68 30.1 ± 4.6

69 1.8 ± 0.3

70 9.6 ± 1.3

71 -


42

3.2.7 Live Cell Imaging

Another attempt to investigate the cellular effects of the newly identified inhibitors

was to use live cell imaging. A HeLa cell line that stably expresses green fluorescent

protein (GFP) tagged Kif18A49 was used in order to detect the localization of Kif18A

and additionally the time in mitosis (time from NEBD to anaphase onset) was

determined (Figure 17). In a first experiment cells synchronized in S-phase with

thymidine were treated with 0.5% DMSO or 25 µM of the three most potent newly

identified inhibitors 53, 55, and 59 or BTB-1. Small molecule 58 was used as toxicity

control since its EC50 value in the alamar blue assay is comparable to BTB-1. 52 and

54 displayed a high toxicity at 25 µM and, were therefore used at different

concentrations (10 µM, 5 µM and 1 µM, experiments are not shown).

0.5%

DM

SO

25 µ

M B

TB-1

25 µ

M 5

3

25 µ

M 5

5

25 µ

M 5

8

25 µ

M 5

9

50 µ

M B

TB-1

50 µ

M 5

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50 µ

M 5

8

50 µ

M 5

9

5 µM

54

0

100

200

300

400

500

NE

BD

to

An

ap

ha

se

on

se

t in

min

Figure 17: Schematic representation of live cell assay. Cells were synchronized in S-phase using thymidine. 6 Hours after release 0.5% DMSO or 5/25/50 µM inhibitors were added. No effect on the time in mitosis was observed. Average and standard deviation of two independent experiments are shown for 25 µM concentration. For 50 µM and 5 µM concentrations only one experiment was carried out and the standard deviation is shown.


43

Nevertheless, none of the small molecules interfered with the localization of Kif18A

and no significant increase in time in mitosis compared to the DMSO control was

detected (Figure 17). The experiment was repeated using 50 µM inhibitor

concentration, except for 54 and 52, where 5 µM was applied. Due to toxic effects of

55 at 50 µM and 52 at 5 µM concentration both molecules were excluded from

analysis. Even the higher concentrations of the inhibitors did not result in a

detectable delocalization of Kif18A. Despite that, 54 led to an increased time in

mitosis at 5 µM concentration compared to the DMSO control (Figure 17). BTB-1

showed a slight increase in mitotic timing (Figure 17). Since no effect on the Kif18A

localization was observed, this experiment was only carried out once. In summary,

the inhibitors are not efficient enough to inhibit Kif18A in cells, since the localization

of Kif18A was not altered upon compound treatment.

3.2.8 Cellular Thermal Shift Assay

As another approach to elucidate the cellular effects of BTB-1 and the most promising

inhibitor 53 a cellular thermal shift assay (CETSA) was performed. In this assay cell

lysate is applied and therefore the small molecules do not have to pass the cell

membrane.159 This assays relies on the thermal stabilization of the target protein

upon Inhibitor binding. Mitotic HeLa cell extract was prepared and treated with 0.5%

DMSO as solvent control or compounds. After incubation and thermal treatment the

samples were analyzed by SDS-PAGE followed by western blot analysis. First, the

temperature at which Kif18A denatures was determined in the presence of 100 µM

BTB-1, 53 or 0.5% DMSO. Unfortunately, no stabilization of Kif18A due to inhibitor

addition was observed. No intensity differences of the samples were detectable

(Figure 18). The DMSO control revealed that Kif18A is destabilized at temperatures

between 50°-54°C (Intensity decrease, Figure 18) and therefore this temperature

range was used to evaluate if higher inhibitor concentrations are able to stabilize

Kif18A.


44

Figure 18: Western blot analysis of temperature gradient from 46°C to 62°C in the presence of 0.5% DMSO, 100 µM BTB-1 or 53. In line 10 (DMSO control 52°C) was less lysate loaded. No stabilization of Kif18A was observed. CycB, Cdc27 and tubulin served as mitotic markers and loading controls.

BTB-1 and 53 were applied in 500 µM, 200 µM, 100 µM and 50 µM concentration,

but still no stabilization of Kif18A was observed (Figure 19). Since BTB-1 as well as 53

are not able to stabilize Kif18A even at concentrations up to 500 µM, it is possible

that these inhibitors are not able to bind efficiently to Kif18A in a cellular

environment.

Figure 19: Western blot analysis of compound titration of BTB-1 and 53 from 50°C to 54°C. The first and last line was loaded with random lysate. No stabilization of Kif18A was observed. CycB, Cdc27 and tubulin served as mitotic markers and loading control.


45

3.2.9 Immunofluorescence Imaging for Kif18A Localization

In order to analyze in detail the effect of BTB-1 and derivatives 53, 54, and 59 on cells

and the architecture of the mitotic spindle, immunofluorescence imaging was

performed. The same HeLa cell line as described for live cell imaging (chapter 3.2.7)

was used.49 Cells synchronized in S-phase using a high concentration of thymidine

were released after nine hours and then treated with the solvent control DMSO or

30/50 µM of BTB-1 or derivatives 53, 54, and 59 (Figure 20). Thirty minutes after

compound addition, cells were chemically fixed and tubulin (red) and DNA (blue)

structures were visualized by immunostaining and treatment with the dye Hoechst

33342, respectively. Since initial immunofluorescence analysis of derivative 52

revealed that this small molecule caused unspecific cytotoxic effects on both dividing

and non-dividing cells, it was excluded from further microscopic studies. The

immunofluorescence images revealed that neither 53 nor 59 significantly affected

spindle structures or alignment of chromosomes at 30/50 µM concentration

(Figure 20, only 50 µM is shown). Additionally, the plus-end accumulation of

GFP-Kif18A at microtubules – which as shown previously depends on the motor

activity of Kif18A44-45 – was not affected by the treatment with 53 or 59.


46


47

Figure 20: The upper panel shows the assay scheme for cell synchronization and compound treatment. First, cells were synchronized in S-phase using thymidine. Nine hours after the release, cells were treated with DMSO as solvent control, 50 nM Nocodazole (Noc) or 30/50 µM BTB-1, 53, 54, 59 followed by formalin fixation and immunostaining. Representative immunofluorescence images of cells treated with DMSO, 50 nM Nocodazole (Noc) or 30/50 µM compounds BTB-1, 53, 54, 59 are shown. For each condition, merged images with DNA (blue), GFP-Kif18A (green) and microtubules (red) are shown on the right. In grey is shown from left to right DAPI (DNA), Kif18A, and tubulin (scale bar = 5 µm).

These, taken together with the normal spindle structures, suggest that treatment

with derivative 53 or 59 does not result in efficient Kif18A inhibition in cells. HeLa

cells treated with 50 µM BTB-1 revealed severe defects in spindle morphology and

chromosome alignment, while 30 µM BTB-1 showed a mild effect (Figure 20),

consistent with previous reports106. Similarly, HeLa cells treated with 30 µM 54

showed multiple spindle poles, highly disorganized and fragmented microtubule

structures and no detectable alignment of chromosomes comparable to the effect of

50 nM Nocodazole160-161 (Figure 20). Since the observed phenotypes did not correlate

with Kif18A depletion (elongated spindles with hyperstable microtubules) 42, 46, it can

be speculated that Kif18A is not the relevant binding partner of BTB-1 or 54 in cells.


48

3.2.10 Tubulin Polymerization Assay and IC50 Determination

The spindle phenotype induced by 54 was reminiscent of the phenotype caused by

low doses of Nocodazole, a microtubule destabilizing compound (Figure 20). To

analyze if the small molecule collection or BTB-1 target microtubules, a

turbidity-based in vitro microtubule polymerization assay was performed.162-163 The

ability of tubulin to polymerize in the presence of GTP at elevated temperatures in a

glutamate buffer and a turbidity based readout was used (Figure 21).

Figure 21: Schematic representation of tubulin polymerization. α-(light green) and β-(dark green or brown) tubulin heterodimers polymerize to microtubules. After GTP hydrolysis the destabilized plus-end depolymerizes. Upon exchange of GDP to GTP in the tubulin dimers repolymerization occurs.

α- and β- tubulin heterodimers are able to polymerize in the presence of GTP. The

GTP cap consists of tubulin dimers in the GTP bound state and stabilizes the highly

dynamic plus-ends of microtubules.164-166 After GTP hydrolysis, depolymerization,

often called catastrophe, takes place. This process can be stopped by addition of

GTP-bound tubulin dimers. After a certain time, the polymerization and

depolymerization processes reach a dynamic equilibrium as long as enough GTP is

present. The microtubule polymerization efficiency in the presence of the solvent


49

control DMSO was normalized to 100% and 1 µM Nocodazole was used as positive

control. A small molecule selection was used at 50 µM concentration (Figure 22).

Figure 22: Screening results of the tubulin polymerization assay. DMSO was used as solvent control and its activity was set to 100%. 1 µM Nocodazole was used as positive control and the small molecules as well as BTB-1 were tested at 50 µM concentration. Average of three independent experiments and standard deviations are shown.

Notably, 52 and 61 showed the highest inhibitory effect on microtubule

polymerization at 50 µM followed by BTB-1, 54, 60, 63 and 64 (Figure 22). BTB-1, 53,

54, and 59 were selected for further analysis due to their high potency against Kif18A

(chapter 3.2.4) and 52 due to its high cytotoxicity (chapter 3.2.6).

Determination of the IC50 values showed that 52 has an IC50 value of 1.2 ± 0.1 µM,

indicating that the high cytotoxicity of the compound might be due to its potent

inhibitory effect on microtubule polymerization (Figure 23). BTB-1 and 54 showed a

moderate effect on microtubule polymerization with IC50 values of 27.5 ± 3.3 µM and

9.2 ± 1.0 µM, respectively. 59 showed an IC50 value of 209.4 ± 37.3 µM on microtubule

polymerization. Small molecule 53 did not show any significant effect on the tubulin

polymerization up to a concentration of 1 mM. Thus, these studies revealed that the

introduction of bulky substituents in phenyl moiety I at para position to the linker

(54), especially the introduction of a nitro group (52), drastically increased the

undesired inhibitory effect on microtubule polymerization.

0%

20%

40%

60%

80%

100%

120%


50

10 -7 10 -6 10 -5 10 -4 10 -3

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52

53

54

59

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% T

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Figure 23: Dose-response curves of BTB-1, 52, 54, and 59 on microtubule polymerization in vitro with 52 being the most potent inhibitor. Derivative 53 did not show an effect on the tubulin polymerization up to 1 mM Average of three independent experiments and standard deviations are shown.

3.2.11 Structure Activity Relationships and Outlook

A small compound selection 52-71 was synthesized in one to two steps using BTB-1

as lead compound. The described synthesis route started from commercially

available building blocks and allowed the robust and cost-efficient assembly of the

small molecule collection. Several BTB-1 analogs, which inhibit the ATPase activity of

Kif18A in the micromolar range were identified and enabled the establishment of

SARs (Figure 24).

