Targeted therapy of cancer by pharmacological activation of mtor

Abstract

The invention relates to the treatment of triple negative breast cancer (TNBC) with an activator of mTOR (Mammalian Target of Rapamycin). It has been found that migration of TNBC cells is significantly inhibited by an activation of mTOR, in particular of mTORC1, so that the early steps of metastasis, i.e. the migration in the microenvironment of the tumour, can be addressed in a targeted manner. The treatment according to the invention is preferably accompanied by chemotherapy or immunotherapy.

Description

Targeted therapy of TNBC through pharmacological activation of mTOR

[0001] Priority is claimed for European patent application No. 24219 968.5.

[0002] The invention relates to the treatment of triple-negative breast cancer (TNBC) with an activator of mTOR (mammalian target of rape formation), specifically mTOR complex 1 (mTORC1). It has been found that the migration of TNBC cells is significantly inhibited by activation of mTOR, particularly mTORC1, thus allowing the early stages of metastasis, i.e., migration within the tumor microenvironment, to be specifically targeted. The treatment according to the invention is preferably administered concurrently with chemotherapy or immunotherapy.

[0003] The invention is defined in the patent claims. The dependent claims define preferred embodiments.

[0004] Triple-negative breast cancer (TNBC) is diagnosed in 15-20% of all breast cancer patients and is characterized by a particularly poor prognosis. A major reason for the frequent and aggressive progression of the disease is its rapid and efficient metastasis, in contrast to other breast cancer subtypes. This is all the more significant because, in most cases, and especially in breast cancer, cancer patients die not from the primary tumor, but from the metastases. The precise processes leading to the efficient spread of TNBC are not yet fully understood in many areas, which hinders the development of targeted therapies (AP Schneider et al., Linacre Quarterly 81, 244-277 (2014); T Ovcaricek et al. in Radiology and Oncology (2011). doi:10.2478 / vl0019-010-0054-4; Center for Cancer Registry Data - Robert Koch Institute. https: / / www.krebsdaten.de / Krebs / DE / Content / Krebsarten / Brustkrebs / brust-krebs_node.html; S. Dawood, Drugs (2010). doi: 10.2165 / 11538150).

[0005] Unlike other subtypes, such as hormone receptor-positive or HER2-positive breast cancer, current treatment for TNBC, according to current guidelines, is primarily based on the administration of chemotherapy. These drugs act non-specifically and can cause significant side effects in healthy tissue.

[0006] There is an urgent need for new, targeted therapies for TNBC that specifically target tumor cells without harming healthy tissue. In particular, approaches that specifically address early metastasis of TNBC and prevent the spread of the disease to other tissues are lacking. Currently, there are no pharmacologically effective cellular targets for the therapeutic treatment of TNBC due to the absence of expression of known oncological targets, such as the estrogen receptor or HER2.

[0007] Therefore, there is currently no efficient targeted therapy available for TNBC.

[0008] Although numerous studies are exploring new targeted therapeutic approaches, such as the use of PARP, angiogenesis and mTOR inhibitors or EGFR-directed treatment, no effective personalized therapy has yet been developed for this type of cancer.

[0009] Only with so-called immune checkpoint inhibitors, in combination with current neoadjuvant chemotherapy, or with PARP inhibitors in later stages of the disease, can the prognosis be improved to some extent in certain cases (ME Robson et al., Ann. Oncol. (2019). doi: 10.1093 / annonc / mdz012; LA Emens et al., Ann. Oncol. (2020). doi: 10.1016 / j.annonc.2020.08.2244; P. Schmid et al., in Journal of Clinical Oncology (2020). doi: 10.1200 / JCO.19.00368; ANJ Tutt et al., N. Engl. J. Med. (2021), doi:10.1056 / nejmoa2105215; A. Bardia et al. al., N. Engl. J. Med. (2021), doi:10.1056 / nejmoa2028485).

[0010] Initial clinical trials are also investigating epigenetic agents such as HDAC inhibitors for the treatment of TNBC. Although these agents are considered to have some therapeutic potential to reduce the high metastatic potential of TNBC by diminishing the high mesenchymal properties of the cancer cells (SY Park et al., Cancers (2019). doi: 10.3390 / cancersl 1070965), current agents target only general cellular epigenetic processes and are not very tumor-specific (Y. Cheng et al., Signal Transduct. Target. Ther. 4, 1-39 (2019)).

[0011] Therefore, current cytotoxic therapy for triple-negative breast cancer is not targeted and tumor-specific.

