Selective inhibitor of the IKK / NF-κB pathway induced by genotoxic stress

Abstract

To provide new classes of pathway tailored inhibitors, which interfere only with a stimulus-specific NF-κB activation, while leaving other modes of NF-κB activation intact.SOLUTION: The invention relates to chemical compounds and their use as a medicament in treatment of a disease associated with genotoxic stress-induced IKK / NF-κB (NF-kappaB) activation, preferably a cancer indicating genotoxic stress-induced IKK / NF-κB (NF-kappaB) activation. The invention further relates to a pharmaceutical composition comprising the compound of the invention for the treatment of a subject afflicted by a disease associated with genotoxic stress-induced IKK / NF-κB activation.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to compounds useful as medicaments in the treatment of diseases associated with IKK / NF-κB (NF-kappa B) activation induced by genotoxic stress and their use. Preferably, it relates to use in the treatment of cancer patients showing IKK / NF-κB activation induced by genotoxic stress. The present invention further relates to pharmaceutical compositions comprising the compounds of the invention for the treatment of patients suffering from diseases associated with IKK-NF-κB activation induced by genotoxic stress.

Background Art

[0002] Adaptation to change is important for the survival of organisms. Environmental, chemical and physical as well as microbiological changes threaten normal tissue function and cellular homeostasis and represent a cause of stress to growth and physiological functions. The main response to stress is cell signaling that affects cell function by altering gene expression programs. The NF-κB (nuclear factor kappa light chain enhancer of activated B cells) system is a major player in the cell stress response. NF-κB is a widely present and rapidly inducible transcription factor (TF). Hundreds of target genes regulated by NF-κB have been identified. Most of those target genes are involved in the regulation of the immune system and inflammation, cell cycle, proliferation and cell death. In addition to its prominent functions in growth and stress response, uncontrolled NF-κB is the cause of a number of diseases, most importantly chronic inflammation, autoimmune diseases and cancer.

[0003] Stimulators that induce NF-κB activation include pro-inflammatory cytokines, PAMPs (pathogen-associated molecular patterns), immune receptors and various types of cell stress, such as gamma irradiation (IR). The activated NF-κB pathway regulates various cellular effects by transcriptional regulation of non-coding RNA (ribonucleic acid) or target genes encoding proteins that control cell survival and proliferation, adhesion and matrix remodeling, lymphocyte activation, host defense or immunity and inflammation.

[0004] NF-κB is a family of TFs that includes five members, p65 / RelA, RelB, c-Rel, p105 / p50, and p100 / p52, which form combinatorial homodimers and heterodimers (Hayen & Ghosh; 2012). Structurally, all NF-κB subunits are characterized by a Rel homology domain (RHD) composed of an N-terminal domain (NTD), followed by a dimerization domain (DID) and a nuclear localization signal (NLS). The RHD facilitates most of the important functions, such as dimerization with another subunit, nuclear translocation, DNA binding, and binding to IκB proteins.

[0005] Rel proteins are further characterized by the presence of a C-terminal transcriptional activation domain (TAD) required for transcription initiation. The precursor proteins p105 and p100 are the gene products of NFKB1 and NFKB2, respectively. Through ubiquitination and proteasomal processing, p105 and p100 give rise to the mature NF-κB subunits p50 and p52, respectively.

[0006] Various post-translational modifications (PTMs) of NF-κB subunits, such as phosphorylation and acetylation, induce conformational changes, thereby affecting ubiquitination, stability, protein-protein interactions, and regulation of target gene expression (Christian et al.; 2016).

[0007] Inactive NF-κB dimers are sequestered in the cytoplasm by association with IκB proteins. Simultaneously with the activation of the IKK / NF-κB pathway, the IKK complex phosphorylates IκBα, thereby marking it for lysine 48-linked (K48) ubiquitination and subsequent proteasomal degradation (Scheidereit, 1998, Hayden & Ghosh; 2008, Scheidereit; 2006). The released NF-κB dimers then translocate to the nucleus and control the transcription of target genes.

[0008] The IκB (inhibitor of nuclear factor κB) protein is an inhibitor of NF-κB and represents a molecular switch by retaining NF-κB from nuclear translocation. The IκB protein contains, as a distinctive structural feature, ankyrin repeat domains (ARDs) that facilitate binding to NF-κB dimers.

[0009] IκBα, IκBβ, and IκBε sequester NF-κB dimers in the cytoplasm by masking the NLS, and activation of NF-κB requires release from IκBs. Release of NF-κB from the major inhibitor, IκBα, requires phosphorylation at two N-terminal serines (S, Ser) at positions S32 and S36. Phosphorylation is important but not sufficient to release NF-κB from IκBα, and proteolysis of IκBα as an additional step is essential.

[0010] Activation of the IKK complex is a fundamental mechanism of NF-κB signaling. The prototype complex consists of two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ / NEMO (NF-κB essential modifier) (Hinz & Scheidereit; 2014) (Hinz & Scheidereit; 2014). When stimulated, the IKK complex phosphorylates IκBα on essential serine residues. As a result of this phosphorylation, the E3 ligase SCF βTrCP attacks the K48-linked ubiquitin chain on IκBα. This degradation signal leads to the degradation of IκBα by the 26S proteasome, resulting in the release of the free NF-κB heterodimer.

[0011] Activation of the IKK complex requires various signaling events. Most of the IKK / NF-κB pathway involves upstream signaling leading to ubiquitin-mediated autophosphorylation of the kinase TAK1 (TGFβ-activated kinase 1). Autophosphorylation of TAK1 is activated by recruitment of the TAK1 / TAB2 / 3 complex to K63-linked ubiquitin chains. Activated TAK1 phosphorylates IKKα and IKKβ at S176 and S177, respectively, in the activation loop (Zhang et al.; 2014). The IKK complex not only phosphorylates the IκB protein but also phosphorylates the NF-κB subunit p65.

[0012] Classical IKK / NF-κB activation is strongly activated by inflammatory stimuli such as cytokines, e.g., IL-1 (interleukin-1) and TNFα (tumor necrosis factor α), and Toll-like receptor agonists (Zhang et al.; 2014). When a ligand binds to their cell membrane-bound receptors, the signal is transmitted to the cytoplasm. Here, adapter proteins recruit signaling components such as kinases and ubiquitin ligases to the receptor complex. Classical NF-κB activation is mediated by a complex interplay of various ubiquitin chain linkages and protein recruitments, which ultimately results in polyubiquitination of IKKγ and phosphorylation of IKKα / β by TAK1.

[0013] The activated IKK complex phosphorylates p65 at S536 and IκBα at S32 and S36, which causes proteasomal degradation. The released active p65 / p50 heterodimer translocates to the nucleus and regulates the transcription of target genes. IκBα is resynthesized as a negative feedback loop, which attenuates NF-κB activation. Another negative feedback loop represents the expression of the deubiquitinating enzyme A20. A20 deubiquitinates RIP1 (receptor-interacting protein 1) by cleaving the attached K63 ubiquitin chains.

[0014] Non-canonical (or alternative) NF-κB signaling depends on proteasomal processing of the precursor p100, which results in the formation of p52. A feature of non-canonical signaling is the requirement for de novo protein synthesis and, in contrast to canonical NF-κB signaling, a distinct, slow kinetics. The central components for activation of non-canonical NF-κB are NF-κB-inducing kinase (NIK) and IKKα.

[0015] NIK is constitutively degraded under steady-state conditions by the proteasome through a mechanism involving ubiquitination mediated by the TRAF3 (tumor necrosis factor (TNF) receptor-associated factor-3)-TRAF2-cIAP (cellular inhibitor of apoptosis) destruction complex. The triggers for non-canonical NF-κB signaling are ligands of some members of the TNF receptor superfamily, including LT-β (lymphotoxin β), BAFF (B cell-activating factor belonging to the TNF family), CD40, RANK (receptor activator of nuclear factor κB), TNFR2, Fn14, etc. When the receptor is stimulated, TRAF3 is degraded, resulting in the accumulation of NIF intracellularly. The accumulated NIK phosphorylates IKKα at the T-loop serine and then phosphorylates p100 within its C-terminus. The phosphorylated precursor molecule is modified by K48-linked ubiquitin chains, triggering proteasomal processing of p100.

[0016] As a result of non-canonical NF-κB signaling, p52 is produced, which preferentially binds to RelB. The activated NF-κB heterodimer p52 / RelB translocates to the nucleus, binds to its consensus sequences, and regulates specific immunological processes such as secondary lymphoid tissue formation, B cell survival and maturation, dendritic cell activation, and bone metabolism (Sun; 2012). However, the pathological mechanisms can cause disordered NIK stabilization or IKKα activation. As a result, the non-canonical NF-κB pathway is constitutively activated, which has been associated with the development of numerous severe diseases, such as autoimmune, inflammatory, and malignant lymphomas such as Hodgkin lymphoma.

[0017] Genotoxic stress induces a complex cellular process called the DNA damage response (DDR). The DDR regulates cell fate decisions such as cell cycle arrest, DNA repair, senescence, quiescence, apoptosis, or other types of cell death, depending on the extent of genotoxic stress. Double-strand breaks in DNA induce a signaling cascade from the nucleus to the cytoplasm, ultimately causing IKK activation in a similar manner to cytokine-induced NF-κB activation (Stilmann et al.; 2009).

[0018] NF-κB activation induced by genotoxic stress is mediated by a bifurcated pathway (Figure 1). Two independent molecular sensors, ATM (ataxia telangiectasia mutated gene) and PARP1 [poly(ADP-ribose) polymerase-1], recognize DNA damage and initiate the DDR. Both PARP1 and ATM perform various functions in the DDR, from the initiation of the stress response to the promotion of DNA damage repair. The most prominent substrate of the kinase ATM is the tumor suppressor protein p53, which exerts its antiproliferative function through the regulation of its target genes. A minor degree of DNA damage results in a reversible cell cycle arrest until the damage is eliminated. Irreparable DNA damage induces a more extensive cellular response. To protect the organism against malignant transformation, diseased cells irreversibly enter a non-amplifying state called cellular senescence or undergo apoptosis (Shiloh & Ziv; 2013).

[0019] Induction of DSBs leads to the activation of ATM by autophosphorylation and causes the synthesis of poly(ADP-ribose) (PAR) by PARP1, which is thought to have a scaffolding function. Subsequently, activation of PARP1 leads to the formation of a nuclear signalosome containing the sensor proteins ATM and PARP1, as well as SUMO (small ubiquitin-related modifier, a ubiquitin-like factor), the E3-ligase PIASy (protein inhibitor of activated STATγ), LRP16 / MACROD1, and the IKK complex subunit IKKγ (Stilmann et al.; 2009; Wu et al., 2015).

[0020] Simultaneously with the induction of genotoxic stress, IKKγ translocates into the nucleus by interacting with the nuclear importer importin 3 and is incorporated into the signalosome by binding to auto-PARylated PARP1. IKKγ is phosphorylated by ATM and SUMOylated by PIASy.

[0021] Subsequently, IKKγ translocates to the cytoplasm and is most likely incorporated into the newly formed IKK holocomplex. At the same time, phosphorylated ATM translocates to the cytoplasm in a Ca 2+ -dependent manner and initiates the formation of cytoplasmic signalosomes (Hinz et al.; 2010). ATM activates TRAF6, leading to Ubc-13-mediated K63-linked polyubiquitination, which serves as a scaffold for the recruitment of cIAP1 and TAB2-TAK1 and subsequent activation of TAK1, and leads to linear ubiquitination of IKKγ, which is achieved by the linear ubiquitin chain assembly complex (LUBAC). Depending on the cellular context and the type of stimulant, additional regulatory components (ELKS, XIAP, or RIP1) have been proposed to participate in the activation of this pathway. Finally, cIAP1-dependent monoubiquitination of IKKγ essentially requires nuclear and cytoplasmic signalosome formation for the activation of the IKK complex, degradation of IκBα, and subsequent activation of NF-κB (Hinz et al.; 2010).

[0022] The IKK / NF-κB pathway induced by genotoxic stress is a major regulator of pro-survival signaling in cells due to either physiologically occurring DNA damage or DNA damage induced by therapy. Thus, IKK / NF-κB activation induced by genotoxic stress and by DDR affects the outcome of many conditions, including development, genetic diseases, aging, and cancer.

[0023] Malignant NF-κB activation is associated with inflammation that promotes carcinogenesis, which is a driving force for tumor formation by maintaining a proliferative environment as a result of inflammatory cytokine secretion (Hanahan & Weinberg; 2011). Furthermore, NF-κB can affect cell proliferation, angiogenesis, and metastasis through transcriptional regulation of target genes (Baud & Karin; 2009). Constitutive activation of NF-κB has been found in certain human cancers and tumor cell lines derived from hematopoietic and lymphoid malignancies, such as multiple myeloma, acute myeloid leukemia, T-cell lymphoma, and tumor cell lines derived from Hodgkin lymphoma. Similarly, elevated NF-κB activation has been observed in melanoma cells, lung cancer cells, bladder cancer cells, breast cancer cells, and pancreatic adenocarcinoma cells.

[0024] Also, the promotion of carcinogenesis by NF-κB is associated with attenuated cell death signaling. TNFα-induced NF-κB activation plays a role in the regulation of anti-apoptotic gene expression and consequently in the inhibition of apoptosis. Similarly, the genotoxic stress-activated NF-κB pathway has been shown to regulate the expression of anti-apoptotic proteins, such as cIAP1 and cIAP2. In addition, NF-κB regulates the expression of A1 / Bfl-1, which potently inhibits etoposide-induced cell death by inhibiting the mitochondrial release of cytochrome c. Importantly, based on its anti-apoptotic activity, NF-κB activation by genotoxic stress is thought to strongly contribute to cancer therapy resistance, and thus inhibition of NF-κB signaling may result in chemosensitization (Lim et al.; 2012).

[0025] Activation of the NF-κB pathway is thought to be a driving force in carcinogenesis and cancer therapy resistance mechanisms, so pharmacological inhibition has been suggested as a useful context-dependent adjuvant for chemotherapy treatment. Proteasome inhibitors were the first NF-κB pathway inhibitors used. However, proteasome inhibitors have uncertain molecular specificity and target both classical and non-classical NF-κB pathways because both signaling cascades rely on degradation or processing functions. Dose-limiting toxic effects of patient treatment with proteasome inhibitors include peripheral neuropathy, thrombocytopenia, neutropenia, anemia, fatigue, and diarrhea. General IKK / NF-κB pathway inhibition causes systemic toxicity and severe side effects due to its pleiotropic functions (Baud & Karin; 2009).

[0026] Cancer is associated with uncontrolled cell proliferation, and cancer treatment focuses on stopping unwanted cell division and growth by inducing DNA damage through treatment with radiotherapy or chemotherapy. Thus, DNA-damaging cancer treatments such as chemotherapy and radiotherapy trigger the activation of the IKK / NF-αB pathway induced by genotoxic stress as part of the DNA damage response (DDR). As a result, the IKK / NF-κB pathway is considered a potential target for a new type of cancer treatment in addition to other conditions such as aging, genetic diseases, reperfusion injury, stroke, neurodegeneration, and oxidative stress-induced DNA damage. Nevertheless, general inhibition of the IKK / NF-κB pathway causes broad immunosuppression and severe side effects due to the pleiotropic functions of IKK and NF-κB and thus cannot be applied as a treatment strategy to patients.

[0027] WO 2007 / 097981 A2 describes α-carboline as an IKK inhibitor for the treatment of cancer. No pathway-specific inhibition of IKK is described at all. Hsu MJ et al. (Biochemical Pharmacology, Elsevier, US, Volume 70, Number 1, July 1, 2005) describe the use of molecules similar to those disclosed herein for the general treatment of cancer.

[0028] WO 2011 / 011186 A2 discloses a group of inhibitors similar to the compounds of the present invention for use in the treatment of cancer. Du Hongtao et al. (Bioorganic & Medicinal Chemistry Letters, Pergamon, Amsterdam, NL, Vol. 26, No. 16, July 1, 2016) describe the synthesis and biological evaluation of N9-substituted harmine derivatives as potential anti-cancer agents.

[0029] Lin Yi-Chien et al. (European Journal of Medicinal Chemistry, Vol. 110, January 7, 2016) disclose the synthesis of novel 3,9-substituted [α]-carboline derivatives with high cytotoxic activity against colorectal cancer cells and their structure-activity relationship. EP 1634881 A1 describes β-carboline-based molecules used in cancer therapy in combination with radiotherapy.

[0030] Chen et al. (International Journal of Cancer, Vol. 114, No. 5, May 1, 2005) describe the anti-tumor and neurotoxic effects of novel harmine derivatives and their structure-activity relationship analysis. Lamchouri et al. (Research on Chemical Intermediates, Vol. 39, No. 5, August 15, 2012) study the quantitative structure-activity relationship of anti-tumor and neurotoxic [β]-carboline alkaloids.

[0031] Zhang et al. (European Journal of Medicinal Chemistry, Vol. 65, pp. 21-31) describe the synthesis of N2-alkylated quaternary [β]-carbolines as novel anti-cancer agents and their structure-activity relationship. Willemann et al. (Bioorganic & Medicinal Chemistry, Vol. 17, No. 13, July 1) disclose the synthesis and cytotoxic activity of 5,6 - aromatic heterocyclic - appended pyridine - 2,4 - diamines.

[0032] Rocca et al. (ChemMedChem, Vol. 11, No. 16, first published on March 23, 2016) describe the hit identification of novel bidirectional binders for h - telo / c - myc G - quadruplexes by a combination of pharmacophore - based virtual screening and docking improvement. The use of the identified molecules for cancer therapy is suggested. Almerico et al. (Journal of Molecular Graphics and Modelling, Vol. 42, March 19, 2013) describe potential inhibitors of the A3 adenosine receptor.

[0033] Silva et al. (Chemical and Pharmaceutical Bulletin, 2012, pp. 1372 - 1379) describe the synthesis, antitumor activity, antileishmanial activity, and antitrypanosomal activity of benzo〔4,5〕quinazolin - 6 - ones having an N′-(substituted benzylidene) carbohydrazide group and an N - alkylcarboxamide group at the C - 2 position. Lamkanfi et al. (The Journal of Cell Biology, Vol. 173, No. 2, April 17, 2006) summarize the mechanism of caspase - mediated activation of NF - κB. Jin et al. (Cancer Research, Vol. 69, No. 5, February 10, 2009) show that cIAP1, cIAP2, and XIAP act cooperatively via a non - redundant pathway that regulates genotoxic stress - induced nuclear factor B activation.

[0034] None of the cited documents disclose a compound that is specifically used as a medicament in the treatment of subjects suffering from cancer that exhibits IKK / NF-κB activation induced by genotoxic stress or in the treatment of subjects in which genotoxic cancer therapy induces IKK / NF-κB activation. In the current state of the art, there is no description at all of a compound that specifically acts on the IKK / NF-κB pathway induced by genotoxic stress, does not directly inhibit IKK, and leaves other pathways that result in IKK / NF-κB activation unaffected. Furthermore, the compounds of the present invention have not been described at all in the prior art.

[0035] Therefore, there is a need to develop a new class of pathway-dependent inhibitors, novel inhibitors that interfere only with agonist-specific NF-κB activation and leave other forms of NF-κB activation intact. Considering the important role of NF-κB in cancer treatment resistance mechanisms, there is an urgent need to develop targeted therapies aimed at the pro-IKK / NF-κB pathway induced by genotoxic stress. To the best of the inventors' knowledge, no NF-κB inhibitor specific for this pathway has been reported to date. In light of the prior art, it remains an important task in the art to provide additional means for the treatment of diseases associated with IKK / NF-κB activation induced by genotoxic stress.

Summary of the Invention

[0036] In view of the prior art, the technical problem underlying the present invention is to provide additional means for the treatment of diseases associated with IKK / NF-κB activation induced by genotoxic stress. This problem is solved by the features of the independent claims. Preferred embodiments of the invention are provided by the dependent claims.

[0037] The present invention relates to a compound of formula I which is used as a medicament in the treatment of diseases associated with IKK / NF-κB activation induced by genotoxic stress:

[0038]

Chemical formula

[0039] [In the above formula R1 is H or O; R2 is 0 to 4, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carboxyl, alkoxycarbonyl, amine, or R2 is alkoxyamine, alkoxyamide, for example

[0040] [Chemical formula]

[0041] (OCH2CONHC2H4NC4H8O) or OC2H4OC2H4NH2; R3 is 0 to 4, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or alternatively, two (adjacent) R3 substituents optionally form a 5- or 6-membered aromatic ring structure, and the aromatic ring structure may optionally contain 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably 2 O atoms; X1, X2, X3 are N or C atoms, preferably C atoms; Ring A is a 5- or 6-membered aromatic ring structure, and the ring structure may optionally contain 0, 1 or 2 heteroatoms selected from O and / or N, preferably forming a pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl ring, Here, the ring structure is optionally substituted with 0 to 3 substituents, which may be the same or different, selected from H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, such as CO-phenyl, carboxyl, alkoxycarbonyl, amine, aryl, such as phenyl (optionally substituted with halogen, C1-C3 alkyl, alkoxy, amine), alkoxyamine, such as CONHC3H6OCH3; The bond z may or may not be present. When the bond z is absent: The C atom of the bond z of ring C is substituted with R3, and X3 of ring A is H, OH, halogen, preferably Br, Cl or F, alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, or amine substituted with C1-C7. It relates to a compound represented by.

[0042] In a preferred embodiment, the compound of formula I is characterized in that at least one of the 0 to 4 R2 is not H. In a preferred embodiment, the compound of formula I is characterized in that X3 is a C atom. In a preferred embodiment, the compound of formula I is characterized in that R1 = O atom. In a preferred embodiment, the compound of formula I is characterized in that ring A is a 5- or 6-membered aromatic heterocyclic structure containing one or two heteroatoms.

[0043] In a preferred embodiment, the present invention is a compound of formula I used as a medicament in the treatment of diseases related to IKK / NF-κB activation induced by genotoxic stress, wherein ring A is

[0044]

Chemical formula

[0045] It relates to a compound having an aromatic heterocyclic structure selected from the group consisting of.

[0046] Preferably, the present invention relates to a compound of formula I for use as a medicament in the treatment of a subject suffering from cancer exhibiting IKK / NF-κB activation induced by genotoxic stress.

[0047] Quite surprisingly, a compound has been discovered that selectively inhibits the pathway leading to IKK / NF-κB activation, which is activated upon genotoxic stress such as DNA damage. All attempts to identify such compounds have been unsuccessful to date, and NF-κB pathway inhibition has mainly been carried out by direct targeting of the IKK complex.

[0048] The NF-κB pathway has numerous actions and functions, and since all of them are IKK-dependent, direct inhibition of the IKK complex or other downstream molecules causes unacceptable side effects including broad immunosuppression, has a high risk of infection, and may allow cancer cells to escape immunosurveillance. These drawbacks of known NF-κB pathway inhibitors are overcome by the method of the present invention, particularly by the compounds described herein under the formulas I, I-a, I-b, II, II-a, II-b, III, III-a, IV, IV-a, V, VI, VII.

[0049] In the prior art, general IKK inhibitors have been described. In contrast, the compounds of the present invention are pathway-selective inhibitors of IKK / NF-κB and do not act directly on IKK. Previously described IKK inhibitors are direct IKK inhibitors and do not distinguish between IKK-NF-κB signaling induced by genotoxic stress and numerous other pathways that activate NF-κB through IKK.

[0050] The compounds of formula I mainly inhibit the activation of the IKK / NF-κB pathway induced by genotoxic stress, but do not inhibit (or only very slightly inhibit and do not significantly inhibit, i.e., do not inhibit as much as the IKK / NF-κB pathway induced by genotoxic stress) alternative pathways of NF-κB activation, including classical and non-classical pathways. Thus, the inhibition exhibited by these compounds is "specific to the IKK / NF-κB pathway induced by genotoxic stress" in such a way that this pathway is inhibited more than other alternative IKK / NF-κB pathways. This selective inhibition has the advantage that it can eliminate or reduce side effects resulting from the inhibition of alternative NF-κB activation pathways, including classical and non-classical NF-κB activation. For this reason, treatment with compounds of the general formula as described herein becomes tolerable over extended periods of days, weeks or even years, thereby providing a novel clinical situation that exceeds the means known in the state of the art. Thus, novel dosing regimens are possible.

[0051] To the best of the inventors' knowledge, the compounds described herein are defined by a novel technical effect, namely the inhibition of IKK / NF-κB activation induced by genotoxic stress.

[0052] The advantageous effects of the compounds of the invention are mediated through the inhibition of the activation of the IKK / NF-κB pathway, which inhibition occurs in response to genotoxic stress or double-strand breaks (DSBs) through unique protein-protein interactions (nuclear PARP1 signalosome), post-translational modifications (SUMOylation and phosphorylation of IKKγ), transport processes (cytoplasmic ATM import) or functional interference by other special components of the NF-κB signaling cascade induced by DNA DSBs or genotoxic stress, which are not involved in or do not share with the activation of any other NF-κB pathway.

[0053] Surprisingly, the compounds of the formula described herein are effective in inhibiting genotoxicity-induced NF-κB activation at sub-micromolar concentrations, while no inhibition of the classical NF-κB pathway was detected. A surprising and significant advantage of treatment with the compounds disclosed herein is that they inhibited both the nuclear export of ATM and the formation of the PARP1 signalosome without affecting enzyme activity. This indicates that the inhibition of the downstream signaling cascades of ATM and PARP1 by the compounds of the present invention is specific for NF-κB activation and does not interfere with the activation of other ATM substrates such as the tumor suppressor protein p53, and is thus a significant advantage of the present invention for preventing the occurrence of side effects resulting from the interference with other functions of ATM and PARP1. Another advantage of treatment with the compounds of formula I is the decrease in the expression of anti-apoptotic genes, which leads to an increase in apoptosis, which is beneficial, for example, in the case of cancer.

[0054] Accordingly, the technical effects achieved by the compounds of the present invention enable the treatment of a new patient population, for example, the treatment of patient populations previously sensitive to off-target side effects of NF-κB inhibition, or in particular the treatment of patients with cancerous conditions that are resistant to DNA-damaging cancer treatments. This specific patient population presents a major challenge to medical practitioners, and the compounds described herein are a powerful solution to this challenge.

[0055] In a preferred embodiment, the present invention relates to a compound of formula I-a for use as a medicament in the treatment of diseases associated with IKK / NF-κB activation induced by genotoxic stress:

[0056]

Chemical formula

[0057] 〔In the above formula R1 is H or an O atom; R2 is one of 0 to 4, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or R2 is alkoxyamine, alkoxyamide, for example

[0058] [Chemical formula]

[0059] or OC2H4OC2H4NH2; R3 is one of 0 to 4, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or two (adjacent) R3 substituents can optionally form a 5- or 6-membered aromatic ring structure, and the aromatic ring structure can optionally contain 0, 1 or 2 heteroatoms, preferably O or N atoms, and more preferably can contain 2 O atoms, R4 is one of 0 to 2, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, aryl; X1, X2 are N or C atoms, preferably C; X3 is an N atom; X4 is an N or C atom, preferably only 1 X4 is N; The bond z may or may not exist. When the bond z does not exist: The C atom of the bond z in ring C is substituted by R3, and X3 in ring A is substituted by H, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, aryl) relates to.