Figure 24: Scheme of SAR for Kif18A inhibition. Green colored groups are essential for activity. Yellow groups are tolerated but cause some activity loss. Red groups are not tolerated and cause complete loss of inhibitory activity.


51

Comparison of identically substituted sulfone (52 and 59) and sulfoxide (64 and 67)

derivatives highlight the importance of the sulfone functionality for the inhibitory

activity of the compounds against Kif18A (Figure 24). Consistently, the replacement

of the sulfone linker of BTB-1 with an ether linkage (71) resulted in an almost

complete loss of inhibitory activity (Figure 24). Additional SAR studies revealed that

the introduction of substituents in para position to the linker at phenyl moiety II was

not tolerated whether they were electron withdrawing (62 and 63) or donating

(57, 58, 66, 60, 61, 68 and 69). Likewise, the sterical expansion of the aromatic

phenyl moiety II by replacing it with naphthalene (56) resulted in a complete loss of

activity (Figure 24). Shifting the NO2 group from ortho (BTB-1) to meta (70) position

to the sulfone linker within phenyl moiety I caused compound inactivity. In contrast,

the replacement of chloride (BTB-1) at R1 of phenyl moiety I with hydrogen (59) or

different electron withdrawing groups like fluoride (53), NO2 (52), or CF3 (54) showed

no drastic effect on compound activity (Figure 24). Since 53 and 59 – the most potent

derivatives – did not interfere with the localization of Kif18A to the plus-ends of

microtubules in mitotic cells, it might be that treatment with these small molecule

inhibitors did not result in efficient Kif18A inhibition. One possible explanation could

be that the compounds 53 and 59 are not cell membrane permeable, but the cellular

effects received from structurally related compounds BTB-1 and 54 argues against

this idea. Another possibility could be that association of Kif18A with binding partners

or posttranslational modifications render Kif18A resistant to the binding of 53 and

59. Remarkably, BTB-1 and 54 revealed a cellular phenotype reminiscent of low doses

of Nocodazole leading to the conclusion that they might interact with tubulin.

Additional in vitro studies confirmed that these compounds potently inhibited

microtubule polymerization (Figure 25). For BTB-1 and 54 it is difficult to determine

whether the altered Kif18A localization in cells treated with these compounds is due

to their direct interaction with Kif18A or caused by the undesired effect on

microtubule dynamics, which per se has an influence on Kif18A plus–end

accumulation at microtubules45. The in vitro microtubule polymerization assays

revealed 52 as the most potent inhibitor (Figure 25). Therefore, it is likely that the

high cytotoxicity of 52 is caused by its severe effect on microtubule polymerization.


52

Figure 25: Results of the SAR studies. The upper panel shows molecules sorted based on their inhibitory effect on microtubule polymerization, decreasing activity from left to right with 52 being the most potent inhibitor for microtubule polymerization. In the lower panel, compounds are sorted based on their inhibitory activity towards Kif18A with the most potent compound BTB-1 shown on the right side.

All these data suggest the introduction of electron withdrawing sterically demanding

groups like NO2 (52) or CF3 (54) at R1 of phenyl moiety I shift the inhibitory activity

towards microtubules as well as the combination of a NO2 group at R1 and a methyl

group at the para position of phenyl moiety II (61). In contrast to that the introduction

of small substituents at R1 like hydrogen (59) or fluoride (53) influence the activity in

favor of Kif18A inhibition (Figure 25).

The insights of the SAR studies might provide an important guideline for future design

and synthesis of more potent Kif18A inhibitors. In order to establish even more

powerful SARs the exact binding of BTB-1 and its analogs to Kif18A has to be resolved.

One possibility to achieve this would be the crystallization of Kif18A with bound

inhibitor. The structure would reveal the interactions of the small molecules with the

amino acids of Kif18A and allow the design of inhibitors that fit to the binding surface.

Such compounds would be invaluable tools for basic research, because Kif18A seems

to have different functions at different times during mitosis and small molecules

could be applied at specific time points. Further, the inhibitor would have the


53

potential to open up new strategies in the treatment of mitosis-related diseases such

as Kif18A overexpressing tumors.

Summary

54

4. Summary

Kinesins are essential for the correct distribution of the genetic material into the

daughter cells during mitosis.4 The functional dissection of individual kinesins

involved in chromosome segregation is a challenging task, because of the complexity

and dynamicity of mitosis. Due to their fast and often reversible mode of action,

membrane permeable small molecules inhibitors are ideal tools to dissect the

different functions of kinesins.

Paprotrain was discovered in 2010 by Tcherniuk et al.109 as inhibitor for the kinesin

Mklp2, whereas a small molecule named SH1, a 5-chloro-2-(3,4-dichlorophenyl)-7-

(trifluoromethyl)quinoxaline, was identified and characterized in the laboratory of

Prof. Thomas U. Mayer in 2009 122. For the design of new Mklp2 inhibitors, SH1 served

as the lead compound. The main drawback of SH1 was its low solubility in aqueous

media. Thus, the aim was to develop new derivatives with enhanced solubility. After

the establishment of a synthetic pathway towards SH1 using glyoxales, several

compounds bearing polar substituents (7-26) as well as their precursors (1-6) were

synthesized (chapter 3.1.3). In a second approach to enhance the water solubility the

phenyl moiety of SH1 was exchanged to a thiophene (27-40, chapter 3.1.4).134

Moreover, SH1 analogs linked to an enzymatic cleavable carbohydrate residue were

synthesized (47-51, chapter 3.1.5).138 All different synthetic approaches to enhance

the water solubility of SH1 derivatives were successful. Characterization of the

biological activity of the synthesized compounds revealed that only compounds

barely soluble in aqueous media were active (chapter 3.1.6). Furthermore, addition

of the detergent Triton X-100 suppressed the inhibitory activity of SH1 towards Mklp2

in vitro. Collectively, these results suggest, that aggregation of SH1 and its derivatives

is necessary for their inhibitory activity (chapter 3.1.7 and 3.1.8).

For the kinesin Kif18A, a small molecule named BTB-1 was identified by Catarinella et

al. in 2009 106 as a potent inhibitor. BTB-1 inhibited 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

Summary

55

BTB-1 analogs. For the synthesis of the small molecule collection, the main reaction

utilized was a nucleophilic aromatic substitution under basic conditions followed by

an oxidation (52-63 and 64-69, chapter 3.2.3). The screening for Kif18A inhibition

identified five active compounds (52-55 and 59), which showed IC50 values in the low

micromolar range (<10 µM, chapter 3.2.4). Further, the selectivity of the newly found

two most potent inhibitors (53 and 59) towards Kif18A was investigated. Both

showed a higher selectivity towards Kif18A compared to BTB-1 (chapter 3.2.5). Next,

the cellular effects of the novel small molecules were investigated. The cellular

cytotoxicity was determined, followed by studies to detect Kif18A localization in cells

(chapters 3.2.6-3.2.8). Fluorescence imaging revealed that the newly identified

inhibitors did not alter the localization of Kif18A to the plus-ends of microtubules.

They might be not efficient enough to inhibit Kif18A in cells, due to binding partners

or posttranslational modifications of Kif18A. Instead some compounds showed a

severe effect on the microtubule network comparable to low doses of the

microtubule poison Nocodazole (BTB-1 and 54, chapter 3.2.9). An in vitro microtubule

polymerization assay confirmed that some compounds interfered with microtubule

polymerization (BTB-1, 52 and 54, chapter 3.2.10). Most importantly, the undesired

effect on microtubule polymerization was separated from the desired Kif18A

inhibition with the help of in-depth SAR studies (chapter 3.2.11). The insights of the

SAR studies provide a powerful guideline for future design and synthesis of highly

potent Kif18A inhibitors, which would be invaluable tools for basic research and could

open up new strategies in the development of novel drugs for mitosis-related

diseases, like Kif18A overexpressing tumors.

Zusammenfassung

56

5. Zusammenfassung

Mitotische Kinesine sind wichtig für die korrekte Verteilung des genetischen

Materials in die beiden Tochterzellen während der Mitose.4 Sie haben verschiedene

Funktionen zu unterschiedlichen Zeitpunkten des Zellzyklus. Spezifische

niedermolekulare Inhibitoren sind daher ideale Werkzeuge, um die verschiedenen

Funktionen der Kinesine zu analysieren, da sie in einer sehr schnellen und oft

reversiblen Art wirken.

Paprotrain wurde 2010 von Tcherniuk et al.109 als Inhibitor für das Kinesin Mklp2

entdeckt, während die niedermolekulare Substanz SH1, ein 5-Chlor-2-(3,4-

dichlorphenyl)-7-(trifluormethyl)chinoxalin, im Labor von Prof. Thomas U. Mayer im

Jahr 2009122 identifiziert und charakterisiert wurde. Für das Design neuer Mklp2

Inhibitoren diente SH1 als Leitstruktur. Der größte Nachteil von SH1 liegt in seiner

schlechten Wasserlöslichkeit, weshalb neue Inhibitoren mit erhöhter

Wasserlöslichkeit synthetisiert werden sollten. Nach der Etablierung eines

Synthesewegs zur Darstellung von SH1 mittels Glyoxalen, wurden verschiedene

Verbindungen mit polaren Substituenten (7-26) sowie deren Vorstufen (1-6)

hergestellt (Kapitel 3.1.3). In einem weiteren Ansatz, um die Löslichkeit zu erhöhen,

wurde der Phenylrest von SH1 durch Thiophen ersetzt (27-40, Kapitel 3.1.4).134

Außerdem wurden SH1 Analoga verbunden mit einem enzymatisch spaltbaren

Kohlenhydratrest synthetisiert (47-51, Kapitel 3.1.5).138 Die verschiedenen

strukturellen Modifikationen von SH1 führten erfolgreich zur Erhöhung der

Wasserlöslichkeit. Die Charakterisierung der biologischen Aktivität zeigte jedoch,

dass nur in wässrigen Medien schwer lösliche Verbindungen, aktiv waren

(Kapitel 3.1.6). In in vitro Untersuchungen mit Triton X-100 wies SH1 keine Inhibition

von Mklp2 auf, was zu dem Schluss führt, dass die Aggregation von SH1 notwendig

für seine hemmende Aktivität ist (Kapitel 3.1.7 und 3.1.8).

