Selective inhibitors of gentotoxic steress-induced IKK/NF-κB pathways

Abstract

A method is for treatment of a disease associated with genotoxic stress-induced inhibitor of nuclear factor-κB kinase/nuclear factor kappa-light chain enhancer of activated B cells (IKK/NF-κB) signaling. The method can include administering a compound to a subject having a cancer exhibiting genotoxic stress induced IKK/NF-κB activation.

Claims

1. A method of treatment of a cancer in a subject in need thereof comprising: (a) identifying the subject as having a cancer exhibiting genotoxic stress-induced IKK/NF-κB signaling activation that causes growth of the cancer, (b) selectively blocking activation of IKK/NF-κB in response to the genotoxic stress upstream of the IKK complex in the subject without blocking NF-κB activation upon signaling caused by other pathways in the subject by administering to the subject a compound according to Formula I, ##STR00206## wherein R1=H, O; R2=from 0-4, can be the same or different, H, OH, halogen, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or wherein R2 is alkoxyamine, alkoxyamide; R3=from 0-4, can be the same or different, H, OH, halogen, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or wherein two (adjacent) R3 substituents can form an optionally aromatic cyclic structure of 5 or 6 members, optionally comprising 0, 1, or 2 heteroatoms; X1 and X2=N or C; X3=C; ring A is an aromatic cyclic structure of 5 or 6 members, optionally comprising 0, 1, or 2 heteroatoms selected from the group consisting of O and N, wherein said cyclic structure is optionally substituted with 0-3 substituents that can be the same or different, selected from the group consisting of H, OH, halogen, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, alkoxycarbonyl, amine, aryl, (optionally substituted with halogen, C1-C3 alkyl, alkoxy, amine) and alkoxyamine; the bond z may be present or not present, wherein when bond z is not present: the C of bond z of ring C is potentially substituted with R3, and X3 of the A ring is optionally substituted with H, OH, halogen, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine.

2. The method according to claim 1, wherein the compound is, according to Formula II, ##STR00207## wherein R1=H, O; R5=H, halogen, C1-C5, alkyl, alkenyl, alkoxy, amine; R6=H, OH, halogen, C1-C5, alkyl, alkoxy, or alkoxyamine, alkoxyamide; R7=H, halogen, C1-C5, alkyl, or alkoxy; R8=H, halogen, C1-C5, alkyl or alkoxy; R9=H, halogen, C1-C5, alkyl or alkoxy; R10=H, halogen, C1-C5, alkyl or alkoxy; R11=H, halogen, C1-C5, alkyl, alkoxy, or carboxyl; R12=H, halogen, C1-C5, alkyl or alkoxy; or wherein when X1 is C, R9 and R10, R10 and R11, R11 and R12, or R12 and the C in the position of bond z of ring C, form an optionally aromatic cyclic structure of 5 or 6 members, comprising 0, 1, or 2 heteroatoms or forming phenyl; X1=N or C; X3=C; ring A is an aromatic cyclic structure of 5 or 6 members, comprising 0, 1, or 2 heteroatoms selected from the group consisting of 0 and N, wherein said cyclic structure is optionally substituted with 0-3 substituents that can be the same or different, selected from the group consisting of H, OH, halogen, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, aryl, (optionally substituted with halogen, C1-C3 alkyl, alkoxy, amine), and alkoxyamine; the bond z may be present or not present, wherein when bond z is not present: the C in the position of bond z of ring C is substituted with halogen, preferably Cl, Br, F, C1-C7, alkyl, and X3 of the A ring is optionally substituted with H, C1-C5, alkyl, or when X3 is C with H, C1-C5, alkyl, OH, halogen.

3. The method according to claim 1, wherein R1=0.

4. The method according to claim 1, wherein at least one of R2 from 0-4 is not H.

5. The method according to claim 1, wherein ring A is a heteroaromatic cyclic structure of 6 members, comprising 1 or 2 heteroatoms selected from 0 and/or N.

6. The method according to claim 1, wherein ring A is selected from the group consisting of ##STR00208##

7. Compound according to Formula I, ##STR00209## wherein: R1=0; R5=H, halogen, C1-C5 alkyl or alkoxy, or amine; R6=H, OH, halogen, C1-C5 alkyl or alkoxy; R7=H, halogen, C1-C5 alkyl or alkoxy; R8=halogen, C1-C5 alkyl or alkoxy, wherein at least one of R5 to R8 is not H; R9=H, halogen, or C1-C5 alkyl or alkoxy; R10=H, halogen, or C1-C5 alkyl or alkoxy; R11=H, halogen, C1-C5 alkyl, alkoxy or carbonyl; R12=H, halogen, or C1-C5 alkyl or alkoxy; or wherein when X1 is C, R9 and R10, R10 and R11, R11 and R12, or R12 and the C in the position of bond z of ring C, form an optionally aromatic cyclic structure of 5 or 6 members, comprising 0, 1, or 2 heteroatoms or forming phenyl; X1=N or C; X3=C; ring A is a heteroaromatic cyclic structure of 6 members, comprising 1 or 2 heteroatoms selected from 0 and/or N, wherein said cyclic structure is optionally substituted with 0-3 substituents that can be the same or different, selected from the group consisting of H, OH, halogen, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, aryl and alkoxyamine; the bond z may be present or not present, wherein when bond z is not present; and the C in the position of bond z of ring C is substituted with halogen or C1-C5 alkyl, or X3 of the A ring is optionally substituted with H, C1-C5 alkyl, OH or halogen.

8. Compound according to claim 7, wherein the compound is of Formula III, ##STR00210## wherein R1=0; R2=from 0-4, can be the same or different, H, OH, halogen, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, alkoxycarbonyl, amine, or wherein R2 is alkoxyamine or alkoxyamide, wherein at least one of R2 from 0-4 is not H; R3=from 0-4, can be the same or different, H, OH, halogen, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or wherein two (adjacent) R3 substituents form an optionally aromatic cyclic structure of 5 or 6 members, optionally comprising 0, 1, or 2 heteroatoms or forming phenyl; X1=N or C; X3=C; ring A is a heteroaromatic structure of 6 members, comprising 1 or 2 N atom, wherein said cyclic structure is optionally substituted with 0-3 substituents that can be the same or different, selected from the group consisting of H, OH, halogen, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, aryl (optionally substituted with halogen, C1-C3 alkyl, alkoxy, amine), alkoxyamine, or wherein ring A is a heteroaromatic structure selected from the group consisting of: and ##STR00211##

9. Compound according to claim 7, wherein the compound is of Formula VIII, ##STR00212## wherein R1=O; R2=from 0-4, can be the same or different, H, OH, halogen, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or wherein R2 is alkoxyamine, alkoxyamide, wherein at least one of R2 from 0-4 is not H; R3=from 0-4, can be the same or different, H, OH, halogen, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or wherein two (adjacent) R3 substituents can form an optionally aromatic cyclic structure of 5 or 6 members, optionally comprising 0, 1, or 2 heteroatoms or form phenyl; X1=N or C; X4=N or C, whereby at least one X4 is N; R16=can be 0-3, the same or different, H, halogen, C1-C5 alkoxy.

10. Compound according to claim 7, wherein the compound is of Formula IX, ##STR00213## wherein R1=O; R2=from 0-4, can be the same or different, H, OH, halogen, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or wherein R2 is alkoxyamine, alkoxyamide, wherein at least one of R2 from 0-4 is not H; R3=from 0-4, can be the same or different, H, OH, halogen C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or wherein two (adjacent) R3 substituents can form an optionally aromatic cyclic structure of 5 or 6 members, optionally comprising 0, 1, or 2 heteroatoms or form phenyl; X1=N or C; R16=can be 0-3, the same or different, H, halogen, C1-C5, alkyl or alkoxy.

11. Compound according to claim 7, wherein the compound is of Formula X, ##STR00214## wherein R1=O; R2=from 0-4, can be the same or different, H, OH, halogen, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or wherein R2 is alkoxyamine, alkoxyamide, wherein at least one of R2 from 0-4 is not H; R3=from 0-4, can be the same or different, H, OH, halogen C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or wherein two (adjacent) R3 substituents can form an optionally aromatic cyclic structure of 5 or 6 members, optionally comprising 0, 1, or 2 heteroatoms or form phenyl; X1=N or C; R16=can be 0-3, the same or different, H, halogen, C1-C5 alkyl, alkoxy or methoxy.

12. Compound according to claim 7, wherein the compound is of Formula VI, ##STR00215## wherein R1=O; R2=from 0-4, can be the same or different, H, OH, halogen, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or wherein R2 is alkoxyamine, alkoxyamide, wherein at least one of R2 from 0-4 is not H; R3=from 0-4, preferably 0, 1, 2, can be the same or different, H, OH, halogen, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or wherein two (adjacent) R3 substituents can form an optionally aromatic cyclic structure of 5 or 6 members, optionally comprising 0, 1, or 2 heteroatoms or form phenyl; X1=N or C; R16=can be 0-3, the same or different, H, halogen, C1-C5 alkoxy.

13. The method according to claim 1, wherein the compound is ##STR00216##

14. The method according to claim 1, wherein the compound is more effective in inhibiting NF-κB-signaling induced by genotoxic stress compared to inhibiting NF-κB-signaling induced by TNF-alpha and/or IL-1 ß.

15. The method according to claim 1, wherein the disease is associated with genomic instability due to defective DNA-repair mechanisms.

16. The method according to claim 1, wherein said cancer is associated with NF-κB-mediated resistance to therapy-induced tumor cell apoptosis.

17. The method according to claim 1, wherein the compound is administered in combination with one or more genotoxic stress-inducing (DNA damage-inducing) cancer therapies.

18. In vitro method for the inhibition of genotoxic stress-induced NF-κB signaling or inhibition of DNA repair mechanisms comprising the use of a compound according to claim 1.

19. The method of claim 1, wherein the genotoxic stress-induced IKK/NF-κB signaling pathway is activated by a factor selected from the group consisting of a pro-inflammatory cytokine, PAMPS (pathogen associated molecular patterns), engagement of immune receptors and γ-irradiation (γ-IR).

Description

FIGURES

(1) The invention is further described by the following figures. These are not intended to limit the scope of the invention, but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.

(2) Brief description of the figures:

(3) FIG. 1: Simplified model of the genotoxic stress-induced NF-κB signaling cascade.

(4) FIG. 2: MW01 is a specific inhibitor of DNA damage induced p65 nuclear translocation.

(5) FIG. 3: Concentration dependent inhibition of DNA damage-induced NF-κB activation by MW01.

(6) FIG. 4: MW01 does not inhibit NF-κB activation by TNFα stimulation.

(7) FIG. 5: MW01 does not inhibit NF-κB activation by IL-1β stimulation.

(8) FIG. 6: Inhibition of genotoxic stress-induced NF-κB activation by MW01 takes place upstream of TAK1 activation.

(9) FIG. 7: MW01 inhibits genotoxic stress-induced NF-κB activation by blocking the cytoplasmic accumulation of ATM.

(10) FIG. 8: MW01 inhibits the formation of the nuclear PARP1-signalosome.

(11) FIG. 9: MW01 does not inhibit enzymatic activity of ATM.

(12) FIG. 10: MW01 do not inhibit the enzymatic activity of PARP1.

(13) FIG. 11: MW01 inhibits the formation of essential IKKγ post-translational modifications following genotoxic stress.

(14) FIG. 12: Overview of the molecule structures of MW01 and derivatives thereof.

(15) FIG. 13: Structure-activity-relationship analyses of MW01 in comparison to its derivatives.

(16) FIG. 14: Inhibition of genotoxic stress-induced NF-κB activation by PARP inhibitors is cell type dependent.

(17) FIG. 15: Influence of MW01 on the mRNA expression of anti- and pro-apoptotic genes following DNA damage.

(18) FIG. 16: MW01 increases apoptotic cell death after genotoxic stress.

(19) FIG. 17: MW01 significantly increases γH2AX foci per cell in untreated cells.

DETAILED DESCRIPTION OF THE FIGURES

(20) FIG. 1: Upon DNA double strand breaks the sensor proteins ATM and PARP1 are activated. PARP1 undergoes poly(ADP)-ribose (PAR) chain auto-modification, which serve as a scaffold for the recruitment of the IKK complex subunit IKKγ, PIASy, and activated ATM. The formation of the this nuclear PARP1 signalosome leads to posttranslational modifications of IKKγ-SUMOylation by PIASy and phosphorylation by ATM. SUMOylated and phosphorylated IKKγ is transported into cytoplasm where it most likely is incorporated into IKK holocomplexes. Simultaneously, ATM is transported into cytoplasm. After binding to TRAF6 it activates its auto-polyubiquitination with Ubc13-assisted lysine 63-linked ubiquitin chains. These ubiquitin chains serve as a scaffold for the recruitment of important signaling components like TAB2-TAK1, cIAP1 and the IKK complex. Ubiquitin-mediated binding of TAK1 to the cytoplasmic signalosome leads to TAK1 auto-phosphorylation that subsequently leads to a priming IKK phosphorylation by TAK1 and an auto-phosphorylation of the IKK T-loop serines. Convergence of exported SUMOylated IKKγ and the cytoplasmic ATM-TRAF6-dependent axis is required for mono-ubiquitination of IKKγ at Lys285, which in turn is essential for full IKK activation (Hinz et al.; 2010, Stilmann et al.; 2009). As an additional step, LUBAC-dependent M1-linked ubiquitination of IKKγ was shown to be critical for the genotoxic NF-κB pathway. Activation of the IKK complex subsequently leads to the degradation of IκBα and the activation of the NF-κB heterodimer p65/p50 analogously to canonical NF-κB activation.