[0060] In a preferred embodiment, the compound of formula I-a is characterized in that R4 is selected from 0 to 2, identical or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, such as CO-phenyl (optionally substituted with halogen, C1-C3 alkyl, alkoxy, amine), carboxyl, alkoxycarbonyl, amine, aryl, such as phenyl (optionally substituted with halogen, C1-C3 alkyl, alkoxy, amine), alkoxyamine, such as CONHC3H6OCH3.

[0061] In a preferred embodiment, the present invention relates to a compound of formula I-b which is used as a medicament in the treatment of diseases associated with NF-κB activation induced by genotoxic stress:

[0062] [Chemical formula]

[0063] [In the above formula R1 is H or an O atom; R2 is 0 to 4, identical or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or R2 is alkoxyamine, alkoxyamide, such as

[0064] [Chemical formula]

[0065] or OC2H4OC2H4NH2; R3 is 0 to 4, identical or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or Alternatively, two (adjacent) R3 substituents can optionally form a 5- or 6-membered aromatic ring structure, which can optionally contain 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably 2 O atoms, X1 and X2 are N or C, preferably C; R16 is 0 to 3, preferably 0, 1, 2, which may be the same or different, H, halogen, preferably Cl, Br or F, C1-C7, preferably C1-C5 alkyl, alkoxy, preferably methoxy; The bond z may or may not be present. When the bond z is absent: The C atom of the bond z of ring C is substituted by R3, and The C atom of the bond z of ring A is substituted by H, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, aryl) relates to.

[0066] In a preferred embodiment, the compound of formula I-b is characterized in that at least one of the 0 to 4 R2 is not H. In a preferred embodiment, the compound of formula I-b is characterized in that R1 is an O atom.

[0067] In a preferred embodiment, the present invention is a compound of formula II used as a medicament in the treatment of diseases related to NF-κB activation induced by genotoxic stress:

[0068]

Chemical formula

[0069] 〔In the above formula R1 is H or an O atom; R5 is H, halogen, preferably Cl, Br, F, C1-C5, preferably C1-C3 alkyl, alkenyl, alkoxy, amine, most preferably H; R6 is H, OH, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, or alkoxyamine, alkoxyamide, for example

[0070]

Chemical formula

[0071] or OC2H4OC2H4NH2; R7 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; R8 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, most preferably H; R9 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; R10 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; R11 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, carboxyl; R12 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, or alternatively, when X1 is C, R9 and R10, R10 and R11, R11 and R12, or the C atom at the position of the bond z between R12 and ring C can optionally form a 5- or 6-membered aromatic ring structure, which contains 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably 2 O atoms, or form phenyl; X1 and X3 are N or C atoms; Ring A is a 5- or 6-membered aromatic ring structure, which optionally contains 0, 1 or 2 heteroatoms selected from O and / or N atoms, preferably forming a pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl ring, wherein said ring structure may optionally be substituted with 0 to 3 substituents, which may be the same or different, selected from H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, such as CO-phenyl, carboxyl, alkoxycarbonyl, amine, aryl, such as phenyl (optionally substituted with halogen, C1-C3 alkyl, alkoxy, amine), alkoxyamine, such as CONHC3H6OCH3; Bond z may or may not be present, and when bond z is absent: The C atom at the position of bond z of ring C is substituted with halogen, preferably Cl, Br, F, C1-C5, preferably C1-C3 alkyl, and X3 of ring A is optionally substituted with H, C1-C5, preferably C1-C3 alkyl, or X3 is optionally a C atom having H, C1-C5, preferably C1-C3 alkyl, OH, halogen, preferably Br, Cl or F. relates to.

[0072] In a preferred embodiment, the compound of formula II is characterized in that at least one of R5-R8 is not H. In a preferred embodiment, the compound of formula II is characterized in that X3 is a C atom. In a preferred embodiment, the compound of formula II is characterized in that R1 is an O atom. In a preferred embodiment, the compound of formula II is characterized in that ring A is a 5- or 6-membered aromatic heterocyclic structure containing 1 or 2 heteroatoms. Ring A is

[0073]

Chemical formula

[0074] In a preferred embodiment, the present invention is a compound of formula II-a used as a medicament in the treatment of diseases associated with NF-κB activation induced by genotoxic stress:

[0075]

Chemical formula

[0076] 〔In the above formula X1 is a C or N atom, preferably C; R1 is H or an O atom; R5 is H, halogen, preferably Cl, Br, F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, most preferably H; R6 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, or OC2H4OC2H4NH2; R7 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; R8 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, most preferably H; R9 is H, halogen, preferably Cl, Br or F; R10 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; R11 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, carboxyl; R12 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; or Alternatively, when X1 is a C atom, R9 and R10, R10 and R11, R11 and R12, or the C atom at the position of the bond z between R12 and ring C (when the bond z does not exist) can optionally form a 5- or 6-membered aromatic ring structure, and the ring structure contains 0, 1, or 2 heteroatoms, preferably O or N atoms, more preferably 2 O atoms, or can form phenyl; R14 is H, C1-C5, preferably C1-C3 alkyl; The bond z may or may not be present. Here, when the bond z is not present: The C atom at the position of the bond z of ring C is substituted with H, halogen, preferably Cl, Br, F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, and The N atom of the bond z of ring A is substituted with H, C1-C5, preferably C1-C3 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, alkoxycarbonyl, amine, aryl) relates to.

[0077] In a preferred embodiment, the present invention is a compound of formula II-b used as a medicament in the treatment of diseases associated with NF-κB activation induced by genotoxic stress:

[0078]

Chemical formula

[0079] 〔In the above formula X1 is a C or N atom, preferably C; R1 is H or an O atom; R5 is H, halogen, preferably Cl, Br, F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, most preferably H; R6 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, or OC2H4OC2H4NH2; R7 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; R8 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, most preferably H; R9 is H, halogen, preferably Cl, Br or F; R10 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; R11 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, carboxyl; R12 is H, halogen, preferably Br, Cl or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; R16 may be the same or different, and is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; alternatively, when X1 is a C atom, R9 and R10, R10 and R11, R11 and R12, or the C atom at the position of the bond z between R12 and ring C (when the bond z does not exist) can optionally form a 5- or 6-membered aromatic ring structure, and the ring structure contains 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably 2 O atoms, or forms phenyl; R14 is H, C1-C5, preferably C1-C3 alkyl; The bond z may or may not exist, and when the bond z does not exist: The C atom at the position of the bond z of ring C is substituted with H, halogen, preferably Cl, Br, F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, and The C atom of the bond z of ring A is substituted with H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, aryl. relates to.

[0080] In a preferred embodiment, the compound of formula II-b is characterized in that at least one of R5-R8 is not H. In a preferred embodiment, the compound of formula II-b is characterized in that R1 = O.

[0081] A further aspect of the present invention relates to a compound of formula III, preferably its medical use as described herein.

[0082] [Chemical formula]

[0083] In the above formula, the substituents of formula III are as follows: R1 is an H or O atom; R2 is 0 to 4, preferably 0, 1 or 2, the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, alternatively, R2 is alkoxyamine, alkoxyamide, for example

[0084] [Chemical formula]

[0085] or OC2H4OC2H4NH2; R3 is 0 to 4, preferably 0, 1 or 2, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or alternatively, two (adjacent) R3 substituents optionally form a 5- or 6-membered aromatic ring structure, which optionally contains 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably contains 2 O atoms, or forms phenyl; X1 and X3 are N or C; Ring A is a 5-membered aromatic heterocyclic structure containing 1 or 2 N atoms, where X3 must be an N atom, preferably forming a pyrazolyl ring or an imidazolyl ring; alternatively, ring A is a 6-membered aromatic heterocyclic structure containing 1 N atom, preferably forming a pyridyl ring, where the ring structure of ring A is optionally substituted with 0 to 3 substituents, which may be the same or different, selected from H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, such as CO-phenyl, carboxyl, alkoxycarbonyl, amine, aryl, such as phenyl (optionally substituted with halogen, C1-C3 alkyl, alkoxy, amine), alkoxyamine, such as CONHC3H6OCH3.

[0086] In one embodiment, the substituents mentioned in the previous paragraph are characterized in that R2 is not carboxyl, where the remaining substituents are the same as those mentioned in the previous paragraph.

[0087] In a preferred embodiment, the compound of formula III is characterized in that at least one of the 0 to 4 R2s is not H. In a preferred embodiment, the compound of formula III is characterized in that X3 is a C atom. In a preferred embodiment, the compound of formula III is characterized in that R1 is an O atom.

[0088] In a preferred embodiment, the compound of formula III is characterized in that ring A is an aromatic heterocyclic structure selected from the group consisting of the following:

[0089]

Chem.

[0090] wherein the aromatic heterocyclic structure is selected from the group consisting of the following:

[0091] A further aspect of the present invention relates to a compound of formula III-a, and preferably to its medical use as described herein:

[0092]

Chem.

[0093] In the above formula R1 is H or O; R2 is 0 to 4, preferably 0, 1 or 2, identical or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, alternatively, R2 is alkoxyamine, alkoxyamide, for example

[0094]

Chem.

[0095] or OC2H4OC2H4NH2; R3 is 0 to 4, preferably 0, 1 or 2, identical or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or Alternatively, two (adjacent) R3 substituents optionally form a 5- or 6-membered aromatic ring structure, which may optionally contain 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably 2 O atoms, or form phenyl; X1 and X3 are N or C; Ring A is a 5- or 6-membered aromatic heterocyclic structure containing 1 or 2 N atoms, preferably forming a pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl ring, where the ring structure may optionally be substituted with 0 to 3 identical or different substituents selected from H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, such as CO-phenyl, carboxyl, alkoxycarbonyl, amine, aryl, such as phenyl (optionally substituted with halogen, C1-C3 alkyl, alkoxy, amine), alkoxyamine, such as CONHC3H6OCH3.

[0096] In one embodiment, the substituents mentioned in the previous paragraph are characterized in that R2 is not carboxyl, where the remaining substituents are the same as those described in the previous paragraph. In a preferred embodiment, the compound of formula III-a is characterized in that at least one of the 0 to 4 R2 is not H. In a preferred embodiment, the compound of formula III-a is characterized in that R1 is an O atom. In a preferred embodiment, the compound of formula III-a has ring A as follows:

[0097]

Chemical formula

[0098] It is characterized in that it is an aromatic heterocyclic structure selected from the group consisting of:

[0099] A further aspect of the present invention relates to a compound of formula IV, and preferably to its medical use as described herein.

[0100]

Chemical formula

[0101] In the above formula, R1 is H or O; R2 is 0 to 4, preferably 0, 1 or 2, identical or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, alternatively, R2 is alkoxyamine, alkoxyamide, for example

[0102]

Chemical formula

[0103] or OC2H4OC2H4NH2; R3 is 0 to 4, preferably 0, 1 or 2, identical or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or alternatively, two (adjacent) R3 substituents can optionally form a 5- or 6-membered aromatic ring structure, which aromatic ring structure can optionally contain 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably 2 O atoms, or can form phenyl, X1 is a C or N atom; X3 is an N atom; X4 is an N or C atom; R4 can be a group selected from 0 to 2, which may be the same or different, of H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, aryl.

[0104] In a preferred embodiment, the compound of formula IV is characterized in that R4 is 0 to 2 groups, which may be the same or different, selected from H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, such as CO-phenyl (optionally substituted with halogen, C1-C3 alkyl, alkoxy, amine), carboxyl, alkoxycarbonyl, amine, aryl, such as phenyl (optionally substituted with halogen, C1-C3 alkyl, alkoxy, amine), alkoxyamine, such as CONHC3H6OCH3.

[0105] In a preferred embodiment, the present invention relates to a compound of formula IV-a and preferably to its pharmaceutical use as described herein:

[0106]

Chemical formula

[0107] In the above formula, R1 is H or an O atom; R2 is 0 to 4, preferably 0, 1 or 2, groups, which may be the same or different, of H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, alternatively, R2 is an alkoxyamine, alkoxyamide, such as

[0108]

Chemical formula

[0109] or OC2H4OC2H4NH2; R3 is 0 to 4, preferably 0, 1 or 2, identical or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or alternatively, two (adjacent) R3 substituents can optionally form a 5- or 6-membered aromatic ring structure, which aromatic ring structure can optionally contain 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably contain 2 O atoms, or can form phenyl, X1 is a C or N atom, preferably a C atom; X4 is an N or C atom, where at least one X4 is an N atom; R4 can be a group selected from 0 to 2, identical or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, aryl.

[0110] In a preferred embodiment, the compound of formula IV-a is characterized in that R4 is 0 to 2, identical or different, groups selected from H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, such as CO-phenyl (optionally substituted with halogen, C1-C3 alkyl, alkoxy, amine), carboxyl, alkoxycarbonyl, amine, aryl, such as phenyl (optionally substituted with halogen, C1-C3 alkyl, alkoxy, amine), alkoxyamine, such as CONHC3H6OCH3.

[0111] In a preferred embodiment, the present invention relates to a compound of formula V and preferably to its pharmaceutical use as described herein:

[0112] [Chemical formula]

[0113] In the above formula, X1 is a C or N atom; R1 is an H or O atom; R5 is H, halogen, preferably Cl, Br, F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, most preferably H; R6 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, or OC2H4OC2H4NH2; R7 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; R8 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, most preferably H; R9 is H, halogen, preferably Cl, Br or F; R10 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; R11 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, carboxyl; R12 is H, halogen, preferably Br, Cl or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; R13 is halogen, preferably Cl, Br or F; Alternatively, when X1 is a C atom, R9 and R10, R10 and R11, R11 and R12, or R12 and R13 can optionally form a 5- or 6-membered aromatic ring structure, and the ring structure can optionally contain 0, 1 or 2 heteroatoms, preferably O or N, more preferably 2 O atoms, or form phenyl; R14 is H, C1-C5, preferably C1-C3 alkyl; R15 is H, C1-C5, preferably C1-C3 alkyl, carbonyl, CO-aryl, preferably benzoyl, and said benzoyl is optionally substituted by halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy.

[0114] In a preferred embodiment, the present invention relates to a compound of formula VI, and preferably to its medical use as described herein:

[0115]

Chemical formula

[0116] In the above formula, R1 is H or an O atom; R2 is 0 to 4, preferably 0, 1 or 2, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, alternatively, R2 is alkoxyamine, alkoxyamide, for example

[0117]

Chemical formula

[0118] or OC2H4OC2H4NH2; R3 is 0 to 4, preferably 0, 1 or 2, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or alternatively, two (adjacent) R3 substituents can optionally form a 5- or 6-membered aromatic ring structure, and the aromatic ring structure optionally contains 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably contains 2 O atoms, or forms a phenyl. X1 is an N or C atom; R16 may be 0 to 3, preferably 0, 1 or 2, identical or different, H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy.

[0119] In a preferred embodiment, the present invention relates to a compound of general formula VI in which at least one of 0 to 4 R2 is not H, and its medical use as described herein. In a preferred embodiment, the present invention relates to a compound of formula VI in which R1 = O atom, and its medical use as described herein.

[0120] In a preferred embodiment, the present invention relates to a compound of formula VII, and preferably its pharmaceutical use as described herein:

[0121]

Chemical formula

[0122] In the above formula, X1 is a C or N atom; R1 is an H or O atom; R5 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, most preferably H; R6 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, or OC2H4OC2H4NH2; R7 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; R8 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, most preferably H; R9 is H, halogen, preferably Cl, Br or F; R10 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; R11 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, carboxyl; R12 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; R13 is H, halogen, preferably Cl, Br or F; Alternatively, when X1 is a C atom, R9 and R10, R10 and R11, R11 and R12, or R12 and R13 can optionally form a 5- or 6-membered aromatic ring structure, and the ring structure can optionally contain 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably 2 O atoms, or form phenyl; R16 may be the same or different and is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy.

[0123] A further aspect of the present invention relates to a compound of formula VII or a medical use thereof as described herein, wherein the substituent of formula VII is as follows: X1 is a C or N atom; R1 is H or an O atom; R5 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, most preferably H; R6 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, or OC2H4OC2H4NH2; R7 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; R8 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, most preferably H; R9 is H, halogen, preferably Cl, Br or F; R10 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; R11 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, carboxyl; R12 is H, halogen, preferably Br, Cl or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; R13 is H, halogen, preferably Cl, Br or F; Alternatively, when X1 is a C atom, R9 and R10, R10 and R11, R11 and R12, or R12 and R13 optionally form a 5- or 6-membered aromatic ring structure, and the ring structure optionally contains 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably 2 O atoms, or forms phenyl; R16 may be the same or different and is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; Here, when R16 is methyl, ring C is substituted with a single Cl atom.

[0124] Regarding compounds of formula VII and preferably their medical uses as described herein, in a preferred embodiment, at least one of R5-R8 is not H. Regarding compounds of formula VII and preferably their medical uses as described herein, in a preferred embodiment, R1 = O atom.

[0125] In a preferred embodiment, the compound of the present invention is selected from the group of compounds given in Table 1. In a preferred embodiment, the present invention relates to the compounds of Table 1 as a medicament in the treatment of diseases associated with IKK / NF-κB activation induced by genotoxic stress.

[0126]

Table 1-1

Table 1-2

Table 1-3

[0127] In a further embodiment, the present invention relates to a compound used as a medicament in the treatment of diseases associated with IKK / NF-κB activation induced by genotoxic stress, which is selected from the group of compounds provided in Table 1 or Table 2.

[0128]

Table 2-1

Table 2-2

Table 2-3

Table 2-4

[0129] In a preferred embodiment, the present invention relates to a compound used as a medicament according to any one of the above aspects,

[0130]

Chemical formula

[0131] In a further preferred embodiment of the present invention, Formulas I-VII can be defined by Ring Structure B or C as disclosed in FIG. 12(D). Their preferred structures can be incorporated into one or more of Formulas I-VII, while the remaining substituents of the formula preferably remain as those disclosed above.

[0132] In another preferred embodiment of the present invention, the disease to be treated is related to IKK / NF-κB activation induced by genotoxic stress. In another preferred embodiment of the present invention, the disease to be treated is cancer.

[0133] In another preferred embodiment of the present invention, cancer is related to IKK / NF-κB activation induced by genotoxic stress.

[0134] In another preferred embodiment of the present invention, the compound is more effective in inhibiting NF-κB signaling induced by genotoxic stress compared to inhibiting NF-κB signaling induced by TNF-α and / or IL-1β. This feature is related to the functional characteristics of the compounds described herein, which are suitable for the definition of the compounds and for distinguishing them from other compounds described in the prior art.

[0135] Another preferred embodiment of the present invention relates to the treatment of diseases associated with genomic instability resulting from defective DNA repair mechanisms. In a preferred embodiment of the present invention, the defect in the DNA repair mechanism is based on genetic changes or epigenetic mutations in one or more DNA repair genes.

[0136] In another preferred aspect of the present invention, the cancer to be treated is related to NF-κB-mediated resistance to treatment-induced tumor cell apoptosis.

[0137] In another preferred embodiment of the present invention, the compound is administered in combination with one or more other cancer treatments, preferably cancer treatments that induce DNA damage. In a preferred embodiment of the present invention, the compound is administered in combination with radiation therapy. In another preferred embodiment of the present invention, the compound is administered in combination with chemotherapy induced by genotoxic stress.

[0138] Another preferred embodiment of the present invention relates to the use of the compounds of the present invention in an in vitro method, preferably in a cell-based assay, for the inhibition of NF-κB signaling induced by genotoxic stress. In another preferred embodiment of the present invention, the compounds of the present invention are used in an in vitro method, preferably in a cell-based assay, for the inhibition of DNA repair mechanisms. Furthermore, the present invention relates to a pharmaceutical composition for the treatment of a subject suffering from a disease associated with IKK / NF-κB activation induced by genotoxic stress, the composition comprising a compound of the present invention and a pharmaceutically acceptable carrier substance.

[0139] 〔Further Embodiments of the Present Invention〕 In a preferred embodiment, the present invention is a compound of formula I which is used as a medicament in the treatment of a disease associated with IKK / NF-κB activation induced by genotoxic stress, wherein R1 is H or an O atom; R2 is 0 to 4, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or R2 is alkoxyamine, alkoxyamide, for example

[0140]

Chemical formula

[0141] or OC2H4OC2H4NH2, wherein at least one of the 0 to 4 R2s is not H; R3 is 0 to 4, identical or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or alternatively, two (adjacent) R3 substituents can optionally form a 5- or 6-membered aromatic ring structure, which can optionally contain 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably 2 O atoms, X1, X2, X3 are N or C atoms, preferably C atoms; ring A is a 5- or 5-membered aromatic heterocyclic structure containing 1 or 2 heteroatoms selected from O and / or N atoms, preferably forming a pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl ring, more preferably selected from the group consisting of

[0142]

Chemical formula

[0143] wherein the ring structure can optionally be substituted with 0 to 3 substituents, identical or different, selected from H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, such as CO-phenyl, carboxyl, alkoxycarbonyl, amine, aryl, such as phenyl (optionally substituted with halogen, C1-C3 alkyl, alkoxy, amine), alkoxyamine, such as CONHC3H6OCH3; bond z may or may not be present, where when bond z is absent: the C atom of bond z in ring C is substituted with R3, and X3 in ring A is substituted with H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine relates to the compound of formula I.

[0144] In a preferred embodiment, the present invention is a compound of formula I, which is used as a medicament in the treatment of diseases associated with IKK / NF-κB activation induced by genotoxic stress, and R1 is H or an O atom; R2 is 0 to 4, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or R2 is alkoxyamine, alkoxyamide, for example

[0145]

Chemical formula

[0146] or OC2H4OC2H4NH2; R3 is 0 to 4, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or alternatively, two (adjacent) R3 substituents can optionally form a 5- or 6-membered aromatic ring structure, which may optionally contain 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably 2 O atoms, X1, X2 are N or C atoms, preferably C atoms; X3 is a C atom; Ring A is a 5- or 6-membered aromatic heterocyclic structure containing 1 or 2 heteroatoms selected from O and / or N atoms, preferably forming a pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl ring, more preferably the following

[0147]

Chemical formula

[0148] selected from the group consisting of, wherein said ring structure is optionally H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, such as CO-phenyl, carboxyl, alkoxycarbonyl, amine, aryl, such as phenyl (optionally substituted with halogen, C1-C3 alkyl, alkoxy, amine), alkoxyamine, such as CONHC3H6OCH3, and may be substituted with 0 to 3 identical or different substituents selected from; the bond z may or may not be present, where when the bond z is absent: the C atom on the bond z of ring C is substituted with R3, and X3 of ring A is H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, substituted with amine relates to the compounds of formula I.

[0149] In a preferred embodiment, the present invention is a compound of formula I used as a medicament in the treatment of diseases associated with IKK / NF-κB activation induced by genotoxic stress, R1 is an O atom; R2 is 0 to 4 identical or different H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or R2 is alkoxyamine, alkoxyamide, such as

[0150]

Chemical formula

[0151] or OC2H4OC2H4NH2; R3 is 0 to 4, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or alternatively, two (adjacent) R3 substituents can optionally form a 5- or 6-membered aromatic ring structure, and the aromatic ring structure can optionally contain 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably can contain 2 O atoms, X1, X2, X3 are N or C atoms, preferably C atoms; ring A is a 5- or 6-membered aromatic heterocyclic structure containing 1 or 2 heteroatoms selected from O and / or N atoms, preferably forms a pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl ring, more preferably the following

[0152]

Chemical formula

[0153] selected from the group consisting of, where the ring structure can optionally be substituted with 0 to 3 substituents, which may be the same or different, selected from H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, such as CO-phenyl, carboxyl, alkoxycarbonyl, amine, aryl, such as phenyl (optionally substituted with halogen, C1-C3 alkyl, alkoxy, amine), alkoxyamine, such as CONHC3H6OCH3; bond z may or may not be present, and where bond z is absent: the C atom of bond z of ring C is substituted with R3, and X3 of ring A is substituted with H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine Relates to the compound of formula I.

[0154] In a preferred embodiment, the compound of formula I is a compound of formula I which is used as a medicament in the treatment of diseases associated with IKK / NF-κB activation induced by genotoxic stress, wherein R1 is an O atom; R2 is 0 to 4, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or R2 is alkoxyamine, alkoxyamide, for example

[0155]

Chemical formula

[0156] or OC2H4OC2H4NH2, wherein at least one of the 0 to 4 R2s is not H; R3 is 0 to 4, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or alternatively, two (adjacent) R3 substituents can optionally form a 5- or 6-membered aromatic ring structure, which aromatic ring structure can optionally contain 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably can contain 2 O atoms, X1, X2, X3 are N or C atoms, preferably C atoms; ring A is a 5- or 6-membered aromatic heterocyclic structure containing 1 or 2 heteroatoms selected from O and / or N atoms, preferably forming a pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl ring, more preferably the following

[0157]

Chemical formula

[0158] Selected from the group consisting of, wherein said ring structure is optionally H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, such as CO-phenyl, carboxyl, alkoxycarbonyl, amine, aryl, such as phenyl (optionally substituted with halogen, C1-C3 alkyl, alkoxy, amine), alkoxyamine, such as CONHC3H6OCH3, and may be substituted with 0 to 3 identical or different substituents selected from; The bond z may or may not be present, where when the bond z is absent: The C atom of the bond z of ring C is substituted with R3, and X3 of ring A is H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, substituted with an amine Relates to the compounds of formula I.

[0159] In a preferred embodiment, the present invention is a compound of formula I used as a medicament in the treatment of diseases associated with IKK / NF-κB activation induced by genotoxic stress, R1 is an O atom; R2 is 0 to 4, identical or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or R2 is alkoxyamine, alkoxyamide, such as

[0160]

Chemical formula

[0161] Or OC2H4OC2H4NH2, where at least one of the 0 to 4 R2s is not H; R3 is 0 to 4, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or alternatively, two (adjacent) R3 substituents can optionally form a 5- or 6-membered aromatic ring structure, and the aromatic ring structure can optionally contain 0, 1 or 2 heteroatoms, preferably O or N atoms, and more preferably can contain 2 O atoms, X1 and X2 are N or C atoms, preferably C atoms; X3 is a C atom; Ring A is a 5- or 6-membered aromatic heterocyclic structure containing 1 or 2 heteroatoms selected from O and / or N atoms, preferably forming a pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl ring, and more preferably selected from the group consisting of the following

[0162]

Chemical formula

[0163] and may optionally be substituted with 0 to 3 substituents, which may be the same or different, selected from H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, such as CO-phenyl, carboxyl, alkoxycarbonyl, amine, aryl, such as phenyl (optionally substituted with halogen, C1-C3 alkyl, alkoxy, amine), alkoxyamine, such as CONHC3H6OCH3; The bond z may or may not be present. Here, when the bond z is absent: The C atom on the bond z of ring C is substituted with R3, and X3 of ring A is substituted with H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine Relates to the compound of formula I.