[0012] There is a desire to develop a treatment with few side effects that is specifically targeted to the tumor and addresses specific pathophysiological changes in the cancer cells. Previous efforts to establish a targeted therapy for triple-negative breast cancer in clinical trials have not shown significant positive results. This is particularly true for approaches aimed at reducing the high metastatic potential of this highly malignant disease.

[0013] It is an object of the invention to improve the treatment of triple negative breast cancer (TNBC), in particular the mesenchymal stem-like or mesenchymal subtype (Lehmann, BD et al., J Clin Invest. 2011 Jul; 12 l(7):2750-67.). Furthermore, the therapy of other tumors, particularly those with high mesenchymal stem cell properties, is to be improved, such as ductal pancreatic carcinoma (PDAC) (especially the "squamous / basal-like" subtype; Bailey, P. et al., Nature. 2016 Mar 3;531(7592):47-52), melanoma (especially the invasive, dedifferentiated phenotype; Tirosh, I. et al., Science. 2016 Apr 8;352(6282): 189-96), and glioblastoma (GBM) (especially the "mesenchymal subtype" according to the TCGA classification; Verhaak, RG et al., Cancer Cell. 2010 Jan 19; 17(1):98-110.), colorectal carcinoma (CRC) (especially the mesenchymal subtype; Guinney J et al., Nat Med. 2015 Nov;21(l 1): 1350-6.), hepatocellular carcinoma (HCC) (especially HCC with high EMT and TGF-beta signaling according to the molecular subtype Hoshida SI; HoshidaY. et al., Cancer Res. 2009 Sep 15;69(18):7385-92.), non-small cell lung cancer (especially basal-like / mesenchymal adenocarcinoma and squamous cell carcinomas); Cancer Genome Atlas Research Network. Nature. 2014 Jul 31;511(7511):543-50), high-grade serous ovarian cancer (HGSOC) (especially the mesenchymal subtype; Tothill RW, et al., Novel molecular subtypes of serous and endometrioid ovarian cancer linked to clinical outcome. Clin Cancer Res. 2008 Aug 15; 14(16):5198-208.), and prostate cancer (especially the neuroendocrine and basal-like / EMT-high subtypes, as well as prostate cancer subtype 1; You S. et al. al., Cancer Res. 2016;76:4948-58.).

[0014] This problem is solved by the subject matter of the patent claims.

[0015] It was surprisingly found that the migration of TNBC cells is significantly inhibited by activation of mTOR, particularly mTORC1, especially under hypoxic conditions. This opens up a new approach for personalized therapy of triple-negative breast cancer, specifically targeting the early stages of metastasis, i.e., migration within the tumor microenvironment.

[0016] Figure 1 shows experimental results of a wound healing assay under hypoxic conditions using a nutrient-poor medium (RPMI medium without glucose with 5% FCS) with TNBC cells. Figure 1A shows the results using the "High Medium" method (H. Rinderknecht et al., Oxygen 2021, 1, 46-61. https: / / doi.org / 10.3390 / oxygenl010006), and Figure 1B shows the results obtained in the hypoxic chamber. The significant inhibition of migration by pharmacological mTORC1 activation via MHY1485 is clearly visible.

[0017] Figure 2 shows a representative microscopic image of a wound healing assay under hypoxic conditions (method "High Medium") with images at t=0 and t=24h, Figure 2A in MDA-MB-231 cells, Figure 2B in MDA-MB-436 cells.

[0018] Figure 3 shows the cell viability of the cells in the migration assay in the hypoxia model with "High Medium", Figure 3A in MDA-MB-231 cells, Figure 3B in MDA-MB-436 cells.

[0019] Figure 4 shows experimental results of a wound healing assay under hypoxic conditions with nutrient-poor medium (RPMI medium without glucose with 5% FCS) using TNBC cells. The results shown are those obtained using the "High Medium" method (H. Rinderknecht et al., Oxygen 2021, 1, 46-61. https: / / doi.org / 10.3390 / oxygenl010006). The significant inhibition of migration by pharmacological mTORC1 activation via 3-BDO is evident.

[0020] Figure 5 shows experimental results of a wound healing assay under hypoxic conditions with nutrient-poor medium (RPMI medium without glucose with 5% FCS) using ductal pancreatic carcinoma cells. The results are presented using the "High Medium" method (H. Rinderknecht et al., Oxygen 2021, 1, 46-61. https: / / doi.org / 10.3390 / oxygenl010006). The marked inhibition of migration by pharmacological mTORC1 activation via 3-BDO is evident.