(21) FIG. 2: U2OS cells were pre-treated and incubated with DMSO or MW01. DNA damage was induced by administration of etoposide. After 2 h cells were fixed, nuclei were stained with DAPI. p65 and phospho-H2AX, which is indicative for DNA DSB, were stained by immunofluorescence. Images were taken at a confocal Zeiss 710 LSM with a 40× oil objective.

(22) FIG. 3: (A) U2OS cells were pre-treated with increasing concentrations of MW01 in duplicates in a 384 well plate. Then, cells were treated with etoposide, fixed and subjected to IF of p65. Spatial measurement of p65 cytosolic and nuclear localisation was used for calculation of p65 translocation rates. (B) U2OS cells were pre-treated with DMSO, MW01 and irradiated with γ-IR. After 90 min cells were lysed and subjected to SDS-PAGE/WB and EMSA. (C) Cells were pre-treated with different concentrations of MW01 as indicated, treated with γ-IR (C) or etoposide (D) and subjected to WB or EMSA, respectively. LDH in (C) represents the loading control.

(23) FIG. 4: (A) U2OS cells were pre-treated with increasing concentrations of MW01 in triplicates in a 384 well plate. Then, cells were treated with TNFα, fixed and subjected to IF staining of p65. Spatial measurement of p65 cytosolic and nuclear signals was used for the calculation of p65 translocation rates. (B) U2OS cells were pre-treated with MW01 at concentrations of 10 or 20 μM and stimulated with TNFα. Cell lysates were subjected to SDS-PAGE/WB. (C) HEK293 cells were pre-treated with DMSO or MW01 followed by administration of etoposide or TNFα. Cell lysates were used for EMSA.

(24) FIG. 5: (A) U2OS cell were pre-treated with MW01 followed by stimulation with IL-1p. Cell lysates were used for SDS-PAGE/WB and EMSA. (B) Cells pre-treated with increasing concentrations MW01 were stimulated with IL-1p. Cell lysates were used for SDS-PAGE/WB and stained with indicated antibodies.

(25) FIG. 6: (A) U2OS cells were pre-treated with DMSO or MW01 and irradiated. Stimulation of cell with IL-1β served as a positive control for IκBα and p65 phosphorylation. Cells were lysed at indicated time points and used for WB analyses. (B,C) Similar experimental setup as in (A). (D) Experiment as in (A-C), but with other time points analysed and performed with HepG2 cells. PARP1 and LDH serve as loading controls.

(26) FIG. 7: Cytoplasmic accumulation of ATM. (A) Fractionation experiment with separated nuclear extracts (NE) and cytoplasmic extracts (CE). PARP1 and LDH staining served as loading and fractionation controls. Cells were pre-treated with DMSO or MW01 (5 μM) prior to irradiation and cell harvesting at indicated times. NE and CE were subjected to SDS-PAGE/WB procedure. (B) U2OS cells were seeded on cover slips 2 days before treatment. The cells were pre-treated with solvent DMSO alone or with MW01 prior to irradiation, cell fixation and subsequent immunofluorescence staining.

(27) FIG. 8: (A) HepG2 cells were pre-treated with DMSO or MW01 and irradiated. Nuclear cell extracts (NE) were used for PIASy immunoprecipitation (IP). Western blot membranes were incubated with indicated antibodies. (B) Experiment as shown in (A), but with IKKγ IP. (C) Experiment as shown in (B), but done in HEK293 cells. (D) Experiment done as shown in (F), but repeated in MEF cells.

(28) FIG. 9: HepG2 cells were pre-incubated with DMSO, MW01 (5 μM) or the ATM inhibitor Ku55933 (10 μM) and irradiated. After 60 min cells were harvested and processed. Immunochemical staining of Western blotting membranes was done using indicated antibodies.

(29) FIG. 10: (A) MEF cells were pre-treated with the indicated substances, irradiated and cell lysates were used for poly(ADP)-ribose probing using a specific antibody. (B) Experiment performed as described for (A), but using U2OS cells.

(30) FIG. 11: (A) HEK293 cells were pre-incubated with DMSO, MW01 or ATM inhibitor KU55933 and irradiated. Lysates were subjected to SDS-PAGE/WB. The specific IKKγ S85 band (lower band) was identified by induction following irradiation and by sensitivity to ATMi and λ-phosphatase (λ-PP) treatment. The asterisk indicates a non-specific band that was neither inducible nor ATMi sensitive. (B) Experiment was done as shown in (A) using MEF cells. (C) HEK293 cells were pre-treated with DMSO or MW01, irradiated and lysed. Lysates were used to immunoprecipitate IKKγ using an IKKγ antibody.

(31) FIG. 12: (A) Molecule structures of MW01 and tested derivatives. (B) Systematic nomenclature of ring systems in the molecule structure of MW01 (Markgraf et al.; 2005). (C) Molecule structures of MW01 and tested derivatives D01-D18. (D) Variations of preferred ring C structures of Formulae I-VII, and preferred ring B structures of Formulae I-VII.

(32) FIG. 13: (A) U2OS cells were pre-treated with DMSO, MW01 or its derivatives MW01A1-MW01C5 at concentrations of 10 μM for 2 h prior to etoposide treatment. After incubation with etoposide cells were harvested, lysed and subjected to SDS-PAGE/WB. Phospho-5536 p65 signal intensities as well as p65 signal intensities were detected using a CCD camera and band intensities were used for densitometrical analyses. DMSO and etoposide treated control was set to 1. Four independent experiments were performed and statistical outliers were identified and eliminated using Grubb's test. The deviation is displayed as the standard error of the mean (SEM). (B) NFkB/293/GFP-luc cells were pre-treated with DMSO, MW01 or its derivatives MW01D01-MW01D18 at concentrations of 10 μM for 1.5 h prior to etoposide treatment. After incubation for 4.5 hours NF-κB-dependent luciferase expression was measured by detection of chemoluminescence. DMSO and etoposide treated control was set to 1. Twelve independent experiments were performed.

(33) FIG. 14: (A) U2OS cells were pre-treated with different concentrations of olaparib prior to co-treatment with etoposide. Whole cell lysis was performed after 90 min and cleared lysates were used for WB with indicated antibodies. (B) U2OS cells were pre-treated with DMSO, olaparib or the ATM inhibitor Ku55933 and co-treated with etoposide. Cells were harvested after 45 min and subjected to SDS-PAGE/WB. Membranes were stained with the indicated antibodies. The experiment of (B) was performed simultaneously to the experiments of (A) and (E) to control functionality of olaparib in inhibiting PAR chain formation. (C) Densitometrical analyses of (A) and two further independent experiments done as shown in (A). (D) U2OS cells were pre-treated with the substances as indicated prior to irradiation. After 90 min mRNA was isolated, transcribed into cDNA and was used for expression analyses of the indicated genes using quantitative real-time PCR (qRT-PCR). (E) HepG2 cells were pre-treated with the PARP inhibitors olaparib, 3-AB and EB-47 prior to γ-irradiation. Cells were harvested after 15 and 90 min and subjected to SDS-PAGE/WB. Membranes were stained with the indicated antibodies. Error bars equal SEM.

(34) FIG. 15: (A), (B) U2OS cells were pre-treated with DMSO or MW01 and irradiated. After 8 h mRNA was isolated and reversely transcribed into cDNA. The obtained cDNA was used to perform qRT-PCR using gene specific exon-exon-spanning primers for mRNA of anti-apoptotic (A) and pro-apoptotic genes (B).

(35) FIG. 16: (A) U2OS cells were pre-treated with DMSO or MW01 and irradiated. Cells were lysed 8 h post γ-IR and were used for western blotting and immunochemical staining. (B) HEK293 cells were pre-treated with DMSO or MW01 and irradiated. After 48 h cells were fixed and stained with crystal violet. Dissolved crystal violet was used for absorbance measurement in a visible light spectrophotometer at a wavelength of 595 nm. (C) HEK293 cells were pre-treated with DMSO or MW01 and irradiated. The percentage of viable cells within the population was calculated by exclusion of annexin V and propidium-iodide positive cells measured by flow cytometry. (D) MEF cells were pre-treated with DMSO or MW01 and irradiated. Cells were used for annexin V staining 24 h and 48 after γ-IR. Percentage of annexin V positive cell population was analysed using a flow cytometer. (E) Experiment done as shown in (D) but with HT1080 cells processed 8 h after irradiation. Statistical significance was calculated using students t-test.

(36) FIG. 17: U2OS cells grown on coverslips were incubated 30 min with DMSO or MW01 (5 μM). Then, cells were γ-irradiated (5 Gy) or mock irradiated (mock IR). After 5 hours, cells were fixed and subjected to immunofluorescence staining procedure. DNA damage-indicating γH2AX foci and nuclei n≥480 nuclei per condition) were counted for the calculation of average foci per nucleus. Significance was calculated using student's t-test.

EXAMPLES

(37) The invention is further described by the following examples. These are not intended to limit the scope of the invention, but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.

(38) Methods Employed in the Examples

(39) RNA Isolation

(40) For RNA isolation cells were washed with ice-cold PBS. Isolation of RNA then was performed according to manufacturer's instructions (Qiagen, RNeasy RNA isolation KIT). Integrity of isolated RNA was ensured by measuring the ratio of 28s and 18s ribosomal RNA at a Bioanalyzer using a RNA testing chip (Agilent RNA 6000 Nano Kit) according to manufacturer's instructions.

(41) Determination of Nucleic Acids Concentration

(42) Using a UV light spectrophotometer DNA/RNA concentration was measured at OD260. Protein or chemical contaminations were checked by measurement of ratios of 0D260/280 and OD260/230. Further analyses were performed on samples with 0D260/280 ratios of about 2.

(43) Reverse Transcriptase-PCR and Quantitative Real-Time PCR (c/RT-PCR)

(44) In order to generate complementary DNA (cDNA) 500-1000 ng total RNA was transcribed using the iScript cDNA synthesis Kit (Promega) following manufacturer's instructions.

(45) To quantitate specific mRNA (messenger RNA) species in samples RNA was isolated, RNA concentration was measured and mRNA was transcribed into cDNA. The amount of mRNA transcripts of certain genes within a sample was quantified by employing gene specific primers and using a C-1000 Thermal cycler (Biorad). The expression of genes of interest was normalised against two or three reference genes (HRPT1, RPL_13a and B2M) using the CFX manager software. The fold induction of mRNA was calculated over untreated sample levels by the ΔΔ-Ct method.

(46) Cell Culture

(47) All cell lines were cultured in media supplemented with 10% FCS and penicillin/streptomycin (100 U/ml and 100 μg/ml) in 95% relative humidity and 5% CO2 atmosphere. U2OS and HEK293 cells were cultured in DMEM, mouse embryonic fibroblast were cultured in DMEM Glutamax, and HepG2 cells were cultured in RPMI 1640 medium (all obtained from Gibco). For passaging, cells were washed with PBS, trypsinised with trypsin/EDTA solution at 37° C. until detachment from the plate and suspended in the corresponding medium. Splitting ratios were between 1:3 to 1:5 (U2OS, HepG2) and 1:10 to 1:15 (MEF and HEK293). For cryo-conservation in liquid nitrogen cells were trypsinised at 37° C., suspended in medium and pelleted by centrifugation at 320×g for 5 min. Afterwards, cells were resuspended in freezing medium (corresponding medium supplemented with 20% FCS, 10% DMSO and penicillin/streptomycin) and were frozen in freezing boxes containing isopropanol in a −80° C. freezer. Cells were transferred to liquid nitrogen at the following day. Thawing of cells was done in at 37° C. in a water bath. Partially-frozen cells were pipetted dropwise to 37° C. pre-warmed medium and centrifuged for 5 min at 300×g. Finally, cells were resuspended in fresh complete medium.

(48) For the activation of the canonical NF-κB pathway cells were treated with recombinant human TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 20-30 min at 37° C.

(49) Genotoxic stress was applied by ionizing irradiation of cells with a Cs137 source (0629 Irradiator, STS Braunschweig), or by inhibition of the topoisomerase II enzyme by administration of etoposide at concentrations between 20-50 μM for 2 h.

(50) Immunofluorescence Staining and Confocal Microscopy

(51) For immunofluorescence staining 0.95×10.sup.5 cells were seeded in 6 well plates onto autoclaved cover slips. Cellular confluency dictated to beginning of the experiment (2-3 days from seeding). After conduction of experiments cells were washed with PBS and fixed with 4% PFA/double-distilled H.sub.2O (ddH2O) for 10 min at RT. Following two additional washing steps cells were incubated with a solution containing 0.12% glycine/0.2% saponin in PBS for 10 min and then blocked with a solution containing 10% FCS/0.2% saponin in PBS for 1 h. Primary antibody incubation was performed overnight at 4° C. (1:500 diluted in 0.2% saponin in PBS). The next day, cover slips were washed five times with a solution containing 0.2% saponin in PBS. Fluorophor-coupled secondary antibodies (1:1000 diluted in 0.2% saponin in PBS) were incubated for 1 h (hour) at RT. Nuclei were stained using 0.2 mg/ml DAPI in PBS for 5 or by directly mounting with DAPI/Mowiol. Finally, the cover slips were washed five times with 0.2% saponin in PBS and two times with ddH2O. Confocal microscopy was performed using a Zeiss 710 LSM with a 40× or a 63× oil objective.