[0164] A further aspect of the present invention relates to a compound of formula I and preferably to its medical use as described herein:

[0165]

Chemical formula

[0166] In the above formula R1 is an O atom; R2 is 0 to 4, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, alkoxycarbonyl, amine, or R2 is alkoxyamine, alkoxyamide, for example

[0167]

Chemical formula

[0168] or OC2H4OC2H4NH2, where at least one of the 0 to 4 R2s is not H; R3 is 0 to 4, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxy, alkoxycarbonyl, amine, or alternatively, two (adjacent) R3 substituents can optionally form a 5- or 6-membered aromatic ring structure, which aromatic ring structure can optionally contain 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably can contain 2 O atoms, X1, X2 are N or C atoms, preferably C atoms; Ring A is a 5- or 6-membered aromatic ring structure, which ring structure optionally contains 1 or 2 heteroatoms selected from O and / or N atoms, preferably forms a pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl ring, more preferably the following

[0169]

Chem.

[0170] selected from the group consisting of, wherein when ring A has a 5-membered ring structure, X3 is an N atom; when ring A has a 6-membered ring structure, X3 is a C atom; wherein said ring structure may optionally be substituted with 0 to 3 identical or different substituents selected from H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, such as CO-phenyl, carboxyl, alkoxycarbonyl, amine, aryl, such as phenyl (optionally substituted with halogen, C1-C3 alkyl, alkoxy, amine), alkoxyamine, such as CONHC3H6OCH3; bond z may or may not be present, wherein when bond z is absent: the C atom of bond z of ring C is substituted with R3, and X3 of ring A is substituted with H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine.

[0171] In a further aspect of the present invention, regarding the compounds of formula II and their medical uses as described herein,

[0172]

Chem.

[0173] In the above formula R1 is an O atom; R5 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkenyl, alkoxy, amine, most preferably H; R6 is H, OH, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, or alkoxyamine, alkoxyamide, for example

[0174]

Chemical formula

[0175] or OC2H4OC2H4NH2; R7 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; R8 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, most preferably H; wherein at least one of R5-R8 is not H; R9 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; R10 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy; R11 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, carboxyl; R12 is H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy, or alternatively, when X1 is a C atom, R9 and R10, R10 and R11, R11 and R12, or the C atom at the position of the bond z between R12 and ring C can optionally form a 5- or 6-membered aromatic ring structure, the ring structure containing 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably 2 O atoms, or forming phenyl; X1, X3 are N or C atoms; Ring A is a 5- or 6-membered aromatic ring structure, which contains 1 or 2 heteroatoms selected from O and / or N, preferably forms a pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl ring, and more preferably the following

[0176]

Chemical formula

[0177] selected from the group consisting of. Here, when ring A is a 5-membered ring structure, X3 is an N atom; when ring A is a 6-membered ring structure, X3 is a C atom; wherein said ring structure is optionally substituted with 0 to 3 identical or different substituents selected from H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, such as CO-phenyl, carboxyl, alkoxycarbonyl, amine, aryl, such as phenyl (optionally substituted with halogen, C1-C3 alkyl, alkoxy, amine), alkoxyamine, such as CONHC3H6OCH3; Bond z may or may not be present. Here, when bond z is absent: The C atom at the position of bond z of ring C is potentially substituted with halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, and X3 of ring A is optionally substituted with H, C1-C5, preferably C1-C3 alkyl, OH, halogen, preferably Br, Cl or F, or when X3 is a C atom, it is substituted with H, C1-C5, preferably C1-C3 alkyl, OH, halogen, preferably Br, Cl or F.

[0178] A further aspect of the present invention relates to the compounds of formula III, and preferably their medical uses as described herein,

[0179]

Chemical formula

[0180] Here, the substituents in Formula III are as follows: R1 is an O atom; R2 is 0 to 4, preferably 0, 1 or 2, identical or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, alkoxycarbonyl, amine, or R2 is alkoxyamine, alkoxyamide, for example

[0181]

Chemical formula

[0182] or OC2H4OC2H4NH2, where at least one of the 0 to 4 R2s is not H; R3 is 0 to 4, preferably 0, 1 or 2, identical or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or alternatively, two (adjacent) R3 substituents optionally form a 5- or 6-membered aromatic ring structure, which optionally contains 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably 2 O atoms, or form phenyl; X1, X3 are N or C atoms, preferably C atoms; Ring A is a 5-membered aromatic heterocyclic structure containing 1 or 2 N atoms, where X3 must be N, preferably a pyrazolyl or imidazolyl ring, preferably

[0183]

Chemical formula

[0184] is formed, or ring A is a 6-membered aromatic heterocyclic structure containing one or two N atoms, where X3 must be a C atom, preferably selected from the following

[0185] [Chemical formula]

[0186] and is selected from the group consisting of where the ring structure of ring A may optionally be substituted with 0 to 3 identical or different substituents selected from H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, such as CO-phenyl, carboxyl, alkoxycarbonyl, amine, aryl, such as phenyl (optionally substituted with halogen, C1-C3 alkyl, alkoxy, amine), alkoxyamine, such as CONHC3H6OCH3.

[0187] A further aspect of the present invention relates to a compound of formula III-a and its medical use as described herein.

[0188] [Chemical formula]

[0189] In the above formula R1 is an O atom; R2 is 0 to 4, preferably 0, 1 or 2, identical or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, alkoxycarbonyl, amine, alternatively, R2 is an alkoxyamine, alkoxyamide, such as

[0190] [Chemical formula]

[0191] or OC2H4OC2H4NH2, where at least one of 0 to 4 R2s is not H; R3 is 0 to 4, preferably 0, 1 or 2, identical or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or alternatively, two (adjacent) R3 substituents can optionally form a 5- or 6-membered aromatic ring structure, which can optionally contain 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably can contain 2 O atoms, or form phenyl; X1, X3 are N or C atoms; Ring A is a 5- or 6-membered aromatic heterocyclic structure containing 1 or 2 N atoms, preferably forming a pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl ring, more preferably the following

[0192] [Chemical formula]

[0193] selected from the group consisting of, where the ring structure can optionally be substituted with 0 to 3 identical or different substituents selected from H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, such as CO-phenyl, carboxyl, alkoxycarbonyl, amine, aryl, such as phenyl (optionally substituted with halogen, C1-C3 alkyl, alkoxy, amine), alkoxyamine, such as CONHC3H6OCH3.

[0194] A further aspect of the present invention relates to the compounds of formula VIII, and preferably to their medical use as described herein,

[0195] [Chemical formula]

[0196] In the above formula R1 is an O atom; R2 is 0 to 4, preferably 0, 1 or 2, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, alternatively, R2 is alkoxyamine, alkoxyamide, for example

[0197]

Chemical formula

[0198] or OC2H4OC2H4NH2, where at least one of the 0 to 4 R2s is not H; R3 is 0 to 4, preferably 0, 1 or 2, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or alternatively, two (adjacent) R3 substituents can optionally form a 5- or 6-membered aromatic ring structure, which aromatic ring structure can optionally contain 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably 2 O atoms, or can form phenyl; X1 is an N or C atom, preferably a C atom; X4 is an N or C atom, whereby at least one X4 is an N atom; R16 is 0 to 3, preferably 0, 1 or 2, which may be the same or different, H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy.

[0199] A further aspect of the present invention relates to the compounds of formula IX, and preferably their medical uses as described herein,

[0200]

Chem.

[0201] In the above formula R1 is an O atom; R2 is 0 to 4, preferably 0, 1 or 2, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, alternatively, R2 is alkoxyamine, alkoxyamide, for example

[0202]

Chem.

[0203] or OC2H4OC2H4NH2, where at least one of 0 to 4 R2s is not H; R3 is 0 to 4, preferably 0, 1 or 2, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or alternatively, two (adjacent) R3 substituents can optionally form a 5- or 6-membered aromatic ring structure, which optionally contains 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably contains 2 O atoms, or can form phenyl; X1 is an N or C atom, preferably a C atom; R16 is 0 to 3, preferably 0, 1 or 2, which may be the same or different, H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy.

[0204] A further aspect of the present invention relates to a compound of formula X, and preferably to its medical use as described herein.

[0205]

Chemical formula

[0206] In the above formula R1 is an O atom; R2 is 0 to 4, preferably 0, 1 or 2, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, alternatively, R2 is alkoxyamine, alkoxyamide, for example

[0207]

Chemical formula

[0208] or OC2H4OC2H4NH2, where at least one of 0 to 4 R2s is not H; R3 is 0 to 4, preferably 0, 1 or 2, which may be the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or alternatively, two (adjacent) R3 substituents can optionally form a 5- or 6-membered aromatic ring structure, which optionally contains 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably contains 2 O atoms, or can form phenyl; X1 is an N or C atom, preferably a C atom; R16 is 0 to 3, preferably 0, 1 or 2, which may be the same or different, H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy.

[0209] In a preferred embodiment, the present invention relates to a compound of formula VI, and its medical uses as described herein,

[0210]

Chemical formula

[0211] In the above formula R1 is an O atom; R2 is 0 to 4, preferably 0, 1 or 2, the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, alternatively, R2 is alkoxyamine, alkoxyamide, for example

[0212]

Chemical formula

[0213] or OC2H4OC2H4NH2, where at least one of the 0 to 4 R2s is not H; R3 is 0 to 4, preferably 0, 1 or 2, the same or different, H, OH, halogen, preferably Br, Cl or F, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, amine, or alternatively, two (adjacent) R3 substituents can optionally form a 5- or 6-membered aromatic ring structure, which optionally contains 0, 1 or 2 heteroatoms, preferably O or N atoms, more preferably 2 O atoms, or can form phenyl; X1 is N or C; R16 is 0 to 3, preferably 0, 1 or 2, identical or different, H, halogen, preferably Cl, Br or F, C1-C5, preferably C1-C3 alkyl, alkoxy, preferably methoxy.

[0214] In a preferred embodiment, the compound of the present invention is selected from the group of compounds given in Table 1 and / or Table 3. In a preferred embodiment, the present invention relates to a compound as described in Table 1 and / or Table 3 for use as a medicament in the treatment of diseases associated with IKK / NF-κB activation induced by genotoxic stress.

[0215] [Table 3-1] [Table 3-2]

[0216] In a further embodiment, the present invention relates to a compound for use as a medicament in the treatment of diseases associated with IKK / NF-κB activation induced by genotoxic stress, said compound being selected from the group of compounds given in Table 1, Table 2, Table 3 and / or Table 4.

[0217] [Table 4] [Brief Description of the Drawings]

[0218]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Mode for Carrying Out the Invention

[0219] 〔Detailed Description of the Invention〕 All cited patent documents and non-patent documents are incorporated herein by reference in their entirety. The present invention relates to compounds used as medicaments in the treatment of diseases associated with genotoxic stress, preferably diseases associated with IKK / NF-κB (NF-kappa B) activation induced by genotoxic stress, and their use.

[0220] Regarding the compounds described herein, the term "alkyl" refers to a branched or straight-chain saturated hydrocarbon group having 1 to 7 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, and the like. Preferred alkyl groups have 1 to 7 carbon atoms, more preferably 1 to 4 carbon atoms. Any one or more alkyl groups described herein may be "substituted alkyl", in which case one or more hydrogen atoms are substituted with substituents such as halogen, cycloalkyl, alkoxy, amino, hydroxyl, aryl, or carboxyl.

[0221] The term "alkenyl" refers to a linear, branched, or cyclic hydrocarbon arrangement and combinations thereof, preferably containing 2 to 7 carbon atoms, more preferably 2 to 4 carbon atoms, formed by removing one hydrogen atom from an alkene, such as those that produce ethenyl and the like.

[0222] The term "alkynyl" refers to a linear, branched, or cyclic hydrocarbon arrangement and combinations thereof, preferably containing 2 to 7 carbon atoms, more preferably 2 to 4 carbon atoms, formed by removing one hydrogen atom from an alkyne, such as those that produce ethynyl and the like.

[0223] The term "cycloalkyl" refers to an arrangement derived from cycloalkane by removal of a hydrogen atom, thereby preferably forming cycloalkyl such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0224] The term "alkoxy" preferably refers to a linear, branched, or cyclic hydrocarbon arrangement containing 1 to 7 carbon atoms, more preferably 1 to 4 carbon atoms, and combinations thereof, and containing an oxygen atom at the bonding site (e.g., O-alkyl). An example of an "alkoxy group" is represented by the formula -OR, where R is an alkyl group which can optionally be an alkyl group substituted with an alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group. Suitable alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, sec-butoxy, cyclohexyloxy, and the like.

[0225] The term "alkylthio" preferably refers to a structure containing a carbon-bonded sulfhydryl or sulphydryl containing 1 to 7 carbon atoms, more preferably 1 to 4 carbon atoms (-C-SH or R-SH, where R is alkyl), which contains an S atom at the bonding site (e.g., S-alkyl). Alkylthio can be represented as RS(O) when n = 0. n The groups RS(O) where n = 1 or 2 n refer to sulfoxide and sulfone respectively, and these groups are also substituents of the compounds of the present invention.

[0226] The term "acyl" is derived from the removal of one or more hydroxyl groups from an oxo acid containing an oxygen atom double-bonded to an alkyl group, and refers to a structure forming -RC(=O). Thus, acyl includes a carbonyl which refers to a group of the formula -C(=O)-. Groups containing a carbonyl include any substituent containing a carbon-oxygen double bond (C=O), such as amides, carboxy groups, esters, ureas, carbamates, carbonates, and ketones and aldehydes, for example substituents based on -COR or -RCHO, where R is alkyl, heteroalkyl, hydroxyl, or a secondary, tertiary, or quaternary amine.

[0227] "Alkoxycarbonyl" refers to a carbonyl group substituted with an alkoxy (e.g., -C(=O)OR), where R represents an alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl or similar moiety that may be optionally substituted.

[0228] The term "aryl" refers to any carbon-based aromatic group, including, without limitation, benzene, etc. The term "aromatic" also includes "heteroaryl groups", which are defined as aromatic groups containing at least one heteroatom in the ring of the aromatic group. Examples of heteroatoms include, without limitation, nitrogen, oxygen, sulfur. An aryl group can be substituted by one or more groups including alkyl, alkynyl, alkenyl, aryl, halide, nitro, amino, ester, ketone, aldehyde, hydroxy, carboxylic acid, or alkoxy, or the aryl group can be unsubstituted.

[0229] The term "amine" refers to a group of the formula -NRR', where R and R' can independently be hydrogen or an alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, alkyl halide or heterocycloalkyl group as described above. The term "amide" (amide / amido) is represented by the formula -C(O)NRR', where R and R' can independently be hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, alkyl halide or heterocycloalkyl group. A suitable amide group is the acetamide group.

[0230] A 5- or 6-membered ring structure containing arbitrarily one or more N or O atoms preferably relates to cycloalkyl, cycloalkane and non-aromatic heterocycles (e.g., morpholine, piperidine, piperazine, thiomorpholine, tetrahydrofuran), aromatic ring structures such as phenyl, naphthalene, aromatic heterocycles such as furan, pyrrole, oxazole, thiophene, thiazole, pyrazole, imidazole, and pyridine, pyrazine, pyrimidine, pyran, thiopyran, oxazine, azepine, thiepine, oxetane, etc. The 5- or 6-membered ring structure preferably forms a pyrazolyl, imidazolyl, pyridyl, pyrimidyl, pyridazyl, pyrazinyl ring.

[0231] "Carbonyl" refers to a group of the formula -C(O)-. Carbonyl-containing groups include any substituent containing a carbon-oxygen double bond (C=O), such as acyl groups, amides, carboxy groups, esters, ureas, carbamates, carbonates, and ketones and aldehydes, such as substituents based on -COR or -RCHO, where R is aliphatic, heteroaliphatic, alkyl, heteroalkyl, hydroxyl, or secondary, tertiary or quaternary amine, phenyl, substituted phenyl (e.g., phenyl substituted with halogen, C1-C3 alkyl, alkoxy, amine), carboxyl, alkoxycarbonyl, amine, aryl.

[0232] The term "alkylamino" refers to an alkyl group as defined above in which at least one hydrogen atom is replaced by an amino group.

[0233] "Aminocarbonyl" alone or in combination means a carbonyl (carbamoyl) group substituted with an amino group, where the amino group is optionally mono- or disubstituted, e.g., substituted with alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, alkanoyl, alkoxycarbonyl, aralkoxycarbonyl, etc. The aminocarbonyl group can be -N(R)-C(O)-R (where R is a substituent or H) or -C(O)-N(R).

[0234] "Carboxyl" refers to the -COOH group. Substituted carboxyl refers to -COOR, where R is aliphatic, heteroaliphatic, alkyl, heteroalkyl, or carboxylic acid or ester.

[0235] The term "hydroxyl" is represented by the formula -OH.

[0236] The term "hydroxyalkyl" refers to an alkyl group in which at least one hydrogen atom is substituted with a hydroxyl group. The term "alkoxyalkyl group" is defined as an alkyl group in which at least one hydrogen atom is substituted with the above alkoxy group.

[0237] The term "aralkyl" refers to an aryl group in which the alkyl group defined above is bonded to the aryl group defined above. An example of an aralkyl group is the benzyl group.

[0238] A group that may be optionally substituted, such as "optionally substituted alkyl", when substituted, refers to a group having 1 to 5 substituents, typically 1, 2 or 3 substituents, selected from alkoxy, optionally substituted alkoxy, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, aryl, carboxyalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, halogen, optionally substituted heteroaryl, optionally substituted heterocyclyl, hydroxy, sulfonyl, thiol and thioalkoxy, for example an alkyl group. In particular, examples of the optionally substituted alkyl group include haloalkyl groups, such as fluoroalkyl groups, and non-limitingly, the trifluoromethyl group. Those potential optional substituents apply to all groups in the formulas disclosed herein when an optional substituent is mentioned. Preferred optional substituents are hydroxyl, alkyl, alkoxy, carbonyl, alkoxycarbonyl, NO2, amine.

[0239] Certain examples of the compounds of the present disclosure contain one or more asymmetric centers; thus, those compounds can exist in different stereoisomeric forms. Accordingly, the compounds and compositions can be provided as individual pure optical isomers or as mixtures of stereoisomers, such as racemic mixtures. In certain embodiments, the compounds disclosed herein are synthesized or purified to be in a substantially enantiopure form, such as an enantiomeric excess of 90%, 95%, 97%, or greater than 99%, for example, in an enantiopure form. The dotted line at the position of the double bond represents any double bond that may or may not be present.

[0240] Protected derivatives of the disclosed compounds are expected to be useful, for example, in the synthesis of the disclosed compounds. A variety of protecting groups suitable for use with the disclosed compounds are disclosed in Greene & Wuts Protective Groups in Organic Synthesis; 3rd Edition; John Wiley & Sons, New York, 1999. Generally, the protecting group is removed under conditions that do not affect the remainder of the molecule. Such methods are well known in the art and include acid hydrolysis, hydrogenolysis, and the like.

[0241] The compounds of the present invention exist in various polymorphs and can exist, for example, as amorphous and crystalline polymorphs. All polymorphs of the compounds of the present invention are within the scope of the present invention and constitute additional embodiments of the present invention.

[0242] The compounds of the present invention may include deuterium substitution of hydrogen. This substitution can result in improved metabolic stability in certain situations (Nature Reviews Drug Discovery 15, 219-221 (2016)).

[0243] The substituents and substitution patterns of the compounds described herein can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and can be readily synthesized by conventional known techniques and further by the methods described in the present disclosure.

[0244] The present invention further relates to pharmaceutically acceptable salts of the compounds described herein. The term "pharmaceutically acceptable salts" refers to salts or esters of the compounds described herein prepared by conventional methods, such as basic salts of inorganic acids and organic acids, for example, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid, tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid, maleic acid, salicylic acid, benzoic acid, phenylacetic acid, mandelic acid, etc. Any compound referred to herein can be administered instead as its pharmaceutically acceptable salt. Acidic salts of inorganic bases and organic bases are also included, including, but not limited to, sodium, potassium, ammonium, triethylamine, etc.

[0245] "Pharmaceutically acceptable salts" also include free acids, bases, and zwitterionic forms. Descriptions of suitable pharmaceutically acceptable salts can be found in Handbook of Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH (2002). For therapeutic use, the salts of the compounds are those in which the counterion is pharmaceutically acceptable. However, salts of acids and bases that are not pharmaceutically acceptable can also find use, for example, in the preparation and purification of pharmaceutically acceptable compounds.

[0246] Another aspect of the disclosure includes a pharmaceutical composition prepared for administration to a subject, which comprises a therapeutically effective amount of one or more compounds disclosed herein. In one aspect, the pharmaceutical composition is useful for the treatment of pain. The therapeutically effective amount of the disclosed compounds will depend on the route of administration, the species of the subject, and the physical characteristics of the subject to be treated. Specific factors to be considered include the severity of the disease stage, the disease stage, body weight, diet, and concomitant drug therapy. The relevance of those factors to determining the therapeutically effective amount of the disclosed compounds is recognized by those skilled in the art.

[0247] A pharmaceutical composition for administration to a subject can include, in addition to a specific molecule, at least one additional pharmaceutically acceptable additive, such as a carrier, bulking agent, diluent, buffer, preservative, surfactant, etc. The pharmaceutical composition can further include one or more additional active ingredients, such as an antibacterial agent, an anti-inflammatory agent, an anesthetic, etc. Pharmaceutically acceptable carriers useful in those formulations are conventional. Remington’s Pharmaceutical Sciences, E.W. Martin, Mack Publishing Co., Easton, PA, 19th Edition (1995) describes compositions and formulations suitable for drug delivery of the compounds disclosed herein.

[0248] Generally, the nature of the carrier will depend on the particular mode of administration to be used. For example, parenteral formulations generally contain as excipients injectable solutions that include pharmaceutically and physiologically acceptable liquids such as water, physiological saline, balanced salt solutions, glucose solution, glycerol, etc. In the case of solid compositions (e.g., powder, pill, tablet or capsule dosage forms), conventional non-toxic solid carriers can include, for example, pharmaceutical mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered can include small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, pH buffering agents, etc., such as sodium acetate or sorbitan monolaurate.

[0249] In accordance with the various therapies of the present disclosure, the compounds can be delivered to a subject in a manner that is not inconsistent with conventional methodologies relevant to the management of the disease for which a treatment or prevention method is sought. According to the disclosure herein, a prophylactically or therapeutically effective amount of a compound and / or another biologically active agent is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, suppress, and / or ameliorate a particular disease or condition or one or more symptoms thereof.

[0250] "Administering" or "administer" a compound shall be construed to mean providing the compound, a prodrug of the compound or a pharmaceutical composition as described herein. The compound or composition can be administered to a subject patient by another person (e.g., intravenously) or it can be self-administered by the patient (e.g., a tablet).

[0251] Any reference to a compound used as a medicament in the treatment of a medical condition relates to a method of treating said condition which comprises administering to a subject in need thereof the compound or a composition comprising said compound, or the use of the compound or a composition comprising said compound in the treatment of said condition.

[0252] The dosage can be varied by the attending physician so as to maintain the desired concentration at the target site (e.g., the lung or the systemic circulation). Higher or lower concentrations can be selected based on the mode of administration, e.g., transdermal, rectal, oral, pulmonary or nasal administration as compared to intravenous or subcutaneous administration. The dosage can also be adjusted based on the release rate of the formulation being administered, e.g., the release rate of an aerosol in the lung versus a powder, a sustained release oral formulation versus an infusion granule or a transdermal administration formulation.

[0253] The present invention also relates to a method of treating a subject suffering from various medical conditions disclosed herein. The treatment method preferably comprises administering to a subject in need of treatment a therapeutically effective amount of a compound of the present disclosure.

[0254] In the context of the present invention, the term "pharmaceutical" refers to a drug, a formulation or a pharmaceutical product used for diagnosing, treating, managing or preventing a disease. It refers to any substance or combination of substances represented as having the property of treating or preventing a disease. This term includes any substance or combination of substances that can be utilized or administered for any purpose of restoring, modifying or improving a physiological function by exerting a pharmacological, immunological or metabolic action, or of performing a medical diagnosis. The term "pharmaceutical" includes biological agents, small molecule drugs, or physical substances that act on physiological processes.

[0255] According to the present invention, the term "treatment" refers to a therapeutic intervention that reduces the signs or symptoms of a disease or pathological condition after the onset of signs or symptoms has begun. As used herein, the term "ameliorate" when used with respect to a disease or pathological condition refers to any observable beneficial therapeutic effect. A beneficial effect can be demonstrated, for example, by a delay in the onset of clinical symptoms of a disease in a susceptible subject, a decrease in the severity of some or all of the clinical symptoms of the disease, a slow progression of the disease, an improvement in the overall health or well-being of the patient, or other parameters well known in the art that are specific to a particular disease.

[0256] The present invention encompasses both therapeutic and prophylactic treatment of a subject. A "prophylactic" treatment is a treatment administered to a subject who shows no signs of a disease or only initial signs thereof, for the purpose of reducing the risk of developing pathology.

[0257] The term "disease" refers to a specific abnormal condition, which, from the perspective of the present invention, refers to a disorder of the structure or function that affects part or all of an organism. It refers to any condition that causes pain, dysfunction, suffering or death in the affected person, and includes injury, physical impairment, disorder, syndrome, infection, individual symptoms, deviant behavior, as well as atypical variations in structure and function. A disease is associated with a malfunction of the body's normal homeostatic functions. A disease can be acquired, congenital, chronic, acute, genetic, idiopathic, hereditary / inherited. Another equivalent term in the context of the present invention is illness, disorder, medical condition, syndrome or pre-disease. A disease can be localized, disseminated, systemic.

[0258] When used in connection with the present invention, the term "genotoxic stress" refers to ROS that induce damage to genetic material, including any given substance, compound, environmental signal, environmental substance, irradiation, and / or cellular metabolite, such as nucleic acids of all kinds, including DNA and RNA. The genome is potentially exposed to genotoxic events that are harmful during each cell division cycle. This endogenous source of DNA damage results from normal errors during cell metabolism or DNA replication and recombination. In addition, exposure of cells and organisms to exogenous genotoxic substances, such as ultraviolet light, oxidative stress, chemical mutagens, causes various nucleotide modifications and DNA strand breaks. To overcome such attacks on the genome, cells have evolved response systems that induce cell cycle arrest and give sufficient time to repair the damage sustained. Genotoxic stress induces DNA damage, which in turn leads to activation of DNA repair. The genotoxic stress response system includes DNA repair, activates appropriate DNA repair pathways, or induces apoptosis if repair is not possible. DNA damage in the form of mutations or genomic instability results from genotoxic stress caused by exposure to toxic substances, such as cytotoxic drugs administered as anti-cancer agents, ultraviolet sunlight, background ionizing radiation, chemicals in food and the environment, and highly reactive molecules produced intracellularly during metabolism. Similar types of DNA damage occur in response to various agents, inducing mutations, base and nucleotide elimination, dimer formation, strand breaks, cross-linking, chromosomal abnormalities. Some of these types of damage accumulate in nuclear or mitochondrial DNA with aging (e.g., point mutations, single-strand breaks, DNA cross-links, additions / deletions, oxidative damage, and methylated bases).