[0021] Figure 6 shows the low activity of mTOR, especially mTORC 1, in MDA-MB-231 cells (left) compared to MCF-7 cells (right).

[0022] Figure 7 shows the reduction in viability in MDA-MB-231 cells during prolonged incubation under severely nutrient-reduced conditions (glucose-free without the addition of FCS and under hypoxia) as a bar chart.

[0023] Figure 8 shows the reduction of viability in MDA-MB-231 cells upon prolonged incubation using an optimized pharmacologically potent inhibitor of the FKBP1A-mTORC1 protein-protein interaction and an mTORC1 activator acting via this interaction (see claim 15) in nutrient-rich cell culture medium under normoxia.

[0024] Figure 9 shows the bioinformatic inverse correlation of FKBP1A (FK506-binding protein aa) and S6 kinase.

[0025] Figure 10 shows the experimental results of TGF-beta quantification in the cell culture supernatant of MDA-MB-231 cells after incubation with 3-BDO or DMSO as a control. The results of quantification using a TGF-beta-specific ELISA are shown. The reduced secretion of TGF-beta in the 3-BDO-treated cells is clearly visible.

[0026] Figure 11 shows the binding sites predicted by molecular docking, BS I (dark gray), BS II (light gray), and BS III (medium gray), of mTORC 1 activator 3-BDO to FKBP1A. Figure 10A shows an overview of the three binding sites: BS I lies within the rapamycin binding site, BS II lies partially within the rapamycin binding site and interacts with domain C (amino acid residues 80 to 108), and BS III occupies a shallow pocket behind the rapamycin binding site. Figures 10B, 10D, and 10F show the individual binding modes in BS I, BS II, and BS III, respectively, in more detail. Figures 10C and 10E show the binding modes in BS I and BS II, respectively, overlapping with the crystallized binding mode of rapamycin. A homologous domain C has also been described for FKBP38 (FK506-binding protein 38, FKBP8), which is structurally related to FKBP1A and inhibits mTORC1 via an interaction with the FRB domain (X. Bai et al., Science. 2007 Nov 9;318(5852):977-80. doi: 10.1126 / science.1147379. PMID: 17991864). According to the molecular docking studies performed, it can be assumed that 3-BDO binds to the domain C of FKBP38 analogously to FKBP1A as described above. This also applies to other FKBPs that have a homologous domain C.

[0027] Figure 12 shows the binding mode of MHY1485 in FKBP1A predicted by molecular docking. Figure 12A provides an overview, with Figure 12B showing a more detailed representation. MHY1485 interacts extensively with domain C of FKBP1A. Figure 12C shows the overlap of the predicted binding modes of MHY1485 and 3-BDO in BS II, and Figure D shows the overlap of MHY1485 with rapamycin, although this overlap is small. Analogous to 3-BDO, the molecular docking studies suggest that MHY1485 binds to the homologous domain C of FKBP38, as described above for FKBP1A (X. Bai et al., Science. 2007 Nov). 9;318(5852):977-80. doi: 10.1126 / science.l147379. PMID: 17991864). This also applies to other FKBPs that have a homologous domain C.

[0028] Unlike MHY1485, 3-BDO also binds to the PPIase center of FKBP1A, the binding site of rapamycin, and covers deeper regions of domain C.

[0029] The aim of this invention is to develop active substances that address the physiological binding sites of FKBP1A or other members of the FKBP family on the FRB of mTOR as broadly as possible and inhibit binding. Figure 13 shows, in FKBP1A (dark gray, amino acid residues 80-108), the regions of both domain C (white) and adjacent regions that are also bound by individual derivatives (light gray, amino acid residues Arg 19, Tyr27, Phe37-Arg58 and Trp60-Glu62) and additionally functionally support or mediate the disruption of protein binding of FKBP1A or other FKBPs to the FRB of mTORC1.