Für das Kinesin Kif18A wurde ein niedermolekulares Molekül namens BTB-1 als

potenter Inhibitor von Catarinella et al. 2009106 identifiziert. BTB-1 inhibierte die

ATPase-Aktivität von Kif18A in vitro und führte bei zellulärer Anwendung zu einer

Erhöhung der Anzahl mitotischer Zellen.106 Dennoch wiesen Zellen, die mit einer

hohen Konzentration von BTB-1 behandelt wurden, keine verlängerten Spindeln auf,

Zusammenfassung

57

die charakteristisch für die Depletion von Kif18A sind. Daher sollte ein Syntheseweg

etabliert werden, um eine Sammlung von BTB-1-Analoga zu synthetisieren und

Struktur-Aktivitäts-Beziehungen aufzustellen. Zur Synthese der BTB-1 Analoga wurde

als Hauptreaktion eine nukleophile aromatische Substitution unter basischen

Bedingungen verwendet, gefolgt von einer Oxidation (52-63 und 64-69,

Kapitel 3.2.3). Im Screening zur Kif18A Inhibition wurden fünf aktive Verbindungen

(52-55 und 59) identifiziert, die IC50-Werte im niedrigen mikromolaren Bereich

zeigten (<10 µM, Kapitel 3.2.4). Ferner wurde die Selektivität der zwei potentesten

Inhibitoren (54 und 60) gegenüber Kif18A im Vergleich zu anderen mitotischen

Kinesinen untersucht und beide zeigten eine erhöhte Selektivität gegenüber Kif18A

im Vergleich zu BTB-1 (Kapitel 3.2.5). Als nächstes wurden die zellulären Effekte der

neuen Inhibitoren untersucht. Zuerst wurde die Zytotoxizität bestimmt, gefolgt von

Studien zur Lokalisation von Kif18A in Zellen (Kapitel 3.2.6-3.2.8).

Fluoreszenz-Bildgebung zeigte, dass die neu identifizierten Inhibitoren nicht die

Lokalisierung von Kif18A zu den Plus-Enden der Mikrotubuli veränderten. Trotz der

Inhibition der ATPase-Aktivität von Kif18A in vitro sind die neu identifizierten

Inhibitoren möglicherweise nicht effizient genug, um Kif18A in Zellen zu hemmen.

Dies könnte durch mögliche Bindungspartner oder posttranslationale Modifikationen

von Kif18A verursacht werden. Stattdessen zeigten einige Verbindungen eine starke

Wirkung auf das Mikrotubuli-Netzwerk, vergleichbar mit niedrigen Dosen des

Spindelgifts Nocodazol (BTB-1 und 54, Kapitel 3.2.9). Ein in vitro

Mikrotubuli-Polymerisationsversuch bestätigte, dass einige Verbindungen die

Mikrotubuli-Polymerisation störten (BTB-1, 52 und 54, Kapitel 3.2.10). Jedoch wurde

mit Hilfe intensiver Struktur-Aktivitäts-Studien die unerwünschte Wirkung auf die

Mikrotubuli-Polymerisation von der gewünschten Kif18A Inhibition getrennt (Kapitel

3.2.11). Die Erkenntnisse der Struktur-Aktivitäts-Studien bieten einen sehr guten

Leitfaden für das zukünftige Design und die Synthese von hochwirksamen

Kif18A Inhibitoren, die nicht nur wertvolle Werkzeuge für die Grundlagenforschung

wären, sondern auch neue Strategien in der Entwicklung innovativer Medikamente

für Mitose-Erkrankungen, wie Kif18A überexprimierende Tumoren, darstellen

könnten.104-105

Materials and Methods

58

6. Materials and Methods

6.1 General

Chemicals, reagents and solvents

All solvents, reagents and fine chemicals are commercially available (Sigma‐Aldrich,

Acros, Merck, Fluka, Roth, TCI, MCAT, ABCR, or Fluorochem) and are used without

further purification. The used petroleum ether (PE) had a boiling point range of

35-80°C.

NMR‐Spectroscopy

1H‐, 13C‐ and 19F‐NMR spectra were recorded either on a Bruker Avance III 400 or

Bruker Avance 600 spectrometer in the NMR facility of the University of Konstanz.

Chemical shifts are given in the δ‐scale and referenced to residual non deuterated

solvent. A BBFOplus probe with actively shielded z‐gradient was used with its inner

(BB‐) coil tuned to 19F. The following abbreviations were used for spin‐systems:

s (singlet); d (doublet); t (triplet); q (quartet). In case of multiple splittings, identifiers

in the fashion of dt (doublet of a triplet) were used or m for undistinguishable

multiplets. The spectra were processed using MestReNova.

Elementary Analysis

The elementary analysis was performed by the University of Konstanz micro

analytical laboratory.

Melting Points

Melting points were measured on a Gallenkamp Melting Point Apparatus and are

uncorrected.

Chromatography

For TLC, pre‐coated plastic sheets with fluorescence indicator Polygram Sil G/UV254

by Macherey‐Nagel with a coating thickness of 0.2 mm were used. The substances

were detected under 254 or 366 nm light. Flash column chromatography

(200 - 300 mbar) was performed on Macherey‐Nagel silica gel 60 M (particle size:

40 - 63 μm). Technical grade solvents were distilled prior to use.


59

6.2 Synthesis of SH1 Analogs

General Methods for Glyoxal Synthesis (Method A)

The glyoxales were prepaired according to literature procedures. 131-133 The

corresponding acetophenone and 1.5 equivalents (equiv.) of selenium (IV) oxide

(SeO2) were heated to reflux in dioxane / water mixture (1 / 0.03) overnight. The

formed black Se precipitate was removed by filtration and the solution was

evaporated to dryness. The received solid was suspended in water, heated to reflux

and the solvent was removed. The subsequent purification is described for each

compound.

General Method for Preparation of Substituted Quinoxalines (Method B)

The corresponding phenylenediamine and glyoxal in equimolar stoichiometry were

dissolved in EtOH and heated to reflux. Upon cooling the formed precipitate was

collected and further purified as indicated.

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

First, the corresponding quinoxaline-6-carboxylic acid is converted into its acid

chloride by dissolving it in an excess of thionylchloride (25 mL) and heating to reflux.

After removing the solvent, the residue was suspended in CH2Cl2 (25 mL) and cooled

to 0°C. First 1.2 equiv. Et3N followed by slow addition of 1.1 equiv. amine/alcohol.

The reaction mixture was allowed to warm to ambient temperature after complete

addition. If not otherwise mentioned water (20 mL) was added and the aqueous

phase was extracted two times with CH2Cl2 (20 mL). The combined organic layers

were washed with water, dried over MgSO4 and the solvent was removed. Further

purification was carried out as mentioned for the corresponding compound.


60

3,4-Dichlorophenylglyoxal (1)

C8H6Cl2O3

M: 221.04 g mol-1

3,4-Dichloroacetophenone (2.0 g, 10.6 mmol) was converted into the glyoxal

according to Method A. After evaporation of the water the white precipitate was

recrystallized from benzene to yield 3,4-dichlorophenylglyoxal monohydrate as white

crystals (1.28 g, 40.4 mmol, 55% yield).

1H NMR (400 MHz, DMSO-d6) δ: 8.18 (d, J = 1.9 Hz, 1H), 7.96 (dd, J = 8.4, 1.9 Hz, 1H),

7.79 (d, J = 8.4 Hz, 1H), 5.92 (d, J = 5.8 Hz, 1H).

3,4-Difluorophenylglyoxal (2)

C8H6F2O3

M: 188.13 g mol-1

3,4-Difluoroacetophenone (10.0 g, 64 mmol) was converted into the glyoxal

according to Method A. Instead of water the yellow oil was dissolved in CH2Cl2 and

heated to reflux. Upon cooling a precipitate was formed and removed by filtration.

The solvent was removed and water was added. After heating to reflux the water was

removed and the received solid was purified by column chromatography

(EtOAc/n-hexane : 7/3). After recrystallization from benzene/n-hexane

3,4-difluorophenylglyoxal hydrate was received as white crystals (7.29 g, 39 mmol,

61% yield).

1H NMR (400 MHz, Chloroform-d) δ: 8.04 – 7.91 (m, 2H), 7.29 (q, J = 8.9 Hz, 1H), 6.27

(d, J = 10.2 Hz, 1H), 5.00 (d, J = 10.2 Hz, 1H).

19F NMR (376 MHz, Chloroform-d) δ: -125.87 (d, J = 20.8 Hz), -134.93 (d, J = 20.9 Hz).


61

Phenylglyoxal (3)

C8H8O3

M: 152.15 g mol-1

Acetophenone (5.0 g, 42 mmol) was converted into the glyoxal according to Method

A. The resulting oil was suspended in water and heated to reflux. Afterwards the

solvent was removed and the received solid was purified by recrystallization from

benzene/PE to yield the phenylglyoxal hydrate (2.7 g, 18 mmol, 43% yield).

1H NMR (400 MHz, DMSO-d6): 8.07 (dt, J = 7.2, 1.4 Hz, 2H), 7.68 – 7.58 (m, 1H), 7.52

(dd, J = 8.4, 7.0 Hz, 2H), 6.72 (s, 1H), 5.68 (t, J = 7.1 Hz, 1H).

4-Bromophenylglyoxal (4)

C8H7BrO3

M: 231.05 g mol-1

Bromoacetophenone (1.0 g, 5.0 mmol) was converted into the glyoxal according to

Method A. The received solid was recrystallized from H2O to yield

4-bromophenylglyoxal hydrate as colorless solid (0.3 g, 1.3 mmol. 26% yield).

1H NMR (400 MHz, DMSO-d6) δ: 8.06 – 7.95 (m, 2H), 7.78 – 7.69 (m, 2H), 6.84 (d,

J = 7.0 Hz, 2H), 5.62 (t, J = 7.0 Hz, 1H).

4-Nitrophenylglyoxal (5)

C8H7NO5

M: 197.03 g mol-1

4-Nitroacetophenone (1.0 g, 6 mmol) was converted into the glyoxal according to

Method A. After removal of the solvent, the received yellow solid was purified with

column chromatography (EtOAc/PE : 1/1) to yield 4-nitrophenylglyoxal hydrate

(1.01 g, 5.1 mmol, 85%)


62

1H NMR (400 MHz, DMSO-d6) δ: 8.43 – 8.24 (m, 4H), 7.04 (d, J = 6.7 Hz, 2H), 5.66 (s,

1H).

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

C6H5ClO3S

M: 192.61 g mol-1

5-Chloro-1-acetylthiophene (0.5 g, 3.2 mmol) was converted into the glyoxal

according to Method A. After filtration the solvent was removed and no water was

added. The crude product was directly used without further purification.

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

C15H8ClF3N2

M: 308.69 g mol-1

3-Chloro-5-(trifluoromethyl)benzene-1,2-diamine (0.5 g, 3.2 mmol) was converted to

5-Chloro-2-phenyl-7-(trifluoromethyl)quinoxaline according to Method B. After

excessive washing of the received solid with EtOH 5-chloro-2-phenyl-7-

(trifluoromethyl)quinoxaline was received as pink solid (0.64 g, 2.1 mmol, 65% yield).