[0259] NF-κB (nuclear factor kappa-light-chain enhancer of activated B cells) is a protein complex that unrestrictedly controls DNA transcription, cytokine production and survival, cell differentiation and proliferation. NF-κB is found in almost all cells and is involved in the cellular response to stimuli such as stress, cytokines, free radicals, heavy metals, ultraviolet light, oxidized LDL, bacterial and viral antigens. NF-κB plays an important role in controlling the immune response to infection and also plays various important roles in adaptive and innate immunity. Dysregulation of NF-κB has been associated with cancer, inflammatory and autoimmune diseases, septic shock, viral infections and inappropriate immune system development. NF-κB is also involved in the processes of synaptic plasticity and memory. All proteins of the NF-κB family share a Rel homology domain at their N-terminus.

[0260] Subfamilies of NF-κB proteins such as RelA, RelB and c-Rel have a transcriptional activation domain at their C-terminus. In contrast, NF-κB1 and NF-κB2 proteins are synthesized as large precursors p105 and p100, which are processed into the mature NF-κB subunits p50 and p52, respectively. The processing of p105 and p100 is mediated by the ubiquitin / proteasome pathway and involves the selective degradation of their C-terminal regions containing ankyrin repeats. The generation of p52 from p100 is a tightly regulated process, while p50 is produced from the constitutive processing of p105. The p50 and p52 proteins are proposed to have no intrinsic ability to activate transcription and to act as transcriptional repressors when κB elements bind as homodimers. Indeed, this confounds the elucidation of p105-knockout studies, which are studies that genetically remove a repressor (p50 homodimer) similar to IκB (full-length p105) in addition to a transcriptional activator (RelA-p50 heterodimer).

[0261] NF-κB is important for controlling cellular responses. This is because it belongs to the category of "rapid-acting" primary transcription factors, i.e., transcription factors that exist in the cell in an inactive state and do not require new protein synthesis to become activated. This makes NF-κB an acute response factor to harmful cellular stimuli. The known inducers of NF-κB activity are very diverse and include reactive oxygen species (ROS), tumor necrosis factor α (TNFα), interleukin 1β (IL-1β), bacterial lipopolysaccharide (LPS), isoproterenol, cocaine, and ionizing radiation. Stimulation by many bacterial products and diverse cell surface receptors causes NF-κB activation and rapid changes in gene expression. The identification of Toll-like receptors (TLRs) as specific pattern recognition molecules and the finding that stimulation of TLRs causes NF-κB activation have enhanced the inventors' understanding of how diverse pathogens activate NF-κB. For example, the inventors' research has identified TLR4 as the receptor for the LPS component of Gram-negative bacteria.

[0262] In unstimulated cells, NF-κB dimers are sequestered in the cytoplasm by a family of inhibitors called IκB (κB inhibitor), which are proteins containing multiple copies of a sequence called ankyrin repeats. By their ankyrin repeat domains, IκB proteins mask the nuclear localization signals (NLSs) of NF-κB proteins and keep them trapped in an inactive state in the cytoplasm. IκB has six or more ankyrin repeat sequences following an N-terminal regulatory domain and a PEST domain near the C-terminus. The IκB family consists of Iκα, Iκβ, IκBε, and Bcl-3, but the most well-studied and important IκB protein is IκBα.

[0263] Due to the presence of ankyrin repeats in the C-terminal portion, p105 and p100 also function as IκB proteins. The C-terminal half of p100 is often called IκBδ, which also functions as an inhibitor. The degradation of IκBδ that occurs in response to developmental stimuli, such as those transmitted through LTβR, enhances the activation of NF-κB dimers in the NIK-dependent non-canonical pathway.

[0264] The activation of NF-κB is initiated by signal-induced degradation of IκB proteins. This occurs mainly through the activation of kinases called "IKK" or IκB kinases. Thus, the term "IKK / NF-κB activation" as used in this patent application refers to the activation of NF-κB through the activation of IKK.

[0265] IKK is composed of a heterodimer of catalytic IKKα and IKKβ subunits and a "master" regulatory protein called NEMO (NF-κB essential modulator) or IKKγ. When activated by a signal, the IκB kinase phosphorylates two serine residues located in the IκB regulatory domain. Concurrent with the phosphorylation of those serines (e.g., serines 32 and 36 in human IκBα), the IκB inhibitor molecule is modified by a process called ubiquitination and undergoes degradation by the proteasome. The degradation of IκB frees the NF-κB complex, which then translocates freely into the nucleus, where it "turns on" the expression of specific genes having adjacent DNA binding sites for NF-κB. The subsequent activation of those genes by NF-κB then elicits a given physiological response, such as an inflammatory or immune response, a cell survival response, or cell proliferation. NF-κB activates the expression of IκBα, which is its own repressor. The newly synthesized IκBα then re-suppresses NF-κB, thereby forming an auto-feedback loop that results in oscillations, dampening, and downstream regulation of NF-κB activity levels.

[0266] According to the present invention, IKK / NF-κB activation induced by genotoxic stress relates to a signaling pathway induced through the occurrence of genotoxic stress, which pathway leads to the activation of IKK and consequently the activation of NF-κB. Genotoxic stress triggers two corresponding signaling axes and activates the IκB kinase (IKK) complex in a similar manner to the classical NF-κB signaling cascade. The first axis is initiated by poly(ADP-ribose) polymerase-1 (PARP-1), a DNA strand break sensor, which constructs a transient nucleoplasmic complex and initiates PIASy-mediated SUMOylation and phosphorylation of nuclear IKKγ mediated by the mutated ataxia telangiectasia gene (ATM). The modified IKKγ returns to the cytoplasm and integrates into the newly formed IKK complex. At the same time, ATM translocates to the cytoplasm, binds to TRAF6, and triggers its K63-linked polyubiquitination. Activated TRAF6 recruits cIAP1 and TAB2-TAK1, resulting in TAK1 activation and IKKβ phosphorylation. However, the final activation of the IKK complex requires cIAP1-dependent IKKγ monoubiquitination at lysine 285, which depends on the formation of the nuclear PARP1 signalosome and the activation of the cytoplasmic signaling axis by ATM-dependent activation of TRAF6.

[0267] Diseases associated with IKK / NF-κB activation induced by genotoxic stress include, but are not limited to, cancer, particularly colon cancer, gastric cancer, breast cancer, melanoma, myelodysplastic syndrome, acute myeloid leukemia (AML), tumors due to increased PARP-1 expression, such as Ewing sarcoma, malignant lymphoma, early stage colorectal carcinogenesis, hematopoietic cell cancer, atypical and typical endometrial hyperplasia, breast cancer, uterine cancer, lung cancer, and ovarian cancer, which occur during the progression of a disease, in an established disease, or as a result of chemotherapy or radiotherapy for a disease. Non-cancer diseases and conditions associated with IKK / NF-κB activation induced by genotoxic stress include, but are not limited to, type I diabetes, type II diabetes, stroke, subarachnoid hemorrhage (SAH), reperfusion injury, particularly kidney and heart reperfusion injury, atherosclerosis, progeria, and aging.

[0268] One of ordinary skill in the art can identify a subject having a cancer that exhibits IKK / NF-κB activation induced by genotoxic stress by using standard analytical means. To identify the subject of the present invention to be treated, there are numerous assays for identifying NF-κB activation induced by genotoxic stress in tumor specimens from cancer patients, some of which are shown below. The following methods are examples of assays for identifying a subject having a cancer that exhibits IKK / NF-κB activation induced by genotoxic stress, and should not be construed as an exhaustive (complete) list of such assays.

[0269] The following five protein modifications indicate that IKK / NF-κB activation was induced by genotoxic stress, such as DNA double-strand breaks (DSBs) (which can be caused, for example, by chemotherapeutic agents or radiation exposure): phospho-Ser 139 γH2A.X, phospho-Ser 1981-ATM, phospho-Ser 85 IKKγ, monoubiquitination of IKKα at Lys 285, and phospho-Ser 536-RelA (references for those modifications can be found in Hinz et al. (2009) Mol Cell). The indicated protein modifications can be assayed by established methods using commercially available antibodies, for example, by Western blot analysis or other antibody-based techniques. An additional method for detecting those modifications is mass spectrometry technology.

[0270] 1) Phospho-Ser 139 γH2A.X: Phosphorylation of H2A.X at residue Ser-139 by the PI3K-like kinases ATM, ATR, and DNA-PK is an early information readout for the cellular response to the generation of DSBs by chemotherapeutic agents or radiation exposure. 2) Phospho-Ser 1981-ATM: This modification indicates activation of ATM by DSBs generated by chemotherapeutic agents or radiation exposure. 3) Phospho-Ser 85 IKKγ: According to current findings, this modification is detected only in cells with DSBs and indicates the presence of DSB-activated ATM. It promotes NF-κB activation by DSBs. 4) Monoubiquitination of IKKγ at Lys 285: Ubiquitination of this residue occurs more highly in genotoxic stress (i.e., DSB)-induced NF-κB compared to cytokine-induced NF-κB. 5) Phospho-Ser 536-RelA: This modification indicates NF-κB activation through multiple activation pathways not limited to the pathway induced by genotoxic stress.

[0271] Subjects who are suitable for the application of the compounds of the present invention and their use as medicaments and who are suffering from cancers showing IKK / NF-κB activation induced by genotoxic stress include subjects having any cancer type that is being treated or has been previously treated with chemotherapy or radiation that induces DNA damage.

[0272] The use of the compounds of the present invention can, in some cases, be mainly used as an "add-on" drug in genotoxic therapies (chemotherapy, radiotherapy) to increase the lethality of cancer / tumor cells by suppressing NF-κB-dependent protection against apoptosis. Thus, a broad spectrum of malignant tumors whose treatment success rate can be improved will be obtained.

[0273] The PARP1-PIASy-ATM-IKKγ complex and the ATM-TRAF6 axis are expected to be activated by chemotherapy and / or radiotherapy in a number of different cancer types. The above assays can be used to confirm the activation of the NF-κB pathway induced by genotoxic stress by each of the standard chemotherapy or radiotherapy protocols in a particular disease. The assays can also be used for treatment-resistant cancers to determine when to apply the compounds of the invention. The assays can be applied prior to any treatment of cancers expected to have high levels of unrepaired DNA damage (e.g., when mutations in DNA repair genes have been demonstrated).

[0274] In a preferred embodiment, the present invention relates to cancer as a disease to be treated. The cancer according to the present invention refers to any type of cancer, neoplasm or malignant tumor found in mammals, including leukemia, lymphoma, sarcoma, melanoma and carcinoma. Examples of cancers are breast cancer, pancreatic cancer, colon cancer, lung cancer, non-small cell lung cancer, ovarian cancer and prostate cancer.

[0275] In the context of the present invention, leukemia includes, but is not limited to, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, leukemic leukemia, basophilic leukemia, blast leukemia, bovine leukemia, chronic myelogenous leukemia, cutaneous leukemia, fetal leukemia, eosinophilic leukemia, Gross leukemia, hairy cell leukemia, hemocytoblastic leukemia, hemocytoblast leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphangitic leukemia, lymphatic leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, microgranuloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelogenous leukemia, myelogranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacytic leukemia, plasmacytoid leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling leukemia, stem cell leukemia, sub-leukemic leukemia, and undifferentiated cell leukemia.

[0276] According to the present invention, lymphomas include, but are not limited to, Hodgkin and non-Hodgkin lymphomas (B-cell and T-cell lymphomas), diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma also known as mucosa-associated lymphoid tissue (MALT) lymphoma, nodal marginal zone B-cell lymphoma, and splenic marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia), hairy cell leukemia, primary central nervous system (CNS) lymphoma, precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma, cutaneous T-cell lymphoma (mycosis fungoides, Sézary syndrome, etc.), indolent, chronic, acute, and lymphoma subtypes including adult T-cell leukemia / lymphoma, angioimmunoblastic T-cell lymphoma, extranodal natural killer cell / T-cell lymphoma, nasal type, enteropathy-type intestinal T-cell lymphoma (EATL), anaplastic large cell lymphoma (ALCL), and unspecified peripheral T-cell lymphoma.

[0277] Sarcomas as defined in the context of the present invention include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanoma, myxosarcoma, osteosarcoma, Abernethy sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, sarcoma botryoides, chloroma, fetal sarcoma, Wilms tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin sarcoma, idiopathic multiple pigmented sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T cells, Jensen sarcoma, Kaposi sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymal sarcoma, parosteal sarcoma, reticuloendothelial sarcoma, Rous sarcoma, serum sarcoma, synovial sarcoma, and telangiectatic sarcoma.

[0278] The melanomas according to the present invention include, for example, acral lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, malignant lentigo melanoma, malignant melanoma, nodular melanoma, subungual melanoma, and superficial spreading melanoma, but are not limited thereto.

[0279] The carcinomas defined by the present invention include squamous cell carcinoma, carcinoma of the breast, adenoid cystic carcinoma, acinar cell carcinoma, cystadenoma, adenoid cystic carcinoma, adenocarcinoma, adrenocortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basal cell carcinoma tumor, basaloid tumor, basal spiny cell carcinoma, bronchioloalveolar carcinoma, bronchial carcinoma, bronchogenic carcinoma, encephaloid carcinoma, cholangiocarcinoma, choriocarcinoma, colloid carcinoma, acne carcinoma, corpuscular carcinoma, cribriform carcinoma, armor-like carcinoma, skin cancer, cylindric carcinoma, columnar cell carcinoma, ductal carcinoma, dural carcinoma, embryonal carcinoma, encephaloid carcinoma, epidermoid carcinoma, adenoid epithelioma, exophytic carcinoma, ulcerating carcinoma, fibrosarcoma, gelatinous carcinoma, colloid carcinoma, giant cell carcinoma, giant cell carcinoma, adenocarcinoma, downstream membrane cell carcinoma, hair matrix carcinoma, hematoid carcinoma, Hurthle cell carcinoma, hepatocellular carcinoma, hyaline carcinoma, adrenoid carcinoma, infantile embryonal carcinoma, intraepithelial carcinoma, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher carcinoma, Kulchitzky cell carcinoma, large cell carcinoma, lenticular carcinoma, liposarcoma, lymphoepithelial carcinoma, medullary carcinoma, medullary carcinoma, melanotic carcinoma, soft ulcerous tumor, mucinous carcinoma, mucous carcinoma, mucinous cell carcinoma, mucoepidermoid carcinoma, mucous carcinoma, mucinous carcinoma, mucinous tumor carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, osteoid carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, spiny cell carcinoma, pasty carcinoma, renal cell carcinoma of the kidney, supplementary cell carcinoma, sarcomatoid carcinoma, Schneider carcinoma, Skills carcinoma, scrotal carcinoma, signet ring cell carcinoma, simple carcinoma, small cell carcinoma, solanoid carcinoma, oval cell carcinoma, spindle cell carcinoma, spongy carcinoma, squamous cell carcinoma, squamous epithelial cell carcinoma, filamentous carcinoma, telangiectatic carcinoma, telangiectatic carcinoma, transitional cell carcinoma, nodular carcinoma, nodular carcinoma, verrucous carcinoma, and choriocarcinoma, but are not limited thereto.

[0280] Additional cancers according to the present invention include, but are not limited to, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, essential thrombocythemia, Waldenström macroglobulinemia, small cell lung tumors, primary brain tumors, gastric cancer, colon cancer, malignant pancreatic islet tumors, malignant carcinoids, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, esophageal cancer, urothelial cancer, hypercalcemia of malignancy, cervical cancer, endometrial cancer, adrenocortical cancer, and prostate cancer.

[0281] In the context of the present invention, the term "DNA damage" refers to changes in the chemical structure of DNA, such as DNA strand breaks, base deletions from the DNA backbone, or chemically modified bases. Damage to DNA that occurs naturally can result from metabolic or hydrolytic processes. Metabolism releases compounds that damage DNA, including, among others, reactive oxygen species, reactive nitrogen species, reactive carbonyl species, lipid peroxides, and alkylating agents, while hydrolysis cleaves chemical bonds in DNA. Most DNA damage can be repaired by DNA repair, but such repair is not 100% effective. Unrepaired DNA damage accumulates in non-replicating cells, such as cells in the adult mammalian brain or muscle, and can cause aging. In replicating cells, such as cells lining the inner surface of the colon, errors occur during replication of past damage in the template strand of DNA or during repair of DNA damage. These errors can lead to mutations or epigenetic mutations. Both types of these mutations will be replicated and passed on to subsequent cell generations. These mutations can alter gene function or regulation of gene expression and may contribute to the progression to cancer. If DNA damage cannot be repaired, inhibition of transcription or replication, mutagenesis, and / or cytotoxicity may occur. In humans, DNA damage has been shown to be involved in various genetically inherited diseases, aging, and carcinogenesis.

[0282] All eukaryotic cells have evolved a multifaceted response to counteract the potentially harmful effects of DNA damage. When DNA damage or replication stalling is sensed, cell cycle checkpoints are activated to halt the progression of the cell cycle, providing time for repair before the damage is passed on to daughter cells. In addition to checkpoint activation, the DNA damage response induces transcriptional programs, enhances DNA repair pathways, and, in cases of severe damage, initiates apoptosis. All of these processes are carefully coordinated so that the genetic material is accurately maintained, replicated, and segregated within the cell.

[0283] The term "DNA repair," as used in the context of the present invention, refers to several cellular processes or pathways for restoring information lost after DNA damage. These processes and pathways include, but are not limited to, cell cycle checkpoints such as the G1 checkpoint, S checkpoint, G2-M checkpoint, and DNA repair pathways such as direct reversal, base excision repair, nucleotide excision repair, DNA mismatch repair, and double-strand break repair. The DNA repair rate depends on various factors such as cell type, cell age, and extracellular environment.

[0284] Cells that continue to accumulate large amounts of DNA damage, or cells whose DNA has incurred damage that can no longer be effectively repaired, may undergo various cellular processes such as an irreversible resting state known as senescence, apoptosis, a programmed cell death program, other cell death programs such as necrosis, non-apoptotic programmed cell death or necroptosis, and chaotic cell division that can lead to the formation of cancerous tumors.

[0285] In the context of the present invention, the term "DNA repair gene" refers to all genes involved in the control or regulation of DNA repair mechanisms or pathways. These include, but are not limited to, for base excision repair (BER): UNG, SMUG1, MBD4, TDG, OGG1, MUTYH (MYH), NTHL1 (NTH1), MPG, NEIL1, NEIL2, NEIL3, APEX1 (APE1), APEX2, LIG3, XRCC1, PNKP, APLF (C2ORF13); for poly(ADP-ribose) polymerase (PARP) enzymes that bind to DNA: PARP1 (ADPRT), PARP2 (ADPRTL2), PARP3 (ADPRTL3); for direct reversal of damage: MGMT, ALKBH2 (ABH2), ALKBH3 (DEPC1); for repair of DNA-topoisomerase crosslinks: TDP1, TDP2 (TTRAP); for mismatch excision repair (MMR): MSH2, MSH3, MSH6, MLH1, PMS2, MSH4, MSH5, MLH3, PMS1, PMS2L3; for nucleotide excision repair (NER): XPC, RAD23B, CETN2, RAD23A, XPA, DDB1, DDB2 (XPE), RPA1, RPA2, RPA3, TFIIH, ERCC3 (XPB), ERCC2 (XPD), GTF2H1, GTF2H3, GTF2H4, GTF2H5 (TTDA), CDK7, CCNH, MNAT1, ERCC5 (XPG), ERCC1, ERCC4 (XPF), LIG1; NER-related ERCC8 (CSA), ERCC6 (CSB), UVSSA (KIAA1530), XAB2 (HCNP), MMS19; for homologous recombination: RAD51, RAD51B, RAD51D, DMC1, XRCC2, XRCC3, RAD52, RAD54L, RAD54B, BRCA1, SHFM1 (DSS1), RAD50, MRE11A, NBN (NBS1), RBBP8 (CtIP), MUS81, EME1 (MMS4L), EME2, GIYD1 (SLX1A), GIYD2 (SLX1B), GEN1;For Fanconi anemia: FANCA, FANCB, FANCC, BRCA2 (FANCD1), FANCD2, FANCE, FANCF, FANCG (XRCC9), FANCI (KIAA1794), BRIP1 (FANCJ), FANCL, FANCM, PALB2 (FANCN), RAD51C (FANCO), BTBD12 (SLX4) (FANCP), FAAP20 (C1orf86), FAAP24 (C19orf40); for non - homologous end joining: XRCC6 (Ku70), XRCC5 (Ku80), PRKDC, LIG4, XRCC4, DCLRE1C (Artemis), NHEJ1 (XLF, Cernunnous); for regulation of nucleotide pools: NUDT1 (MTH1), DUT, PRM2B (p53R2); for DNA polymerases (catalytic subunits): POLB, POLG, POLD1, POLE, PCNA, REV3L (POLZ), MAD2L2 (REV7), REV1L (REV1), POLH, POLI (RAD30B), POLQ, POLK (DINB1), POLL, POLM, POLN (POL4P); for editing and processing of nucleases: FEN1 (DNase IV), FAN1 (MTMR15), TREX1 (DNase III), TREX2, EXO1 (HEX1), APTX (aprataxin), SPO11, ENDOV; for ubiquitination and modification: UBE2A (RAD6A), UBE2B (RAD6B), RAD18, SHPRH, HLTF (SMARCA3), RNF168, SPRTN (C1orf124), RNF8, RNF4, UBE2V2 (MMS2), UBE2N (UBC13); for chromatin structure and modification: H2AFX (H2AX), CHAF1A (CAF1), SETMAR (METNASE); for gene deletions in diseases associated with sensitivity to DNA - damaging agents: BLM, WRN, RECQL4, ATM, TTDN1 (C7orf11); for other identified genes with known or suspected DNA repair functions: DCLRE1A (SNM1), DCLRE1B (SNM1B), RPA4, PRPF19 (PSO4), RECQL (RECQ1), RECQL5, HELQ (HEL308), RDM1 (RAD52B), OBFC2B (SSB1);For other stored DNA damage response genes, ATR, ATRIP, MDC1, RAD1, RAD9A, HUS1, RAD17 (RAD24), CHEK1, CHEK2, TP53, TP53BP1 (53BP1), RIF1, TOPBP1, CLK2, PER1 are included.;

[0286] In one embodiment of the present invention, the compound is preferably more effective in inhibiting NF-κB signaling induced by genotoxic stress compared to the inhibition of NF-κB signaling induced by TNF-α (TNFα) and / or IL-1β.

[0287] According to the present invention, TNFα, i.e., tumor necrosis factor alpha, is a cell signaling protein (cytokine) involved in systemic inflammation and is one of the cytokines that make up the acute phase response. TNFα regulates immune cells, induces fever, apoptotic cell death, cachexia, inflammation, inhibits tumor formation and viral replication, and can respond to sepsis via IL1- and IL6-producing cells. Dysregulation of TNFα production is thought to be related to various human diseases including Alzheimer's disease, cancer, major depression, psoriasis, and inflammatory bowel disease (IBD). TNFα can bind to two receptors, TNFR1 (TNF receptor type 1; CD120a; p55 / 60) and TNFR2 (TNF receptor type 2; CD120b; p75 / 80). TNFR signaling induces the activation of multiple intracellular signaling pathways, including the activation of NF-κB.

[0288] In the context of the present invention, IL-1β is also known, among other names, as "leukocytic pyrogen", "leukocyte endogenous mediator", "monocyte factor", "lymphocyte activating factor", and it is a cytokine protein encoded by the IL1B gene in humans. Il-1β is a member of the interleukin-1 family of cytokines. This cytokine is produced as a precursor protein by activated macrophages, which is proteolytically processed to its active form by caspase 1 (CASP1 / ICE). This cytokine is an important mediator of the inflammatory response and is involved in various cellular activities including cell proliferation, differentiation, and apoptosis.

[0289] In the context of the present invention, the term "NF-κB signaling induced by TNFβ and / or IL-1β" refers to the activation of the classical or canonical NF-κB signaling pathway that is activated when stimulated with TNFα and / or IL-1β. In the classical signaling pathway, NF-κB / Rel proteins are bound and inhibited by IκB proteins. Proinflammatory cytokines such as TNFα and IL-1β, LPS, growth factors, and antigen receptors induce a signaling cascade leading to the activation of the IKK complex (IKKβ, IKKα, and NEMO) that phosphorylates the IκB protein. Phosphorylation of IκB results in its ubiquitination and proteasomal degradation, releasing the NF-κB / Rel complex. The active NF-κB / Rel complex is further activated by post-translational modifications (phosphorylation, acetylation, glycosylation, ubiquitination), translocates to the nucleus, where, alone or together with other transcription factors such as AP-1, Ets, and Stat, they induce the expression of target genes.

[0290] The present invention also relates to the treatment of diseases associated with genomic instability due to defective DNA repair mechanisms.

[0291] The term "genomic instability" as used in the context of the present invention refers to a high frequency of mutations within the genome of a cell lineage. Such mutations can include changes in nucleic acid sequences, chromosomal rearrangements or aneuploidy. Genomic instability occurs in bacteria. In multicellular organisms, genomic instability is central to carcinogenesis and occurs in a variety of cancer types. A person skilled in the art can readily identify cancers associated with genomic instability by routine testing. Other diseases other than cancers associated with genomic instability include neurodegenerative diseases such as amyotrophic lateral sclerosis, neuromuscular diseases, and myotonic dystrophy.

[0292] Many neurological and neurodegenerative disorders are associated with hereditary or acquired defects in DNA repair pathways, or genomic instability due to excessive genotoxic oxidative stress. This has been established for many such diseases, including xeroderma pigmentosum, Cockayne syndrome, sulfur-deficient brittle hair disease (trichothiodystrophy), Down syndrome, triple A syndrome, spinocerebellar ataxia with axonal neuropathy 1, Huntington's disease, Alzheimer's disease, Parkinson's disease, Down syndrome and amyotrophic lateral sclerosis, Huntington's disease, various spinocerebellar ataxias, Friedreich's ataxia, myotonic dystrophy types 1 and 2, ataxia telangiectasia, ataxia telangiectasia-like disorder, Nijmegen breakage syndrome and Alzheimer's disease. Xeroderma pigmentosum, Cockayne syndrome, sulfur-deficient brittle hair disease, Down syndrome and triple A syndrome have defects in the DNA nucleotide excision repair pathway, and spinocerebellar ataxia with axonal neuropathy 1, Huntington's disease, Alzheimer's disease, Parkinson's disease, Down syndrome and amyotrophic lateral sclerosis result in or are associated with increased oxidative stress and the inability of the base excision repair pathway to handle the resulting DNA damage. Huntington's disease, various spinocerebellar ataxias, Friedreich's ataxia and myotonic dystrophy types 1 and 2 often have abnormal expansions of repetitive DNA sequences, presumably due to genomic instability, and ataxia telangiectasia, ataxia telangiectasia-like disorder, Nijmegen breakage syndrome and Alzheimer's disease have defects in genes involved in the repair of DNA double-strand breaks.

[0293] In cancer, genomic instability can occur before or as a result of transformation. Genomic instability refers to any abnormal change in the three-dimensional structure of DNA that can cause any of the following: accumulation of extra copies of DNA or chromosomes, chromosomal translocations, chromosomal inversions, chromosomal deletions, single-strand breaks in DNA, double-strand breaks in DNA, intercalation of foreign substances into DNA double helices, or loss of DNA or misexpression of genes, but is not limited to these. The unpredictable nature of these events is also a major cause of the heterogeneity observed among tumor cells.