(52) Crystal Violet Staining

(53) For crystal violet staining, cells were washed with ice-cold PBS and fixed with 4% PFA in PBS for 15 min under a fume hood. After washing with PBS, cells were stained with 0.1% crystal violet for 20 min at RT. Afterwards, cells were washed again three times with PBS and were air dried. Cells were incubated with 10% acetic acid for 20 min while shaking. Then, 0.25 ml of stain was diluted 1:4 in ddH2O and absorbance was measured at 595 nm using a spectrophotometer against 10% acetic acid as blank.

(54) Flow Cytometry

(55) Cells were washed with ice-cold PBS and detached from growing dishes using Trypsin/EDTA solution. Detached cells were centrifuged at 300×g for 5 min. Detection of early apoptotic cells was performed by staining with annexin V-FITC antibody according to manufacturer's instructions (eBioscience Annexin V-FITC Apoptosis detection Kit). Necroptotic and late apoptotic cells were stained by addition of propidium iodide (final concentration 1 μg/ml) prior to measurements.

(56) Cell Harvesting

(57) Tissue culture plates of interest were washed with ice-cold PBS. The cells were scraped in PBS using cell scrapers and the cell suspension was transferred to 1.5 ml reaction tubes. Cells were pelleted by centrifugation at 20,000×g for 15 s at 4° C. The supernatant was discarded and cells were snap frozen or lysed directly.

(58) Whole Cell Lysis

(59) Cell pellets were resuspended in 3 volumes of Baeuerle lysis buffer on ice and lysed for 20 min while shaking moderately at 4° C. Samples were centrifuged at 20,000×g for 10 min at 4° C. and the supernatant, representing the whole cell protein extract, was transferred into a new 1.5 ml reaction tube.

(60) Subcellular Fractionation

(61) For the preparation of nuclear and cytoplasmic fractions, cells were lysed with buffer A (supplemented with 1 mM DTT, 10 mM NaF, 20 mM β-glycerophosphate, 250 nM NaVO3, complete protease inhibitor cocktail (Roche) and 50 nM calyculin A. Lysates were adjusted to a final concentration of 0.2% NP-40, vortexed for 10 s and spun down. The supernatant, representing the cytoplasmic extract (CE), was transferred into a new 1.5 ml reaction tube. The pellet was washed with buffer A, was resuspended with buffer C and shaken for 20 min at 4° C. Following 10 min of centrifugation at 14,000 rpm, the supernatant, representing the nuclear extract (NE), was transferred into a new reaction cap.

(62) Determination of Protein Concentration

(63) To determine protein concentration of cell lysates, 1-2 μl of protein extracts were mixed with 1 ml Bradford reagent diluted 1:5 with ddH2O. Absorbance was measured in a spectrophotometer at a wavelength of 595 nm against a lysis buffer reference and was compared to a BSA standard curve.

(64) Immunoprecipitation

(65) Following cell lysis the protein concentration of samples was determined. For input controls, 40 μg lysate were mixed with 6×SDS-buffer and denatured by heating to 95° C. for 4 min. Approximately 1500 μg protein lysate was used for pulldown and samples volumes were equalled with lysis buffer. Lysates were precleared with 30 μl sepharose A or sepharose G beads (depending on the antibody type used for pulldown) for 30 min, and centrifuged for 5 min at 1,500×g. The supernatant was transferred to a new reaction tube. Primary antibody (2-2.5 μg) was added to the cleared lysate for immunoprecipitation overnight while rotating at 4° C. The next day 30 μl sepharose beads per sample were used for immobilisation of antibodies. Following 4 washes with IP wash buffer precipitated proteins were eluted by mixing with 3×SDS-buffer and heating to 95° C. for 4 min.

(66) Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

(67) For preparation of cell lysates for SDS-PAGE 20-40 μg of protein lysates were mixed with 6× reaction buffer and heated to 95° C. for 4 min. After boiling samples were loaded into a polyacrylamide gel. Gels were casted consisting of a separating gel and a stacking gel. The concentration of acrylamide within the separating gels was depending on the experiment and the desired separation between certain molecular weights, but generally ranged between 8% and 12%.

(68) TABLE-US-00005 Stacking gel Tris-HCl, pH 6.8 125 mM Acrylamide .sup. 5% SDS 0.1% APS (ammonium persulphate) 0.1% TEMED 0.1% Separation gel Tris-HCl, pH 8.8 375 mM Acrylamide 8-12%  SDS 0.1% APS 0.075%  TEMED 0.05% 

(69) After sample loading a voltage of 80 V was applied to allow protein concentration at the border line of stacking and separating gel. Afterwards, voltage was increased to 140 V and proteins were separated for circa 2 h.

(70) Western Blotting

(71) Proteins separated by SDS-PAGE (6.3.5) were immobilised by Western blotting (WB) to methanol-activated PVDF membrane using transfer buffer and a semi-dry blotting apparatus. Proteins were transferred to membranes by applying a constant current of 80 mA per 6×9 cm membrane for 90 min. For the transfer of small proteins (<30 kDa) the blotting time was reduced to 30 min.

(72) Immunochemical Detection of Proteins on Membranes

(73) After transfer of proteins on PVDF membranes unspecific binding of antibodies was blocked by incubation of membranes in 5% skim milk powder in TBST buffer (or 3% BSA in TBST for phosphorylation-specific antibodies) for 1 h at RT. Membranes were incubated overnight at 4° C. with a solution of primary antibody in 5% skim milk powder in TBST or 3% BSA in TBST (phosphorylation-specific antibodies) diluted 1:1000. The next day membranes were washed three times with TBST for 5 min. Then, membranes were incubated for 1 h with a HRP-coupled secondary antibody (1:10000) directed against the FC-part of the used corresponding primary antibody. After three times of washing with TBST and once with PBS for 5 min, chemiluminescent photon emission was detected using a CCD camera system (Fusion Solo). Enhanced chemiluminescence (ECL) solution (Millipore) was used as HRP substrate.

(74) Membranes were stripped to allow subsequent probing with multiple antibodies using Restore PLUS WB Stripping buffer (Thermo Scientific) for 35 min at RT. After extensive washing with TBST, membranes were blocked again with 5% skim milk powder in TBST for 1 h and were incubated with the next primary antibody overnight.

(75) H2K/NF-κB Oligonucleotide Preparation

(76) Oligonucleotides were ordered as high-performance liquid chromatography (HPLC) purified BamHI ends. For annealing 5 μg of each strand were incubated for 10 mins at 90° C. in 50 μl annealing buffer resulting in a final concentration of 200 ng/μl. Hybridized oligonucleotides were allowed to cool down over night in the thermal block and stored at −20° C. afterwards. Annealing of oligonucleotides was analysed in a 12% polyacrylamide gel by comparing 1 μg of hybridized oligonucleotides with 1 μg single strand oligonucleotides.

(77) Radioactive Labelling and Purification of NF-κB Oligonucleotides

(78) For radioactive labelling of the H2K/NF-κB probe, the reaction recipe was followed and the mixture was incubated for 15 minutes (min) at 25° C. The purification of radioactive labelled NF-κB probe was done using the QIAquick Nucleotide Removal Kit (Qiagen) according to the manufacturer's instructions. Radioactive labelling was measured using a scintillation counter. Radioactive probe was stored at −20° C.

(79) Labelling Recipe:

(80) H2O 10.2 μl

(81) DNA-Oligonucleotide (200 ng) 1.0 μl

(82) 10× Klenow buffer 2.5 μl

(83) dCTP, dGTP, dTTP (2 mM each) 1.8 μl

(84) α[32P] dATP 7.5 μl (3 MBq)

(85) DNA Pol I (Klenow fragment, 5 U/μl) 0.2 μl (1 U)

(86) Electro Mobility Shift Assay (EMSA)

(87) Nuclear or whole cell lysates were incubated with a .sup.32P-labeled NF-κB DNA-consensus

(88) sequence. The shift mixture was prepared following the shift mixture recipe:

(89) Shift mixture for EMSA (H2K/NF-κB)

(90) total lysate 3-5 μg

(91) 2× shift buffer 10.0 μl

(92) BSA (10 ng/μl) 1.0 μl

(93) DTT (100 mM) 0.4 μl

(94) Poly dl-dC (2 μg/μl) 1.0 μl

(95) .sup.32P-labeled oligonucleotide 45,000 cpm

(96) ddH2O ad 20 μl

(97) The shift mixture was incubated for 30 min at 37° C. before the samples were loaded onto an

(98) EMSA gel:

(99) EMSA gel recipe (native polyacrylamide gel)

(100) ddH2O 44 ml

(101) 10×TBE 6 ml

(102) Acrylamide (30%) 10 ml

(103) APS (10%) 450 μl

(104) TEMED 45 μl

(105) For electrophoresis, a current of 26 mA was applied for 2 h. After drying the gel onto a Whatman paper, signals were visualised on an autoradiography film (GE Healthcare) after overnight incubation at −80° C. in a radiography cassette. All work using radioactive substances were done at a monitored work space suitable for radioactive work.

(106) Results of the Examples

(107) Identification of MW01 by High Content Screening

(108) In order to identify specific inhibitors of the DNA damage-induced NF-κB pathway, a differential screening assay was designed. The primary screening for inhibitors of genotoxic stress-induced NF-κB signaling utilised a library of compounds from ChemBioNet and donated compounds of academic chemists. DNA damage was applied by application of etoposide. All compounds which inhibited p65 nuclear translocation were taken for subsequent counter screening. For the counter screening administration of TNFα was used to induce canonical NF-κB signaling. All substances inhibiting TNFα-induced canonical NF-κB activation were discarded from the list of potential DNA damage-pathway specific substances.

(109) Based on its IC.sub.50 value of 0.46 μM, its percentage in activity change of 120% (as recorded in IC.sub.50 determination assay) and the calculated Hill coefficient of −0.9, compound MW01 was chosen for further analyses. MW01 was identified as a shown to specifically selective inhibition of genotoxic stress induced IKK/NF-κB activation, as it inhibited NF-κB activation in response to etoposide stimulation, but not after TNFα stimulation.

(110) Validation of Compound MW01 as a DNA Damage-Specific NF-κB Inhibitor

(111) MW01 Inhibits NF-κB Activation Upon Genotoxic Stress

(112) The small molecule MW01 was identified as the most promising genotoxic stress-specific NF-κB inhibitor by differential discrimination but needed further validation with material from another provider. Therefore, a fresh stock of lead compound MW01 was obtained from vendors, solved in DMSO and tested for reproducible inhibition of etoposide-induced p65 nuclear translocation using IF staining of p65 (FIG. 2). Additionally, γH2AX foci as a sensitive marker for DNA DSB were visualised. As performed in the differential screening, pre-treatment of cells with MW01 inhibited p65 translocation upon DNA DSB-induction by the administration of etoposide.

(113) The measured IC.sub.50 curves of MW01 indicated that MW01 inhibited the nuclear translocation of p65 upon etoposide stimulation in a concentration dependent manner (FIG. 3A) and a concentration of 5 μM was sufficient for maximal inhibition of p65 nuclear translocation.

(114) In addition to etoposide treatment, γ-irradiation of cells was used as an alternative way to induce DNA damage in further experiments. The pre-treatment of cells with MW01 inhibited the γ-IR-induced NF-κB DNA binding activity and p65 phosphorylation at S536 (FIG. 3B), thus showing etoposide-independent inhibition of NF-κB. As observed for etoposide treatment, the pre-treatment with MW01 also led to a concentration dependent inhibition of p65 S536 phosphorylation following γ-IR (FIG. 3C). In addition to p65 nuclear translocation and p65 S536 phosphorylation, etoposide-induced NF-κB DNA binding activity was also inhibited by MW01 pre-treatment in a concentration dependent manner (FIG. 3D).

(115) Taken together, the concentration-dependent inhibition of p65 S536 phosphorylation by MW01 (FIG. 3C) is in perfect agreement with the corresponding results on the observed inhibition of p65 nuclear translocation (FIG. 3A).

(116) Hence, the analysis of p65 nuclear translocation, p65 S536 phosphorylation and of NF-κB DNA binding activity, validates MW01 as genuine inhibitor of genotoxic stress-induced NF-κB activation.

(117) MW01 does not Inhibit Canonical NF-κB Activation

(118) Canonical NF-κB signaling is initiated by the binding of extracellular ligands to their cell membrane bound receptors, which initiate an intracellular signaling cascade ultimately activating the IKK complex and consequently NF-κB. MW01 was tested in experiments using TNFα to stimulate NF-κB activity in order to confirm specificity for the genotoxic stress-induced NF-κB activation.

(119) MW01 did not interfere with p65 nuclear translocation rates at different concentrations (FIG. 4A). In addition, pre-treatment of cells with MW01 at concentrations of 10 μM and 20 μM had no effect on p65 S536 phosphorylation (FIG. 4B). Furthermore, pre-treatment of cells with MW01 did not interfere with induced NF-κB DNA binding activity upon TNFα stimulation (FIG. 4C).

(120) This experiment was performed in HEK293 cells and substantiated the cell line independent inhibitory effect of MW01.

(121) Genotoxic stress-induced and IL-1β stimulated NF-κB activation share the ubiquitin E3 ligase TRAF6 as an important signaling module. Upon activation, TRAF6 is auto-modified with K63-linked ubiquitin chains, which serve as a scaffold for the recruitment of TAK1 via its adaptor protein TAB2 (Hinz et al.; 2010).