1H NMR (400 MHz, DMSO-d6) δ: 9.47 (s, 1H), 8.31 – 8.30 (m, 3H), 8.05 (d, J = 1.8 Hz,

1H), 7.57 – 7.62 (m, 3H).

19F NMR (376 MHz, Chloroform-d) δ: –62.74 (s).

CHN (calc/found %): C(58.36/58.05), H(2.61/2.67), N(9.08/9.14).

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

C15H8Cl2N2O2

M: 319.14 g mol-1


63

3,4-Diaminobenzoic acid (5.0 g, 22.6 mmol) was converted into

2-(3,4-dichlorophenyl)quinoxaline-6-carboxylic acid using Method B. The crude

product was recrystallized from dioxane yielding 2-(3,4-dichlorophenyl)quinoxaline-

6-carboxylic acid as off-white solid (6.8 g, 21.2 mmol, 94% yield).

1H NMR (400 MHz, DMSO-d6, 72°C) δ: 13.19 (s, 1H), 9.65 (s, 1H), 8.72 – 8.46 (m, 2H),

8.39 – 8.12 (m, 3H), 7.83 (d, J = 8.3 Hz, 1H).

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

C15H8F2N2O2

M: 286.24 g mol-1

3,4-Diaminobenzoic acid (578 mg, 3.4 mmol) was converted into

2-(3,4-difluorophenyl)quinoxaline-6-carboxylic acid using Method B without heating.

The resulting solid was washed with EtOH to yield 2-(3,4-difluorophenyl)quinoxaline-

6-carboxylic acid as off-white solid (680 mg, 2.5 mmol, 75% yield).

1H NMR (400 MHz, DMSO-d6) δ: 13.36 (s, 1H), 9.65 (s, 1H), 8.57 (s, 1H), 8.33 (t, J =

10.0 Hz, 1H), 8.28 – 8.16 (m, 3H), 7.66 (q, J = 10.0, 8.6 Hz, 1H).

19F NMR (376 MHz, DMSO-d6) δ: –135.22 (d, J = 22.2 Hz), –137.29 (d, J = 22.2 Hz).

2-Phenylquinoxaline (16)

C14H10N2

M: 206.25 g mol-1

1,2-Phenylenediamine (0.3 g, 2.0 mmol) was converted into 2-phenylquinoxaline

using Method B. After cooling to ambient temperature the resulting black precipitate

was removed. The filtrate was concentrated and 2-phenylquinoxaline was received

as off-white solid (0.4 g, 2.0 mmol, 97% yield).


64

1H NMR (400 MHz, DMSO-d6) δ: 9.58 (s, 1H), 8.35 – 8.33 (m, 2H), 8.13 (td, J = 8.3, 1.7

Hz, 2H), 7.63– 7.55 (m, 3H), 7.89 – 7.84 (m, 2H).


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

C15H7BrClF3N2

M: 387.58 g mol-1

3-Chloro-5-(trifluoromethyl)benzene-1,2-diamine (0.18 g, 0.9 mmol) was converted

to 2-(4-bromophenyl)-5-chloro-7-(trifluoromethyl)quinoxaline according to Method

B. After excessive washing with EtOH 2-(4-bromophenyl)-5-chloro-7-

(trifluoromethyl)quinoxaline was received as colorless solid (0.24 g, 0.62 mmol, 72%

yield).

1H NMR (400 MHz, DMSO-d6) δ: 9.78 (s, 1H), 8.44 – 8.42 (m, 1H), 8.36 – 8.34 (m, 3H),

7.83 – 7.81 (m, 2H), 8.28 – 8.16 (m, 3H), 7.66 (q, J = 10.0, 8.6 Hz, 1H).

19F NMR (376 MHz, DMSO-d6) δ: –61.15 (s).


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

C12H7ClN2S

M: 246.71 g mol-1

1,2-Phenylenediamine (0.34 g, 3.2 mmol) was converted into 2-phenylquinoxaline

and the crude 1-(5-Chlorothiophen-2-yl)-2,2-dihydroxyethan-1-one (6) using Method

B. The pure product was received after excessive washing with EtOH (0.40 g, 1.6

mmol, 51% yield).


65

1H NMR (400 MHz, Chloroform-d) δ: 9.53 (s, 1H), 8.10 (d, J = 4.1 Hz, 1H), 8.08-8.04

(m, 1H), 8.02-7.97 (m, 1H), 7.87-7.75 (m, 2H), 7.30 (d, J = 4.1 Hz, 1H)


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

C13H5Cl2F3N2S

M: 349.15 g mol-1

3-Chloro-5-(trifluoromethyl)benzene-1,2-diamine (0.67 g, 3.2 mmol) was converted

to 5-chloro-2-(5-chlorothiophen-2-yl)-7-(trifluoromethyl)quinoxaline using crude 1-

(5-Chlorothiophen-2-yl)-2,2-dihydroxyethan-1-one (6) according to Method B. The

crude product was recrystallized from EtOH and received as crystals (0.55 g, 1.6

mmol).

1H NMR (400 MHz, DMSO-d6) δ: 9.63 (s, 1H), 8.34 (m, 1H), 8.21 (d, J = 1.9 Hz, 1H),

8.17 (d, J = 4.1 Hz, 1H), 7.31 (dd, J = 0.5, 4.1 Hz, 1H).

19F NMR (376 MHz, DMSO-d6) δ: –61.27 (s).


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

C17H12Cl2N2O2

M: 347.20 g mol-1

2-(3,4-dichlorophenyl)quinoxaline-6-carboxylic acid (0.5 g, 1.6 mmol) was converted

into ethyl 2-(3,4-dichlorophenyl)quinoxaline-6-carboxylate using Method C. The

crude product was recrystallized from toluene and washed with hexane to yield ethyl


66

2-(3,4-dichlorophenyl)quinoxaline-6-carboxylate as white crystal needles (0.32 g,

0.9 mmol, 59% yield).

1H NMR (400 MHz, Chloroform-d) δ: 9.36 (s, 1H), 8.84 (d, J = 1.8 Hz, 1H), 8.45 – 8.33

(m, 2H), 8.18 (d, J = 8.7 Hz, 1H), 8.07 (dd, J = 8.4, 2.1 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H),

4.49 (q, J = 7.2 Hz, 2H), 1.47 (t, J = 7.2 Hz, 3H).

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

C19H17Cl2N3O

M: 374.27 g mol-1


into 2-(3,4-dichlorophenyl)-N,N-diethylquinoxaline-6-carboxamide using Method C.

After warming to ambient temperature no water was added. Instead, the solvent was

removed and the crude product was dissolved in EtOAc and filtered over a SiO2-pad.

The solvent was removed and the product was washed wit n-hexane yielding

2-(3,4-dichlorophenyl)-N,N-diethylquinoxaline-6-carboxamide as white solid (0,45 g,


1H NMR (400 MHz, DMSO-d6) δ: 9.67 (s, 1H), 8.58 (d, J = 2.1 Hz, 1H), 8.34 (dd, J = 8.5,

2.1 Hz, 1H), 8.20 (d, J = 8.6 Hz, 1H), 8.05 (d, J = 1.7 Hz, 1H), 7.87 (d, J = 8.5 Hz, 1H),

7.84 (dd, J = 8.6, 1.8 Hz, 1H), 3.38 (broad, 4H), 1.15 (broad, 6H).

13C NMR (101 MHz, DMSO-d6) δ: 168.55, 149.23, 144.44, 141.00, 140.77, 138.83,

136.36, 133.44, 132.12, 131.33, 129.76, 129.17, 128.94, 127.56, 125.87, 99.50, 42.93,

14.03, 12.78.



67

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

C20H18Cl2N4O

M: 401.29 g mol-1


into (2-(3,4-dichlorophenyl)quinoxalin-6-yl)(4-methylpiperazin-1-yl)methanone

using Method C. The combined organic layers were additionally washed with 0.1 M

aq. HCl and finally with water. After drying over MgSO4 the solvent was removed, the

crude product was washed with EtOAc and n-hexane to yield the desired product as

off-white solid (0.42 g, 1.1 mmol, 53% yield).

1H NMR (400 MHz, Chloroform-d) δ: 9.34 (s, 1H), 8.38 (d, J = 2.1 Hz, 1H), 8.20 (d, J =

8.6 Hz, 1H), 8.15 (d, J = 1.8 Hz, 1H), 8.06 (dd, J = 8.4, 2.1 Hz, 1H), 7.85 (dd, J = 8.6, 1.9

Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 3.75 (broad, 4H), 2.53 (broad, 4H), 2.39 (s, 3H).

13C NMR (101 MHz, Chloroform-d) δ: 168.92, 150.37, 143.59, 142.54, 141.34, 137.21,

136.33, 135.27, 133.96, 131.35, 130.51, 129.62, 129.57, 127.91, 126.65, 55.18, 46.00.


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

C19H15Cl2N3O2

M: 388.25 g mol-1

2-(3,4-Dichlorophenyl)quinoxaline-6-carboxylic acid (0.5 g, 1.6 mmol) was converted

into (2-(3,4-dichlorophenyl)quinoxalin-6-yl)(morpholino)methanone using Method

C. The combined organic layers were additionally washed with 0.1 M aq. HCl and

finally with water. After drying over MgSO4 the solvent was removed, the crude

product was washed with n-hexane to yield the desired product as off-white solid

(0.41 g, 1.1 mmol, 68% yield).


68

1H NMR (400 MHz, Chloroform-d) δ: 9.34 (s, 1H), 8.38 (d, J = 2.1 Hz, 1H), 8.21 (d, J =

8.6 Hz, 1H), 8.15 (d, J = 1.8 Hz, 1H), 8.06 (dd, J = 8.4, 2.1 Hz, 1H), 7.85 (dd, J = 8.6, 1.9

Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 3.67 (broad, 8H).


136.28, 135.31, 133.97, 131.36, 130.60, 129.62, 129.57, 128.00, 126.65, 67.03.


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

C19H17F2N3O

M: 341.36 g mol-1

2-(3,4-Difluorophenyl)quinoxaline-6-carboxylic acid (0.5 g, 1.7 mmol) was converted

into 2-(3,4-difluorophenyl)-N,N-diethylquinoxaline-6-carboxamide using Method C.

The crude product was dissolved in EtOAc and filtered through a short SiO2 pad.

Afterwards the solvent was removed to yield the desired product (0.30 g, 0.9 mmol,

53% yield).

1H NMR (400 MHz, DMSO-d6) δ: 9.63 (s, 1H), 8.39 (ddd, J = 11.8, 7.8, 1.9 Hz, 2H), 8.26

– 8.20 (m, 1H), 8.16 (d, J = 8.5 Hz, 1H), 8.04 (d, J = 1.4 Hz, 1H), 7.82 (dd, J = 8.6, 1.7 Hz,

1H), 7.72 – 7.61 (m, 1H), 3.51 (broad, 4H), 3.25 (broad, 6H).