[0294] Other diseases associated with genomic instability include progeroid syndromes (PS) and potentially associated NF-κB-dependent lesions, including tumors. Examples of PS include Werner syndrome (WS), Bloom syndrome (BS), Rothmund-Thomson syndrome (RTS), Cockayne syndrome (CS), xeroderma pigmentosum (XP), trichothiodystrophy (TTD), combined xeroderma pigmentosum-Cockayne syndrome (XP-CS), restrictive dermopathy (RD), and Hutchinson-Gilford progeria syndrome (HGPS).

[0295] In the context of the present invention, the term "defective" refers to something in a cellular mechanism, such as a DNA repair system or a DNA damage response system, that has a problem or defect that prevents it from functioning properly.

[0296] In the context of the present invention, the term "modification" refers to any kind of change, alteration, or adjustment that is made such that some original state is changed or altered when used in the context of the present invention. Thus, genetic modification refers to a change occurring on genetic material, including a change occurring in the nucleotide sequence of a nucleic acid molecule. Epigenetic variation refers to a change in the epigenetic state of a nucleic acid molecule (e.g., a DNA molecule) that does not change the nucleotide sequence of the molecule. Epigenetic variation can occur on nucleic acids or chromatin, which includes histones and histone modifications. Epigenetic modifications or variations include, but are not limited to, acetylation, methylation, ubiquitination, phosphorylation, SUMOylation, ribosylation, and citrullination.

[0297] The term "resistance" in the context of the present invention refers to a decrease in the effectiveness of drugs such as antibacterial, anthelmintic or antitumor drugs when treating a disease or condition. This term is used, for example, with respect to pathogens or cancer cells that have "acquired" resistance to a drug or to another treatment or mechanism directed against the pathogen or cancer cell. Antibacterial resistance and antitumor drug resistance are said to be multi-drug resistance when an organism or cancer cell is resistant to multiple drugs.

[0298] According to the present invention, cancer treatment resistance refers to the development of resistance by cancer cells via various mechanisms to treatments such as chemotherapy, radiotherapy, radiation therapy, cell therapy and targeted therapy. These mechanisms include specific genetic and epigenetic changes in cancer cells and / or the microenvironment in which the cancer cells are present. Also, activation of various signaling pathways including the NF-κB pathway can contribute to the development of cancer treatment resistance. The term "NF-κB-mediated resistance to apoptosis" when used in the context of the present invention refers to a cellular mechanism in which the genotoxic stress-activated NF-κB pathway inhibits the induction of apoptosis. NF-κB activation in response to DNA-damaging cancer treatment is the main cellular mechanism of inducible tumor cell resistance.

[0299] Cancers associated with NF-κB-mediated resistance to treatment-induced tumor cell apoptosis in the context of the present invention include, but are not limited to, BRCA1 or BRCA2 mutant ovarian cancer, breast cancer, cervical cancer, gastric cancer, pancreatic cancer or prostate cancer.

[0300] The compounds of the invention described herein can include different types of carriers depending on whether they are administered in solid, liquid or aerosol form and whether they need to be sterile for the route of administration such as injection. The invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrathoracically, intratracheally, intranasally, intravitreally, intravaginally, rectally, topically, intratumorally, intramuscularly, subcutaneously, subconjunctivally, intravesically, intramucosally, epicardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation (e.g., aerosol inhalation), by injection, infusion, continuous infusion, topical perfusion bath, directly to target cells, via catheter, in cream, in lipid composition (e.g., liposome), by other methods known to those skilled in the art or by any combination of the foregoing (see, e.g., Remington’s Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, which is incorporated herein by reference).

[0301] In the present invention, the term “cancer treatment” refers to any type of cancer treatment including, but not limited to, surgery, chemotherapy, radiation therapy, irradiation therapy, hormone therapy, targeted therapy, cell therapy, cancer immunotherapy, monoclonal antibody therapy.

[0302] Administration of the compounds can be carried out individually as monotherapy or in combination with one or more other cancer treatments. In the context of the present invention, the term “in combination” indicates that an individual to whom a compound according to the invention is administered receives another cancer treatment which does not necessarily occur simultaneously, in a single pharmacological composition or via the same route. Thus, “in combination” refers to treating an individual suffering from cancer with multiple cancer treatments. Combination administration includes concurrent therapy or co-therapy, and the treatments can be carried out within minutes of each other, at the same time, on the same day, in the same week, or in the same month.

[0303] DNA damage-induced cancer treatment in the meaning of the present invention includes irradiation therapy and chemotherapy, and includes, but is not limited to, actions by overwhelming the ability of cells to repair DNA damage to cause cell death.

[0304] In this regard, chemotherapy refers to a category of cancer treatment that uses one or more anti-cancer agents (chemotherapeutic drugs) as part of a standardized chemotherapy regimen. Chemotherapy may be carried out for curative purposes (almost always involving a combination of drugs) or for palliative purposes such as life extension or symptom relief (palliative chemotherapy). Chemotherapy is one of the main categories of medical oncology (the medical specialty dedicated to the drug treatment of cancer). Chemotherapeutic drugs are used to treat cancer and are administered in one or more cycles over a period of several days to several weeks, combining two or more agents. Such drugs are toxic not only to cells with a high growth rate, such as cancer itself, but also to the gastrointestinal tract (causing nausea and vomiting), the bone marrow (causing various cytopenias), and the hair (causing alopecia).

[0305] Chemotherapeutic agents include, but are not limited to, actinomycin, all-trans retinoic acid, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine.

[0306] Irradiation or radiotherapy in the context of the present invention relates to a treatment approach that uses ionizing radiation or ultraviolet-visible (UV / Vis) radiation as part of cancer treatment to generally control or kill malignant cells such as cancer cells or tumor cells. Radiotherapy can be curative in many cancer types when they are localized to one area of the body. It can also be used as part of adjuvant therapy to prevent the recurrence of tumors after surgery to remove primary malignant tumors (e.g., early breast cancer). Radiotherapy is synergistic with chemotherapy and is used before, during, and after chemotherapy in sensitive cancers. Radiotherapy is commonly applied to cancerous tumors due to its ability to suppress cell proliferation. Ionizing radiation functions by damaging the DNA of cancerous tissue, resulting in cell death. Radiotherapy can be used systemically or locally.

[0307] Radiotherapy functions by damaging the DNA of cancerous cells. This DNA damage is caused by one of two types of energy: photons or charged particles. This damage is direct or indirect ionization of the atoms that make up the DNA strand. Indirect ionization occurs as a result of the ionization of water, forming free radicals including hydroxyl radicals, which damage the DNA. In photon therapy, most of the radiation effects are mediated by free radicals. Cells have mechanisms to repair single-strand DNA damage and double-strand DNA damage. However, double-strand DNA breaks are much more difficult to repair and can lead to dramatic chromosomal abnormalities and gene deletions. Targeted double-strand breaks increase the likelihood that cells will undergo cell death.

[0308] The radiation dose used in photon radiotherapy is measured in grays (Gy) and varies depending on the type and stage of cancer to be treated. In curative treatment, typical doses for solid epithelial tumors range from 60 to 80 Gy, and lymphomas are treated with 20 to 40 Gy. Prophylactic (adjuvant) doses are typically about 45 to 60 Gy in 1.8 to 2 Gy fractions (breast cancer, head and neck cancers).

[0309] Various types of radiation therapy are known, including conventional external beam therapy, stereotactic radiation therapy (radiosurgery), virtual simulation, three-dimensional conformal radiation therapy, and intensity-modulated radiation therapy (IMRT), volumetric modulated arc therapy (VMAT), particle therapy, Auger therapy, brachytherapy, intraoperative radiation therapy, radioisotope therapy, and deep inspiration breath-hold.

[0310] External beam therapy includes X-rays, gamma rays, and charged particles, and can be applied as low-dose rate or high-dose rate depending on the overall treatment approach.

[0311] In internal radiation therapy, radioactive substances can be conjugated to one or more monoclonal antibodies. For example, radioactive iodine can be used for thyroid malignancies. In prostate cancer, brachytherapy with high-dose rate (HDR) or low-dose rate (LDR) can be combined with IR.

[0312] According to the present invention, chemotherapy that induces DNA damage includes the administration of chemotherapeutic agents such as, but not limited to, anthracyclines such as daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, mitoxantrone; inhibitors of topoisomerase I such as irinotecan (CPT-11) and topotecan; inhibitors of topoisomerase II including etoposide, teniposide, and talaporoside; platinum-based agents such as carboplatin, cisplatin, and oxaliplatin; and other chemotherapeutic agents such as bleomycin.

[0313] The pharmaceutical composition can be administered to a subject by various mucosal administration modes including oral, rectal, intraocular, intranasal, intrapulmonary, or transdermal delivery, or by topical delivery to other surfaces. Optionally, the composition can be administered by non-mucosal routes including intramuscular, intraocular, subcutaneous, intravenous, intraarterial, intra-articular, intraperitoneal, intrathecal, intraventricular, or parenteral routes.

[0314] The compositions of the present disclosure can include pharmaceutically acceptable carrier substances required to approximate physiological conditions, such as pH adjusters and buffers, tonicity agents, wetting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. In the case of solid compositions, for example, conventional non-toxic pharmaceutically acceptable excipients including pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. can be used.

[0315] The present disclosure also encompasses kits, packages, and multi-container units that include the pharmaceutical compositions, active ingredients, and / or means for administering them described herein, which are used for the prevention and treatment of diseases and other conditions in mammalian subjects.

[0316] The present invention will be further described with reference to the following drawings. These are not intended to limit the scope of the present invention, but rather represent preferred embodiments of aspects of the present invention provided for a more detailed description of the present invention described herein.

[0317] 〔Detailed Description of the Drawings〕 Figure 1: When the DNA double strand is cleaved, the sensor proteins ATM and PARP1 are activated. PARP1 undergoes self-modification with poly(ADP) ribose (PAR) chains and serves as a scaffold for the recruitment of the IKK complex subunit IKKγ, PIASy, and activated ATM. The formation of this nuclear PARP1 signalosome causes post-translational modifications such as IKKγ-SUMOylation by PIASy and phosphorylation by ATM. SUMOylated and phosphorylated IKKγ is transported to the cytoplasm where it is presumably incorporated into the IKK holo-complex. At the same time, ATM translocates to the cytoplasm. After binding to TRAF6, it activates its own ubiquitination by Ubc13-assisted Lys63-linked ubiquitin chains. These ubiquitin chains function as scaffolds for recruiting important signaling components such as TAB2-TAK1, cIAP1, and the IKK complex. Ubiquitin-mediated binding of TAK1 to the cytoplasmic signalosome causes TAK1 autophosphorylation, which subsequently leads to priming IKK phosphorylation by TAK1 and autophosphorylation of the serine residue in the IKKβ T-loop. The convergence of the exported SUMOylated IKKγ and the cytoplasmic ATM-TRAF6-dependent axis is required for the monoubiquitination of IKKγ at position Lys285 and thus for complete IKK activation (Hinz et al., 2010; Stilman et al., 2009). As an additional step, LUBAC-dependent M1-linked ubiquitination of IKKγ has been shown to be essential for the genotoxic NF-κB pathway. Activation of the IKK complex, similar to classical NF-κB activation, results in the degradation of IκBα and the activation of the NF-κB heterodimer p65 / p50.

[0318] Figure 2: U2OS cells were pretreated and incubated with DMSO or MW01. DNA damage was induced by the administration of etoposide. Cells were fixed after 2 hours and nuclei were stained with DAPI. p65 and phospho-H2AX, which are indicators of DNA DSBs, were stained by immunofluorescence. Images were taken under a confocal Zeiss 710 LSM with a 40x objective lens.

[0319] Figure 3: (A) U2OS cells were pretreated with increasing concentrations of MW01 in a 384-well plate in two ways. The cells were then treated with etoposide, fixed, and subjected to IF for p65. Spatial measurements of cytoplasmic and nuclear localization of p65 were used for calculation of the p65 translocation rate. (B) U2OS cells were pretreated with DMSO, MW01, and irradiated with γ-IR. After 90 minutes, the cells were lysed and subjected to SDS-PAGE / WB and EMSA. (C) Cells were pretreated with different concentrations of MW01 as indicated, treated with γ-IR (C) or etoposide (D), and subjected to WB or EMSA analysis, respectively. LDH in (C) represents the loading control.

[0320] Figure 4: (A) U2OS cells were pretreated with increasing concentrations of MW01 in a 384-well plate in three ways. The cells were then treated with TNFα, fixed, and subjected to IF staining for p65. Spatial measurements of cytoplasmic and nuclear signals of p65 were used for calculation of the p65 translocation rate. (B) U2OS cells were pretreated with MW01 at concentrations of 10 or 20 μM and sensitized with TNFα. The cell lysates were subjected to SDS-PAGE / WB analysis. (C) HEK293 cells were pretreated with DMSO or MW01 and then administered etoposide or TNFα. The cell lysates were used for EMSA.

[0321] Figure 5: (A) After U2OS cells were pretreated with MW-01, they were sensitized with IL-1β. The cell lysates were used for SDS-PAGE / WB and EMSA. (B) Cells pretreated with increasing concentrations of MW01 were stimulated with IL-1β. The cell lysates were used for SDS-PAGE / WB and stained with the indicated antibodies.

[0322] Figure 6: (A) U2OS cells were pretreated with DMSO or MW01 and irradiated. Stimulation of the cells with IL-1β served as a positive control for phosphorylation of IκBα and p65. The cells were lysed at the indicated time points and used for WB analysis. (B, C) The same experimental setup as (A). (D) The same experiment as (A - C), but analyzed at different time points and performed using HepG2 cells. PARP1 and LDH serve as loading controls.

[0323] Figure 7: Intracellular accumulation of ATM. (A) Fractionation experiments using isolated nuclear extracts (NE) and cytoplasmic extracts (CE). PARP1 and LDH staining served as loading and fractionation controls. Cells were pretreated with DMSO or MW01 (5 μM) prior to cell harvesting at the indicated times of irradiation. NE and CE were subjected to SDS-PAGE / WB. (B) U2OS cells were seeded on cover glasses 2 days prior to treatment. Cells were pretreated with solvent DMSO only or with MW01, then irradiated, fixed, and then immunostained.

[0324] Figure 8: (A) HepG2 cells were pretreated with DMSO or MW01 and irradiated. Nuclear cell extracts (NE) were used for PIASy immunoprecipitation (IP). Western blot membranes were incubated with the indicated antibodies. (B) Experiment as shown in (A) using IKKγ IP. (C) Experiment as shown in (B) but performed in HEK293 cells. (D) Experiment was performed as shown in (A) but repeated in MEF cells.

[0325] Figure 9: HepG2 cells were preincubated with DMSO, MW01 (5 μM), or the ATM inhibitor Ku55933 (10 μM) and irradiated. After 60 minutes, cells were harvested and processed. Immunochemical staining of Western blotting membranes was performed using the indicated antibodies.

[0326] Figure 10: (A) MEF cells were pretreated with the indicated substances, irradiated, and cell lysates were used for poly(ADP) ribose probing using specific antibodies. (B) Experiment was performed as described for (A) but using U2OS cells.

[0327] Figure 11: (A) HEK293 cells were pre-incubated with DMSO, MW01 or the ATM inhibitor KU55933 and irradiated. The lysates were subjected to SDS-PAGE / WB analysis. The specific IKKγ S85 band (lower band) was identified by induction after irradiation and sensitivity to ATMi and γ-phosphatase (γ-PP) treatment. Asterisks indicate non-specific bands that are neither inducible nor ATMi-sensitive. (B) Experiments were performed using MEF cells as shown in (A). (C) HEK293 cells were pretreated with DMSO or MW01, irradiated and lysed. Using the lysates, IKKγ was immunoprecipitated with an IKKγ antibody.

[0328] Figure 12: (A) Molecular structures of MW01 and the derivatives tested. (B) Systematic nomenclature of the ring systems in the MW01 molecular structure (Markgraf et al.; 2005). (C) Molecular structures of MW01 and the derivatives D01 - D18 tested. (D) Variations of the preferred ring C structures of Formulas I - VII and the preferred ring B structures of Formulas I - VII.

[0329] Figure 13: (A) Prior to etoposide treatment, U2OS cells were pretreated with DMSO, MW01 or its derivatives MW01A1 - MW01C5 at a concentration of 10 μM for 2 hours. After incubation with etoposide, the cells were harvested, lysed and subjected to SDS-PAGE / WB analysis. The phospho-S536 p65 signal intensity and the p65 signal intensity were detected using a CCD camera and the band intensities were used for densitometry analysis. The DMSO and etoposide treatment controls were set to "1". Four independent experiments were performed and statistical outliers were identified and removed using the Grubb test. Deviations are shown as the standard error of the mean (SEM). (B) Prior to etoposide treatment, NF-κB / 293 / GFP-luc cells were pretreated with DMSO, MW01 or its derivatives MW01D01 - MW01D18 at a concentration of 10 μM for 1.5 hours. After 4.5 hours of incubation, NF-κB-dependent luciferase expression was measured by detection of chemiluminescence. The controls treated with DMSO and etoposide were set to 1. Twelve independent experiments were performed.

[0330] Figure 14: (A) U2OS cells were pretreated with different concentrations of olaparib before co-treatment with etoposide. After 90 minutes, complete cell lysis was performed, and the clarified lysates were used for WB analysis using the indicated antibodies. (B) U2OS cells were pretreated with DMSO, olaparib, or the ATM inhibitor Ku55933 and co-treated with etoposide. After 45 minutes, the cells were harvested and subjected to SDS-PAGE / WB. The membranes were stained using the indicated antibodies. To control the functionality of olaparib in the inhibition of PAR strand formation, the experiment in (B) was performed simultaneously with the experiments in (A) and (E). (C) Densitometry analysis of (A) and two additional independent experiments performed as shown in (A). (D) U2OS cells were pretreated with the substances indicated before irradiation. After 90 minutes, mRNA was isolated, transcribed into cDNA, and used for expression analysis of the indicated genes using quantitative real-time PCR (qRT-PCR). (E) HepG2 cells were pretreated with the PARP inhibitors olaparib, 3-AB, and EB-47 before irradiation. Cells were harvested at 15 minutes and 90 minutes and subjected to SDS-PAGE / WB. The membranes were stained using the indicated antibodies. Error bars are equal to SEM.

[0331] Figure 15: (A) (B) U2OS cells were pretreated with DMSO or MW01 before irradiation. After 8 hours, mRNA was isolated and reverse transcribed into cDNA. The resulting cDNA was used to perform qRT-PCR using gene-specific exon-exon primers for anti-apoptosis mRNA (A) and pro-apoptosis genes (B).

[0332] Figure 16: (A) U2OS cells were pretreated with DMSO or MW01 and irradiated. Cells were lysed 8 hours after γIR and used for Western blotting and immunochemical staining. (B) HEK293 cells were pretreated with DMSO or MW01 and irradiated. After 48 hours, the cells were fixed and stained with crystal violet. The dissolved crystal violet was used for absorbance measurement with a visible light spectrophotometer at a wavelength of 595 nm. (C) HEK293 cells were pretreated with DMSO or MW01 and irradiated. The viable cell rate within the population was calculated by exclusion of annexin V- and propidium iodide-positive cells by flow cytometry. (D) MEF cells were pretreated with DMSO or MW01 and irradiated. At 24 hours and 48 hours after γ-IR, the cells were used for annexin V staining. The ratio of the annexin V-positive cell population was analyzed using a flow cytometer. (E) The same as shown in (D) but using HT1080 for the experiment, and the cells were measured by flow cytometry. (D) MEF cells were pretreated with DMSO or MW01 and irradiated. At 24 hours and 48 hours after γ-IR, the cells were used for annexin V staining. (E) The same as shown in (D) but using HT1080 cells for the experiment, and the HT1080 cells were treated 8 hours after irradiation. Statistical significance was calculated using Student's t-test.

[0333] Figure 17: (A) U2OS cells grown on glass slides were incubated with DMSO or MW01 (5 μM) for 30 minutes. The cells were then γ-irradiated (5 Gy) or sham-irradiated (sham IR). After 5 hours, the cells were fixed and subjected to immunofluorescence staining. γH2AX foci indicating DNA damage and nuclei (n ≥ 480 nuclei per condition) were counted for calculation of the average number of foci per nucleus. Significance was calculated using Student's t-test.

Example

[0334] The present invention will be further described by the following examples. These are not intended to limit the scope of the present invention, but represent preferred embodiments of the aspects of the present invention provided for a more detailed description of the present invention described herein. 〔Method used in the examples〕

[0335] Isolation of RNA For RNA isolation, cells were washed with ice-cold PBS. Then RNA was isolated according to the manufacturer's instructions (Qiagen, RNeasy RNA isolation KIT). The integrity of the isolated RNA was ensured by measuring the ratio of 28s and 18s ribosomal RNA on a bioanalyzer using an RNA test chip (Agilent RNA 6000 Nano Kit) according to the manufacturer's instructions.

[0336] Determination of nucleic acid concentration The DNA / RNA concentration was measured at OD260 using a UV spectrophotometer. Contamination with proteins or chemicals was confirmed by measuring the OD260 / 280 ratio and the OD260 / 230 ratio. Further analysis was performed on samples with an OD260 / 280 ratio of approximately 2.

[0337] Reverse transcriptase PCR and quantitative real-time PCR (qRT-PCR) To generate complementary DNA (cDNA), 500 - 1000 ng of total RNA was transcribed into cDNA using the iScript cDNA synthesis kit (Promega) according to the manufacturer's instructions. To quantify specific mRNA (messenger RNA) species in the samples, RNA was isolated, quantified, and the mRNA was transcribed into cDNA. The amount of mRNA transcripts of a specific gene in the samples was quantified by using gene-specific primers and a C-1000 thermal cycler (Biorad). The expression of the target gene was normalized to two or three reference genes (HRPT1, RPL_13a, and B2M) using CFX manager software. The induction rate of mRNA was calculated by the comparative Ct method ΔΔ-Ct method relative to the untreated sample level.

[0338] Cell culture All cell lines were cultured in medium supplemented with 10% FCS and penicillin / streptomycin (100 U / mL and 100 μg / mL) under conditions of 95% relative humidity and 5% CO2 atmosphere. U2OS cells and HEK293 cells were cultured in DMEM, mouse embryonic fibroblasts were cultured in DMEM Glutamax, and HepG2 cells were cultured in RPMI 1640 medium (all obtained from Gibco). For subculture, cells were washed with PBS, trypsinized at 37 °C with trypsin / EDTA solution until the cells detached from the plate, and then suspended in the corresponding medium. The splitting ratio was between 1:3 and 1:5 (U2OS, HepG2) and between 1:10 and 1:15 (MEF and HEK293). For cryopreservation in liquid nitrogen, cells were trypsinized at 37 °C, suspended in medium, and pelleted by centrifugation at 320×g for 5 minutes. Then, the cells were resuspended in cryopreservation medium (the corresponding medium supplemented with 20% FCS, 10% DMSO, and penicillin / streptomycin), and frozen in a freezing box containing isopropanol in an -80 °C freezer. The next day, the cells were transferred to liquid nitrogen. Thawing of the cells was performed at 37 °C in a thermostatic bath. A portion of the still-frozen cells was added dropwise with a pipette into pre-warmed medium at 37 °C and centrifuged at 300×g for 5 minutes. Finally, the cells were resuspended in fresh complete medium.

[0339] For activation of the classical NF-κB pathway, cells were treated with recombinant human TNFα (10 ng / mL) or IL-1β (10 ng / mL) at 37 °C for 20 - 30 minutes. Genotoxic stress was applied by ionizing irradiation of the cells with a Cs137 source (OB29 Irradiator, STS Braunshweig) or by inhibition of topoisomerase II enzyme by administration of etoposide at a concentration of 20 - 50 μM for 2 hours.

[0340] Immunofluorescence staining and confocal microscopy For immunofluorescence staining, 0.95×10 5Cells were seeded onto 6-well plates with autoclaved cover glasses. The cell confluence was adjusted at the start of the experiment (2 - 3 days after seeding). After the experiment, the cells were washed with PBS and fixed at RT for 10 min using 4% PFA / double-distilled water (ddH2O). After two additional washing steps, the cells were incubated with a solution containing 0.12% glycine / 0.2% saponin in PBS for 10 min and then blocked for 1 h using a solution containing 10% FCS / 0.2% saponin in PBS. Primary antibody incubation was performed overnight at 4°C (diluted 1:500 with 0.2% saponin in PBS). The next day, the cover glasses were washed 5 times with a solution containing 0.2% saponin in PBS. The fluorophore-conjugated secondary antibody (diluted 1:1000 with 0.2% saponin in PBS) was incubated at room temperature (RT) for 1 h (1 h). Nuclear staining was performed for 5 min using 0.2 mg / mL DAPI in PBS or by directly mounting DAPI / Mowiol. Finally, the cover glasses were washed 5 times with 0.2% saponin in PBS and then 2 times with ddH2O. Confocal microscopy was performed using a Zeiss 710 LSM equipped with a 40x or 63x objective lens.

[0341] Crystal violet staining For crystal violet staining, the 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, the cells were stained with 0.1% crystal violet at room temperature for 20 min. Then, the cells were rewashed 3 times with PBS and air-dried. The cells were incubated with 10% acetic acid while shaking. Next, 0.25 mL of the stain was diluted 1:4 in ddH2O, and the absorbance at 595 nm was measured against 10% acetic acid as a blank using a spectrophotometer.

[0342] Flow cytometry The cells were washed with ice-cold PBS and detached from the culture dish using trypsin / EDTA solution. The detached cells were centrifuged at 300×g for 5 minutes. Detection of early apoptotic cells was performed by staining with annexin V-FITC antibody according to the manufacturer's instructions (eBioscience Annexin V-FITC apoptosis detection kit). Necrotic cells and late apoptotic cells were stained by addition of propidium iodide (final concentration 1 μg / mL) before measurement.

[0343] Cell harvesting The target tissue culture plate was washed with ice-cold PBS. Cells were scraped in PBS using a cell scraper and the cell suspension was transferred to a 1.5 mL reaction tube. The cells were pelleted by centrifugation at 20,000×g for 15 seconds at 4°C. The supernatant was discarded and the cells were either snap-frozen or directly lysed.

[0344] Whole cell lysis The cell pellet was resuspended on ice in 3 volumes of Baeuerle lysis buffer and lysed for 20 minutes with gentle shaking at 4°C. The sample was centrifuged at 20,000×g for 10 minutes at 4°C and the supernatant corresponding to the total cell protein extract was transferred to a new 1.5 mL reaction tube.

[0345] Intracellular fractionation For preparation of nuclear and cytoplasmic fractions, cells were lysed in 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]. The cell lysate was adjusted to a final concentration of 0.2% NP-40, vortexed for 10 seconds and centrifuged. The supernatant corresponding to the cytoplasmic extract (CE) was transferred to a new 1.5 mL reaction tube. The pellet was washed with buffer A, resuspended in buffer C and incubated for 20 minutes at 4°C. After centrifugation for 10 minutes at 14,000 rpm, the supernatant corresponding to the nuclear extract (NE) was transferred into a new reaction cap.