(122) Therefore, MW01 was analysed to investigate, whether they would interfere with IL-1β-induced NF-κB activation.

(123) Pre-treatment with both compounds neither inhibited p65 S536 phosphorylation nor NF-κB DNA binding activity following IL-1β stimulation (FIG. 5A). Furthermore, MW01 was tested for their impact on IKK activation and p65 S536 phosphorylation at high concentrations up to 100 μM, but had no effect on the phosphorylation state of IKK or p65 (FIG. 5B).

(124) In summary, MW01 did not inhibit canonical NF-κB signaling induced by either TNFα or IL-1β stimulation and thus showed specificity for the DNA damage-induced NF-κB pathway.

(125) MW01 Inhibits the Nuclear-to-Cytoplasmic Signal Transduction that is Required for DNA Damage-Induced NF-κB Activation

(126) Inhibition of Genotoxic Stress-Induced NF-κB Activation by MW01 Takes Place Upstream of TAK1 Activation

(127) TNFα and IL-1β-induced NF-κB activation is dependent on signaling cascades involving TAK1 and IKK activation by phosphorylation upstream of IκBα and p65 phosphorylation. Signaling dynamics of MW01 pre-treated cells were analysed to rule out the possibility that the compounds inhibit NF-κB activation downstream of TAK1 in a genotoxic stress-dependent manner (FIG. 6).

(128) Cells were pre-treated with MW01, γ-irradiated and harvested at indicated time points in a time course experiment (FIG. 6A). Pre-treatment of cells with MW01 led to the complete inhibition of IκBα phosphorylation at 45 min and 60 min after γ-IR.

(129) Similarly, p65 S536 phosphorylation at 45 min and 60 min after γ-IR was inhibited by MW01. Given that IκBα phosphorylation is a consequence of IKK activation, the phosphorylation state of IKK was analysed in the next step (FIG. 6B). The pre-treatment of cells with MW01 also abolished IKK phosphorylation 90 min after irradiation.

(130) The kinase TAK1 is located upstream of IKK in the pathway and is similarly activated by phosphorylation. MW01 pre-treatment strongly inhibited TAK1 phosphorylation at 45 and 60 min following irradiation (FIG. 6C). This result was also true in HepG2 cells. TAK1 and p65 phosphorylation were abolished by pre-treatment with MW01, although ATM was phosphorylated as a consequence of γ-IR (FIG. 6D). Of note, the repeated inhibition TAK1 and p65 phosphorylation in HepG2 cells indicated general, cell line independent inhibitory function of MW01 on genotoxic stress-activated NF-κB signaling.

(131) Taken together, these results strongly suggest that the inhibited step within the genotoxic stress-initiated NF-κB signaling cascade upstream of TAK1 activation.

(132) MW01 Inhibits Genotoxic Stress-Induced NF-κB Activation by Blocking the Cytoplasmic Accumulation of ATM

(133) The DNA DSB-activated kinase ATM is mainly localised in the nucleus, but translocates into cytoplasm upon DNA damage. Hinz et al. (2010) showed that the accumulation of activated ATM within the cytoplasm and membrane fractions leads to the activation and subsequent autoubiquitination of TRAF6 with K63-linked ubiquitin chains. The polyubiquitin chains serve as a scaffold for the recruitment of signaling components, including TAK1 and the IKK complex (Hinz et al.; 2010). Thus, this nuclear-to-cytoplasmic signaling cascade leads to the activation of the IKK complex by a mechanism that requires the cytoplasmic translocation of ATM.

(134) To analyse the impact of MW01 on the DNA damage-induced ATM accumulation in the cytoplasm following γ-IR, fractionation experiments were performed. Pre-treatment of cells with MW01 did not affect detection of phosphorylated ATM in nuclear extracts at 45 min and 90 min after irradiation (FIG. 7 A) (compare lanes 2-3 with lanes 5-6). However, detection of activated ATM species in the cytoplasmic extracts was abolished in MW01 pre-treated samples (compare lanes 8-9 with lanes 11-12). The analysis of total ATM amounts revealed that pre-treatment of cells with MW01 inhibited the accumulation of ATM in the cytoplasm at 45 min after irradiation (FIG. 7 A) (compare lane 8 with lane 11). Same results were obtained when U2OS instead of HepG2 cells were tested (data not shown). Furthermore, the effect of MW01 on ATM relocalisation upon DNA DSB was analysed by IF (FIG. 7 B). Irradiation led to strong ATM translocation into the cytoplasm and to the cellular periphery. In contrast, in MW01 treated samples ATM was predominantly localized within the nucleus. The results of the IF imaging strongly support those observed in the fractionation experiments.

(135) The results of the fractionation experiments indicated that the targeted signaling step by MW01 is at the level of ATM cytoplasmic translocation and possibly upstream. Therefore, ATM-mediated TRAF6-autoubiquitination was not further analysed. Hence, considering the results obtained for IL-1 n-stimulated NF-κB activation (FIG. 5 B), it is plausible that TRAF6 activation is not targeted directly, but abolished by both compounds as a result of the inhibited cytoplasmic accumulation of ATM

(136) MW01 Inhibits DNA Damage-Induced NF-κB Activation Downstream of PARP1 and ATM Activation

(137) The Formation of the Nuclear PARP1-Signalosome is Inhibited by MW01

(138) The formation of a nuclear IKKγ-PIASy-PARP1-ATM signalosome is important to trigger the genotoxic stress-induced NF-κB signaling cascade. The formation of this signalosome requires PARP1, whose enzymatic activity is activated by DNA DSB to attach poly-(ADP)-ribose (PAR) chains onto its substrates and onto itself. These polymers serve as a scaffold for the recruitment of the remaining components of the signalosome (Stilmann et al.; 2009). The influence of MW01 on signalosome formation was analysed by interaction studies using co-immunoprecipitations (Co-IP). The immunoprecipitation of PIASy led to the γ-irradiation-induced Co-IP of phosphorylated ATM-51981 species, which was lost after pre-treatment with MW01 (FIG. 8 A). Next, the interaction between PARP1 and IKKγ was analysed by immunoprecipitation of IKKγ. PARP1 was co-immunoprecipitated with IKKγ from not-irradiated and γ-irradiated cell lysates. Importantly, PARP1 was not co-immunoprecipitated with IKKγ after pre-treatment with MW01 (FIG. 8 B). That MW01 abrogated Co-IP of PARP-1 with IKKγ was also observed in HEK293 cells (data not shown), indicating cell type independent mode of action.

(139) Next, HEK293 cells were used to examine the effect of MW01 treatment on the IKKγ-PIASy-interaction. The PIASy co-immunoprecipitation with IKKγ was inducible and dependent on γ-irradiation, but the interaction was abolished when cells were treated with MW01 (FIG. 8 C). In addition, murine MEF were used for further species and cell type independent generalisation of these findings (FIG. 8 D). As seen in HepG2 or HEK293 cells, MW01 pre-treatment led to the abrogation of the interaction of IKKγ with PARP1, p-ATM S1981 or PIASy, whereas the interactions were shown for DMSO pre-treated samples. The species independent inhibition of the signalosome formation by MW01 indicates that the mode of action is based on a general and conserved mechanism.

(140) MW01 does not Inhibit the Enzymatic Activity of ATM

(141) Activation of the cellular DDR to DNA DSB is strongly dependent on the activity of the serine-kinase ATM. Activated ATM can phosphorylate a plethora of substrates within the mammalian cell and regulates cell cycle arrest, DNA repair or apoptosis (Shiloh and Ziv; 2013). Similarly, it is an essential component of the genotoxic stress-mediated NF-κB signaling pathway (Hinz et al.; 2010).

(142) Therefore, the enzymatic activity of ATM was analysed in cells pre-treated with MW01 after γ-irradiation. MW01 was tested in comparison to the ATM inhibitor KU55933 in order to show that the phosphorylation of the different substrates indeed is ATM dependent. The treatment of cells with KU55933 inhibited the ATM auto-phosphorylation and the phosphorylation of the ATM substrates p53BP1, p53 and KAP1. Despite pre-treatment of cells with MW01 had mild effects on the phosphorylation state of p53bp1, no effects on the phosphorylation state of ATM and the other substrates p53 and KAP1 were observed compared to the solvent and the ATMi control. In addition, MW01 pre-treatment did not lead to impaired phosphorylation of the ATM substrate histone H2AX after etoposide treatment as already shown in FIG. 2. Importantly, MW01 treatment drastically reduced p65 S536 phosphorylation level (FIG. 9).

(143) Taken together, the analyses of ATM auto-phosphorylation and substrate phosphorylation show that the enzymatic activity of ATM is not affected by pre-treatment of cells with MW01.

(144) MW01 does not Inhibit the Enzymatic Activity of PARP1.

(145) Stilmann and colleagues described that the enzymatic activity of PARP1 was essential for PARP1 signalosome formation and recruitment of other signaling components to initiate the DNA damage-induced NF-κB signaling cascade (Stilmann et al.; 2009). Therefore, the influence of MW01 on PARP1 enzymatic function was analysed. Upon γ-irradiation, a strong band was detected using a PAR chain specific antibody in DMSO and MW01 pre-treated samples in MEF and U2OS cells (FIG. 10 A-B). In contrast, pre-treatment of cells with the PARP inhibitors EB-47, 3-AB (FIG. 10 A) or the clinically approved drug Olaparib (Mullard; 2014) led to the inhibition of PAR chain formation (FIG. 10 A+B). Thus, it was shown that MW01 did not interfere with activation of PARP1 enzymatic activity in human and murine cells.

(146) MW01 Inhibits the Formation of Essential Post-Translational Modifications of IKKγ Following Genotoxic Stress

(147) The formation of the PARP1 signalosome upon irradiation is a prerequisite for DNA damage-induced NF-κB signaling, because IKKγ needs to be subjected to at least 3 different PTMs. Following DNA DSB, IKKγ is SUMOylated by PIASy within the PARP1 signalosome (Stilmann et al.; 2009). Then, ATM phosphorylates IKKγ at Serine 85 (Z. H. Wu et al.; 2006). As a consequence of the activated signaling cascade IKKγ is mono-ubiquitinated by cIAP1 (Hinz et al.; 2010).

(148) In order to analyse the influence of MW01 on the ATM-dependent IKKγ phosphorylation at S85, cells were pre-treated with the compounds prior to irradiation. MW01 pre-treatment as well as the inhibition of ATM abolished the phosphorylation of IKKγ at S85 in human (FIG. 11 A) and in murine cells (FIG. 11 B).

(149) MW01 pre-treatment abolished IKKγ S85 phosphorylation as well as the inhibition of ATM. Treatment of lysates with A-protein phosphatase prior to subjection to SDS-PAGE was used as an additional control to show that the detected bands indeed were phosphorylation dependent.

(150) Next, the IKKγ mono-ubiquitination, which is a prerequisite for IKK complex activation (Hinz et al.; 2010), was analysed by immunoprecipitation of IKKγ. The characteristic band of the IKKγ mono-ubiquitinated species (Hinz et al.; 2010) was only detected in the DMSO pre-treated and irradiated sample. Pre-treatment of cells with MW01 led to the abolishment of the IKKγ mono-ubiquitination (FIG. 11 C).

(151) In conclusion, the pre-treatment of cells with MW01 inhibited the formation of essential IKKγ post-translational modifications that are required for DNA damage-induced NF-κB activation.

(152) Structure-Activity-Relationship Analyses of MW01

(153) Different derivatives of MW01 were obtained (FIG. 12 A) and tested for their ability to inhibit genotoxic stress-induced phosphorylation of p65 at S536. Densitometry of western blot bands was used to quantify signal intensities of the p65 S536 phosphorylation and total p65 amount. The signal intensities of the p65 S536 phosphorylation were normalised to the signal intensities of total p65 and the quotients were normalised to DMSO/etoposide and compared with MW01/etoposide co-treated samples (FIG. 13). The MW01 derivatives MW01C2, MW01C3, and MW01C4 showed lowest p-p65/p65 ratios as a consequence of strongly inhibited NF-κB activation following etoposide co-treatment. Compared to MW01, these compounds are derivatives in which the hydroxyl group was exchanged with a small substitute either fluoride, chloride or a methyl group, respectively. Furthermore, MW01C3 and MW01C4 differed in the substitution of the aromatic ring system V (FIG. 12 B) regarding the two methoxy groups that are missing. The methoxy groups did not seem to be essential for the inhibitory function of the derivatives, but potentially may have an impact on their solubility.

(154) In comparison to the exchange of the hydroxyl group, also the presence of a methoxy group at the vicinal carbon atom in ring system I in MW01C1 resulted in a highly potent derivative.

(155) Hence, by analysing structure-activity-relationships the hydroxyl group of MW01 was identified as the position suitable for structural or covalent modifications that could maintain the inhibitory function. Furthermore, the hydroxyl group is suitable for different reactions such as nucleophilic substitution.

(156) In Contrast to MW01, PARP1 Inhibitors Block NF-κB Activation after Genotoxic Stress in a Cell Type Dependent Manner.