13C NMR (101 MHz, DMSO-d6) δ: 168.58 , 151.07 (dd, J = 250.5, 12.6 Hz), 149.93 (dd,

J = 246.1, 12.9 Hz), 149.45 , 144.35 , 140.97 , 140.58 , 138.64 , 133.42 (dd, J = 6.0, 3.5

Hz), 129.67 , 128.89 , 125.87 , 124.77 (dd, J = 7.0, 3.3 Hz), 118.33 (d, J = 17.5 Hz),

116.57 (d, J = 18.7 Hz), 42.91 , 14.02 , 12.78 .

19F NMR (376 MHz, DMSO-d6) δ: -135.76 (d, J = 22.3 Hz), -137.42 (d, J = 22.3 Hz).



69

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

C20H18F2N4O

M: 368.39 g mol-1


into (2-(3,4-difluorophenyl)quinoxalin-6-yl)(4-methylpiperazin-1-yl)methanone using

Method C. The crude product was dissolved in EtOAc and filtered through a short SiO2

pad. Afterwards the solvent was removed to yield the desired product (0.23 g,


1H NMR (400 MHz, DMSO-d6) δ: 9.65 (s, 1H), 8.61 – 8.35 (m, 2H), 8.33 – 8.12 (m, 3H),

8.08 (d, J = 2.3 Hz, 1H), 7.84 (ddd, J = 13.2, 8.5, 1.9 Hz, 1H), 7.75 – 7.60 (m, 1H), 3.69

(broad, 4H), 2.36 (broad, 4H), 2.21 (s, 3H).

13C NMR (151 MHz, DMSO-d6) δ: 167.62 , 151.13 (dd, J = 250.6, 12.7 Hz), 149.96 (dd,

J = 245.9, 12.5 Hz), 149.60 , 144.40 , 141.24 , 137.40 , 133.40 (dt, J = 5.7, 2.9 Hz),

129.71 , 129.31 , 127.15 , 126.93 , 124.89 – 124.78 (m), 118.39 (d, J = 17.5 Hz), 116.64

(d, J = 18.7 Hz), 47.08 , 45.57 , 41.53 .

19F NMR (376 MHz, DMSO-d6) δ: -135.65 (d, J = 22.5 Hz), -137.38 (d, J = 22.1 Hz).

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

C23H23FN4O3

M: 422.46 g mol-1


into the acid chloride as described in Method C. Afterwards an excess of morpholine

(25 mL) was added and the reaction mixture was heated to reflux. After 3h the

solvent was removed and the residue was dissolved in CH2Cl2. Unsoluble solid was


70

removed by filtration and the filtrate was washed once with 1 M aq HCl and twice

with H2O, dried over MgSO4 and the solvent was removed. The crude product was

dissolved in EtOAc and filtered through a SiO2 pad. The solvent was removed and the

resulting solid was washed with n-hexane to yield (2-(4-fluoro-3-

morpholinophenyl)quinoxalin-6-yl)(morpholino)methanone as yellow solid (0.17 g,


1H NMR (400 MHz, Chloroform-d) δ: 9.31 (s, 1H), 8.15 (dd, J = 8.5, 2.4 Hz, 1H), 8.10

(d, J = 1.6 Hz, 1H), 8.04 – 7.87 (m, 3H), 7.78 (ddd, J = 21.6, 8.6, 1.8 Hz, 1H), 7.07 (t, J =

8.6 Hz, 1H), 3.94 – 3.87 (m, 4H), 3.66 (broad, 8H), 3.30 – 3.13 (m, 4H).

13C NMR (101 MHz, Chloroform-d) δ: 169.22, 155.77 (d, J = 246.9 Hz), 151.32 (d, J =

2.4 Hz), 143.77 , 142.74 , 140.76 , 137.13 , 135.94 , 130.30 , 129.22 , 123.93 (d, J = 3.0

Hz), 118.75 (d, J = 3.7 Hz), 115.52 (d, J = 22.9 Hz), 66.98 (d, J = 5.6 Hz), 50.56 (d, J =

4.0 Hz).


6.3 BTB-1 Analogs Synthesis

General Methods for the Preparation of Sulfones and Sulfoxides

The sulfones and sulfoxides were synthesized according to known literature

procedures.106, 151-152 To a solution of the corresponding phenol, thiophenol or

thiophenolate and substituted nitrobenzene, an equimolar amount of base for the

thiophenol or 0.3 equiv. for the thiophenolate was added and heated to reflux. EtOH

and 0.1 M aqueous NaOH was used as the solvent if not otherwise mentioned. After

complete consumption of the starting material the solvent was removed and a 1/1

mixture of water and EtOAc was added. The aqueous phase was extracted twice with

EtOAc. The combined organic layers were dried over MgSO4 and the solvent was

removed in vacuum to yield the crude sulfide.

Method D The sulfide was dissolved in 5 mL acetic acid and an excess of 0.7 mL

30% H2O2 was added dropwise if not otherwise mentioned. After

complete addition the reaction mixture was heated to reflux. If


71

necessary another equivalent of H2O2 was added. After complete

reaction the solvent was removed in vacuum.

Method E The sulfide was dissolved in CH2Cl2 cooled to 0°C and 1 equiv. of m-

CPBA solution in CH2Cl2 was added drop wise. Saturated NaHCO3

solution was added for neutralization and the organic phase was

separated and the solvent removed to yield the crude sulfoxide.

1-Chloro-2-nitro-4-(phenylsulfonyl)benzene167 (70)

C12H8ClNO4S

M: 297.71 g mol-1

AlCl3 (1.56 g, 11.7 mmol) was suspended in benzene (5 mL) and 4-chloro-3-

nitrobenzenesulfonyl chloride (2.5 g, 9.8 mmol) was added. The mixture was stirred

for 24 h at ambient temperature and hydrolyzed with water (10 mL). The mixture was

extracted three times with EtOAc (15 mL). The organic layer was washed two times

with 1 M NaOH (5 mL) and with brine (5 mL). After drying over MgSO4 the solvent

was removed followed by column chromatographic purification (PE/EtOAc : 4/1) and

recrystallization from EtOAc/n-hexane to yield 1-chloro-2-nitro-4-

(phenylsulfonyl)benzene as white crystals (1.36 g, 4.5 mmol, 46% yield).

m.p: 124°C.

1H NMR (400 MHz, Chloroform-d) δ: 8.41 (d, J = 2.1 Hz, 1H), 8.05 (dd, J = 8.4, 2.1 Hz,

1H), 7.96 (m, 2H), 7.71 (d, J = 8.5 Hz, 1H), 7.68 – 7.61 (m, 1H), 7.61 – 7.53 (m, 2H).


131.67, 129.95, 128.11, 125.07.



72

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

C12H8N2O6S

M: 308.26 g mol-1

2,4-Dinitro-1-(phenylsulfonyl)benzene was synthesized according to Method D using

2,4-dinitrochlorobenzene (288 mg, 1.4 mmol) and sodium thiophenolate (188 mg, 1.4

mmol). After pruficiation by column chromatography (PE/EtOAc : 3/1) and

recrystallization from EtOAc/hexane 2,4-dinitro-1-(phenylsulfonyl)benzene was

received as off-white crystalls (110 mg, 0.3 mmol , 25% yield).

m.p.: 153°C

1H NMR (400 MHz, Chloroform-d) δ: 8.58 (d, J = 1.3 Hz, 2H), 8.53 (t, J = 1.3 Hz, 1H),

8.07 – 7.94 (m, 2H), 7.75 – 7.66 (m, 1H), 7.65 – 7.56 (m, 2H)

13C NMR (101 MHz, Chloroform-d) δ 150.57, 148.88, 140.10, 139.15, 134.86,

133.47, 129.63, 128.84, 126.93, 120.31.


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

C12H8N2O5S

M: 292.27 g mol-1

2,4-Dinitro-1-(phenylsulfinyl)benzene was prepared according to Method E using

thiophenole (0.4 mL, 3.4 mmol) and 2,4-dinitrochlorobenzene (705 mg, 3.4 mmol).

The crude sulfoxide was purified by column chromatography (PE/EtOAc : 9/1)

followed by recrystallization from EtOH to yield 2,4-dinitro-1-(phenylsulfinyl)benzene

as yellow crystalles (370 mg, 1.3 mmol, 38% yield).

m.p.: 108°C


73

1H NMR (400 MHz, Chloroform-d) δ: 9.04 (d, J = 1.9 Hz, 1H), 8.86 – 8.76 (m, 2H),

7.77 – 7.68 (m, 2H), 7.51 – 7.40 (m, 3H).

13C NMR (101 MHz, Chloroform-d) δ: 151.24, 149.58, 144.96, 144.06, 132.41,

129.78, 129.31, 128.24, 126.94, 120.81.


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

C12H8FNO4S

M: 281.26 g mol-1

4-Fluoro-2-nitro-1-(phenylsulfonyl)benzene was synthesized according to Method D

using 1-chloro-4-fluoro-2-nitrobenzene (500 mg, 2.8 mmol) and sodium

thiophenolate (380 mg, 2.8 mmol). After column chromatography (n-hexane/EtOAc :

3/1) 4-fluoro-2-nitro-1-(phenylsulfonyl)benzene was received as a white solid (50 mg,


m.p.: 126°C

1H NMR (400 MHz, Chloroform-d) δ 8.44 – 8.37 (m, 1H), 7.99 – 7.92 (m, 2H), 7.69 –

7.62 (m, 1H), 7.61 – 7.54 (m, 2H), 7.51 – 7.43 (m, 2H).

19F NMR (376 MHz, Chloroform-d) δ: -99.24.

13C NMR (151 MHz, Chloroform-d) δ: 164.92 (d, J = 261.8 Hz), 149.77 (d, J = 8.8 Hz),

140.26 , 134.21 (d, J = 9.4 Hz), 133.96 , 130.94 (d, J = 4.1 Hz), 129.20 , 128.24 ,

119.55 (d, J = 21.4 Hz), 113.08 (d, J = 27.2 Hz).



74

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

C13H8F3NO4S

M: 331.27 g mol-1

2-Nitro-1-(phenylsulfonyl)-4-(trifluoromethyl)benzene was prepared following

Methode D using 1-chloro-2-nitro-4-(trifluoromethyl)benzene (321 mg, 1.4 mmol)

and sodium thiophenolate (188 mg, 1.4 mmol). After column chromatography

(PE/EtOAc : 3/1) 2-nitro-1-(phenylsulfonyl)-4-(trifluoromethyl)benzene was

recrystallized from EtOAc/n-hexane and recieved as white crystalls (120 mg, 0.3

mmol, 26% yield).

m.p.: 142°C

1H NMR (400 MHz, Chloroform-d) δ 8.52 (d, J = 8.2 Hz, 1H), 8.08 – 8.01 (m, 1H), 8.01

– 7.95 (m, 3H), 7.73 – 7.65 (m, 1H), 7.64 – 7.55 (m, 2H).