[0030] Figure 14 shows the disruption of the interaction between mTOR (dark gray) and FKBP1A (gray) by the predicted binding mode of MHY1485 (light gray) in FKBP1A projected onto the crystal structure of the mTOR / FKBP1A complex (PDB ID: 8ERA). An analogous disruption is shown in paragraphs

[0026] and

[0027] Due to the analogous binding of the homologous domain C of FKBP38, this is also to be expected for the mTORC1 / FKBP38 complex (X. Bai et al., Science. 2007 Nov 9;318(5852):977-80. doi: 10.1126 / science.l147379. PMID: 17991864). The same applies to other members of the FKBP family with homologous domain C. In particular, it has been shown for FKBP5 and also FKBP4 that they can form inhibitory interactions with mTORC1 comparable to FKBP1A (März AM, Fabian AK, Kozany C, Bracher A, Hausch F. Large FK506-binding proteins shape the pharmacology of rapamycin. Mol Cell Biol. 2013 Apr;33(7): 1357-67).

[0031] Figure 15 shows how MHY1485 (black), in the same conformation as in Figure 14, binds into a pocket formed by TGFBR1 (Transforming Growth Factor Receptor 7; dark gray) and FKBP1A (light gray) and could thus act as a “molecular glue”, thereby enhancing the interaction of TGFBR1 with FKBP1A, but possibly also other members of the FKBP family mentioned in this report (“protein-protein interaction stabilizer”).

[0032] Figure 16 shows possible molecular derivatives of 3-BDO that are thought to be more likely than 3-BDO to disrupt mTORC1 / FKBPlA or mTORC1 / FKBP38 interactions.

[0033] Figure 17 shows possible molecular derivatives of MHY1485 that are thought to be more likely than MHY1485 to disrupt mTORC1 / FKBPlA or mTORC1 / FKBP38 interactions.

[0034] Figure 18 is a graphical representation of the novel "mode of action" (MDO) disclosed according to the invention.

[0035] Figure 19 shows the reduction of metastasis in the TNBC xenograft zebrafish model after incubation with MHY1485. MDA-MB-231 cells were incubated for three hours with 20 pM MHY1485 or DMSO was incubated as a control and then injected into the perivitelline space. After three days, metastasis was quantified using confocal microscopy.

[0036] Figure 20 shows the low expression of FKBP1A in primary tumors and high expression in metastatic tumor cells of the tumor microenvironment; immunohistological analysis of FKBPIA expression in breast cancer tissue. The gray arrow indicates migrating tumor cells in the extracellular matrix of the tumor with high FKBPIA expression, the black arrows indicate cell clusters of the primary tumor with low FKBPIA expression (Source: Protein Atlas (Uhlen, M. et al. Tissue-based map of the human proteome. 2015. Science, 347(6220), 1260419)).

[0037] Figure 21 shows the reduction in cell viability in 3D-TNBC organoids after incubation with MHY1485. MDA-MB-231 cells were incubated for five days with 10 pM and 20 pM MHY1485 or DMSO as a control. After two days, the medium was changed and the substance was added again. The cells were then lysed, and the cellular ATP level was determined using CellTiter-Glo (Promega). The figure shows the relative luminescence, which is proportional to the relative cellular ATP content.

[0038] Further work has now focused particularly on the in

[0029] Based on the findings presented, active substances were developed which, starting from the binding poses predicted in the docking of the previous MHY1485 derivatives, not only engage in more and stronger interactions with FKBP1A, but also cover a larger area of ​​domain C and thus disrupt the interactions between FKBP1A and mTORC1 more efficiently. Docking of the further MHY1485 derivatives showed that they bind to FKBP1A exactly as intended by the design, and by exploiting specific interactions with FKBP1A, the predicted binding energy was significantly more advantageous compared to the previous derivatives. The biological evaluation of these new derivatives showed that those with higher predicted binding strength also had an effect at lower concentrations. Furthermore, those derivatives that had a similar predicted binding strength to 3-BDO, but covered domain C better, had a greater effect than 3-BDO.Thus, there is a clear correlation between predicted binding strength and coverage of domain C in docking with the biological effect of the derivatives.