(157) Damage to DNA is a major threat to survival of cells and induces the DNA damage response that regulates cell fate. It has been shown in literature that the DNA damage-sensing protein PARP1 has multiple functions in the DDR. It is important for the accomplishment of single strand break repair, regulation of transcription and participation in NF-κB mediated pro-survival signaling (Gibson and Kraus; 2012). Stilmann et al. (2009) described by loss-of-function studies that the genotoxic stress-activated NF-κB pathway is dependent on PARP1-dependent PAR chain formation as a scaffold for signalosome component recruitment. Consequently, the application of PARP1 inhibitors inhibited the signaling cascade. In that study the authors used the pharmacological PARP1 inhibitors 3-AB and EB-47. The treatment of HepG2 cells with 3-AB inhibited PAR chain formation and NF-κB DNA binding activity after γ-irradiation. In addition, the study showed that MEF cells treated with 3-AB or EB-47 have abrogated PAR chain formation and NF-κB binding activity after etoposide administration (Stilmann et al.; 2009).

(158) In order to compare MW01 with PARP inhibitors, it was tested, whether inhibition of PARP1 by the clinically approved drug olaparib would inhibit signalosome formation and consequently inhibit the phosphorylation of p65. U2OS cells were pre-treated with increasing concentrations of olaparib ranging from 0.63 μM to 10.0 μM. Then, cells were co-treated with etoposide, harvested 90 min after etoposide application, and were analysed for their phosphorylation state of p65 at S536. No significant decrease in p65 S536 phosphorylation could be detected in comparison to the DMSO/etoposide co-treated controls (FIG. 14 A). Olaparib-mediated inhibition of PAR chain formation was ensured by a control experiment (FIG. 14 B). Therein, cells were pre-treated with DMSO, 3 μM olaparib or 10 μM of the ATM inhibitor Ku55933. Cells were harvested 45 min after application of etoposide and tested for PAR chain formation. The type of experiment as shown in FIG. 14 A was repeated in two independent biological replicates. The signal intensities of S536 phosphorylated p65 and total p65 of all three experiments were quantified by densitometry. The results are displayed as the signal intensity ratio in FIG. 14 C. Comparison of the olaparib/etoposide co-treated samples to the DMSO/etoposide co-treated samples showed that olaparib treatment, despite the inhibition of PAR chain formation, did not influence p65 S536 phosphorylation in U2OS cells.

(159) In order to investigate the influence of PARP1 inhibition by olaparib on general NF-κB activation, qRT-PCR analyses of NF-κB target genes were done.

(160) The relative normalised mRNA levels of NFKBIA (encodes IκBα), TNFAIP3 (encodes A20) and CXCL8 (encodes IL-8) were strongly increased in the irradiated DMSO samples compared to all samples, which were not irradiated. Pre-treatment of cells with olaparib did not change the target gene expression after irradiation compared to the DMSO control sample. In contrast, pre-treatment of cells with either MW01 or the ATM inhibitor KU55933 led to the complete inhibition of NFKBIA, TNFAIP3, and CXCL8 mRNA induction upon irradiation (FIG. 14 D).

(161) Next, it was investigated, if p65 was phosphorylated despite the inhibition of PARP1 by olaparib in HepG2 cells (FIG. 14 E). Expectedly, the pre-treatment of HepG2 cells with the PARP inhibitors olaparib, 3-AB and EB-47 abolished the PAR chain formation 15 min after irradiation, which was detected in the DMSO/IR treated sample as shown in FIG. 14 D. Inhibition of PAR chain formation led to decreased phosphorylation of p65 at S536 in the olaparib and in the 3-AB treated sample 90 min after irradiation. Interestingly, p-p65 S536 phosphorylation was detected in the EB-47 treated sample despite the inhibition of PAR chain formation.

(162) The results shown in FIG. 14 indicate a cell type specific impact of olaparib- and EB-47-mediated PARP1 inhibition on the phosphorylation of p65 at S536. In line with this, inhibition of PARP1-dependent PAR chain formation by 3-AB did not abolish NF-κB DNA binding activity in HEK293 (data not shown, personal communication with Dr. Michael Stilmann).

(163) Collectively, in contrast to MW01, inhibition of PARP1-dependent PAR chain formation by PARP inhibitors (3-AB, EB-47, and olaparib) inhibit p65 activation after genotoxic stress in a cell type dependent manner.

(164) Radio-Sensitisation of Cells by MW01 Mediated Inhibition of DNA Damage-Induced NF-κB

(165) Cellular apoptosis is a fine tuned mechanism depending on the processing of anti- and pro-apoptotic signals and the anti-apoptotic functions of NF-κB have already been described in literature (Kucharczak et al.; 2003). In order to show that the inhibition of NF-κB by MW01 led to the upregulation of apoptotic signaling, induction of expression of anti-apoptotic gene products was analysed by quantitative real-time PCR. The pre-treatment of U2OS cells with MW01 did not significantly change the mRNA expression of the genes BIRC3 (encodes cIAP2), XIAP or BCL2L1 (encodes BCL-X.sub.L) in comparison to the DMSO control. The γ-IR of cells led to a nearly two-fold induction of BIRC3 mRNA in the irradiated control, but BIRC3 mRNA was down-regulated in MW01 pre-treated cells. Like BIRC3 mRNA, the mRNA of XIAP and BCL2L1 was induced by irradiation. The pre-treatment of cells with MW01 moderately inhibited the expression of XIAP and fully inhibited the expression of BCL2L1. The strongest effect on anti-apoptotic gene regulation was detected on TNFAIP3 (encodes A20), as shown in above FIG. 14D. Pre-treatment of cells with MW01 led to abolished mRNA expression of TNFAIP3 after γ-IR in comparison to the irradiated controls.

(166) Next, the mRNA expression of the pro-apoptotic genes BBC3 (encodes PUMA) and PMAIP1 (encodes NOXA) was analysed. BBC3 mRNA expression was not influenced by pre-treatment of cells with MW01. After irradiation of cells the BBC3 mRNA expression was induced 4-fold in the positive control. The pre-treatment with MW01 only led to a slightly reduced expression, which was still 3-fold induced (FIG. 15 B).

(167) The mRNA expression of PMAIP1 was already increased by pre-treatment with MW01. In the MW01 pre-treated sample PMAIP1 mRNA expression was further elevated after irradiation, but was not changed in the irradiated samples (FIG. 15 B).

(168) In order to analyse the influence of MW01 on apoptotic cell death after genotoxic stress in more detail, apoptotic marker were examined. One of these markers is the caspase-3-dependent cleavage of PARP1. The pre-incubation of U2OS cells with MW01 led to a slight increase of PARP1 cleavage in resting cells. After irradiation of cells a marginal increase of PARP1 cleavage was detected in the irradiated control sample. In contrast, MW01 pre-incubation strongly increased the cleavage of PARP1 (FIG. 16 A).

(169) Using crystal violet staining it was analysed, if the pre-treatment with the compounds of cells prior to γ-irradiation exerted an influence on the cell number. The pre-treatment of cells with MW01 already reduced the cell number in comparison to the DMSO treated samples. After irradiation of cells the pre-treatment with MW01 had a significant effect on further reduction in cell number compared to the DMSO/IR control (FIG. 16 B).

(170) To test whether the reduction in cell number was caused by reduced proliferation, the percentage of viable cells after compound treatment and irradiation was measured by exclusion of annexin V and/or propidium-iodide staining positive cells. Similar to the result of the crystal violet staining MW01 pre-treatment exerted an effect on non-irradiated cells. The percentage of viable cells was slightly reduced compared to the DMSO control. However, after irradiation around 14% less viable cells were measured in the DMSO sample and 17% less viable cells were measured in the MW01 sample (FIG. 16 C).

(171) The sensitising effect of MW01 on cells was tested in MEF cells with a low irradiation dose of 2 Gy, an amount cells are able to repair. Cells were pre-treated with MW01, irradiated and cells were analysed by annexin V staining 24 or 48 h after irradiation. After 24 h, the treatment of cells with MW01 led to an increase of annexin V positive cells of about 10%. This is in line with the results displayed in FIG. 15, indicating increased apoptotic signaling in cells treated with MW01 even without IR. Annexin V staining of cells was further increased after 2 Gy of γ-irradiation in MW01 treated samples (circa 34%), but was only marginally increased in the DMSO control. Comparing the annexin V positive cells of MW01 treated and MW01/γ-IR co-treated samples, a sensitising effect for low γ-irradiation dose-induced apoptosis of about 12% was found (FIG. 16 D).

(172) In addition, the sensitising effect of NF-κB inhibition was tested in HT1080 cells. After pre-treatment with DMSO or MW01, cells were irradiated with a dose of 10 Gy and analysed by annexin V staining. The co-treatment of cells with MW01 led to a significant increase in annexin V staining compared to the irradiated control. The population of early apoptotic cells was roughly doubled (FIG. 16 E).

(173) Considering the results of this section, co-treatment of cells with MW01 in combination with the induction of DNA DSBs led to an increase in the percentage of apoptotic cells compared to single treatments.

(174) MW01 Inhibits DNA Repair Mechanisms that are NF-κB Independent

(175) U2OS cells were grown on coverslips and incubated 30 min with DMSO or MW01 (5 μM). Then, cells were γ-irradiated (5 Gy) or mock irradiated (mock IR). After 5 hours, cells were fixed and subjected to immunofluorescence staining procedure. DNA damage-indicating γH2AX foci and nuclei (n≥480 nuclei per condition) were counted for the calculation of average foci per nucleus. Significance was calculated using student's t-test.

(176) Treatment of cells with MW01 led to a significant increase in γH2AX foci per cell in untreated (non-irradiated) cells, indicating that MW01 inhibited, in addition to the genotoxic stress-induced IKK/NF-κB signaling pathway, other DNA repair mechanisms occurring in steady state.

(177) Examples of Chemical Compounds of the Invention:

(178) When in the final step of the synthesis of a compound an acid such as trifluoroacetic acid or acetic acid was used, for example when trifluoroacetic acid was employed to an acid-labile protecting group (e.g. a t-Bu group) or when a compound was purified by chromatography using an eluent which contained such an acid, in some cases, depending on the work-up procedure, for example the details of a freeze-drying process, the compound was obtained partially or completely in the form of a salt of the acid used, for example in the form of the acetic acid salt, formic acid salt or trifluoroacetic acid salt or hydrochloric acid salt. Likewise starting materials or intermediates bearing a basic center like for example a basic nitrogen were either obtained and used as free base or in salt form like, for example, a trifluoroacetic acid salt, a hydro bromic acid salt, sulfuric acid salt, or a hydrochloric acid salt.

Abbreviations Used

(179) Acetonitrile ACN

(180) Aqueous Aq.

(181) tert-Butyl t-Bu

(182) dibenzylidenacetone dba

(183) Dichloromethane DCM

(184) 4-Dimethyaminopyridine DMAP

(185) N,N-Dimethylformamide DMF

(186) Dimethylsulfoxide DMSO

(187) Ethanol EtOH

(188) Ethyl acetate EtOAc

(189) Formic Acid FA

(190) High performance liquid chromatography HPLC

(191) Methanol MeOH

(192) N-Methyl-2-pyrrolidone NMP

(193) Room temperature 20° C. to 25° C. RT

(194) Saturated sat.

(195) Triethanolamine TEA

(196) Tetrahydrofuran THF

(197) Trifluoroacetic acid TFA

(198) LCMS (method 1): Instrument: Agilent Technologies 6220 Accurate Mass TOF LC/MS linked to Agilent Technologies HPLC 1200 Series; Column: Thermo Accuore RP-MS; Particle Size: 2.6 μM Dimension: 30×2.1 mm; Eluent A: H.sub.2O with 0.1% FA Eluent B: ACN with 0.1% FA; Gradient: 0.00 min 95% A, 0.2 min 95% A, 1.1 min 1% A, 2.5 min Stop time, 1.3 min Post time; Flow rate: 0.8 ml/min; UV-detection: 220 nm, 254 nm, 300 nm.

(199) LCMS (method 2): Instrument: Agilent Technologies 6120 Quadrupole LC/MS linked to Agilent Technologies HPLC 1290 Infinity; Column: Thermo Accuore RP-MS; Particle Size: 2.6 μM Dimension: 30×2.1 mm; Eluent A: H.sub.2O with 0.1% FA Eluent B: ACN with 0.1% FA; Gradient: 0.00 min 95% A, 0.2 min 95% A, 1.1 min 1% A, 2.5 min Stop time, 1.3 min Post time; Flow rate: 0.8 ml/min; UV-detection: 220 nm, 254 nm, 300 nm.

(200) Preparative HPLC (method 1): Instrument: Waters preparative HPLC-System composed of: binary gradient module 2545, UV detector 2489, waters prep inject, and waters fraction collector III; Column: Macherey-Nagel VP 250/21 Nucleodor 100-7 C18ec; Eluent A: H.sub.2O with 0.1% TFA Eluent B: ACN with 0.1% TFA; Gradient: 0.00 min 85% A, 2.00 min 85% A, 22.00 min 15% A, 25.00 min 15% A, 26.00 min 0% A, 28.00 min 0% A, 29.00 min, 85% A 30.00 min 85% A, 30.10 min stop; Flow rate: 30 ml/min; UV-detection: 254 nm.

(201) Preparative HPLC (method 2): Instrument: Waters preparative HPLC-System composed of: binary gradient module 2545, UV detector 2489, waters prep inject, and waters fraction collector III; Column: Macherey-Nagel VP 250/21 Nucleodor 100-7 C18ec; Eluent A: H.sub.2O with 0.1% TFA Eluent B: ACN with 0.1% TFA; Gradient: 0.00 min 70% A, 2.00 min 70% A, 22.00 min 10% A, 25.00 min 10% A, 26.00 min 0% A, 28.00 min 0% A, 29.00 min, 70% A 30.00 min 70% A, 30.10 min stop; Flow rate: 30 ml/min; UV-detection: 254 nm.