19F NMR (376 MHz, Chloroform-d) δ: -63.33.

13C NMR (101 MHz, Chloroform-d) δ: 148.73 , 139.61 , 138.23 , 136.63 (q, J = 34.9

Hz), 134.54 , 132.77 , 129.50 , 129.70 – 129.26 (m), 128.69 , 122.26 (q, J = 3.7 Hz),

122.03 (q, J = 273.8 Hz).


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

C10H6ClNO4S2

M: 303.73 g mol-1

2-((4-Chloro-2-nitrophenyl)sulfonyl)thiophene was synthesized according to Method

D using thiophene-2-thiol (163 mg, 1.4 mmol) and 1,4-dichloro-2-nitrobenzene (269

mg, 1.4 mmol) and 1 M aq. NaOH as base. After column chromatography (PE/EtOAc

: 3/1) 2-((4-Chloro-2-nitrophenyl)sulfonyl)thiophene was received as off-white

crystalls (290 mg, 0.95 mmol, 68% yield).


75

m.p.: 134°C


1H), 7.78 (dd, J = 5.0, 1.3 Hz, 1H), 7.71 (dd, J = 8.4, 2.0 Hz, 1H), 7.69 (d, J = 1.9 Hz,

1H), 7.17 (dd, J = 4.9, 3.9 Hz, 1H).


133.58, 132.70, 132.46, 128.15, 124.95.


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

C16H10ClNO4S

M: 347.77 g mol-1

2-((4-Chloro-2-nitrophenyl)sulfonyl)naphthalene was prepared according to Method

D using 2-thionaphthalene (224 mg, 1.4 mmol) and 1,4-dichloro-2-nitrobenzene (269

mg, 1.4 mmol). The crude sulfone was purified by column chromatography (n-

hexane/EtOAc : 3/1) followed by recrystallization from EtOAc/ n-hexane to yield 2-

((4-chloro-2-nitrophenyl)sulfonyl)naphthalene as off-white solid (30 mg, 0.08 mmol,

6% yield).

m.p.: 155°C

1H NMR (400 MHz, Chloroform-d) δ: 8.60 (d, J = 8.5 Hz, 1H), 8.35 (s, 1H), 8.23 (d, J =

2.0 Hz, 1H), 7.99 (dd, J = 8.5, 2.0 Hz, 1H), 7.95 – 7.89 (m, 1H), 7.88 – 7.78 (m, 2H),

7.65 – 7.52 (m, 3H), 1.55 (s, 2H, H2O).


132.71, 129.97, 129.08, 128.55, 128.34, 128.06, 127.91, 127.55, 125.59, 121.66.



76

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

C16H10ClNO3S

M: 331.77 g mol-1

2-((4-Chloro-2-nitrophenyl)sulfinyl)naphthalene was synthesized following Methode

E using 2-thionaphthalene (224 mg, 1.4 mmol) and 1,4-dichloro-2-nitrobenzene (269

mg, 1.4 mmol). The crude sulfoxide was received as yellow oil which crystallizes upon

cooling. The yellow crystalls were washed with n-hexane to yield the pure 2-((4-

chloro-2-nitrophenyl)sulfinyl)naphthalene (306 mg, 0.9 mmol, 66% yield).

m.p.: 126°C

1H NMR (600 MHz, Chloroform-d) δ: 8.59 (d, J = 8.5 Hz, 1H), 8.35 (d, J = 1.8 Hz, 1H),

8.21 (d, J = 2.1 Hz, 1H), 7.98 (dd, J = 8.5, 2.1 Hz, 1H), 7.93 – 7.88 (m, 1H), 7.85 (d, J =

8.7 Hz, 1H), 7.83 – 7.79 (m, 1H), 7.59 (dd, J = 8.7, 1.9 Hz, 1H), 7.58 – 7.54 (m, 2H).


134.60, 132.64, 129.92, 129.03, 128.50, 128.30, 128.01, 127.85, 127.50, 125.53,

121.63.


4-Chloro-2-nitro-1-tosylbenzene (57)

C13H10ClNO4S

M: 311.74 g mol-1

4-Chloro-2-nitro-1-tosylbenzene was synthesized according to Method D using 4-

methylbenzenethiol (174 mg, 1.4 mmol) and 1,4-dichloro-2-nitrobenzene (269 mg,

1.4 mmol). After column chromatography (n-hexane/EtOAc : 3/1) the product was

received as light brown solid (30 mg, 0.09 mmol, 7%).


77

m.p.: 117°C


7.71 (dd, J = 8.5, 2.1 Hz, 1H), 7.68 (d, J = 2.1 Hz, 1H), 7.40 – 7.32 (m, 2H), 2.44 (s,

3H).


132.78, 132.58, 130.00, 128.58, 125.01, 21.85.


4-Chloro-1-((4-methoxyphenyl)sulfonyl)-2-nitrobenzene (58)

C13H10ClNO5S

M: 327.74 g mol-1

4-Chloro-1-((4-methoxyphenyl)sulfonyl)-2-nitrobenzene was synthesized according

to Methode D using 4-methoxybenzenethiol (196 mg, 1.4 mmol) and 1,4-dichloro-2-

nitrobenzene (269 mg, 1.4 mmol). After column chromatography (PE/EtOAc : 3/1) the

product was received as off-white solid (54 mg, 0.16 mmol, 12%).

m.p.: 104°C


7.70 (dd, J = 8.5, 2.1 Hz, 1H), 7.66 (d, J = 2.0 Hz, 1H), 7.08 – 6.95 (m, 2H), 3.88 (s,

3H).


132.52, 131.40, 131.06, 124.93, 114.62, 55.91.



78

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

C13H10ClNO4S

M: 311.74 g mol-1

4-Chloro-1-((4-methoxyphenyl)sulfinyl)-2-nitrobenzene was synthesized according

to Method E using 4-methoxybenzenethiol (350 mg, 2.5 mmol) and 1,4-dichloro-2-

nitrobenzene (480 mg, 2.5 mmol). The crude sulfoxide was crystallized from

EtOAc/PE. Due to traces of m-chlorbenzoic acid the crude product was dissolved in

CH2Cl2, washed with sat. K2CO3 solution and dried over MgSO4. After removal of the

solvent 4-chloro-1-((4-methoxyphenyl)sulfinyl)-2-nitrobenzene was received as

yellow solid (560 mg, 1.8 mmol, 72% yield).

m.p.: 118°C


7.95 (dd, J = 8.5, 2.1 Hz, 1H), 7.69 – 7.52 (m, 2H), 6.94 – 6.85 (m, 2H), 3.80 (s, 3H).


135.35, 128.90, 127.71, 125.54, 114.86, 55.66.


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

C12H9NO4S

M: 263.27 g mol-1

According to Methode D thiophenol (1.47 g, 13.3 mmol) and 1-chloro-2-nitrobenzene

(2.1 g 13.3 mmol) were converted to 1-nitro-2-(phenylsulfonyl)benzene. The crude

sulfide was recrystallized from water. After oxidation the crude sulfone was purified

with column chromatography (PE/EtOAc : 1/1) and recrystalized from EtOH to yield

1-nitro-2-(phenylsulfonyl)benzene as white solid (120 mg, 0.4 mmol, 3% yield).

m.p.: 145°C


79

1H NMR (400 MHz, Chloroform-d) δ: 8.41 – 8.33 (m, 1H), 8.03 – 7.94 (m, 2H), 7.84 –

7.70 (m, 3H), 7.68 – 7.60 (m, 1H), 7.60 – 7.52 (m, 2H).


132.63, 131.75, 129.25, 128.39, 124.91.


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

C12H9NO3S

M: 247.27 g mol-1

According to Method E thiophenol (1.47 g, 13.3 mmol) and 1-chloro-2-nitrobenzene

(2.1 g 13.3 mmol) were converted to 1-nitro-2-(phenylsulfinyl)benzene. The crude

sulfide was recrystallized from water. After oxidation the crude sulfoxide was purified

with column chromatography (EtOAc/PE : 1/1) and recrystallized from EtOH to yield

1-nitro-2-(phenylsulfinyl)benzene as yellow crystalls(1.7 mmol, 427 mg, 13% yield).

m.p.: 88°C

1H NMR (400 MHz, Chloroform-d) δ: 8.59 (dd, J = 7.9, 1.4 Hz, 1H), 8.26 (dd, J = 8.1,

1.2 Hz, 1H), 8.01 (m), 7.78 – 7.65 (m, 3H), 7.49 – 7.36 (m, 3H).


131.57, 129.41, 126.92, 126.45, 125.46.


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

C13H11NO5S

M: 293.29 g mol-1


80

4-Methoxybenzenethiol (0.89 g, 6.3 mmol) and 1-chloro-2-nitrobenzene (1.00 g, 6.3

mmol) were dissolved in EtOH. 0.8 g KOH was dissolved in EtOH and was added to

the reaction mixture followed by heating to 70°C. After filtration water was added to

the filtrate and extracted twice with 30 mL CH2Cl2. The organic layer was washed with

30 mL of 2M aq. KOH and dried over Na2SO4. After removal of the solvent the crude

sulfide was oxidized to the corresponding sulfone following Method D. The crude

product was washed with EtOAc yielding 1-((4-methoxyphenyl)sulfonyl)-2-

nitrobenzene as a white solid (100 mg, 0.34 mmol, 5% yield).

m.p: 148°C

1H NMR (400 MHz, Chloroform-d) δ: 8.35 – 8.25 (m, 1H), 7.99 – 7.87 (m, 2H), 7.78 –

7.64 (m, 3H), 7.09 – 6.94 (m, 2H), 3.87 (s, 2H).


131.81, 131.40, 131.01, 124.69, 114.51, 55.87.


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

C13H11NO4S

M: 277.29 g mol-1

4-Methoxybenzenethiol (1.78 g, 12.7 mmol) and 1-chloro-2-nitrobenzene (2.00 g,

12.7 mmol) were dissolved in EtOH. 0.8 g KOH was dissolved in EtOH and was added

to the reaction mixture followed by heating to 70°C. After filtration water was added

to the filtrate and extracted twice with 30 mL CH2Cl2. The organic layer was washed

with 30 mL of 2 M aq. KOH and dried over Na2SO4. After removal of the solvent the

crude sulfide was oxidized to the corresponding sulfoxide following Method E. The

sulfoxide was purified via cholumn chromatography (EtOAc/PE : 2/1) yielding 1-((4-

methoxyphenyl)sulfinyl)-2-nitrobenzene as yellow crystals (70 mg, 0.25 mmol, 2%).

m.p: 94°C


81

1H NMR (400 MHz, Chloroform-d) δ: 8.57 (dd, J = 7.9, 1.2 Hz, 1H), 8.24 (dd, J = 8.1,

1.0 Hz, 1H), 8.05 – 7.95 (m, 1H), 7.70 – 7.64 (m, 1H), 7.63 – 7.57 (m, 2H), 6.87 (dd, J

= 9.4, 2.5 Hz, 2H), 3.77 (s, 2H).