[0346] Determination of protein concentration For measuring the protein concentration of the cell lysate, 1 - 2 μL of the protein extract was mixed with 1 mL of Bradford reagent diluted 1:5 with ddH2O. The absorbance was measured at a wavelength of 595 nm against the lysis buffer reference using a spectrophotometer and compared to the BSA calibration curve.

[0347] Immunoprecipitation After cell lysis, the protein concentration of the sample was measured. For the input control, 40 μg of the lysate was mixed with 6× SDS buffer and denatured by heating at 95 °C for 4 minutes. Approximately 1500 μg of the protein lysate was used for the pull - down, and the sample volume was made equivalent to the lysis buffer. The lysate was pre - clarified for 30 minutes using 30 μL of Sepharose A or Sepharose G beads (depending on the antibody species used for the pull - down) and centrifuged at 1,500 g for 5 minutes. The supernatant was transferred to a new reaction tube. The primary antibody (2 - 2.5 μg) was added to the clarified lysate, and immunoprecipitation was carried out overnight with rotation at 4 °C. The next day, 30 μL of Sepharose beads per sample were used for antibody immobilization. After 4 washes in the IP, the precipitated proteins were eluted by mixing with 3× SDS buffer and heating at 95 °C for 4 minutes.

[0348] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) For the preparation of cell lysates for SDS - PAGE, 20 - 40 μg of the protein lysate was mixed with 6× reaction buffer and heated at 95 °C for 4 minutes. After boiling, the samples were loaded onto a polyacrylamide gel. A gel consisting of a separating gel and a stacking gel was poured. The acrylamide concentration in the separating gel varied from experiment to experiment and depending on the desired separation between a certain molecular weight range, but generally was in the range of 8% - 12%.

[0349] · Stacking gel Tris - HCl, pH 6.8 125 mM Acrylamide 5% SDS 0.1% APS (ammonium persulfate) 0.1% TEMED 0.1% · Gel for separation Tris-HCl, pH 8.8 375 mM Acrylamide 8 - 12% SDS 0.1% APS 0.075% TEMED 0.05%

[0350] After applying the sample, a voltage of 80 V was applied to concentrate the protein at the boundary between the stacking gel and the separation gel. Then, the voltage was increased to 140 V and the protein was separated for about 2 hours.

[0351] Western blotting The protein separated by SDS-PAGE (6.3.5) was immobilized on a methanol-activated PVDF membrane by Western blotting (WB) using a transfer buffer and a semi-dry blotting apparatus. The protein was transferred to the membrane by applying a constant current of 80 mA per 6×9 cm membrane for 90 minutes. In the case of transferring small proteins (<30 kDa), the blotting time was shortened to 30 minutes.

[0352] Immunochemical detection of proteins on membranes After the transfer of the protein onto the PVDF membrane, non-specific binding of the antibody was blocked by incubating the membrane in 5% non-fat dry milk powder in TBST buffer (or 3% BSA in TBST in the case of phosphorylation-specific antibodies) at room temperature for 1 hour. The membrane was incubated overnight at 4°C with the primary antibody solution in 5% non-fat dry milk powder in TBST diluted 1:1000 or 3% BSA in TBDT (in the case of phosphorylation-specific antibodies). The next day, the membrane was washed 3 times with TBST for 5 minutes each. Next, the membrane was incubated for 1 hour with the HRP-conjugated secondary antibody (1:10000) against the Fc portion of the corresponding primary antibody used. After washing 3 times with TBST and 1 time with PBS, each for 5 minutes, chemiluminescent photon emission was detected using a CCD camera system (Fusion Solo). Enhanced chemiluminescence (ECL) solution (Millipore) was used as the HRP substrate.

[0353] The membrane was stripped using Restore PLUS WB buffer (Thermo Scientific) at room temperature for 35 minutes, enabling subsequent probing with multiple antibodies. After thorough washing with TBST, the membrane was re-blocked with 5% non-fat dry milk in TBST for 1 hour and incubated overnight with the following primary antibody.

[0354] Preparation of H2K / NF-κB oligonucleotides Oligonucleotides were ordered as HPLC-purified BamHI ends. For annealing, 5 μg of each strand was incubated at 90 °C for 10 minutes in 50 μL of annealing buffer to a final concentration of 200 ng / μL. The hybridized oligonucleotides were cooled overnight in a thermal block and then stored at -20 °C. The annealing of oligonucleotides was analyzed on a 12% polyacrylamide gel by comparing 1 μg of hybridized oligonucleotides with 1 μg of single-stranded oligonucleotides.

[0355] Radioactive labeling and purification of NF-κB oligonucleotides For the radiolabeling of the H2K / NF-κB probe, the mixture was incubated at 25 °C for 15 minutes according to the reaction recipe. Purification of the radiolabeled NF-κB probe was performed using the QIAquick Nucleotide Removal Kit (Qiagen) according to the manufacturer's instructions. The radiolabeling was measured using a scintillation counter. The radiolabeled probe was stored at -20 °C.

[0356] · Labeling recipe: H2O 10.2 μL DNA-oligonucleotide (200 ng) 1.0 μL 10×Klenow buffer 2.5 μL dCTP, dGTP, dTTP (each 2 mM) 1.8 μL α[32P]-dATP 7.5 μL (3 MBq) DNA Pol I (Klenow fragment, 5 U / μL) 0.2 μL (1 U)

[0357] Electrophoretic mobility shift assay (EMSA) The nuclear lysate or whole cell lysate was 32 incubated with a P-labeled NF-κB DNA consensus sequence. A shift mixture was prepared according to the following shift mixture recipe.

[0358] · Shift mixture for EMSA (H2K / NF-κB) 3 - 5 μg of whole lysate 10.0 μL of 2× shift buffer 1.0 μL of BSA (10 ng / μL) 0.4 μL of DTT (100 mM) 1.0 μL of poly dI-dC (2 μg / μL) 32 45,000 cpm of P-labeled oligonucleotide ddH2O to 20 μL

[0359] After incubating the shift mixture at 37 °C for 30 minutes, the sample was loaded onto an EMSA gel. · EMSA gel recipe (native polyacrylamide gel) 44 mL of ddH2O 6 mL of 10× TBE 10 mL of acrylamide (30%) 450 μL of APS (10%) 45 μL of TEMED

[0360] For electrophoresis, a current of 26 mA was applied for 2 hours. The gel was dried on Whatman paper and incubated at -80 °C overnight, and then the signal was visualized on an autoradiography film (GE Healthcare). All work using radioactive substances was carried out in a monitoring work area suitable for radioactive work.

[0361] 〔Results of the Example〕 Identification of MW01 by high-content screening To identify specific inhibitors of the NF-κB pathway induced by DNA damage, a differential screening assay was designed. For the primary screening of inhibitors of NF-κB signaling by genotoxic stress, a compound library from ChemBioNet and donated compounds from academic chemists were utilized. DNA damage was induced by the application of etoposide. All compounds that inhibited p65 nuclear translocation were adopted for subsequent counter screening. For counter screening, classical NF-κB signaling was induced using the administration of TNFα. All substances that inhibited TNFα-induced classical NF-κB activation were discarded from the list of candidates for DNA damage pathway-specific substances.

[0362] Its IC of 0.46 μM 50 value, its activity change rate of 120% (IC 50 as recorded in the measurement assay), and based on the calculated Hill coefficient of -0.9, compound MW01 was selected for further analysis. MW01 was identified as showing specific selective inhibition of IKK / NF-κB activation induced by genotoxic stress as it inhibited NF-κB activation in response to etoposide stimulation but not after TNFα stimulation.

[0363] Verification of compound MW01 as a DNA damage-specific NF-κB inhibitor MW01 inhibits NF-κB activation by genotoxic stress The small molecule MW01 was identified as the most promising genotoxic stress-specific NF-κB inhibitor by differential discrimination, but further validation with substances from other providers was needed. Therefore, a fresh stock solution of the lead compound MW01 was obtained from the supplier, dissolved in DMSO, and tested for reproducible inhibition of etoposide-induced p65 nuclear translocation using IF staining of p65 (Figure 2). In addition, γH2AX foci as a sensitivity marker for DNA DSBs were visualized. Pretreatment of cells with MW01, as performed in differential screening, inhibited p65 translocation that occurred simultaneously with DNA DSB induction by etoposide administration.

[0364] The measured IC of MW01 50 The curve showed that MW01 inhibited the nuclear translocation of p65 in a concentration-dependent manner by etoposide stimulation (Figure 3A), indicating that a concentration of 5 μM was sufficient for maximal inhibition of p65 nuclear translocation.

[0365] In addition to etoposide treatment, γ-irradiation of cells was used as an alternative method to induce DNA damage in additional experiments. Pretreatment of cells with MW01 inhibited γ-IR-induced NF-κB DNA binding activity and phosphorylation of p65 at S536 (Figure 3B), indicating NF-κB inhibition independent of etoposide. As observed for etoposide treatment, pretreatment with MW01 also resulted in concentration-dependent inhibition of p65 S536 phosphorylation after γ-IR (Figure 3C). In addition to nuclear translocation of p65 and phosphorylation of p65 at S536, etoposide-induced NF-κB DNA binding activity was also inhibited by MW01 pretreatment in a concentration-dependent manner (Figure 3D).

[0366] Overall, the concentration-dependent inhibition of p65 S536 phosphorylation by MW01 (Figure 3C) was in complete agreement with the corresponding results for the observed inhibition of p65 nuclear translocation (Figure 3A).

[0367] Therefore, the analysis of p65 nuclear translocation, p65 S536 phosphorylation, and NF-κB DNA binding activity confirms MW01 as a true inhibitor of genotoxic stress-induced NF-κB activation.

[0368] MW01 does not inhibit classical NF-κB activation The classical NF-κB signaling pathway is initiated by the binding of an extracellular ligand to its cell membrane-bound receptor, which initiates an intracellular signaling cascade that ultimately activates the IKK complex, resulting in the activation of NF-κB. To confirm the specificity of MW01 for genotoxic stress-induced NF-κB activation, MW01 was tested in experiments stimulating NF-κB activation using TNFα.

[0369] MW01 did not affect the p65 nuclear translocation rate at various concentrations (Figure 4A). Moreover, pretreatment of cells with MW01 at concentrations of 10 μM and 20 μM did not interfere with the induced NF-κB DNA binding activity even when stimulated with TNFα (Figure 4C).

[0370] This experiment was also conducted in HEK293 cells, demonstrating the cell line-independent inhibitory effect of MW01.

[0371] Genotoxic stress-induced NF-κB activation and the same activation stimulated by IL-1β share the ubiquitin E3 ligase TRAF6 as an important signaling module. When activated, TRAF6 is auto-modified by K63-linked ubiquitin chains, which serve as a scaffold for the recruitment of TAK1 via the adapter protein TAB2 (Hinz et al.; 2010).

[0372] Therefore, MW01 was analyzed to examine whether it inhibits IL-1β-induced NF-κB activation.

[0373] Pretreatment with both compounds did not inhibit either p65 S536 phosphorylation or NF-κB DNA-binding activity after IL-1β stimulation (Figure 5A). Furthermore, we examined their effects on IKK activation and p65 S536 phosphorylation at high concentrations up to 100 μM of MW01, but it had no effect on the phosphorylation status of IKK or p65 (Figure 5B).

[0374] In summary, MW01 did not inhibit classical NF-κB signaling induced by either TNFα or IL-1β, thus indicating its specificity for the NF-κB pathway induced by DNA damage.

[0375] MW01 inhibits the signal transduction from the nucleus to the cytoplasm, which is required for DNA damage-induced NF-κB activation Inhibition of genotoxic stress-induced NF-κB activation by MW01 occurs downstream of TAK1 activation NF-κB activation induced by TNFα and IL-1β is dependent on a signaling cascade involving the activation of TAK1 and IKK by phosphorylation downstream of the phosphorylation of IκBα and p65. To rule out the possibility that the compound inhibits NF-κB activation downstream of TAK1 in a genotoxic stress-dependent manner, we analyzed the kinetics of signal transduction in cells pretreated with MW01 (Figure 6).

[0376] Cells were pretreated with MW01, γ-irradiated, and harvested at the indicated time points in time-course experiments (Figure 6A). Pretreatment of cells with MW01 resulted in complete inhibition of IκBα phosphorylation at 45 and 60 minutes after γ-IR.

[0377] Similarly, p65 S536 phosphorylation at 45 and 60 minutes after γ-IR was inhibited by MW01. Considering that ΙκΒα phosphorylation is a result of IKK activation, we analyzed the phosphorylation status of IKK in the next step (Figure 6B). Pretreatment of cells with MW01 also abolished IKK phosphorylation 90 minutes after irradiation.

[0378] Kinase TAK1 is located upstream of IKK in this pathway and is similarly activated by phosphorylation. MW01 pretreatment strongly inhibited TAK1 phosphorylation at 45 and 60 minutes after irradiation (Figure 6C). This result also applied to HepG2 cells. TAK1 and p65 phosphorylation disappeared with pretreatment with MW01, but ATM was phosphorylated as a result of γ-IR (Figure 6D). In particular, the repeated inhibition of TAK1 and p65 phosphorylation in HepG2 cells indicates the cell line-independent general inhibitory function of MW01 against NF-κB signaling activated by genotoxic stress.

[0379] Collectively, these results strongly suggest that this inhibited step is upstream of TAK1 activation in the NF-κB signaling cascade initiated by genotoxic stress.

[0380] MW01 inhibits genotoxic stress-induced NF-κB activation by blocking the cytoplasmic accumulation of ATM DNA DSB-activated kinase ATM is mainly present in the nucleus but translocates to the cytoplasm upon DNA damage. Hinz et al. showed that the accumulation of activated ATM in the cytoplasm causes the activation of TRAF6 and subsequent auto-ubiquitination of TRAF6 by K63-linked ubiquitin chains. The ubiquitin chains serve as a scaffold for the recruitment of signaling components including TAK1 and the IKK complex (Hinz et al.; 2010). Thus, this nucleus-cytoplasm signal transduction cascade causes the activation of the IKK complex by a mechanism that requires the cytoplasmic translocation of ATM.

[0381] To analyze the effect of MW01 on cytoplasmic ATM accumulation induced by DNA damage after γ-IR, fractionation experiments were performed. Pretreatment of cells with MW01 had no effect on the detection of phosphorylated ATM in nuclear extracts at 45 and 90 minutes after irradiation (Figure 7A) (compare lanes 2-3 with lanes 5-6). However, the detection of activated ATM species in cytoplasmic extracts was abolished in MW01-pretreated samples (compare lanes 8-9 with lanes 11-12). Analysis of the total ATM amount clearly showed that pretreatment of cells with MW01 inhibited cytoplasmic ATM accumulation at 45 minutes after irradiation (Figure 7A) (compare lane 8 with lane 11). The same results were obtained when U2OS was tested instead of HepG2 cells (data not shown). Furthermore, the effect of MW01 on ATM relocalization by DNA DSB was analyzed by IF (Figure 7B). Irradiation caused the translocation of ATM to the cytoplasm and pericentrosome. In contrast, in samples treated with MW01, ATM was mainly localized in the nucleus. The results of IF imaging strongly supported the results observed in fractionation experiments.

[0382] The results of the fractionation experiments indicate that the target signaling step by MW01 is at the level corresponding to ATM cytoplasmic translocation and probably downstream thereof. Therefore, considering the results observed for TRAF6-stimulated NF-κB activation mediated by ATM (Figure 5B), it is reasonable that TRAF6 activation is not directly targeted but abolished as a result of inhibited cytoplasmic ATM accumulation by both compounds.

[0383] MW01 suppresses NF-κB activation induced by DNA damage downstream of PARP1 and ATM activation The formation of nuclear PARP1-signalosome is suppressed by MW01 The formation of the nuclear IKKγ-PIASy-PARP1-ATM signalosome is important for activating the genotoxic stress-induced NF-κB signaling cascade. The formation of this signalosome requires PARP1, whose enzymatic activity is activated by DNA DSBs to attach poly(ADP)-ribose (PAR) chains to its substrates and itself. These polymers serve as scaffolds for the recruitment of the remaining components of the signalosome (Stilman et al.; 2009). The effect of MW01 on signalosome formation was analyzed by interaction studies using co-immunoprecipitation (Co-IP). Immunoprecipitation of PIASy resulted in γ-irradiation-induced Co-IP of phosphorylated ATM-S1981 species, which disappeared after pretreatment with MW01 (Figure 8A). The interaction between PARP1 and IKKγ was analyzed by immunoprecipitation of IKKγ. PARP1 immunoprecipitated IKKγ from both unirradiated and γ-irradiated cell lysates. Importantly, PARP1 did not co-immunoprecipitate with IKKγ after pretreatment with MW01 (Figure 8B). The result that MW01 abolished the Co-IP of IKKγ and PARP-1 was also observed in HEK293 cells (data not shown), indicating that the mode of action is cell type-independent.

[0384] Next, using HEK293 cells, the effect of MW01 treatment on the IKKγ-PIASy interaction was examined. PIASy immunoprecipitation with IKKγ was inducible and dependent on γ-irradiation, but the interaction was abolished when the cells were treated with MW01 (Figure 8C). In addition, mouse MEFs were used to generalize these findings to other species and cell types independent of them (Figure 8D). As seen in HepG2 or HEK293 cells, pretreatment with MW01 abolished the interaction of IKKγ with PARP1, p-ATM S1981, or PIASy, while such interactions were observed for DMSO-pretreated samples. The species-independent inhibition of signalosome formation by MW01 indicates that the mode of action is based on a general and conserved mechanism.

[0385] MW01 does not inhibit the enzymatic activity of ATM Activation of the cellular DDR in response to DNA DSBs strongly depends on the activation of the serine kinase ATM. Activated ATM can phosphorylate a large number of substrates in mammalian cells and can control cell cycle arrest, DNA repair, or apoptosis (Shiloh & Ziv; 2013). Similarly, it is an essential component of the genotoxic stress-mediated NF-κB signaling pathway (Hinz et al.; 2010).

[0386] Therefore, the enzymatic activity of ATM was analyzed in cells pretreated with MW01 after γ-irradiation. To demonstrate that the phosphorylation of various substrates is indeed ATM-dependent, MW01 was tested in comparison with the ATM inhibitor KU55933. Treatment of cells with KU55933 inhibited ATM autophosphorylation and the phosphorylation of the ATM substrates p53BP1, p53, and KAP1. Although pretreatment of cells with MW01 had a mild effect on the phosphorylation status of p53bp1, no effect was observed on the phosphorylation status of ATM and the other substrates p53 and KAP1 compared to the vehicle and ATMi controls. In addition, as already shown in Figure 2, MW01 pretreatment did not cause damage to the phosphorylation of the ATM substrate histone H2AX after etoposide treatment. Importantly, MW01 treatment significantly decreased the p65 S536 phosphorylation level (Figure 9).

[0387] Collectively, the analysis of ATM autophosphorylation and substrate phosphorylation indicates that the enzymatic activity of ATM is not affected by pretreatment of cells with MW01.

[0388] MW01 does not inhibit the enzymatic activity of PARP1 Stilmann and co-workers have described that the enzymatic activity of PARP1 is essential for PARP1 signalosome formation and is required for the recruitment of another signaling component to initiate the DNA damage-induced NF-κB signaling cascade (Stilmann et al.; 2009). Therefore, the effect of MW01 on PARP1 enzyme function was analyzed. Upon γ-irradiation, intense bands were detected using PAR-chain specific antibodies in samples pretreated with DMSO and MW01 in MEF cells and U2OS cells (Figures 10A - B). In contrast, pretreatment of cells with the PARP inhibitors EB-47, 3-AB (Figure 10A) or the clinically approved drug olaparib (Mullard; 2014) caused inhibition of PAR-chain formation (Figures 10A + B). Thus, it was demonstrated that MW01 does not interfere with the activation of PARP1 enzymatic activity in human and mouse cells.

[0389] MW01 inhibits the formation of essential post-translational modifications (PTMs) of IKKγ after genotoxic stress PARP1 signalosome formation upon irradiation is a prerequisite for DNA damage-induced NF-κB signaling. This is because IKKα has to undergo at least three different PTMs. After DNA DSB, IKKγ is SUMOylated by PIASy within the PARP1 signalosome (Stilmann et al.; 2009). Subsequently, ATM phosphorylates IKKγ at serine 85 (Z.H. Wu et al.; 2006). As a result of the activated signaling cascade, IKKγ is mono-ubiquitinated by cIAP1 (Hinz et al.; 2010).

[0390] To analyze the effect of MW01 on ATM-dependent IKKγ phosphorylation at S85, cells were pretreated with MW01 prior to irradiation. MW01 pretreatment and inhibition of ATM abrogated (abolished) IKKγ phosphorylation at S85 in human (Figure 11A) and mouse cells (Figure 11B).

[0391] MW01 pretreatment abrogated IKKγ S85 phosphorylation as well as ATM inhibition. Prior to SDS-PAGE, treatment of cell lysates with λ protein phosphatase was used as an additional control to show that the bands actually detected were phosphorylation-dependent.

[0392] Next, IKKγ monoubiquitination, which is required for IKK complex activation (Hinz et al.; 2010), was analyzed by immunoprecipitation of IKKγ. The characteristic band of the IKKγ monoubiquitinated species (Hinz et al.; 2010) was observed only in samples pretreated with DMSO and irradiated. Pretreatment of cells with MW01 caused the disappearance of IKKγ monoubiquitination (Figure 11C).

[0393] In conclusion, pretreatment of cells with MW01 inhibited the formation of essential IKKγ post-translational modifications required for DNA damage-induced NF-κB activation.

[0394] Analysis of the structure-activity relationship of MW01 Various derivatives of MW01 were obtained (Figure 12A), and their ability to inhibit genotoxic stress-induced phosphorylation of p65 at S536 was tested. Western blot band densitometry was used to quantify the signal intensity of p65 S536 phosphorylation and the total amount of p65. The signal intensity of p65 S536 phosphorylation was normalized to the signal intensity of total p65, and the percentage was normalized to DMSO / etoposide and compared to the MW01 / etoposide co-treatment samples (Figure 13). MW01C2, MW01C3, and MW01C4 of the MW01 derivatives showed the lowest p-p65 / p65 ratio as a result of strongly inhibiting NF-κB activation after co-treatment with etoposide. Compared to MW01, these compounds are derivatives in which the hydroxyl group is replaced by a small substituent of either fluorine, chlorine, or a methyl group, respectively. Furthermore, MW01C3 and MW01C4 differ in that there are no substituents on aromatic ring system V with respect to the two methoxy groups (Figure 12B). The methoxy groups did not appear to be essential for the inhibitory function of the derivatives but potentially may affect their solubility.

[0395] Compared to the substitution of the hydroxyl group, the presence of a methoxy group at the adjacent carbon atom in ring system I of MW01C1 also resulted in a very potent derivative.

[0396] Thus, by analyzing the structure-activity relationship, the hydroxyl group of MW01 was identified as a position suitable for structural or covalent modifications that can maintain the inhibitory function. Furthermore, this hydroxyl group is also suitable for another reaction such as nucleophilic substitution.

[0397] Compared with MW01, PARP1 inhibitors inhibit NF-κB activation after genotoxic stress in a cell type-dependent manner Damage to DNA is a major threat to cell survival and induces the DNA damage response that regulates cell fate. The protein PARP1, which is sensitive to DNA damage, has been shown in the literature to have multiple functions in the DDR. It is important for the achievement of single-strand break repair, transcriptional control, and involvement in NF-κB-mediated survival-promoting signaling (Gibson & Kraus; 2012). Stilmann et al. (2009) described by loss-of-function studies that the genotoxic stress-activated NF-κB pathway depends on PARP1-dependent PAR chain formation as a scaffold for the recruitment of signalosome components. As a result, the application of PARP1 inhibitors inhibited the signaling cascade. In that study, the authors used the pharmacological PARP1 inhibitors 3-AB and EB-47. 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 lost PAR chain formation and NF-κB binding activity after etoposide administration (Stilmann et al.; 2009).

[0398] To compare MW01 with PARP inhibitors, we examined whether inhibition of PARP1 by olaparib, a clinically approved drug, inhibits signalosome formation and consequently inhibits phosphorylation of p65. U2OS cells were pretreated with increasing concentrations of olaparib ranging from 0.63 μM to 10.0 μM. The cells were then co-treated with etoposide, and the cells were harvested 90 minutes after etoposide administration and analyzed for their phosphorylation status of p65 at S536. No significant decrease in p65 S536 phosphorylation was detected compared to the DMSO / etoposide co-treatment control (Figure 14A). PAR chain formation inhibition mediated by olaparib was confirmed by a control experiment (Figure 14B). In that case, the cells were pretreated with DMSO, 3 μM olaparib, or 10 μM of the ATM inhibitor Ku55933. Cells were harvested 45 minutes after etoposide administration and tested for PAR chain formation. The experiment as shown in Figure 14A was repeated in two biological replicates. The signal intensities of phosphorylated p65 at S536 and total p65 in all three experiments were quantified by densitometry. The results are shown as signal intensity ratios in Figure 14C. Comparison of olaparib / etoposide co-treatment samples with DMSO / etoposide co-treatment samples revealed that olaparib treatment did not affect p65 S536 phosphorylation in U2OS cells, regardless of inhibition of PAR chain formation.

[0399] To examine the effect of PARP1 inhibition by olaparib on general NF-κB activation, qRT-PCR analysis of NF-κB target genes was performed.

[0400] The relative normalized mRNA levels of NFKBIA (encoding IκBα), TNFAIP3 (encoding A20), and CXCL8 (encoding IL-8) were strongly increased in irradiated DMSO samples compared to all non-irradiated samples. Pretreatment of cells with olaparib did not change target gene expression after irradiation compared to DMSO control samples. In contrast, pretreatment of cells with either MW01 or the ATM inhibitor KU55933 resulted in complete inhibition of NFKBIA, TNFAIP3, and CXCL8 mRNA induction by irradiation (Figure 14D).

[0401] Next, it was examined whether p65 was phosphorylated by olaparib in HepG2 cells regardless of PARP1 inhibition (Figure 14E). As expected, pretreatment of HepG2 cells with the PARP inhibitors olaparib, 3-AP, and EB-47 abolished PAR strand formation at 15 minutes after irradiation, as detected in DMSO / IR-treated samples as shown in Figure 14D. Inhibition of PAR strand formation caused a decrease in phosphorylation of p65 at S536 at 90 minutes after irradiation in olaparib-treated and 3-AB-treated samples. Interestingly, p-p65 S536 phosphorylation was detected in EB-47-treated samples despite inhibition of PAR strand formation.

[0402] The results shown in Figure 14 indicate the cell-type specific effects of olaparib-mediated and EB-47-mediated PARP1 inhibition. Consistent with this, in HEK293 cells, inhibition of PARP1-dependent PAR strand formation did not abolish NF-κB DNA binding activity (data not shown, personal communication with Dr. Michael Stilmann).