(202) ##STR00124##

(203) The synthesis of β-carbolines (e.g. 6-methoxy-9H-pyrido[3,4-b]indole) were performed as described by Laha et al. (Laha, J. K., et al. J. Org. Chem. (2011) 76, 6421-6425).

General Reaction to the 9-benzyl-9H-pyrido[3,4-b]indole Derivatives

(204) ##STR00125##

Example 1: 9-(2-Chlorobenzyl)-6-methoxy-9H-pyrido[3,4-b]indole

(205) ##STR00126##

(206) To a solution of 27.6 mg 6-methoxy-9H-pyrido[3,4-b]indole (0.14 mmol, 1.0 eq.) in 1 ml DMF, 8.91 mg of sodium hydride (0.22 mmol, 1.6 eq.) were added under nitrogen. The mixture was stirred for 20 minutes at RT and a solution of 34.3 mg (0.17 mmol, 1.2 eq.) 2-chlorobenzyl bromide, and 1.7 mg DMAP (0.01 mmol, 0.1 eq.) in 1 ml DMF was added dropwise. After complete addition the reaction mixture was stirred for 3 h at 70° C. Upon completion of the reaction the mixture was diluted with water and sat. solution of NaHCO.sub.3 was added. The water phase was extracted with DCM three times. The combined organic phases were dried over magnesium sulfate and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel chromatography using a gradient of DCM/MeOH as eluent. The fractions containing the product were evaporated under reduced pressure to yield the title compound as a solid.

(207) Yield: 25.9 mg MS (ES+) [M+H]: m/e=323

Example 2: 9-(2-Chlorobenzyl)-7-methoxy-1-methyl-9H-pyrido[3,4-b]indole

(208) ##STR00127##

(209) The title compound was prepared by adapting the procedure described in example 1 with the difference that harmine was used instead of 6-methoxy-9H-pyrido[3,4-b]indole.

(210) Yield: 19.3 mg MS(ES+) [M+H]: m/e=337

Example 3: 3-Methoxy-4-((6-methoxy-9H-pyrido[3,4-b]indol-9-yl)methyl)benzoic Acid

(211) ##STR00128##

(212) The title compound was prepared by adapting the procedure described in example 1 with the difference that Methyl 4-(bromomethyl)-3-methoxybenzoate was used instead of 2-chlorobenzyl bromide.

(213) Yield: 12.2 mg MS(ES+) [M+H]: m/e=363

Example 4: 9-Benzyl-6-methoxy-9H-pyrido[3,4-b]indole

(214) ##STR00129##

(215) The title compound was prepared by adapting the procedure described in example 1 with the difference that 3-methoxybenzyl bromide was used instead of 2-chlorobenzyl bromide.

(216) Yield: 22.4 mg MS(ES+) [M+H]: m/e=319

Example 5: 9-Benzyl-6-methoxy-9H-pyrido[3,4-b]indole

(217) ##STR00130##

(218) The title compound was prepared by adapting the procedure described in example 1 with the difference that benzyl bromide was used instead of 2-chlorobenzyl bromide.

(219) Yield: 16.7 mg MS(ES+) [M+H]: m/e=289

Example 6: 9-(3,4-Dichlorobenzyl)-6-methoxy-9H-pyrido[3,4-b]indole

(220) ##STR00131##

(221) The title compound was prepared by adapting the procedure described in example 1 with the difference that 3,4-dichlorobenzyl bromide was used instead of 2-chlorobenzyl bromide.

(222) Yield: 19 mg MS(ES+) [M+H]: m/e=357/359 dichloro pattern

Example 7: 9-((6-Bromobenzo[d][1,3]dioxol-5-yl)methyl)-6-methoxy-9H-pyrido[3,4-b]indole

(223) ##STR00132##

(224) The title compound was prepared by adapting the procedure described in example 1 with the difference that 5-bromo-6-bromomethyl-1,3-benzodioxole was used instead of 2-chlorobenzyl bromide.

(225) Yield: 11.4 mg MS(ES+) [M+H]: m/e=411/413 bromo pattern

Example 8: 9-(2-Bromo-5-methoxybenzyl)-6-methoxy-9H-pyrido[3,4-b]indole

(226) ##STR00133##

(227) The title compound was prepared by adapting the procedure described in example 1 with the difference that 2-Bromo-5-methoxybenzyl bromide was used instead of 2-chlorobenzyl bromide.

(228) Yield: 3.5 mg MS(ES+) [M+H]: m/e=397/399 bromo pattern

General Reaction to the 9-(2-aroyl)-carbazole Derivatives

(229) ##STR00134##

Example 9: 9-(2-benzoyl)-carbazole. (D08) (CAS: 19264-68-7)

(230) ##STR00135##

(231) The title compound was prepared by adding to a cooled (0° C.) solution of 100 mg carbazole (0.60 mmol, 1.0 eq.) in 5 ml toluene/DMF (1:1), 23.9 mg sodium hydride (0.60 mmol, 1.0 eq.) under nitrogen. After stirring at 0° C. for 30 minutes a solution of 69.4 μl benzoyl chloride (0.60 mmol, 1.0 eq.) in 200 μl toluene was added dropwise. The reaction mixture was stirred for 17 hours at RT and the precipitated solid was filtered and washed with EtOAC. The filtrate was evaporated under reduced pressure. The crude product was purified by silica gel chromatography using a gradient of cyclohexane/EtOAc as eluent. The fractions containing the product were evaporated under reduced pressure to yield the title compound as a solid.

(232) Yield: 107 mg MS(ES+) [M+H]: m/e=272

Example 10: (6-Methoxy-9H-pyrido[3,4-b]indol-9-yl)(phenyl)methanone

(233) ##STR00136##

(234) The title compound was prepared by adding 10 mg 6-methoxy-9H-pyrido[3,4-b]indole (0.05 mmol, 1.0 eq.) in 5 ml toluene/DMF (1:1), 2.02 mg sodium hydride (0.05 mmol, 1.0 eq.) to a cooled (0° C.) solution under nitrogen. After stirring at 0° C. for 30 minutes a solution of 5.9 μl benzoyl chloride (0.05 mmol, 1.0 eq.) in 17 μl toluene was added dropwise. The reaction mixture was stirred for 2 hours at RT and then the solvent was evaporated under reduced pressure. The crude product was purified by silica gel chromatography using a gradient of DCM/MeOH as eluent. The fractions containing the product were evaporated under reduced pressure to yield the title compound. This product was then again purified via preparative HPLC method 1. The fractions containing the product were evaporated and lyophilized to yield a solid. The product was obtained as its trifluoroacetate salt.

(235) Yield: 8.1 mg MS(ES+) [M+H]: m/e=303

Example 11: (6-Methoxy-9H-pyrido[3,4-b]indol-9-yl)(4-methoxyphenyl)methanone

(236) ##STR00137##

(237) The title compound was prepared by adding to a suspension of 20 mg 6-methoxy-9H-pyrido[3,4-b]indole (0.10 mmol; 1.00 eq.) in 2.0 ml ACN, sequentially 41 μl 4-methoxybenzoyl chloride (0.30 mmol; 3.00 eq.), 37.0 mg DMAP (0.30 mmol; 3.00 eq.), and 42 μl TEA (0.30 mmol; 3.00 eq.). The mixture was stirred for 1 hour at RT. Then, the reaction mixture was diluted with 1 ml water, filtered and purified by preparative HPLC method 1. The fractions containing the product were evaporated and lyophilized to yield a solid. The product was obtained as its trifluoroacetate salt.

(238) Yield: 19.2 mg MS(ES+) [M+H]: m/e=333

Example 12: Benzo[d][1,3]dioxol-5-yl(6-methoxy-9H-pyrido[3,4-b]indol-9-yl)methanone

(239) ##STR00138##

(240) The title compound was prepared by adapting the procedure described in example 11 with the difference that piperonyloyl chloride was used instead of 4-methoxybenzoyl chloride.

(241) Yield: 41.5 mg MS(ES+) [M+H]: m/e=347

Example 13: (2-Bromo-5-methoxyphenyl)(6-methoxy-9H-pyrido[3,4-b]indol-9-yl)methanone

(242) ##STR00139##

(243) The title compound was prepared by adapting the procedure described in example 11 with the difference that 2-bromo-5-methoxy benzoyl chloride was used instead of 4-methoxybenzoyl chloride.

(244) Yield: 25.2 mg MS(ES+) [M+H]: m/e=411/413 (bromo pattern)

Example 14: (2-Chloropyridin-3-yl)(6-methoxy-9H-pyrido[3,4-b]indol-9-yl)methanone

(245) ##STR00140##

(246) The title compound was prepared by adapting the procedure described in example 11 with the difference that 2-chloronicotinoyl chloride was used instead of 4-methoxybenzoyl chloride.

(247) Yield: 5.4 mg MS(ES+) [M+H]: m/e=338

Example 15: (6-Methoxy-9H-pyrido[3,4-b]indol-9-yl)(naphthalen-1-yl)methanone

(248) ##STR00141##

(249) The title compound was prepared by adapting the procedure described in example 11 with the difference that 1-naphtoyl chloride was used instead of 4-methoxybenzoyl chloride.

(250) Yield: 17.3 mg MS(ES+) [M+H]: m/e=353

Example 16: 8H-Benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one (CAS 38478-71-6)

(251) ##STR00142##

(252) The title compound was prepared by dissolving 86 mg 9H-pyrido[3,4-b]indol-1-yl trifluoromethanesulfonate (0.272 mmol, 1-00 eq.), 68.5 mg 2-methoxycarbonylphenyl boronic acid (0.381 mmol, 1.40 eq.), 12.5 mg Pd.sub.2(dba).sub.3 (0.014 mmol; 0.05 eq.), 7.1 mg triphenylphosphine (0.027 mmol; 0.10 eq.) in 2.7 ml toluene and 1.8 ml EtOH. The solution was purged with nitrogen for 5 minutes. To the reaction mixture was added 0.9 ml sat. Aq. Na.sub.2CO.sub.3 solution and the mixture was purged for 5 minutes with nitrogen. Then, the solution was stirred at 80° C. for 90 minutes.

(253) The solution was diluted with EtOAc and washed two times with water, dried over magnesium sulfate and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel chromatography using a gradient of DCM/MeOH as eluent. The fractions containing the product were evaporated under reduced pressure to yield the title compound as a solid.

(254) Yield: 30 mg MS(ES+) [M+H]: m/e=271

Example 17: 5,6,11,12-Tetramethoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

(255) ##STR00143##

(256) The title compound was prepared by dissolving 80 mg 6,7-dimethoxy-9H-pyrido[3,4-b]indol-1-yl trifluoromethanesulfonate (0.202 mmol; 1.00 eq.), 67.9 mg 4,5-dimethoxy-2-(methoxycarbonyl)benzeneboronic acid (0.283 mmol; 1.40 eq.), 9.2 mg Pd.sub.2(dba).sub.3 (0.010 mmol; 0.05 eq.), 5.3 mg triphenylphosphine (0.020 mmol; 0.10 eq.) in 2.3 ml toluene and 1.8 ml EtOH. The solution was purged with nitrogen for 5 minutes. To the reaction mixture was added 0.6 ml saturated aqueous sodium carbonate solution and the mixture was purged again for 5 minutes with nitrogen. Then, the solution was stirred at 80° C. for 17 hours. The solution was diluted with ethyl acetate and washed two times by water, dried over magnesium sulfate and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel chromatography using a gradient of DCM/MeOH as eluent. The fractions containing the product were evaporated under reduced pressure to yield the title compound. This product was then further purified via preparative HPLC method 2. The fractions containing the product were evaporated and lyophilized to yield a white solid. The product was obtained as its trifluoroacetate salt.

(257) Yield: 0.4 mg MS(ES+) [M+H]: m/e=391

General Reaction to the methylpyrazolo[3,4-b]indole Derivatives

(258) ##STR00144##

(259) 3-Methylpyrazolo[3,4-b]indoles were synthesized according to a literatureprocedure (Monge, A., et al. Eur. J. Med. Chem. (1991) 26, 179-188).

Example 18: (3-Bromophenyl)(3-methylpyrazolo[3,4-b]indol-8(1H)-yl)methanone

(260) ##STR00145##

(261) The title compound was prepared by adding to a suspension of 25 mg 3-methylpyrazolo[3,4-b]indole (0.146 mmol; 1.00 eq.) in 2.9 ml ACN, sequentially 57.7 μl 3-bromobenzoyl chloride (0.438 mmol; 3.00 eq.), 53.5 mg 4-diemthylaminopyridine (DMAP) (0.438 mmol; 3.00 eq.), and 60.7 μl TEA (0.438 mmol; 3.00 eq.). The mixture was stirred for at least 3 hours at RT. After complete reaction the reaction mixture was diluted with 1 ml water, filtered and purified by preparative HPLC method 1. The fractions containing the product were evaporated and lyophilized to yield a solid. The product was obtained as its trifluoroacetate salt.