131.47, 129.04, 126.44, 125.58, 114.83, 55.70.


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

C13H10N2O6S

M: 322.29 g mol-1

2,4-Dinitro-1-tosylbenzene was synthesized according to Method E using 1-chloro-

2,4-dinitrobenzene (910 mg, 4.5 mmol) and 4-methylbenzenethiol (558 mg, 4.5

mmol). After purification by column chromatography (EtOAc/PE : 1/1) 2,4-dinitro-1-

tosylbenzene was received as an off-white solid (241 mg, 0.8 mmol, 18%).

m.p: 181°C

1H NMR (400 MHz, Chloroform-d) δ: 8.59 – 8.47 (m, 3H), 7.93 – 7.77 (m, 2H), 7.39

(d, J = 8.1 Hz, 2H), 2.46 (s, 2H).


133.31, 130.28, 128.99, 126.84, 120.19, 21.92.


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

C13H10N2O5S

M: 306.29 g mol-1


82

2,4-Dinitro-1-(p-tolylsulfinyl)benzene was synthesized according to Method E using

1-chloro-2,4-dinitrobenzene (910 mg, 4.5 mmol) and 4-methylbenzenethiol (558 mg,

4.5 mmol). After column chromatographic purification (EtOAc/PE : 1/2) and

recrystallization from EtOAc/n-hexane 4-dinitro-1-(p-tolylsulfinyl)benzene was

received as yellow crystals (260 mg, 0.9 mmol, 20%).

m.p: 134°C


8.78 (dd, J = 8.6, 1.9 Hz, 1H), 7.61 – 7.55 (m, 2H), 7.25 – 7.20 (m, 2H), 2.35 (s, 3H).

13C NMR (101 MHz, Chloroform-d) δ 151.49, 149.50, 144.93, 143.27, 140.87,

130.41, 129.23, 128.22, 127.00, 120.79, 21.58.


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

C12H7Cl2NO4S

M: 332.15 g mol-1

4-Chloro-1-((4-chlorophenyl)sulfonyl)-2-nitrobenzene was synthesized according to

Method D using 4-chlorobenzenethiol (750 mg, 5.18 mmol), 1,4-dichloro-2-

nitrobenzene (1.00 g, 5.18 mmol), MeOH as the solvent and 1 M aqueous NaOH as

the base. The crude reaction mixture was evaporated to dryness and the residue

dissolved in CH2Cl2 and washed with H2O. The aqueous layer was extracted twice with

CH2Cl2. The combined organic layers were dried over MgSO4 and the solvent was

removed in order to recieve the crude sulfide as a yellow solid. After oxidation the

crude product was purified by column chromatography (n-hexane/EtOAc : 3/1)

yielding 4-chloro-1-((4-chlorophenyl)sulfonyl)-2-nitrobenzene as a white powder

(850 mg, 2.83 mmol, 55% yield).

m.p.: 134°C


83


7.79 – 7.71 (m, 2H), 7.59 – 7.49 (m, 2H).


132.82, 129.96, 129.79, 129.70, 125.30.


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

C12H7ClN2O6S

M: 342.71 g mol-1

1-((4-Chlorophenyl)sulfonyl)-2,4-dinitrobenzene was synthesized according to

Method D using 1-chloro-2,4-dinitrobenzene (284 mg, 1.4 mmol) and 4-

chlorobenzenethiol (202 mg, 4.5 mmol). Upon cooling after the oxidation with H2O2

1-((4-chlorophenyl)sulfonyl)-2,4-dinitrobenzene crystallizes from glacial acetic acid

(368 mg, 1.1 mmol, 77% yield).

m.p.: 166°C

1H NMR (400 MHz, Chloroform-d) δ: 8.55 – 8.49 (m, 2H), 8.48 (dd, J = 2.0, 0.8 Hz,

1H), 7.92 – 7.78 (m, 2H), 7.53 – 7.47 (m, 2H).


133.49, 130.36, 130.00, 127.07, 120.44.


4-Chloro-2-nitro-1-phenoxybenzene (71)168

C12H8ClNO3

M: 249.65 g mol-1


84

To a solution of phenol (508 mg, 5.4 mmol) in DMSO (30 mL) was added KOH (303

mg, 5.4 mmol) and the reaction mixture was heated to 50°C for 30 min. After cooling

to ambient temperature a solution of 1,4-dichloro-2-nitrobenzene (1.037 g, 5.4

mmol) in DMSO (7 mL) was added and heated to 90°C for 16 h. After addition of H2O

(15 mL) the aqueous phase was extracted three times with EtO2 (50 mL). The organic

layer was washed with H2O and dried over MgSO4. After removal of the solvent 4-

chloro-2-nitro-1-phenoxybenzene was received as brown oil (1.300 g, 5.2 mmol, 96%

yield).


1H), 7.43 – 7.36 (m, 2H), 7.24 – 7.18 (m, 1H), 7.07 – 7.02 (m, 2H), 6.96 (d, J = 8.9 Hz,

1H).


128.23, 125.74, 125.12, 121.62, 119.43.


4-Chloro-1-iodo-2-nitrobenzene (72)154

C6H3ClINO2

M: 283.45 g mol-1

Para-toluenesulfonic acid (1.7 g, 9 mmol) was dissolved in MeCN (12 mL). Upon

addition of 4-chloro-2-nitroaniline (0.52 g, 3 mmol) a yellow salt was formed. The

reaction mixture was cooled to 0°C and a solution of NaNO2 (0.41 g, 6 mmol) and KI

(1.2 g, 7.5 mmol) in water (1.8 mL) was added dropwise. After 10 min the reaction

mixture was allowed to warm to ambient temperature and was stirred overnight. H2O

(50 mL) was added and the pH was brought to 9 by addition of saturated aq. Na2CO3

solution. The aqueouse phase was extracted three times with Et2O (16 mL). The

combined organic phases were washed with saturated aq. NaCl solution, dried over

MgSO4 and the solvent was removed. The crude oil was purified by column

chromatography (n-hexane/CH2Cl2 = 3/1) to yield 4-chloro-1-iodo-2-nitrobenzene as

yellow solid (0.58 g, 2 mmol, 68% yield).


85

1H NMR (400 MHz, DMSO-d6) δ: 8.12 (d, J = 2.4 Hz, 1H), 8.10 (d, J = 8.5 Hz, 1H), 7.52

(dd, J = 8.5, 2.4 Hz, 1H).

13C NMR (101 MHz, DMSO-d6) δ: 154.18, 142.38, 133.88, 133.57, 124.78, 86.33.

4-Chloro-2-nitrobenzenediazonium (74)156

C6H3ClN3O2+

M: 184.56 g mol-1

4-Chloro-2-nitroaniline (8.6 g, 50 mmol) was suspended in 50% aqueous HBF4-

solution (70 mL) and diazotized at 0°C with a aq. NaNO2 solution (3.4 g, 50 mmol).

After 15 min the precipitate was separated and washed with EtOH and Et2O.

Afterwards, the salt was dissolved in MeCN and precipitated upon addition of Et2O to

yield 4-chloro-2-nitrobenzenediazonium tetrafluoroborate as off-white salt (5.2 g, 19

mmol, 38% yield).

6.4 Biochemical and Cellular Assays

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

All kinesin motor domains were expressed and purified as described previously.106

Bacteria of the E.coli strain BL21RIL were transformed with the plasmids coding for

poly-histidine-tagged motor proteins in the presence of selective antibiotics

(ampicillin 100 μg mL-1 and chloramphenicol 34 μg mL-1) and incubated over night at

37°C. Multiple colonies were used to inoculate 25 to 100 mL culture of LB medium,

grown over night at 37°C including antibiotics as above. 10 mL per liter of pre-

inoculum were used to inoculate a new liquid culture (2 to 4 liters) plus antibiotics as

above, which was grown at 37°C until OD600 0.6 units was reached. Afterwards the

expression of the recombinant protein was induced with 0.5 mM IPTG and allowed

for 18 h at 18°C and the bacteria were harvested by centrifugation (4500 x g, 15 min,

4°C). The pellet was frozen (-80°C, 20 min) and thawe, resuspended with 10 mL lysis

buffer (20 mM Tris pH 8.0, 300 mM NaCl, 5 mM imidazole, 0.1% Triton X 100,


86

Complete protease inhibitor Roche) per liter of original culture. The cells were lysed

by high-pressure treatment using the EmulsiFlex-C5 or C3 homogenizer (Avestin) and

then cleared by centrifugation (40000 x g, 30 min, 4°C). The lysate was incubated with

Ni-NTA beads (Qiagen) for 2 h at 4°C on a rotating wheel (0.25 to 0.5 mL resin per

liter of original culture), washed 3 times in batch with 20 mL washing buffer (20 mM

Tris pH 8.0, 300 mM NaCl, 20 mM imidazole, +/- 1 mM NaATP and 1 mM MgCl2) and

finally eluted over a Poly- Prep Chromatography Column (Bio-Rad) in 0.5 to 1 mL

fractions (elution buffer: 20 mM Tris pH 8.0, 300 mM NaCl, 200 mM imidazole). The

fractions containing the recombinant protein were pooled together and dialyzed o/n

at 4°C (dialysis buffer: 10 mM β-mercaptoethanol, 10% glycerol, 25 mM Tris pH 7.4,

300 mM NaCl) then aliquoted, snap-frozen and stored at -80°C.

Polymerization of Taxol Stabilized Microtubules

1 mL of pig brain purified tubulin dimers (10 mg mL-1) was precleared by

ultracentrifugation (186000 g, 4°C, 10 min) and supplemented with 25% glycerol, 1.5

mM GTP and 668 µl BRB80. The mixture was incubated at 37°C for 30 min and

afterwards taxol was added to a final concentration of 40 µM to the mix and the

microtubules were allowed to polymerize for additional 30 min at 37°C. Finally the

microtubules were pelleted by ultra-centrifugation (186000 g, 30 min, 35°C) and the

pellet was resuspended in BRB80 containing 25 μM taxol. The concentration of

polymerized microtubules was determined by measuring the absorbance at 280 nm

of tubulin dimers (resuspended in 6 M guanidine-HCl).