[0403] In summary, compared to MW01, inhibition of PARP1-dependent PAR strand formation by PARP inhibitors (3-AB, EB-47, and olaparib) inhibits p65 activation after genotoxic stress in a cell-type dependent manner.

[0404] Cell radiosensitization by MW01-mediated inhibition of NF-κB induced by DNA damage Cell apoptosis is a finely tuned mechanism that depends on the processing of apoptosis-antagonizing and -promoting signals, and the anti-apoptotic function of NF-κB has already been described in the literature (Kucharczak et al.; 2003). To demonstrate that inhibition of NF-κB by MW01 results in upstream regulation (upregulation) of apoptosis signaling, induction of the expression of anti-apoptotic gene products was analyzed by quantitative real-time PCR. Pretreatment of U2OS cells with MW01 did not significantly alter the mRNA expression of gene BIRC3 (encoding cIAP2), XIAP, or BCL2L1 (encoding BCL-X L ). γ-IR of the cells resulted in an almost 2-fold induction of BIRX3 mRNA in the irradiated control, while BIRC3 mRNA was downregulated in MW01-pretreated cells. Similar to BIRC3 mRNA, MW01 moderately inhibited the expression of XIAP and completely inhibited the expression of BCL2L1. The strongest effect on anti-apoptotic gene regulation was detected in TNFAIP3 (encoding A20), as shown in Fig. 14D. Pretreatment of the cells with MW01 abrogated the mRNA expression of TNFAIP3 after γ-IR compared to the irradiated control.

[0405] Next, the mRNA expressions of apoptosis-promoting genes BBC3 (encoding PUMA) and PMAIP1 (encoding NOXA) were analyzed. BBC3 mRNA expression was not affected by pretreatment of the cells with MW01. After irradiation of the cells, BBC3 mRNA expression was induced more than 4-fold in the positive control. Pretreatment with MW01 resulted in a slightly decreased expression, which was still induced 3-fold (Fig. 15B).

[0406] The mRNA expression of PMAIP1 was already increased by pretreatment with MW01. The mRNA expression of PMAIP1 in the MW01-pretreated sample was further enhanced after irradiation, while there was no change in the irradiated sample (Fig. 15B).

[0407] To further analyze the effect of MW01 on apoptotic cell death after genotoxic stress, apoptosis markers were investigated. One of those markers is the caspase-3-dependent cleavage of PARP1. Pre-incubation of U2OS cells with MW01 resulted in a slight increase in PARP1 cleavage in resting cells. After irradiation of the cells, a slight increase in PARP1 cleavage was detected in the irradiation control samples. In contrast, pre-incubation with MW01 strongly increased the cleavage of PARP1 (Figure 16A).

[0408] Crystal violet staining was used to analyze whether pre-treatment of the cells with the compound before γ-irradiation affected the cell number. Pre-treatment of the cells with MW01 decreased the cell number compared to the previously DMSO-treated samples. After irradiation of the cells, pre-treatment with MW01 had a significant effect on the further decrease in cell number compared to the DMSO / IR control (Figure 16B).

[0409] To investigate whether the decrease in cell number was caused by a reduction in 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 results of crystal violet staining, pre-treatment with MW01 had some effect on non-irradiated cells. The percentage of viable cells was slightly decreased compared to the DMSO control. However, about 14% lower viable cells were measured in the DMSO samples after irradiation, and 17% lower viable cells were measured in the MW01 samples (Figure 16C).

[0410] The sensitizing effect of MW01 on cells was tested in MEF cells using a low radiation dose of 2 Gy (an amount that cells can repair). Cells were pretreated with MW01, irradiated, and analyzed by annexin V staining at 24 or 48 hours after irradiation. After 24 hours, treatment of cells with MW01 resulted in an increase in annexin V-positive cells of approximately 10%. This is consistent with the results shown in Figure 15, indicating that an increase in apoptosis signaling is observed in cells treated with MW01 without using IR. The annexin V staining of cells was further increased (by approximately 34%) after 2 Gy of γ-irradiation in the MW01-treated samples, but only a slight increase was observed in the DMSO control. When comparing annexin V-positive cells between the MW01-treated samples and the MW01 / γ-IR co-treated samples, a sensitizing effect on apoptosis induced by a low γ-irradiation dose of approximately 12% was observed (Figure 16D).

[0411] In addition, the sensitizing effect of NF-κB inhibition was also tested in HT1080 cells. After pretreatment with DMSO or MW01, cells were irradiated at a dose of 10 Gy and analyzed by annexin V staining. Co-treatment of cells with MW01 resulted in a significant increase in annexin V staining compared to the irradiation control. It was almost twice as high in the early apoptotic cell population (Figure 16E).

[0412] Considering the results of this item, co-treatment of cells with MW01 in combination with the induction of DNA DSBs increased the proportion of apoptotic cells compared to single treatment.

[0413] MW01 inhibits DNA repair mechanisms that are NK-κB-independent U2OS cells were grown on glass slides and incubated with DMSO or MW01 (5 μM) for 30 minutes. Then, the cells were γ-irradiated (5 Gy) or sham-irradiated (sham IR). After 5 hours, the cells were fixed and subjected to immunofluorescence staining. γH2AX foci indicating DNA damage and nuclei (n ≥ 480 nuclei per condition) were counted for the calculation of the average number of foci per nucleus. Significance was calculated using the Student's t-test.

[0414] Treatment of cells with MW01 resulted in a significant increase in γΗ2ΑΧ foci per cell in untreated (non-irradiated) cells, indicating inhibition of other DNA repair mechanisms occurring at steady state in addition to the IKK / NF-κΒ signaling pathway induced by genotoxic stress.

[0415] 〔Examples of the compounds of the present invention〕 In the final stage of the synthesis of the compounds, an acid such as trifluoroacetic acid or acetic acid was used. For example, when trifluoroacetic acid is used for an acid-labile protecting group (e.g., t-Bu group), or when the compound is purified by chromatography using an eluent containing such an acid, in some cases, depending on the post-treatment procedure, for example, depending on the details of the lyophilization method, the compound was obtained partially or completely in the form of a salt of the acid used, such as acetate, formate, trifluoroacetate or hydrochloride. Similarly, starting materials or intermediates containing a basic center, such as basic nitrogen, are obtained or used in the form of a free base or in the form of a salt, such as trifluoroacetate, hydrobromide, sulfate or hydrochloride.

[0416] 〔Abbreviations used〕 Acetonitrile ACN Aqueous aq. tert-Butyl t-Bu Dibenzylideneacetone dba Dichloromethane DCM 4-Dimethylaminopyridine DMAP N,N-Dimethylformamide DMF Dimethyl sulfoxide DMSO Ethanol EtOH Ethyl acetate EtOAc Formic acid FA High performance liquid chromatography HPLC Methanol MeOH N-Methyl-2-pyrrolidone NMP Room temperature 20°C to 25°C RT Saturated sat. Triethanolamine TEA Tetrahydrofuran THF Trifluoroacetic acid TFA

[0417] LCMS (Method 1): Instrument: Agilent Technologies 6220 Accurate Mass TOF LC / MS connected to Agilent Technologies HPLC1200 series; Column: Thermo Accuore RP-MS; Particle size: 2.6 μm; Dimensions: 30 × 2.1 mm; Eluent A: 0.1% FA / H2O; Eluent B: 0.1% FA / ACN; Gradient: 95% A at 0.00 min, 95% A at 0.2 min, 1% A at 1.1 min, stop time 2.5 min, post time 1.3 min; Flow rate: 0.8 mL / min; UV detection: 220 nm, 254 nm, 300 nm.

[0418] LCMS (Method 2): Instrument: Agilent Technologies 6120 Quadrupole LC / MS connected to Agilent Technologies HPLC1290 Infinity series; Column: Thermo Accuore RP-MS; Particle size: 2.6 μm; Dimensions: 30 × 2.1 mm; Eluent A: 0.1% FA / H2O; Eluent B: 0.1% FA / ACN; Gradient: 95% A at 0.00 min, 95% A at 0.2 min, 1% A at 1.1 min, stop time 2.5 min, post time 1.3 min; Flow rate: 0.8 mL / min; UV detection: 220 nm, 254 nm, 300 nm.

[0419] Preparative HPLC (Method 1): Instrument: Consisting of Waters preparative HPLC system: Binary gradient module 2545, UV detector 2489, Waters prep injector, and Waters fraction collector III; Column: Macherey-Nagel VP 250 / 21 Nucleodor 100-7 C18ec; Eluent A: 0.1% TFA / H2O; Eluent B: 0.1% TFA / ACN; Gradient: 85% A at 0.00 min, 85% A at 2.00 min, 15% A at 22.00 min, 15% A at 25.00 min, 0% A at 26.00 min, 0% A at 28.00 min, 85% A at 29.00 min, 85% A at 30.00 min, stop at 30.10 min; Flow rate: 30 mL / min; UV detection: 254 nm.

[0420] Preparative HPLC (Method 2): Instrument: Consisting of Waters preparative HPLC system: Binary gradient module 2545, UV detector 2489, Waters prep injector, and Waters fraction collector III; Column: Macherey-Nagel VP 250 / 21 Nucleodor 100-7 C18ec; Eluent A: 0.1% TFA / H2O; Eluent B: 0.1% TFA / ACN; Gradient: 70% A at 0.00 min, 70% A at 2.00 min, 10% A at 22.00 min, 10% A at 25.00 min, 0% A at 26.00 min, 0% A at 28.00 min, 70% A at 29.00 min; 70% A at 30.00 min, stop at 30.10 min; Flow rate: 30 mL / min; UV detection: 254 nm.

[0421]

Chem.

[0422] The synthesis of β-carboline (e.g., 6-methoxy-9H-pyrido[3,4-b]indole) was carried out as described by Laha et al. (Laha, J.K. et al., J. Org. Chem. (2011) 76, 6421-6425).

[0423] General reaction to 9-benzyl-9H-pyrido[3,4-b]indole derivatives:

[0424]

Chem.

[0425] Example 1: 9-(2-Chlorobenzyl)-6-methoxy-9H-pyrido[3,4-b]indole

[0426]

Chem.

[0427] To a solution of 27.6 mg of 6-methoxy-9H-pyrido[3,4-b]indole (0.14 mmol, 1.0 equiv) in 1 mL of DMF was added 8.91 mg of sodium hydride (0.22 mmol, 1.6 equiv) under nitrogen. The mixture was stirred at RT for 20 minutes, and a solution of 34.3 mg (0.17 mmol, 1.2 equiv) of 2-chlorobenzyl bromide and 1.7 mg of DMAP (0.01 mmol, 0.1 equiv) in 1 mL of DMF was added dropwise. After the addition was complete, the reaction mixture was stirred at 70 °C for 3 hours. After completion of the reaction, the mixture was diluted with water and saturated NaHCO3 solution was added. The aqueous phase was extracted 3 times with DCM. The combined organic phases were dried over magnesium sulfate 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 the eluent. The fractions containing the product were evaporated under reduced pressure to give the title compound as a solid. Yield: 25.9 mg, MS(ES+) [M+H]: m / e = 323

[0428] Example 2: 9-(2-Chlorobenzyl)-7-methoxy-1-methyl-9H-pyrido[3,4-b]indole

[0429]

Chem.

[0430] The title compound was prepared by using the procedure described in Example 1, except that harmine was used instead of 6-methoxy-9H-pyrido[3,4-b]indole. Yield: 19.3 mg, MS(ES+) [M+H]: m / e = 337

[0431] Example 3: 3-Methoxy-4-((6-methoxy-9H-pyrido[3,4-b]indol-9-yl)methyl)benzoic acid

[0432]

Chemical formula

[0433] The title compound was prepared by using the procedure described in Example 1, except that methyl 4-(bromomethyl)-3-methoxybenzoate was used instead of 2-chlorobenzyl bromide. Yield: 12.2 mg, MS(ES+) [M+H]: m / e = 363

[0434] Example 4: 9-Benzyl-6-methoxy-9H-pyrido[3,4-b]indole

[0435]

Chemical formula

[0436] The title compound was prepared by using the procedure described in Example 1, except that 3-methoxybenzyl bromide was used instead of 2-chlorobenzyl bromide. Yield: 22.4 mg, MS(ES+) [M+H]: m / e = 319

[0437] Example 5: 9-Benzyl-6-methoxy-9H-pyrido[3,4-b]indole

[0438]

Chemical formula

[0439] The title compound was prepared by using the procedure described in Example 1, except that benzyl bromide was used instead of 2-chlorobenzyl bromide. Yield: 16.7 mg, MS(ES+) [M+H]: m / e=289

[0440] Example 6: 9-(3,4-Dichlorobenzyl)-6-methoxy-9H-pyrido[3,4-b]indole

[0441]

Chemical formula

[0442] The title compound was prepared by using the procedure described in Example 1, except that 3,4-dichlorobenzyl bromide was used instead of 2-chlorobenzyl bromide. Yield: 19 mg, MS(ES+) [M+H]: m / e=357 / 359 dichloro pattern

[0443] Example 7: 9-((6-Bromobenzo[d][1,3]dioxol-5-yl)methyl)-6-methoxy-9H-pyrido[3,4-b]indole

[0444]

Chemical formula

[0445] The title compound was prepared by using the procedure described in Example 1, except that 5-bromo-6-bromomethyl-1,3-benzodioxole was used instead of 2-chlorobenzyl bromide. Yield: 11.4 mg, MS(ES+) [M+H]: m / e=411 / 413 bromo pattern

[0446] Example 8: 9-(2-Bromo-5-methoxybenzyl)-6-methoxy-9H-pyrido[3,4-b]indole

[0447] [Chemical formula]

[0448] The title compound was prepared by using the procedure described in Example 1, except that 2-bromo-5-methoxybenzyl bromide was used instead of 2-chlorobenzyl bromide. Yield: 3.5 mg, MS(ES+) [M+H]: m / e = 397 / 399 bromo pattern

[0449] General reaction to 9-(2-aroyl)carbazole derivatives:

[0450] [Chemical formula]

[0451] Example 9: 9-(2-benzoyl)carbazole (D08) (CAS: 19264-68-7)

[0452] [Chemical formula]

[0453] The title compound was prepared by adding 23.9 mg of sodium hydride (0.60 mmol, 1.0 equiv) under nitrogen to a cooled (0 °C) solution of 100 mg of carbazole (0.60 mmol, 1.0 equiv) in 5 mL of toluene / DMF (1:1). After stirring at 0 °C for 30 minutes, a solution of 69.4 μL of benzoyl chloride (0.60 mmol, 1.0 equiv) in 200 μL of toluene was added dropwise. The reaction mixture was stirred at RT for 17 hours, 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 the eluent. The fractions containing the product were evaporated under reduced pressure to give the title compound as a solid. Yield: 107 mg, MS(ES+) [M+H]: m / e = 272

[0454] Example 10: (6-Methoxy-9H-pyrido[3,4-b]indol-9-yl)(phenyl)methanone

[0455]

Chem.

[0456] The title compound was prepared by adding 2.02 mg of sodium hydride (0.05 mmol, 1.0 equiv) under nitrogen to a cooled solution (0 °C) of 10 mg of 6-methoxy-9H-pyrido[3,4-b]indole (0.05 mmol, 1.0 equiv) in 5 mL of toluene / DMF (1:1). After stirring at 0 °C for 30 minutes, a solution of 5.9 μL of benzoyl chloride (0.05 mmol, 1.0 equiv) in 17 μL of toluene was added dropwise. The reaction mixture was stirred at RT for 2 hours 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 the eluent. The fractions containing the product were evaporated under reduced pressure to give the title compound. The product was then re-purified by preparative HPLC method 1. The fractions containing the product were evaporated and lyophilized to give a solid. The product was obtained as the trifluoroacetate salt. Yield: 8.1 mg, MS(ES+) [M+H]: m / e = 303

[0457] Example 11: (6-Methoxy-9H-pyrido[3,4-b]indol-9-yl)(4-methoxyphenyl)methanone

[0458]

Chem.

[0459] The title compound was prepared by sequentially adding 41 μL of 4-methoxybenzoyl chloride (0.30 mmol; 3.00 eq), 37.0 mg of DMAP (0.30 mmol; 3.00 eq), and 42 μL of TEA (0.30 mmol; 3.00 eq) to a suspension of 20 mg of 6-methoxy-9H-pyrido[3,4-b]indole (0.10 mmol; 1 eq) in 2.0 mL of ACN. The resulting mixture was stirred at RT for 1 h. The reaction mixture was then diluted with 1 mL of water, filtered, and purified by preparative HPLC method 1. The fractions containing the product were evaporated and lyophilized to give a solid. The product was obtained as the trifluoroacetate salt. Yield: 19.2 mg, MS(ES+) [M+H]: m / e = 333

[0460] Example 12: Benzo[d][1,3]dioxol-5-yl(6-methoxy-9H-pyrido[3,4-b]indol-9-yl)methanone

[0461]

Chem.

[0462] The title compound was prepared by using the procedure described in Example 11, except that piperonoyl chloride was used instead of 4-methoxybenzoyl bromide. Yield: 41.5 mg, MS(ES+) [M+H]: m / e = 347

[0463] Example 13: (2-Bromo-5-methoxyphenyl)(6-methoxy-9H-pyrido[3,4-b]indol-9-yl)methanone

[0464]

Chem.

[0465] The title compound was prepared by using the procedure described in Example 11, except that 2-bromo-5-methoxybenzoyl chloride was used instead of 4-methoxybenzoyl chloride. Yield: 25.2 mg, MS(ES+) [M+H]: m / e = 411 / 413 (bromo pattern)

[0466] Example 14: (2-Chloropyridin-3-yl)(6-methoxy-9H-pyrido[3,4-b]indol-9-yl)methanone

[0467]

Chem.

[0468] The title compound was prepared by using the procedure described in Example 11, except that 2-chloronicotinoyl chloride was used instead of 4-methoxybenzoyl chloride. Yield: 5.4 mg, MS(ES+) [M+H]: m / e = 338

[0469] Example 15: (6-Methoxy-9H-pyrido[3,4-b]indol-9-yl)(naphthalen-1-yl)methanone

[0470]

Chem.

[0471] The title compound was prepared by using the procedure described in Example 11, except that 1-naphthoyl chloride was used instead of 4-methoxybenzoyl chloride. Yield: 17.3 mg, MS(ES+) [M+H]: m / e = 353

[0472] Example 16: 8H-Benzo[c]indolo[3.2.1-ij][1,5]naphthyridin-8-one (CAS 38478-71-6)

[0473]

Chem.

[0474] The title compound was prepared by dissolving 86 mg of 9H-pyrido[3,4-b]indol-1-yl trifluoromethanesulfonate (0.272 mmol, 1.00 equiv), 68.5 mg of 2-methoxycarbonylphenylboronic acid (0.381 mmol, 1.40 equiv), 12.5 mg of Pd2(dba)3 (0.014 mmol, 0.05 equiv), and 7.1 mg of triphenylphosphine (0.027 mmol; 0.10 equiv) in 2.7 mL of toluene and 1.8 mL of EtOH. The solution was purged with nitrogen for 5 minutes. 0.9 mL of saturated aqueous Na2CO3 solution was added to the reaction mixture, and the mixture was purged with nitrogen for 5 minutes. The solution was then stirred at 80 °C for 90 minutes. It was diluted with EtOH, washed twice 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 the eluent. The fractions containing the product were evaporated under reduced pressure to give the title compound as a solid. Yield: 30 mg, MS(ES+) [M+H]: m / e = 271

[0475] Example 17: 5,6,11,12-Tetramethoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

[0476]

Chemical Structure

[0477] The title compound was prepared by dissolving 80 mg of 6,7-dimethoxy-9H-pyrido[3,4-b]indole-1-yl trifluoromethanesulfonate (0.202 mmol; 1.00 equiv), 67.9 mg of 4,5-dimethoxy-2-(methoxycarbonyl)phenylboronic acid (0.283 mmol; 1.40 equiv), 9.2 mg of Pd2(dba)3 (0.010 mmol; 0.05 equiv), and 5.3 mg of triphenylphosphine (0.020 mmol; 0.10 equiv) in 2.3 mL of toluene and 1.8 mL of EtOH. The solution was purged with nitrogen for 5 minutes. 0.6 mL of saturated aqueous sodium carbonate solution was added to the reaction mixture, and the mixture was purged with nitrogen again for 5 minutes. The solution was then stirred at 80 °C for 17 hours. It was diluted with ethyl acetate, washed twice 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 the eluent. The fractions containing the product were evaporated under reduced pressure to give the title compound. This product was further purified by preparative HPLC method 2. The fractions containing the product were evaporated under reduced pressure to give a white solid. The product was obtained as the trifluoroacetate salt. Yield: 0.4 mg, MS(ES+) [M+H]: m / e = 391

[0478] General reaction of methyl pyrazolo[3,4-b]indole derivatives

[0479]

Chem.

[0480] 3-Methylpyrazolo[3,4-b]indole was synthesized according to the literature procedure (Monge, A. et al., Eur. J. Med. Chem. (1991) 26, 179-188).

[0481] Example 18: (3-Bromophenyl)(3-methylpyrazolo[3,4-b]indol-8(1H)-yl)methanone

[0482]

Chem.

[0483] The title compound was prepared by sequentially adding 57.7 μL of 3-bromobenzoyl chloride (0.438 mmol; 3.00 eq), 53.7 mg of 4-dimethylaminopyridine (DMAP) (0.438 mmol; 3.00 eq), and 60.7 μL of TEA (0.438 mmol; 3.00 eq) to a suspension of 25 mg of 3-methylpyrazolo[3,4-b]indole (0.146 mmol; 1.00 eq) in 2.9 mL of ACN. The mixture was stirred at RT for at least 3 h. After completion of the reaction, the reaction mixture was diluted with 1 mL of water, filtered, and purified by preparative HPLC method 1. The fractions containing the product were evaporated and lyophilized to give a solid. The product was obtained as its trifluoroacetate salt. Yield: 2.5 mg, MS(ES+)[M+H]: m / e = 354 / 356 bromine pattern

[0484] Example 19: (4-Methoxyphenyl)(3-methylpyrazolo[3,4-b]indol-8(1H)-yl)methanone

[0485]

Chem.

[0486] The title compound was prepared by using the procedure described in Example 18, except that 4-methoxybenzoyl chloride was used instead of 3-bromobenzoyl chloride, and the reaction scale was adjusted to 100 mg of 3-methylpyrazolo[3,4-b]indole (0.584 mmol; 1.00 eq). Yield: 94.6 mg, MS(ES+) [M+H]: m / e = 323

[0487] Example 20: (3-Methylpyrazolo[3,4-b]indol-8(1H)-yl)(phenyl)methanone

[0488]

Chem.

[0489] The title compound was prepared by using the procedure described in Example 18, except that benzoyl chloride was used instead of 3-bromobenzoyl chloride. Yield: 5.5 mg, MS(ES+) [M+H]: m / e = 276

[0490] Example 21: (3-Methylpyrazolo[3,4-b]indole-1,8-diyl)bis(phenylmethanone)

[0491]

Chem.

[0492] The title compound was obtained as a by-product from the synthesis of Example 20. Yield: 7.8 mg, MS(ES+) [M+H]: m / e = 380

[0493] Example 22: (2-Chloropyridin-3-yl)(3-methylpyrazolo[3,4-b]indol-8(1H)-yl)methanone

[0494]

Chem.

[0495] The title compound was prepared by using the procedure described in Example 18, except that 2-chloronicotinoyl chloride was used instead of 3-bromobenzoyl chloride. Yield: 12.1 mg, MS(ES+) [M+H]: m / e = 311 / 313 chlorine pattern

[0496] Example 23: (2-Bromo-6-chlorophenyl)(3-methylpyrazolo[3,4-b]indol-8(1H)-yl)methanone

[0497]

Chem.

[0498] The title compound was prepared by using the procedure described in Example 18, except that 2-bromo-6-chlorobenzoyl chloride was used instead of 3-bromobenzoyl chloride. Yield: 8.7 mg, MS(ES+) [M+H]: m / e = 388 / 390 isotope pattern

[0499] Example 24: 5-(Pyridin-3-yl)phenanthridin-6(5H)-one

[0500]

Chemical Structure

[0501] The title compound was prepared by charging 150 mg of 6(5H)-phenanthridinone (0.77 mmol; 1.00 equivalent), 111 μL of 3-bromopyridine (1.15 mmol; 1.50 equivalent), 106 mg of potassium carbonate (0.77 mmol; 1.00 equivalent), and 2.7 mg of copper(I) iodide (0.04 mmol; 0.05 equivalent) into a flask. 900 μL of NMP (7.7 mmol; 10.0 equivalents) was added to the solid. The mixture was heated to 180 °C and the reaction was stopped at a starting material:product conversion ratio of 1:1. Then, the reaction mixture was diluted with diethyl ether and extracted with water. The aqueous phase was washed 3 times with diethyl ether. The combined organic phases were washed once with water, dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. During the evaporation of the solvent under reduced pressure, the starting material precipitated as a white solid, which was removed by filtration. The filtrate was evaporated to dryness. The crude product was purified by silica gel chromatography using a gradient of cyclohexane / EtOAc as the eluent. The fractions containing the product were evaporated under reduced pressure to obtain the title compound. Yield: 62 mg, MS(ES+) [M+H]: m / e = 273

[0502] General reaction to β-carbolinone derivatives

[0503]

Chem.

[0504] β-Carbolinone derivatives (e.g., 6-methoxy-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one) were synthesized as described in the literature (La Regina, G. et al., Synthesis (2014), 46, 2093-2097).

[0505] Example 25: 12-Methoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

[0506]

Chem.

[0507] The title compound was prepared by dissolving 78.9 mg of 6-methoxy-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one (0.368 mmol, 1.0 equiv) in 3.8 mL of pyridine. The solution was cooled to 4 °C and purged with nitrogen. 439 μL of trifluoromethanesulfonic anhydride (0.737 mmol, 2.0 equiv) was added dropwise to this solution (over about 30 min). The mixture was stirred at RT for 45 min.

[0508] After completion of the reaction, the mixture was poured into water and the aqueous phase was extracted 3 times with EtOAc. 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.

[0509] 73 mg of 6-methoxy-9H-pyrido[3,4-b]indol-1-yl trifluoromethanesulfonate (0.179 mmol, 1.0 eq), 45 mg of (2-(methoxycarbonyl)phenyl)boronic acid (0.251 mmol, 1.4 eq), 8.2 mg of tris(dibenzylideneacetone)dipalladium(0) (0.009 mmol, 0.05 eq) and 4.7 mg of triphenylphosphine (0.018 mmol, 0.1 eq) were dissolved in 1.8 mL of toluene and 1.2 mL of ethanol. The solution was purged with nitrogen and 0.6 mL of saturated aqueous sodium carbonate was added. The resulting mixture was stirred at 80 °C for 90 minutes. After completion of the reaction, the mixture was diluted with EtOAc, the organic phase was washed twice with water, dried over magnesium sulfate and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel chromatography using DCM / MeOH as eluent and then further purified by HPLC using ACN / water. Yield: 18 mg, MS(ES+) [M+H]: m / e = 301

[0510] Example 26: 11-Methoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

[0511]

Chem.