(262) Yield: 2.5 mg MS(ES+) [M+H]: m/e=354/356 bromo pattern

Example 19: (4-Methoxyphenyl)(3-methylpyrazolo[3,4-b]indol-8(1H)-yl)methanone

(263) ##STR00146##

(264) The title compound was prepared by adapting the procedure described in example 18 with the difference that 4-methoxybenzoyl chloride was used instead of 3-bromobenzoyl chloride and that the scale of the reaction was performed for 100 mg 3-methylpyrazolo[3,4-b]indole (0.584 mmol; 1.00 eq.).

(265) Yield: 94.6 mg MS(ES+) [M+H]: m/e=323

Example 20: (3-Methylpyrazolo[3,4-b]indol-8(1H)-yl)(phenyl)methanone

(266) ##STR00147##

(267) The title compound was prepared by adapting the procedure described in example 18 with the difference that benzoyl chloride was used instead of 3-bromobenzoyl chloride.

(268) Yield: 5.5 mg MS(ES+) [M+H]: m/e=276

Example 21: (3-Methylpyrazolo[3,4-b]indole-1,8-diyl)bis(phenylmethanone)

(269) ##STR00148##

(270) The title compound was obtained as a side product from the synthesis of example 20.

(271) Yield: 7.8 mg MS(ES+) [M+H]: m/e=380

Example 22: (2-Chloropyridin-3-yl)(3-methylpyrazolo[3,4-b]indol-8(1H)-yl)methanone

(272) ##STR00149##

(273) The title compound was prepared by adapting the procedure described in example 18 with the difference that 2-chloronicotinoyl chloride was used instead of 3-bromobenzoyl chloride.

(274) Yield: 12.1 mg MS(ES+) [M+H]: m/e=311/313 chloro pattern

Example 23: (2-Bromo-6-chlorophenyl)(3-methylpyrazolo[3,4-b]indol-8(1H)-yl)methanone

(275) ##STR00150##

(276) The title compound was prepared by adapting the procedure described in example 18 with the difference that 2-bromo-6-chlorobenzoyl chloride was used instead of 3-bromobenzoyl chloride.

(277) Yield: 8.7 mg MS(ES+) [M+H]: m/e=388/390 isotope pattern

Example 24: 5-(Pyridin-3-yl)phenanthridin-6(5H)-one

(278) ##STR00151##

(279) The title compound was prepared by adding 150 mg 6(5H)-Phenanthridinone (0.77 mmol; 1.00 eq.), 111 μl 3-Bromopyridin (1.15 mmol; 1.50 eq.), 106 mg potassium carbonate (0.77 mmol; 1.00 eq.), 2.7 mg copper(I)iodide (0.04 mmol; 0.05 eq.) to a flask. To the solids 900 p1 NMP (7.7 mmol; 10.0 eq.) were added. The mixture was heated to 180° C. and the reaction stopped at a conversion ratio, starting material to product 1:1. Then, the reaction mixture was diluted with diethyl ether and extracted with water. The water phase was washed with diethyl ether three times. The combined organic phases were washed with water one time, dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. While evaporating the solvents under reduced pressure starting material precipitated as a white solid and was filtered off. The filtrate was evaporated to dryness. The crude product was purified by silica gel chromatography using a gradient of cyclohexane/EtOAc as eluent. The fractions containing the product were evaporated under reduced pressure to yield the title compound.

(280) Yield: 62 mg MS(ES+) [M+H]: m/e=273

General Reaction to the β-carbolinone Derivatives

(281) ##STR00152##

(282) The β-carbolinone derivate (e.g. 6-methoxy-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one) were synthesized as described in literature (La Regina, G., et al. Synthesis (2014), 46, 2093-2097)

Example 25: 12-methoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

(283) ##STR00153##

(284) The title compound was prepared by dissolving 78.9 mg 6-methoxy-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one (0.368 mmol, 1.0 eq.) in 3.8 ml pyridine. The solution was cooled down to 4° C. and purged with nitrogen. To this solution 439 μl triflic anhydride (0.737 mmol, 2.0 eq.) was added dropwise (≈30 min). The mixture was stirred for 45 minutes at RT.

(285) After complete reaction the mixture was purred into water and the water phase was extracted with EtOAc three times. The combined organic phases were dried over magnesium sulfate and the solvent was evaporated under reduced pressure. The crude product (6-methoxy-9H-pyrido[3,4-b]indol-1-yl trifluoromethanesulfonate) was used in the next step without further purification.

(286) 73 mg 6-methoxy-9H-pyrido[3,4-b]indol-1-yl trifluoromethanesulfonate (0.179 mmol, 1.0 eq.), 45 mg (2-(methoxycarbonyl)phenyl)boronic acid (0.251 mmol, 1.4 eq.), 8.2 mg Tris(dibenzylideneacetone) dipalladium(0) (0.009 mmol, 0.05 eq.) and 4.7 mg triphenylphosphine (0.018 mmol, 0.1 eq.) were dissolved in 1.8 ml toluene and 1.2 ml ethanol. The solution was purged with nitrogen and 0.6 ml of a saturated aqueous sodium carbonate solution was added. The mixture was stirred for 90 minutes at 80° C. After complete reaction the mixture was diluted with EtOAc and the organic phase was washed two times with water, dried over magnesium sulfate and the solvent was evaporated under reduced pressure. The crude product was purified using silica gel chromatography with DCM/MeOH as solvent and was afterwards further purified by HPLC with ACN/water.

(287) Yield: 18 mg MS (ES+) [M+H]: m/e=301

Example 26: 11-methoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

(288) ##STR00154##

(289) The title compound was prepared by adapting the procedure described in example 25 with the difference that 7-methoxy-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one was used instead of 6-methoxy-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one.

(290) Yield: 5 mg MS (ES+) [M+H]: m/e=301

Example 27: 11,12-dimethoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

(291) ##STR00155##

(292) The title compound was prepared by adapting the procedure described in example 25 with the difference that 6,7-dimethoxy-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one was used instead of 6-methoxy-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one.

(293) Yield: 5 mg MS (ES+) [M+H]: m/e=331

General Reaction to the 6-methoxy-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one Derivatives

(294) ##STR00156##

(295) 6-methoxy-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one was synthesized as described in literature (La Regina, G., et al. Synthesis (2014), 46, 2093-2097).

Example 28: (1-iodo-6-methoxy-9H-pyrido[3,4-b]indol-9-yl)(phenyl)methanone

(296) ##STR00157##

(297) The title compound was prepared by dissolving 78.9 mg 6-methoxy-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one (0.368 mmol, 1.0 eq.) in 3.8 ml pyridine. The solution was cooled down to 4° C. and purged with nitrogen. To this solution 439 μl triflic anhydride (0.737 mmol, 2.0 eq.) was added dropwise 30 min). The mixture was stirred for 45 minutes at RT.

(298) After complete reaction the mixture was purred into water and the water phase was extracted with EtOAc three times. The combined organic phases were dried over magnesium sulfate and the solvent was evaporated under reduced pressure. The crude product (6-methoxy-9H-pyrido[3,4-b]indol-1-yl trifluoromethanesulfonate) was used in the next step without further purification.

(299) 100 mg 6-methoxy-9H-pyrido[3,4-b]indol-1-yl trifluoromethanesulfonate (0.289 mmol, 1.0 eq.) and 216 mg sodium iodide (1.44 mmol, 5.0 eq.) were dissolved under nitrogen in 0.7 ml acetonitrile. The solution was cooled down to 0° C. and 50 μl triflic acid (0.578 mmol, 2.0 eq.) were added dropwise 15 min). After complete addition the mixture was stirred at room temperature for 3 h. After complete reaction the mixture was diluted with EtOAc and water and was cooled down to 0° C. The aqueous phase was brought to pH 10 with NaOH (c=10 mol/l, <1 ml), then the phases were separated. The organic phase was washed with sodium thiosulfate solution (w=5%), NaOH solution (c=1 mol/l) and with brine. The organic phase was dried over magnesium sulfate and the solvent was evaporated under reduced pressure. The crude product was purified using silica gel chromatography with cyclohexane/EtOAc/MeOH as solvent to achieve 1-iodo-6-methoxy-9H-pyrido[3,4-b]indole. To a suspension of 20 mg 1-iodo-6-methoxy-9H-pyrido[3,4-b]indole (0.062 mmol, 1.0 eq.) in 1.2 ml ACN, sequentially 21 μl benzoyl chloride (0.19 mmol; 3.0 eq.), 22.6 mg DMAP (0.19 mmol; 3.0 eq.), and 26 μl TEA (0.19 mmol; 3.0 eq.) were added. The mixture was stirred for 72 hours at RT. Afterwards the reaction mixture was diluted with 1 ml water, filtered and purified by preparative HPLC method 1.

(300) Yield: 15 mg MS (ES+) [M+H]: m/e=428

General Reaction to the 6-methoxy-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one Derivatives

(301) ##STR00158##

(302) 6-methoxy-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one was synthesized as described in literature (La Regina, G., et al. Synthesis (2014), 46, 2093-2097).

Example 29: 9-benzoyl-6-methoxy-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one

(303) ##STR00159##

(304) The title compound was prepared by adding to a suspension of 20 mg methoxy-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one (0.093 mmol, 1.0 eq.) in 1.2 ml ACN, sequentially 33 μl benzoyl chloride (0.28 mmol; 3.0 eq.), 34.2 mg DMAP (0.28 mmol; 3.0 eq.), and 34 μl TEA (0.28 mmol; 3.0 eq.) were added. The mixture was stirred for 72 hours at RT. Afterwards the reaction mixture was diluted with 1 ml water and the precipitated product was filtered off. The filtrate contained product and was dried via lyophilization and purified by preparative HPLC method 1.

(305) Yield: 9 mg MS (ES+) [M+H]: m/e=319

General Reaction to (1-methylpyrazolo[3,4-b]indol-8(1H)-yl)(phenyl)methanone Derivatives

(306) ##STR00160##

Example 30: (2-bromophenyl)(5-methoxy-1,3-dimethylpyrazolo[3,4-b]indol-8(1H)-yl)methanone

(307) ##STR00161##

(308) The title compound was prepared by dissolving 100 mg 1-(2-chloro-5-methoxy-1H-indol-3-yl)ethanone (0.45 mmol, 1.0 eq.) and 71 μl monomethyl hydrazine in 1.3 ml ethanol. The solution was kept at reflux for 12 h. After complete reaction the mixture was cooled down and the precipitated product was collected by filtration. The solid compound was washed with ethanol to achieve pure 5-methoxy-1,3-dimethyl-1,8-dihydropyrazolo[3,4-b]indole. To a suspension of 20 mg 5-methoxy-1,3-dimethyl-1,8-dihydropyrazolo[3,4-b]indole (0.093 mmol, 1.0 eq.) in 1.9 ml ACN, sequentially 36 μl 2-bromobenzoyl chloride (0.28 mmol, 3.0 eq.), 34 mg DMAP (0.28 mmol, 3.0 eq.), and 39 μl TEA (0.28 mmol; 3.0 eq.) were added. The mixture was stirred for 6 hours at RT. Afterwards the reaction mixture was diluted with water and the precipitated product was collected by filtration.

(309) Yield: 17 mg MS (ES+) [M+H]: m/e=398/400 isotope pattern

Example 31: (5-methoxy-1-methylpyrazolo[3,4-b]indol-8(1H)-yl)(phenyl)methanone

(310) ##STR00162##

(311) The title compound was prepared by adapting the procedure described in example 30 with the difference that 2-chloro-5-methoxy-indole-3-carbaldehyde was used instead of 1-(2-chloro-5-methoxy-1H-indol-3-yl)ethanone and benzoyl chloride was used instead of 2-bromobenzoyl chloride.

(312) Yield: 15 mg MS (ES+) [M+H]: m/e=306

General Reaction to pyrazolo[3,4-b]indole-1,8-diylbis(phenylmethanone) Derivatives

(313) ##STR00163##

Example 32: (5-methoxy-3-methylpyrazolo[3,4-b]indole-1,8-diyl)bis((2-bromophenyl)methanone)

(314) ##STR00164##

(315) The title compound was prepared by dissolving 200 mg 1-(2-chloro-5-methoxy-1H-indol-3-yl)ethanone (0.90 mmol, 1.0 eq.) and 131 μl hydrazine hydrate in 2.7 ml ethanol. The solution was kept at reflux for 8 h. After complete reaction the mixture was cooled down and the precipitated product was collected by filtration. The solid compound was washed with ethanol to achieve pure 5-methoxy-3-methyl-1,8-dihydropyrazolo[3,4-b]indole. To a suspension of 20 mg 5-methoxy-3-methyl-1,8-dihydropyrazolo[3,4-b]indole (0.099 mmol, 1.0 eq.) in 2 ml ACN, sequentially 35 μl 2-bromobenzoyl chloride (0.30 mmol, 3.0 eq.), 36 mg DMAP (0.30 mmol, 3.0 eq.), and 41 μl TEA (0.30 mmol; 3.0 eq.) were added. The mixture was stirred for 6 hours at RT. Afterwards the reaction mixture was diluted with water and the precipitated product was collected by filtration and washed with ACN.

(316) Yield: 30 mg MS (ES+) [M+H]: m/e=566/568/570 isotope pattern

Example 33: (5-methoxy-3-methylpyrazolo[3,4-b]indole-1,8-diyl)bis(phenylmethanone)

(317) ##STR00165##

(318) The title compound was prepared by adapting the procedure described in example 32 with the difference that benzoyl chloride was used instead of 2-bromobenzoyl chloride.