Malachite Green Assay

Mklp2MD (80 nM), taxol stabilized microtubules (1 µM), Triton X-100 (0.1%), MgATP

(400 µM) and SH-1 (10 µM, 25 µM, 50 µM and 100 µM) were incubated in buffer

containing 20 mM PIPES (pH 6.8), 1 mM MgCl2, 1 mM EGTA, 0.1 mg mL-1 BSA and 0.1

μM taxol at room temperature. In 6 minute intervals 10 µL of reaction mixture were

quenched by addition of 40 μl 1 M HClO4. 50 μl malachite green solution (1.3 M HCl,


87

8.5 mM ammonium molybdate, 363 nM malachite green) were added. After 20

minutes at room temperature absorbance at 650 nm was measured. Relative

absorbance was calculated by subtracting the absorbance after 0 min.

Enzyme Coupled Assay

A mix of MgATP, taxol stabilized microtubules, 200 µM NADH, 5 mM PEP (pH 7), 4 U

mL-1 Pyruvate kinase, 8 U mL-1 Lactate dehydrogenase and assay buffer (10 mM

Imidazole/acetate pH 7.2, 5 mM Mg-acetate, 2 mM EGTA, 20 µM taxol) was

supplemented with DMSO or compounds. The reaction was started by addition of

kinesin motor domain and absorbance was measured every 30 seconds at 340 nm for

10 minutes using the Tecan 500 plate reader. DMSO was used as solvent control and

the activity of the kinesin motor domain was set to 100%. The ATPase activity was

calculated using following equation

[(-∆A340nm min-1/(εNADH × d)]/c(Kif18A)/(60s × min-1)

Enzyme Coupled Assay SH1 Analogs

A mix of 1 mM MgATP, 2µM taxol stabilized microtubules, 40 nM Mklp2MD and the

standard conditions as described above were used. DMSO was applied in 5% or 10%

and the SH-1 analogs at 50 µm.

Enzyme Coupled Assay SH1 Titration with Triton X-100

A mix of 1 µM MgATP, 2 µM or 400 nM taxol stabilized microtubules, 40 nM Mklp2MD

and the standard conditions as described above were used. DMSO was applied at 5%

and SH1 in 25 µM, 50 µM 75 µM and 100 µM concentration.


88

Enzyme Coupled Assay BTB-1 Analogs

A mix of 650 µM MgATP, 3 µM taxol stabilized microtubules, Kif18AMD (20 nM) and

the standard conditions as described above were used.The mix was supplemented

with DMSO (0.5%) or BTB-1 and analogs (5µM).

IC50 (Kif18A)

Increasing concentrations of BTB-1 analogs were used to determine the residual

activity of 20 nM His-Kif18AMD in the presence of 650 μM ATP and 3 μM taxol

stabilized microtubules by enzyme-coupled assay (see above). DMSO was used as

solvent control and the activity of the protein in its presence was set to 100%. The

assays were performed in 384 well plates and the oxidation of NADH over time was

monitored using a plate reader as reported above.

Basal Kif18A ATPase Activity

The ATPase activity of 300 nM Kif18AMD without microtubules was determined using

the enzyme coupled assay (as described above) in the presence of 50 µM BTB-1, 53

and 59 or 0.5% DMSO as solvent control.

Inhibition Mode of 59

Increasing concentrations of 59 and ATP were used in an enzym coupled assay (as

described above) to elucidate the inhibition mode. According to the Michaelis-

Menten kinetics each data set was fit to competitive inhibitory model using the

software Prism 5 (GraphPad).

Cellular Thermal Shift Assay (CETSA)159

In order to prepare a mitotic extract HeLa cells were synchronized using 2mM

thymidine (24 h) and released into 0.5 µM Nocodazole (14 h). The cells were shook


89

off, washed three times with PBS and released in fresh media for 30 min. After

harvesting, the cell suspension was washed three times with PBS supplemented with

Complete protease inhibitor (Roche) and snap frozen in liquid nitrogen. The cell

suspension was freeze-thawed three times and cleared by centrifugation (20000 g,

20 min at 4°C). The supernatant was diluted with PBS containing Complete protease

inhibitor (Roche) and divided into three aliquots, with two aliquots treated with

compounds and one aliquot used as DMSO control. The samples were incubated for

30 min at room temperature and divided into smaller aliquots (25 µl), which were

heated individually at different temperatures (46-62°C in 2°C steps) using the Veriti

thermal cycler followed by cooling to room temperature for 3 min. The treated

lysates were cleared by centrifugation (20000 g, 20 min at 4°C) and analyzed by SDS-

PAGE followed by western blot analysis.

Western Blot Analysis

SDS page electrophoresis and western blot analysis were performed according to

standard protocols.169 The detection of the protein of interest was performed using

specific primary antibodies, commercially available HRP-conjugated secondary

antibodies (Bio-Rad and Dianova) and ECL (100 mM Tris-HCl pH 8.5, 1.25 mM luminol,

225 μM coumaric acid, 0.015% v/v H2O2) reagent.

Antigen Organism Specification/Antibody number

Kif18A (human) Rabbit M. Mayr42

Tubulin Mouse FITC conjugated (SIGMA)

CycB Mouse TUM359

Cdc27 Rabbit TUM 229

Tubulin Polymerization Assay

A mix of 0.8 µM glutamate (pH 6.6), 100 µM MgCl2 and 10 µM tubulin was

supplemented with DMSO (0.5%), compound (50µM) or Nocodazole, which was used

as a control tubulin polymerization inhibitor, and incubated for 15 min at ambient


90

temperature. Afterwards, the mixtures were cooled on an ice bath for 5 min and the

polymerization was started by addition of GTP (final conc. 0.4 mM) and incubated for

30 sec at 30°C. The assays were performed in 384 well plates and the polymerization

was measured turbidimetrically at 350 nm every 20 sec over a time period of 30 min

using the plate VARIOSKAN FLASH from Thermo Scientific. The tubulin polymerization

velocity was determined by linear regression and compared to the DMSO control

which was set to 100%.

IC50 (Tub.Polym.)

The experiments were performed using the conditions described above in quartz

cuvettes with the final assay volume of 150 µL with increasing concentrations of BTB-

1 and its analogs. The polymerization was measured with VarianInc Cary100bio

spectrophotometer for 20 min at 30°C and the depolymerization for 15 min at 15°C.

The tubulin polymerization velocity was determined by linear regression and

compared to the DMSO control which was set to 100%.

Cell Culture

HeLa cells were cultured in Dulbecco’s modified Eagle medium (DMEM)

supplemented with 10% fetal calf serum (Gibco), penicillin (100 U mL-1) and

streptomycin (100 μg mL-1) at 37°C in a humid atmosphere with 5% CO2.

Immunofluorescence

A DeltaVision Core system (Applied Precision) mounted on a IX-71 inverted

microscope (Olympus) equipped with a LED-system, a CoolSnap HQ-2 camera

(Photometrics) and a 40 x 1.35 NA UApo/340 (Olympus) oil objective was used for

image acquistion.

HeLa cells stably expressing GFP labeled Kif18A, as previously described in Häfner et

al.49 were synchronized using 2 mM thymidine (18 hours) and plated on coverslips in

6 well plates (200,000 cells/well). The coverslips were treated with different

concentrations of BTB-1 or its analogs or DMSO as solvent control for 30 min. The


91

cells were then treated and immunostained as described previously to visualize

microtubules and DNA.42

Live Cell Imaging

A 20 x 0.4 NA LD Plan Neo (Zeiss) objective on a Axio Observer Z1 (Zeiss) equipped

with an environmental chamber (Zeiss), Colibri LED Modules and a CoolSnap-ES2

camera (Photometrics) controlled by MetaMorph Software were used for image

acquisition.

SH1 Analogs

For live cell imaging a stably GFP-H2B expressing HeLa cell line was used. The cells

were seeded in a 12 well plate 24 h prior to use in a density of 100000 mL-1 in 2mM

thymidine. After 16 h the cells were released from the thymidine block and after 8 h

the CO2 independent live cell media premixed with compound solution was applied

to the cells. The cells were subsequently imaged for about 18 hours. Five random

positions were determined per well and every 10 minutes a picture was taken of

every position using bright field and the 470 nm (for GFP) fluorescence channel.

BTB-1 Analogs

HeLa cells stably expressing GFP labeled Kif18A, as previously described in Häfner et

al.49 were synchronized using 2 mM thymidine in a density of 200000 mL-1 (18 hours).

The cells were released from the thymidine block and after 6 h the CO2 independent

live cell media premixed with compound solution was applied to the cells. The cells

were subsequently imaged for about 18 hours. Five random positions were

determined per well and every 10 minutes a picture was taken of every position using

bright field and the 470 nm (for GFP) fluorescence channel.

Alamar Blue Assay158 and EC50 Values

HeLa cells were seeded in 96-well plates (4,000 cells/well) and allowed to attach for

24 h. Compounds to be tested were dissolved in a suitable amount of DMSO and

different concentrations were prepared to give final concentrations with a maximum


92

DMSO content of 1%. The cells were incubated for 48 h with different concentrations

of the compound. AlamarBlue (10 µL) was added and the cells were incubated for

another hour. After excitation at 530 nm, fluorescence at 590 nm was measured using

a FL600 Fluorescence Microplate Reader (Bio-TEK). Cell viability is expressed in

percent with respect to a control containing 1% DMSO.

References

93

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Appendix

107

8. Appendix

8.1 Appreviations

ADP adenosine diphosphate

aq. aqueous

ATP adenosine triphosphate

CETSA cellular thermal shift assay

DMAP 4-dimethylaminopyridine

DMF dimethylformamide

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

EC50 half maximal effective concentration

ECA enzyme coupled assay

equiv. equivalents

EtOAc ethyl acetate

EtOH ethanol

FRET Förster resonance energy transfer

G1-phase gap1 phase

G2-phase gap2 phase

GAP GTPase-activating protein

GDP guanosine diphosphate

GFP green fluorescent protein

GTP guanosine triphosphate

HCS high content screening

HTS high throughput screening

IC50 half maximal inhibitory concentration

INCENP inner centromere protein

K-fibers kinetochore microtubules

MAPs microtubule-associated proteins

m-CPBA meta-chloroperoxybenzoic acid

Appendix

108

MeCN acetonitrile

MGA malachite green assay

Mklp2 mitotic kinesin-like protein 2

M phase mitotic phase

MPP1 M-phase phosphoprotein 1

NADH nicotinamide adenine dinucleotide

NEBD nuclear envelope breakdown

PE petroleum ether

PEP phosphoenolpyruvate

PLK1 polo-like kinase 1

PRC1 protein regulator of cytokinesis

p-TsOH para-toluenesulfonic acid

RNAi ribonucleic acid interference

SAC spindle assembly checkpoint

SAR structure activity relationship

SNAr nucleophilic aromatic substitution

S phase synthesis phase

TMEDA Tetramethylethylenediamine

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Novel Small Molecule Inhibitors for the Human Kinesins