[0512] The title compound was prepared by using the procedure described in Example 25, except 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. Yield: 5 mg, MS(ES+) [M+H]: m / e = 301

[0513] Example 27: 11,12-Dimethoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

[0514] [Chemistry]

[0515] The title compound was prepared by using the procedure described in Example 25, except 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. Yield: 5 mg, MS(ES+) [M+H]: m / e = 331

[0516] General reaction to 6-methoxy-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one derivatives

[0517] [Chemistry]

[0518] 6-Methoxy-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one was synthesized as described in the literature (La Regina, G. et al., Synthesis (2014), 46, 2093-2097).

[0519] Example 28: (1-Iodo-6-methoxy-9H-pyrido[3,4-b]indol-9-yl)(phenyl)methanone

[0520] [Chemistry]

[0521] The title compound was prepared by dissolving 78.9 mg of 6-methoxy-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one (0.368 mmol, 1.0 equivalent) in 3.8 mL of pyridine. The solution was cooled to 4 °C and purged with nitrogen. 439 μL of triflic anhydride (0.737 mmol, 2.0 equivalents) was added dropwise to this solution (over about 30 minutes). The mixture was stirred at RT for 45 minutes.

[0522] After completion of the reaction, the mixture was poured into water, and the aqueous phase was extracted three times with EtOAc. 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.

[0523] 100 mg of 6-methoxy-9H-pyrido[3,4-b]indol-1-yl trifluoromethanesulfonate (0.289 mmol, 1.0 equiv) and 216 mg of sodium iodide (1.44 mmol, 5.0 equiv) were dissolved in 0.7 mL of acetonitrile under nitrogen. The solution was cooled to 0 °C, and 50 μL of triflic acid (0.578 mmol, 2.0 equiv) was added dropwise (over about 15 min). After the addition was complete, the mixture was stirred at room temperature for 3 h. After completion of the reaction, the mixture was diluted with EtOAc and water and cooled to 0 °C. The aqueous phase was adjusted to pH 10 with NaOH (c = 10 mol / L, <1 mL), and then phase separation was carried out. The organic phase was washed successively with sodium thiosulfate solution (w ≒ 5%), NaOH (c = 1 mol / L), and brine. The organic phase was dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel chromatography using cyclohexane / EtOAc / MeOH as the eluent to obtain 1-iodo-6-methoxy-9H-pyrido[3,4-b]indole. To a suspension of 20 mg of 1-iodo-6-methoxy-9H-pyrido[3,4-b]indole (0.062 mmol, 1.0 equiv) in 1.2 mL of ACN, 21 μL of benzoyl chloride (0.19 mmol; 3.0 equiv), 22.6 mg of DMAP (0.19 mmol; 3.0 equiv), and 26 μL of TEA (0.19 mmol; 3.0 equiv) were added successively. The mixture was stirred at RT for 72 h. Thereafter, the reaction mixture was diluted with 1 mL of water, filtered, and purified by preparative HPLC method 1. Yield: 15 mg, MS(ES+) [M+H]: m / e = 428

[0524] General reaction to 6-methoxy-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one derivatives

[0525]

Chem.

[0526] 6-Methoxy-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one was synthesized as described in the literature (La Regina, G. et al., Synthesis (2014), 46, 2093-2097).

[0527] Example 29: 9-Benzoyl-6-methoxy-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one

[0528]

Chem.

[0529] The title compound was prepared by sequentially adding 33 μL of benzoyl chloride (0.28 mmol; 3.0 eq), 34.2 mg of DMAP (0.28 mmol; 3.0 eq) and 34 μL of TEA (0.28 mmol; 3.0 eq) to a suspension of 20 mg of 6-methoxy-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one (0.093 mmol, 1.0 eq) in 1.2 mL of ACN. The mixture was stirred at RT for 72 h. Then, the reaction mixture was diluted with 1 mL of water and the precipitate was removed by filtration. The filtrate contained the product, which was dried by lyophilization and purified by preparative HPLC method 1. Yield: 9 mg, MS(ES+) [M+H]: m / e = 319

[0530] (1-Methylpyrazolo[3,4-b]indol-8(1H)-yl)(phenyl)methanone derivative general reaction

[0531]

Chem.

[0532] Example 30: (2-Bromophenyl)(5-methoxy-1,3-dimethylpyrazolo[3,4-b]indol-8(1H)-yl)methanone

[0533]

Chem.

[0534] The title compound was prepared by dissolving 100 mg of 1-(2-chloro-5-methoxy-1H-indol-3-yl)ethanone (0.45 mmol, 1.0 eq) and 71 μL of monomethylhydrazine in 1.3 mL of ethanol. The solution was maintained at reflux for 12 hours. After completion of the reaction, the mixture was cooled and the precipitated product was collected by filtration. The solid compound was washed with ethanol to obtain pure 5-methoxy-1,3-dimethyl-1,8-dihydropyrazolo[3,4-b]indole. To a suspension of 20 mg of 5-methoxy-1,3-dimethyl-1,8-dihydropyrazolo[3,4-b]indole (0.093 mmol, 1.0 eq) in 1.9 mL of ACN were sequentially added 36 μL of 2-bromobenzoyl chloride (0.28 mmol, 3.0 eq), 34 mg of DMAP (0.28 mmol, 3.0 eq) and 39 μL of TEA (0.28 mmol, 3.0 eq). The mixture was stirred at RT for 6 hours. Thereafter, the reaction mixture was diluted with water and the precipitated product was collected by filtration. Yield: 17 mg, MS(ES+) [M+H]: m / e = 398 / 400 isotope pattern

[0535] Example 31: (5-Methoxy-1-methylpyrazolo[3,4-b]indol-8(1H)-yl)(phenyl)methanone

[0536]

Chem.

[0537] The title compound was prepared by using the procedure described in Example 30, except that 2-chloro-5-methoxyindole-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. Yield: 15 mg, MS(ES+) [M+H]: m / e = 306

[0538] General reaction to pyrazolo[3,4-b]indole-1,8-diylbis(phenylmethanone) derivatives

[0539]

Chem.

[0540] Example 32: (5-Methoxy-3-methylpyrazolo[3,4-b]indole-1,8-diyl)bis((2-bromophenyl)methanone)

[0541]

Chem.

[0542] The title compound was prepared by dissolving 200 mg of 1-(2-chloro-5-methoxy-1H-indol-3-yl)ethanone (0.90 mmol, 1.0 eq) and 131 μL of hydrazine hydrate in 2.7 mL of ethanol. The solution was maintained at reflux for 8 h. After completion of the reaction, the mixture was cooled and the precipitated product was collected by filtration. The solid compound was washed with ethanol to give pure 5-methoxy-3-methyl-1,8-dihydropyrazolo[3,4-b]indole. To a suspension of 20 mg of 5-methoxy-3-methyl-1,8-dihydropyrazolo[3,4-b]indole (0.099 mmol, 1.0 eq) in 2 mL of ACN were sequentially added 35 μL of 2-bromobenzoyl chloride (0.30 mmol, 3.0 eq), 36 mg of DMAP (0.30 mmol, 3.0 eq) and 41 μL of TEA (0.30 mmol, 3.0 eq). The mixture was stirred at RT for 6 h. Thereafter, the reaction mixture was diluted with water and the precipitated product was collected by filtration and washed with ACN. Yield: 30 mg, MS(ES+) [M+H]: m / e = 566 / 568 / 570 isotope pattern

[0543] Example 33: (5-Methoxy-3-methylpyrazolo[3,4-b]indole-1,8-diyl)bis(phenylmethanone)

[0544]

Chem.

[0545] The title compound was prepared by using the procedure described in Example 32, except that benzoyl chloride was used instead of 2-bromobenzoyl chloride. Yield: 13 mg, MS(ES+) [M+H]: m / e = 410

[0546] Example 34: (5-Bromo-3-methylpyrazolo[3,4-b]indole-1,8-diyl)bis(phenylmethanone)

[0547]

Chem.

[0548] The title compound was prepared by using the procedure described in Example 32, except 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. Yield: 5 mg, MS(ES+) [M+H]: m / e = 458 Isotope pattern

[0549] Example 35: (5-Bromo-3-methylpyrazolo[3,4-b]indole-1,8-diyl)bis((2-bromophenyl)methanone)

[0550]

Chem.

[0551] The title compound was prepared by using the procedure described in Example 32, except 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. Yield: 14 mg, MS(ES+) [M+H]: m / e = 616 Isotope pattern

[0552] Example 36: (5-Bromo-3-methylpyrazolo[3,4-b]indole-1,8-diyl)bis((4-methoxyphenyl)methanone)

[0553]

Chem.

[0554] The title compound was prepared by using the procedure described in Example 32, except 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 4-methoxybenzoyl chloride was used instead of 2-bromobenzoyl chloride. Yield: 18 mg, MS(ES+) [M+H]: m / e = 518 / 520 isotope pattern

[0555] General reaction to 5-benzyl-5H-pyrimido[5,4-b]indole derivative (Example 37)

[0556]

Chem.

[0557] Example 37: 5-Benzyl-8-methoxy-5H-pyrimido[5,4-b]indol-2-amine

[0558]

Chem.

[0559] The title compound was prepared by dropwise adding a solution of 600 mg of 5-methoxy-3-iodo-1H-indole-2-carbaldehyde (2.00 mmol, 1.0 eq) in 4 mL of dry DMF to a solution of 62.2 mg of sodium hydride (60% in mineral oil) (2.59 mmol, 1.3 eq) in 4 mL of dry DMF at 0 °C. The mixture was stirred at 0 °C for 20 min, and a solution of 946 μL of benzyl bromide (7.97 mmol, 4.0 eq) was added. The resulting suspension was stirred at room temperature for 1 h, and an additional 38.3 mg of sodium hydride (60% in mineral oil) (1.59 mmol, 0.8 eq) and 473 μL of benzyl bromide (3.99 mmol, 2.0 eq) were added. The mixture was stirred at room temperature for an additional 12 h. After completion of the reaction, the mixture was quenched with ice 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 using cyclohexane / EtOAc as the eluent to give pure 1-benzyl-3-iodo-5-methoxy-1H-indole-2-carbaldehyde.

[0560] Step B: A suspension of 300 mg of 1-benzyl-3-iodo-5-methoxy-1H-indole-2-carbaldehyde (0.77 mmol, 1.0 eq), 147 mg of guanidine (1.53 mmol, 2.0 eq), 500 mg of cesium carbonate (1.53 mmol, 2.0 eq), 14.6 mg of copper(I) iodide (0.08 mmol, 0.1 eq) and 1,10-phenanthroline in 2.5 mL of dry DMSO was stirred at 90 °C for 48 h under nitrogen. After completion of the reaction, water and EtOAc were added and the mixture was filtered through Celite filter paper. The aqueous phase was extracted twice with EtOAc. 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 using cyclohexane / EtOAc as the eluent. The product was further purified once again by preparative HPLC method 3. Yield: 36 mg, MS(ES+) [M+H]: m / e = 305

[0561] General reaction to 5-benzyl-5H-pyrimido[5,4-b]indole derivative (Example 38)

[0562]

Chem.

[0563] Example 38: 5-Benzyl-2-chloro-8-methoxy-5H-pyrimido[5,4-b]indole

[0564]

Chem.

[0565] The title compound was prepared by dissolving 15 mg of 5-benzyl-8-methoxy-5H-pyrimido[5,4-b]indol-2-amine (Example 43) (0.05 mmol, 1.0 equivalent) in 0.5 mL of 1,2-dichloroethane. The solution was cooled to -10 °C and a solution of 25 mg of antimony trichloride (0.11 mmol, 2.2 equivalents) in 0.1 mL of 1,2-dichloroethane was added. Then, 27.7 μL of tert-butyl nitrile (0.23 mmol, 4.7 equivalents) was added dropwise. The reaction mixture was stirred at -10 °C for 2 hours and then ice water was added. After completion of the reaction, the mixture was extracted 3 times with EtOAc. The combined organic phases were washed once with water, dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. The product was purified using preparative HPLC Method 1. Yield: 16 mg, MS(ES+) [M+H]: m / e = 324

[0566] General reaction to 5-benzyl-5H-pyrimido[5,4-b]indole derivative (Example 39)

[0567]

Chem.

[0568] Example 39: 5-Benzyl-8-methoxy-5H-pyrimido[5,4-b]indol-2-ol

[0569]

Chem.

[0570] The title compound was prepared by dissolving 15 mg of 5-benzyl-8-methoxy-5H-pyrimido[5,4-b]indol-2-amine (Example 43) (0.05 mmol, 1.0 equivalent) in 0.2 mL of acetic acid. The solution was cooled to 10 °C, and a solution of 10 mg of sodium nitrite (0.15 mmol, 3.0 equivalents) dissolved in 68 μL of water was added. The reaction mixture was stirred for 30 minutes, then 1.5 mL of water was added, and the solution was stirred at 90 °C for 4 hours. After completion of the reaction, the solvent was distilled off under reduced pressure, the residue was taken up in water, and extracted three times with EtOAc. The combined organic phases were dried over sodium sulfate, and the solvent was evaporated under reduced pressure. Yield: 12 mg, MS(ES+) [M+H]: m / e = 306

[0571] General reaction to 5-benzyl-5H-pyrimido[5,4-b]indole derivative (Example 40)

[0572]

Chem.

[0573] 4-Chloro-8-methoxy-5H-pyrimido[5,4-b]indole was obtained from a commercial source.

[0574] Example 40: 5-Benzyl-4-chloro-8-methoxy-5H-pyrimido[5,4-b]indole

[0575]

Chem.

[0576] The title compound was prepared by dissolving 60 mg of 4-chloro-8-methoxy-5H-pyrimido[5,4-b]indole (0.26 mmol, 1.0 eq) in 4 mL of DMF. To this solution were added 16 mg of sodium hydride (60% in oil) (0.41 mmol, 1.6 eq) and 3.1 mg of DMAP (0.03 mmol, 0.1 eq). The mixture was stirred at RT for about 20 minutes, and 53 mg (0.31 mmol, 1.2 eq) of benzyl bromide was added dropwise thereto. After the addition was completed, the reaction mixture was stirred at 70 °C for 18 hours. After completion of the reaction, the solvent was distilled off, and the crude product was purified by preparative HPLC method 1. Yield: 11.6 mg, MS(ES+) [M+H]: m / e = 324

[0577] Example 41: 5-Benzyl-8-methoxy-5H-pyrimido[5,4-b]indol-4-ol

[0578]

Chemical Structure

[0579] The title compound was obtained as a by-product from the synthesis of Example 40. Yield: 14.8 mg, MS(ES+) [M+H]: m / e = 306

[0580] General reaction to phenyl(5H-pyrimido[5,4-b]indol-5-yl)methanone derivatives

[0581]

Chemical Structure

[0582] Example 42: (4-Chloro-8-methoxy-5H-pyrimido[5,4-b]indol-5-yl)(phenyl)methanone

[0583]

Chemical Structure

[0584] The title compound was prepared by sequentially adding 89 μL of benzoyl chloride (0.77 mmol, 3.0 eq), 94 mg of DMAP (0.77 mmol, 3.0 eq) and 107 μL of TEA (0.7 mmol, 3.0 eq) to a suspension of 60 mg of 4-chloro-8-methoxy-5H-pyrimido[5,4-b]indole (0.26 mmol, 1.0 eq) in 5 mL of ACN. The mixture was stirred at RT for 18 h, and an additional 89 μL of benzoyl chloride (0.77 mmol, 3.0 eq) and 107 μL of TEA (0.7 mmol, 3.0 eq) were added. The reaction mixture was then diluted with water and the precipitate was removed. The filtrate was dried in vacuo and the crude product was purified by silica gel chromatography using cyclohexane / EtOAc as eluent. Yield: 14.7 mg, MS(ES+) [M+H]: m / e = 338

[0585] General reaction to 8H-dibenzo[b,f]pyrimido[4,5,6-hi]indolizin-8-one derivatives

[0586]

Chemical formula

[0587] Example 43: 5,6,12-Trimethoxy-8H-dibenzo[b,f]pyrimido[4,5,6-hi]indolizin-8-one

[0588]

Chemical formula

[0589] The title compound was prepared by dissolving 40 mg of 4-chloro-8-methoxy-5H-pyrimido[5,4-b]indole (0.171 mmol, 1.0 eq) and 88 mg of bromotripyrrolidinophosphonium hexafluorophosphate (0.19 mmol, 1.1 eq) in 1.4 mL of 1,4-dioxane under nitrogen. To this solution, 47 μL of trimethylamine was added and the mixture was stirred at 70 °C for 2 h. Thereafter, 27 mg of 4,5-dimethoxy-2-(methoxycarbonyl)phenylboronic acid (0.18 mmol, 1.05 eq), 6.0 mg of bis(triphenylphosphine)palladium(II) dichloride (0.009 mmol, 0.05 eq), 36 mg of sodium carbonate (0.34 mmol, 2.0 eq) and 0.7 mL of water were added. The mixture was stirred at 70 °C for 18 h to give a suspension. After completion of the reaction, the solid product was removed by filtration and washed with water and MeOH. Yield: 41 mg, MS(ES+) [M+H]: m / e = 362

[0590] Example 44: 12-Methoxy-8H-dibenzo[b,f]pyrimido[4,5,6-hi]indolizin-8-one

[0591]

Chemical Structure

[0592] The title compound was prepared using the procedure described in Example 43, except that 2-methoxycarbonylphenylboronic acid was used instead of 4,5-dimethoxy-2-(methoxycarbonyl)phenylboronic acid. Yield: 47 mg, MS(ES+) [M+H]: m / e = 302

[0593] General Reaction for Example 45 (MW01)

[0594]

Chemical Structure

[0595] Example 45: 12-Hydroxy-6,7-dimethoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

[0596]

Chemical formula

[0597] The title compound was prepared by dissolving 2.00 g of L-5-hydroxytryptophan (9.1 mmol, 1.0 equivalent) and 2.10 g of 2-carboxy-3,4-dimethoxybenzaldehyde (10 mmol, 1.1 equivalents) in 9 mL of glacial acetic acid. While blowing a constant stream of air into the liquid, the liquid was refluxed for 6 hours and then refluxed for an additional 18 hours. After completion of the reaction, the solid product was removed by filtration and washed with water and acetic acid. The crude product was crystallized from DMF. Yield: 1.18 g, MS(ES+) [M+H]: m / e = 347

[0598] Further examples of other compounds of the present invention that can be prepared by using synthetic procedures well known to those skilled in the art and by applying the general procedures described above are as follows:

[0599] Example: 12-(2-(2-Aminoethoxy)ethoxy)-6,7-dimethoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

[0600]

Chemical formula

[0601] Example A1: 1-(4-Chlorophenyl)-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

[0602]

Chemical formula

[0603] Example A2: 1-(2-Chlorophenyl)-6,7-dimethoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

[0604] [Chemical formula]

[0605] Example A3: 6,7-Dimethoxy-1-(4-methoxyphenyl)-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

[0606] [Chemical formula]

[0607] Example A4: Methyl 6,7-dimethoxy-8-oxo-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridine-2-carboxylate

[0608] [Chemical formula]

[0609] Example A5: 8-Oxo-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridine-2-carboxylic acid

[0610] [Chemical formula]

[0611] Example A7: N-(3-Methoxypropyl)-8-oxo-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridine-2-carboxamide

[0612] [Chemical formula]

[0613] Example A8: N-Isopropyl-8-oxo-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridine-2-carboxamide

[0614]

Chem.

[0615] Example B1: 2-(4-Methylpiperazine-1-carbonyl)-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

[0616]

Chem.

[0617] Example B2: 13-((Diethylamino)methyl)-12-hydroxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

[0618]

Chem.

[0619] 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

[0620]

Chem.

[0621] Example B4: 12-Butoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

[0622]

Chem.

[0623] Example B5: 12-Ethoxy-6,7-dimethoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

[0624]

Chemical Structure

[0625] Example B6: 6,7-Dimethoxy-8-oxo-N-pentyl-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridine-2-carboxamide

[0626]

Chemical Structure

[0627] Example B7: 6,7-Dimethoxy-12-propoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

[0628]

Chemical Structure

[0629] Example B8: 6,7-Dimethoxy-2-(4-methylpiperazine-1-carbonyl)-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

[0630]

Chemical Structure

[0631] Example C1: 6,7,11-Trimethoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

[0632]

Chemical Structure

[0633] Example C2: 12-Fluoro-6,7-dimethoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

[0634] [Chemical formula]

[0635] Example C3: 12-Methyl-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

[0636] [Chemical formula]

[0637] Example C4: 12-Chloro-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

[0638] [Chemical formula]

[0639] Example C5: 13-Allyl-12-methoxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one

[0640] [Chemical formula]

[0641] References Baud, V. & Karin, M. Is NF-kappaB a good target for cancer therapy? Hopes and pitfalls. Nat Rev Drug Discov 8, 33-40, (2009). Christian, F., Smith, E. L. & Carmody, R. J. The Regulation of NF-kappaB Subunits by Phosphorylation. Cells 5, (2016). Gibson, B. A. & Kraus, W. L. New insights into the molecular and cellular functions of poly(ADP-ribose) and PARPs. Nat Rev Mol Cell Biol 13, 411-424, (2012). Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 144, 646-674, (2011). Hayden, M. S. & Ghosh, S. Shared principles in NF-kappaB signaling. Cell 132, 344-362, (2008). Hayden, M. S. & Ghosh, S. NF-kappaB, the first quarter-century: remarkable progress and outstanding questions. Genes Dev 26, 203-234, (2012). Hinz, M., Stilmann, M., Arslan, S. C., Khanna, K. K., Dittmar, G. & Scheidereit, C. A cytoplasmic ATM-TRAF6-cIAP1 module links nuclear DNA damage signaling to ubiquitin-mediated NF-kappaB activation. Mol Cell 40, 63-74, (2010). Hinz, M., Arslan, S. C. & Scheidereit, C. It takes two to tango: IkappaBs, the multifunctional partners of NF-kappaB. Immunol Rev 246, 59-76, (2012). Hinz, M. & Scheidereit, C. The IkappaB kinase complex in NF-kappaB regulation and beyond. EMBO Rep 15, 46-61, (2014). Kucharczak, J., Simmons, M. J., Fan, Y. & Gelinas, C. To be, or not to be: NF-kappaB is the answer--role of Rel / NF-kappaB in the regulation of apoptosis. Oncogene 22, 8961-8982, (2003). Lim, K. H., Yang, Y. & Staudt, L. M. Pathogenetic importance and therapeutic implications of NF-kappaB in lymphoid malignancies. Immunol Rev 246, 359-378, (2012). Mullard, A. European regulators approve first PARP inhibitor. Nat Rev Drug Discov 13, 877-877, (2014). Scheidereit, C. (1998) Signal transduction: Docking IkappaB kinases. Nature 395, 225-226 Scheidereit, C. IkappaB kinase complexes: gateways to NF-kappaB activation and transcription. Oncogene 25, 6685-6705, (2006). Shiloh, Y. & Ziv, Y. The ATM protein kinase: regulating the cellular response to genotoxic stress, and more. Nat Rev Mol Cell Biol 14, 197-210, (2013). Stilmann, M., Hinz, M., Arslan, S. C., Zimmer, A., Schreiber, V. & Scheidereit, C. A nuclear poly(ADP-ribose)-dependent signalosome confers DNA damage-induced IkappaB kinase activation. Mol Cell 36, 365-378, (2009). Sun, S. C. The noncanonical NF-kappaB pathway. Immunol Rev 246, 125-140, (2012). Wu, C. J., Conze, D. B., Li, T., Srinivasula, S. M. & Ashwell, J. D. Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-kappaB activation [corrected]. Nat Cell Biol 8, 398-406, (2006). Wu, Z., Wang C., Bai, M., Li, X., Mei, Q., Li, X., Wang, Y., Fu, X., Luo, G., & Han, W. (2015) An LRP16-containing preassembly complex contributes to NF-κB activation induced by DNA double-strand breaks. Nucleic Acids Res. 43(6):3167-79 Zhang, J., Clark, K., Lawrence, T., Peggie, M. W. & Cohen, P. An unexpected twist to the activation of IKKbeta: TAK1 primes IKKbeta for activation by autophosphorylation. Biochem J 461, 531-537, (2014).

Claims

1. Formula II: 【Chemical 1】 〔In the above formula R １ is an O atom; R ５ is H, halogen, C1-C5 alkyl, alkenyl, alkoxy, or amine; R ６ is H, OH, halogen, C1-C5 alkyl, alkoxy, or alkoxyamide; R ７ is H, halogen, C1-C5 alkyl, or alkoxy; R ８ is halogen, C1-C5 alkyl, or alkoxy; where R ５ to at least one of R7 is not H; R ９ is H, halogen, C1-C5 alkyl, or alkoxy; R １０ is H, halogen, C1-C5 alkyl, or alkoxy; R １１ is halogen, C1-C5 alkyl, alkoxy, or carboxyl; R １２ is H, halogen, C1-C5 alkyl, or alkoxy; Alternatively, X １ is C, R ９ and R １０ , R １０ and R １１ , R １１ and R １２ , or R １２ and the C atom at the position of the bond z of the ring C optionally form a 5- or 6-membered aromatic ring structure, which contains 0, 1 or 2 heteroatoms, or form phenyl; X １ and X ３ is an N or C atom; Ring A is a 5- or 6-membered aromatic ring structure, which contains 0, 1 or 2 heteroatoms selected from O and / or N atoms. Here, when ring A is a 5-membered aromatic ring structure, X ３ is an N atom, and when ring A is a 6-membered aromatic ring structure, X ３ is a C atom. The ring structure is optionally substituted with 0 to 3 identical or different substituents selected from H, OH, halogen, C1-C7 alkyl, alkenyl, alkynyl, alkoxy, carbonyl, carboxyl, alkoxycarbonyl, aryl (substituted by halogen, C1-C3 alkyl, or -OCH ３ ), and alkoxyamine); the bond z may or may not be present, and when the bond z is not present: The C atom at the position of the bond z of the ring C may be substituted with a halogen or a C1-C7 alkyl, and X of the ring A ３ may be substituted with H, OH, or a halogen, or when X ３ is a C atom, it may be substituted with H, OH, or a halogen. a compound represented by.

2. Formula II-b: [Chemical Formula 2] 〔In the above formula X1 is C; R1 is O; R5 is H, halogen, C1-C5 alkyl, -OCH3, -OCH2CH3, -OCH2CH2CH3, or -OCH2CH2CH2CH3; R6 is H, OH, halogen, C1-C5 alkyl, -OCH3, -OCH2CH3, -OCH2CH2CH3, or -OCH2CH2CH2CH3; R7 is H, halogen, C1-C5 alkyl, -OCH3, -OCH2CH3, -OCH2CH2CH3, or -OCH2CH2CH2CH3; R8 is halogen, C1-C3 alkyl, or alkoxy; wherein at least one of R5 to R7 is not H; R9 to R12 may be the same or different, and are H or -OCH3; R16 may be the same or different, and are H, -C(O)OCH3, -C(O)OH, or aryl (optionally substituted by halogen, C1-C3 alkyl, or -OCH3); the bond z is present.〕 The compound according to claim 1, represented by.

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