(319) Yield: 13 mg MS (ES+) [M+H]: m/e=410

Example 34: (5-bromo-3-methylpyrazolo[3,4-b]indole-1,8-diyl)bis(phenylmethanone)

(320) ##STR00166##

(321) The title compound was prepared by adapting the procedure described in example 32 with the difference that 1-(5-bromo-2-chloro-1H-indol-3-yl)ethan-1-one was used instead of 1-(2-chloro-5-methoxy-1H-indol-3-yl)ethanone and benzoyl chloride was used instead of 2-bromobenzoyl chloride.

(322) Yield: 5 mg MS (ES+) [M+H]: m/e=458 isotope pattern

Example 35: (5-bromo-3-methylpyrazolo[3,4-b]indole-1,8-diyl)bis((2-bromophenyl)methanone)

(323) ##STR00167##

(324) The title compound was prepared by adapting the procedure described in example 32 with the difference that 1-(5-bromo-2-chloro-1H-indol-3-yl)ethan-1-one was used instead of 1-(2-chloro-5-methoxy-1H-indol-3-yl).

(325) Yield: 14 mg MS (ES+) [M+H]: m/e=616 isotope pattern

Example 36: (5-bromo-3-methylpyrazolo[3,4-b]indole-1,8-diyl)bis((4-methoxyphenyl)methanone)

(326) ##STR00168##

(327) The title compound was prepared by adapting the procedure described in example 32 with the difference that 1-(5-bromo-2-chloro-1H-indol-3-yl)ethan-1-one was used instead of 1-(2-chloro-5-methoxy-1H-indol-3-yl) and 4-methoxybenzoyl chloride was used instead of 2-bromobenzoyl chloride.

(328) Yield: 18 mg MS (ES+) [M+H]: m/e=518/520 isotope pattern

General Reaction to 5-benzyl-5H-pyrimido[5,4-b]indole Derivatives (Example 37)

(329) ##STR00169##

Example 37: 5-benzyl-8-methoxy-5H-pyrimido[5,4-b]indol-2-amine

(330) ##STR00170##

(331) The title compound was prepared by adding a solution of 600 mg 5-methoxy-3-iodo-1H-indole-2-carbaldehyde (2.00 mmol, 1.0 eq.) in 4 ml dry DMF dropwise to a solution of 62.2 mg sodium hydride (60% in paraffin oil) (2.59 mmol, 1.3 eq.) in 4 ml dry DMF at 0° C. The mixture was stirred for 20 minutes at 0° C. and a solution of 946 μl benzyl bromide (7.97 mmol, 4.0 eq.) was added. The suspension was stirred for 1 h at room temperature and another 38.3 mg sodium hydride (60% in paraffin oil) (1.59 mmol, 0.8 eq.) and 473 μl benzyl bromide (3.99 mmol, 2.0 eq.). The mixture was further stirred at room temperature for 12 h. After complete reaction the mixture was quenched with iced water and extracted with EtOAc. The organic phase was washed with water and brine, dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel chromatography, with cyclohexane/EtOAc as solvent to achieve clean 1-benzyl-3-iodo-5-methoxy-1H-indole-2-carbaldehyde. Step B: A suspension of 300 mg 1-benzyl-3-iodo-5-methoxy-1H-indole-2-carbaldehyde (0.77 mmol, 1.0 eq.), 147 mg guanidine (1.53 mmol, 2.0 eq.), 500 mg cesium carbonate (1.53 mmol, 2.0 eq.), 14.6 mg copper(I) iodide (0.08 mmol, 0.1 eq.), and 1,10-phenanthroline in 2.5 ml dry DMSO was stirred for 48 h at 90° C. under nitrogen. After complete reaction water and EtOAc were added and the mixture was filtrated by a celite filter. The aqueous phase was extracted with EtOAc two times. The combined organic phases were washed with brine, dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel chromatography, with cyclohexane/EtOAc as solvent. The product was purified once more via preparative HPLC Method 3.

(332) Yield: 36 mg MS (ES+) [M+H]: m/e=305

General Reaction to 5-benzyl-5H-pyrimido[5,4-b]indole Derivatives (Example 38)

(333) ##STR00171##

Example 38: 5-benzyl-2-chloro-8-methoxy-5H-pyrimido[5,4-b]indole

(334) ##STR00172##

(335) The title compound was prepared by dissolving 15 mg 5-benzyl-8-methoxy-5H-pyrimido[5,4-b]indol-2-amine (example 43) (0.05 mmol, 1.0 eq.) in 0.5 ml 1,2-dichloroethane. The solution was cooled down to −10° C. and a solution of 25 mg antimony trichloride (0.11 mmol, 2.2 eq.) in 0.1 ml 1,2-dichloroethane was added. Afterwards 27.7 μl tert-butyl-nitrite (0.23 mmol, 4.7 eq.) were added dropwise. The reaction mixture was stirred for 2 h at −10° C., next iced water was added. After complete reaction the mixture was extracted with EtOAc three times. The combined organic phases were washed with water once, dried over magnesium sulfate and the solvent was evaporated under reduced pressure. The product was purified o via preparative HPLC Method 1.

(336) Yield: 16 mg MS (ES+) [M+H]: m/e=324

General Reaction to 5-benzyl-5H-pyrimido[5,4-b]indole Derivatives (Example 39)

(337) ##STR00173##

Example 39: 5-benzyl-8-methoxy-5H-pyrimido[5,4-b]indol-2-ol

(338) ##STR00174##

(339) The title compound was prepared by dissolving 15 mg 5-benzyl-8-methoxy-5H-pyrimido[5,4-b]indol-2-amine (example 43) (0.05 mmol, 1.0 eq.) in 0.2 ml acetic acid. The solution was cooled down to 10° C. and a solution of 10 mg sodium nitrite (0.15 mmol, 3.0 eq.) in 68 μl water was added. The reaction mixture was stirred for 30 min, next 1.5 ml water was added and the solution was stirred at 90° C. for 4 h.

(340) After complete reaction the solvent was removed under vacuum and the residue was taken up with water and extracted with EtOAc three times. The combined organic phases were dried over sodium sulfate and the solvent was evaporated under reduced pressure.

(341) Yield: 12 mg MS (ES+) [M+H]: m/e=306

General Reaction to 5-benzyl-5H-pyrimido[5,4-b]indole Derivatives (Example 40)

(342) ##STR00175##

(343) 4-chloro-8-methoxy-5H-pyrimido[5,4-b]indole was obtained commercially

Example 40: 5-benzyl-4-chloro-8-methoxy-5H-pyrimido[5,4-b]indole

(344) ##STR00176##

(345) The title compound was prepared by dissolving 60 mg 4-chloro-8-methoxy-5H-pyrimido[5,4-b]indole (0.26 mmol, 1.0 eq.) in 4 ml DMF. To this solution 16 mg sodium hydride (60% in oil) (0.41 mmol, 1.6 eq.) and 3.1 mg DMAP (0.03 mmol, 0.1 eq.) were added. The mixture was stirred for around 20 minutes at RT and 53 mg (0.31 mmol, 1.2 eq.) benzyl bromide was added dropwise. After complete addition the reaction mixture was stirred for 18 h at 70° C.

(346) After complete reaction the solvent was removed and the crude product was purified via preparative HPLC Method 1.

(347) Yield: 11.6 mg MS (ES+) [M+H]: m/e=324

Example 41: 5-benzyl-8-methoxy-5H-pyrimido[5,4-b]indol-4-ol

(348) ##STR00177##

(349) The title compound was obtained as a side product from the synthesis of example 40.

(350) Yield: 14.8 mg MS(ES+) [M+H]: m/e=306

General Reaction to phenyl(5H-pyrimido[5,4-b]indol-5-yl)methanone Derivatives

(351) ##STR00178##

Example 42: (4-chloro-8-methoxy-5H-pyrimido[5,4-b]indol-5-yl)(phenyl)methanone

(352) ##STR00179##

(353) The title compound was prepared by adding to a suspension of 60 mg 4-chloro-8-methoxy-5H-pyrimido[5,4-b]indole (0.26 mmol, 1.0 eq.) in 5 ml ACN, sequentially 89 μl benzoyl chloride (0.77 mmol, 3.0 eq.), 94 mg DMAP (0.77 mmol, 3.0 eq.), and 107 μl TEA (0.7 mmol; 3.0 eq.). The mixture was stirred for 18 hours at RT and another 89 μl benzoyl chloride (0.77 mmol, 3.0 eq.) and 107 μl TEA (0.7 mmol; 3.0 eq.) were added. Afterwards the reaction mixture was diluted with water and the precipitate was removed. The filtrate was dried under vacuum and the crude product was purified by silica gel chromatography, with cyclohexane/EtOAc as solvent.

(354) Yield: 14.7 mg MS (ES+) [M+H]: m/e=338

General Reaction to 8H-dibenzo[b,f]pyrimido[4,5,6-hi]indolizin-8-one Derivatives

(355) ##STR00180##

Example 43: 5,6,12-trimethoxy-8H-dibenzo[b,f]pyrimido[4,5,6-hi]indolizin-8-one

(356) ##STR00181##

(357) The title compound was prepared by dissolving 40 mg 4-chloro-8-methoxy-5H-pyrimido[5,4-b]indole (0.171 mmol, 1.0 eq.) and 88 mg bromotripyrrolidinophosphonium hexafluorophosphate (0.19 mmol, 1.1 eq.) under nitrogen in 1.4 ml 1,4-dioxane. To the solution 47 μl trimethylamine was added and the mixture was stirred for 2 h min at 70° C. Afterwards 27 mg 4,5-Dimethoxy-2-(methoxy carbonyl)benzeneboronic acid (0.18 mmol, 1.05 eq.), 6.0 mg bis(triphenylphosphine)palladium(II) dichloride (0.009 mmol, 0.05 eq.), 36 mg sodium carbonate (0.34 mmol, 2.0 eq.), and 0.7 ml water were added. The mixture was stirred at 70° C. for 18 h, a suspension is formed. After complete reaction, solid product was removed by filtration and washed with water and MeOH.

(358) Yield: 41 mg MS (ES+) [M+H]: m/e=362

Example 44: 12-methoxy-8H-dibenzo[b,f]pyrimido[4,5,6-hi]indolizin-8-one

(359) ##STR00182##

(360) The title compound was prepared by adapting the procedure described in example 43 with the difference that 2-methoxy carbonylphenylboronic acid was used instead of 4,5-Dimethoxy-2-(methoxy carbonyl)benzeneboronic acid.

(361) Yield: 47 mg MS (ES+) [M+H]: m/e=302

General Reaction to Example 45 (MW01)

(362) ##STR00183##

Example 45: 12-hydroxy-6,7-dimethoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

(363) ##STR00184##

(364) The title compound was prepared by dissolving 2.00 g L-5-Hydroxytryptohan (9.1 mmol, 1.0 eq.) and 2.10 g 2-Carboxy-3,4-dimethoxybenzaldehyde (10 mmol, 1.1 eq.) in 9 ml glacial acetic acid. The mixture was kept under reflux for 6 h and another 18 h under reflux with a constant flow of air bubbling through the liquid. After complete reaction, solid product was removed by filtration and washed with water and acetic acid. The crude product was crystallized from DMF.

(365) Yield: 1.18 g MS (ES+) [M+H]: m/e=347

(366) Further examples of the present invention which can be prepared by using synthetic procedure well known to those skilled in the art and by adapting the general procedures described above are:

Example: 12-(2-(2-aminoethoxy)ethoxy)-6,7-dimethoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

(367) ##STR00185##

Example A1: 1-(4-chlorophenyl)-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

(368) ##STR00186##

Example A2: 1-(2-chlorophenyl)-6,7-dimethoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

(369) ##STR00187##

Example A3: 6,7-dimethoxy-1-(4-methoxyphenyl)-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

(370) ##STR00188##

Example A4: methyl 6,7-dimethoxy-8-oxo-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridine-2-carboxylate

(371) ##STR00189##

Example A5: 8-oxo-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridine-2-carboxylic Acid

(372) ##STR00190##

Example A7: N-(3-methoxypropyl)-8-oxo-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridine-2-carboxamide

(373) ##STR00191##

Example A8: N-isopropyl-8-oxo-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridine-2-carboxamide

(374) ##STR00192##

Example B1: 2-(4-methylpiperazine-1-carbonyl)-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

(375) ##STR00193##

Example B2: 13-((diethylamino)methyl)-12-hydroxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

(376) ##STR00194##

Example B3: 2-((6,7-dimethoxy-8-oxo-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-12-yl)oxy)-N-(2-morpholinoethyl)acetamide

(377) ##STR00195##

Example B4: 12-butoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

(378) ##STR00196##

Example B5: 12-ethoxy-6,7-dimethoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

(379) ##STR00197##

Example B6: 6,7-dimethoxy-8-oxo-N-pentyl-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridine-2-carboxamide

(380) ##STR00198##

Example B7: 6,7-dimethoxy-12-propoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

(381) ##STR00199##

Example B8: 6,7-dimethoxy-2-(4-methylpiperazine-1-carbonyl)-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

(382) ##STR00200##

Example C1: 6,7,11-trimethoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

(383) ##STR00201##

Example C2: 12-fluoro-6,7-dimethoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

(384) ##STR00202##

Example C3: 12-methyl-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

(385) ##STR00203##

Example C4: 12-chloro-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

(386) ##STR00204##

Example C5: 13-allyl-12-methoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

(387) ##STR00205##

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