[0039] The above-mentioned derivatives of MHY1485 consist of the core shown in Figure 22, where Ri is selected from linear or branched (CO) alkyl, linear or branched (CMO) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, alkoxyalkyl (Ci-io), alkylaminoalkyl (CO), alkyliminoalkyl (CMO), a ring from the list: aromatic and non-aromatic (hetero-)cycles with three to seven ring members with up to two fused cycles, e.g. B. benzene, furan, thiophene, pyrrole, pyrazole, imidazole, pyran, thiopyran, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrrolidine, pyrazolidine, imidazolidine, tetrazole, piperidine, piperazine, morpholine, thiomorpholine, quinoline, isoquinoline, quinazoline, quinoxaline, chromene, isochromene, indole or Indazole containing an akylamino (C1-3), aminoalkyl (C1-3), alkoxy (C1-3), oxyalkyl (C1-3), alkyl (C1-4), alkenyl (C2-4), Alkynyl (C2-4), amino or oxy is bonded to the core; these residues can be unsubstituted or (multiple times) substituted at any position with linear or branched (Ci-e) alkyl, linear or branched (Ci-e) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, rings from the list: aromatic and non-aromatic (hetero-)cycles with three to seven ring members with up to two fused cycles, e.g. B. Furan, thiophene, pyrrole, pyrazole, imidazole, pyrrolidine, pyrazolidine, imidazolidine or tetrazol, halogens, haloalkyl (Ci-e) or hydrophilic groups such as [hydroxyl, sulfhydryl, unbranched and branched (thio)ethers (Ci-e), amino, azido, hydroxyamino, hydrazino, nitro, nitroso, cyano, isocyano, isocyanato, isothiocyanato, thiocyano, fulminato, semicarbazono, carboxylic acids, as well as their sulfur, selenium and tellurium analogues, imide, sulfonic, sulfinic, selenone, phosphonic and phosphinic acids, as well as those with linear or branched (Ci-e) alkyl,linear or branched (Ci-e) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl substituted esters and amides of the aforementioned acids, oxo or imino]; these residues may in turn be unsubstituted in any position or (also multiple times) substituted with linear or branched (Ci-e) alkyl, linear or branched (Ci-e) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, halogens or hydrophilic groups such as [hydroxyl, sulfhydryl, unbranched and branched (thio-)ether (Ci-e), prim, sec. and tert. Amines, azido, hydroxyamino, hydrazino, nitro, nitroso, cyano, isocyano, isocyanato, isothiocyanato, thiocyano, fulminato, carboxylic acids, and their sulfur, selenium and tellurium analogues, imide, sulfonic, sulfinic, selenone, phosphonic and phosphinic acids, and the esters and amides of the aforementioned acids substituted with linear or branched (Ci-e) alkyl, linear or branched (Ci-e) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenylOxo or Imino]. A (4-nitrophenyl)amino residue is preferred, for example. R2 is selected from a ring in the list: aromatic and non-aromatic (hetero-)cycles with three to seven ring members with up to two fused cycles, e.g. B. Benzene, furan, thiophene, pyrrole, pyrazole, imidazole, pyran, thiopyran, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrrolidine, pyrazolidine, imidazolidine, tetrazole, piperidine, piperazine, morpholine, thiomorpholine, quinoline, isoquinoline, quinazoline, quinoxaline, chromene, isochromene, indole, indole, 2-oxa-5-aza-bicyclo[4.3.0]nonane, 2-oxa-5-aza-bicyclo[4.4.0]decane or indazole], which has an akylamino (C1-3), aminoalkyl (C1-3), alkoxy (C1-3), oxyalkyl (C1-3), alkyl (C1.4), alkenyl (C2-4), alkynyl (C2-4), amino, oxy or directly bonded to the core or halogens, haloalkyl (Ci-e) or hydrophilic groups such as [hydroxyl, sulfhydryl, unbranched and branched (thio-)ether (Ci-e), amino, azido, hydroxyamino, hydrazino, nitro, nitroso, cyano, isocyano, isocyanato, isothiocyanato, thiocyano, fulminato, semicarbazono, carboxylic acids, as well as their sulfur, selenium and tellurium analogues, imide, sulfonic, sulfinous, selenone, phosphonic and phosphinic acids, as well as the esters and amides of the aforementioned acids substituted with linear or branched (Ci-e) alkyl, linear or branched (Ci-e) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, Oxo or Imino]; these residues can be unsubstituted or (multiple) substituted in any position with linear or branched (C1-10) alkyl, linear or branched (C1-10) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, alkoxyalkyl(Ci-io), alkylaminoalkyl (Cn. 10), Alkyliminoalkyl (Ci-io), rings from the list: aromatic and non-aromatic (hetero-)cycles with three to seven ring members with up to two fused cycles, e.g. B. Benzene, furan, thiophene, pyrrole, pyrazole, imidazole, pyran, thiopyran, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrrolidine, py-razolidine, imidazolidine, tetrazole, piperidine, piperazine, morpholine, thiomorpholine, quinoline, isoquinoline, quinazoline, quinoxaline, chromene, isochromene, indole, 2-Oxa-5-aza-bicyclo[4.3.0]nonane, 2-oxa-5-aza-bicyclo[4.4.0]decane or indazole containing an akylamino (C1-3), aminoalkyl (C1-3), alkoxy (Cn 3), oxyalkyl (C1-3), alkyl (C1-4), alkenyl (C2-4), alkynyl (C2-4), amino, oxy or directly bonded, halogens, Haloalkyl (Ci-e) or hydrophilic groups such as [Hydroxyl, Sulfhydryl, unbranched and branched (thio-)ether (Ci-e), Amino, Azido, Hydroxyamino, Hydrazino, Nitro, Nitroso, Cyan, Isocyan, Isocyanato, Isothiocyanato, Thiocyano, Fulminato, Semicarbazono, Carboxylic acids,as well as their sulfur, selenium and tellurium analogues, imide, sulfonic, sulfinic, selenone, phosphonic and phosphinic acids, as well as the esters and amides of the aforementioned acids substituted with linear or branched (Ci-e) alkyl, linear or branched (Ci-e) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, oxo or imino; these residues may in turn be unsubstituted at any position or (also multiple times) substituted with linear or branched (C1-10) alkyl, linear or branched (C1-10) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, alkoxyalkyl (Ci-io), alkylaminoalkyl (C1-10), alkylimi-noalkyl (C1-10), rings from the list: aromatic and non-aromatic (Hetero-)cycles with three to seven ring members with up to two fused cycles, e.g., benzene, furan, thiophene, pyrrole, pyrazole, imidazole, pyran, thiopyran, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrrolidine, pyrazolidine, imidazolidine, tetrazole, piperidine, piperazine, morpholine, thiomorpholineQuinoline, isoquinoline, quinazoline, quinoxaline, chromene, isochromene, indole, 2-oxa-5-aza-bicyclo[4.3.0]nonane, 2-oxa-5-aza-bicyclo[4.4.0]decane or indazole, which are bonded to an acylamine (C1-3), aminoalkyl (C1-3), alkoxy (C1-3), oxyalkyl (C1-3), alkyl (C1-4), alkenyl (C2-4), alkynyl (C2-4), amino, oxy or directly bonded to halogens, haloalkyl (Ci-e) or hydrophilic groups such as [hydroxyl, sulfhydryl, unbranched and branched (thio)ether (Ci-e), amino, azido, hydroxyamino, hydrazino, nitro, nitroso, cyano, isocyano, isocyanato, isothiocyanato, thiocyano, fulminato, Semicarbazono, carboxylic acids, and their sulfur, selenium and tellurium analogues, imide, sulfonic, sulfinic, selenone, phosphonic and phosphinic acids, as well as the esters and amides of the aforementioned acids substituted with linear or branched (Ci-e) alkyl, linear or branched (Ci-e) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl,Oxo or imino]; these residues can in turn be unsubstituted or (multiple times) substituted at any position with linear or branched (C1-10) alkyl, linear or branched (C1-10) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, alkoxyalkyl(C1-10), alkylaminoalkyl (C1-10), alkylimi-noalkyl (C1-10), rings from the list: aromatic and non-aromatic (hetero-)cycles with three to seven ring members with up to two fused cycles, e.g. B. Benzene, furan, thiophene, pyrrole, pyrazole, imidazole, pyran, thiopyran, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrrolidine, pyrazolidine, imidazolidine, tetrazole, piperidine, piperazine, morpholine, thiomorpholine, quinoline, isoquinoline, quinazoline, quinoxaline, chromene, isochromene, indole, 2-Oxa-5-aza-bicyclo[4.3.0]nonane, 2-Oxa-5-aza-, bicyclo[4.4.0]decane or indazole, which are bonded to an acylamine (C1-3), aminoalkyl (C1-3), alkoxy (C1-3), oxyalkyl (C1-3), alkyl (C1-4), alkenyl (C2-4), alkynyl (C2-4), amino, oxy or directly, halogens, haloalkyl (CM) or hydrophilic groups such as [hydroxyl, sulfhydryl, unbranched and branched (thio)ethers (CM), amino, azido, hydroxyamino, hydrazino, nitro, nitroso, cyano, isocyano, isocyanato, isothiocyanato, thiocyano, fulminato, semicarbazono, carboxylic acids, as well as their sulfur, selenium and tellurium analogues, imide, sulfonic, sulfinic, selenone, phosphonic and phosphinic acids, as well as those with linear or branched (CM) alkyl, linear or branched (CM) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl substituted esters and amides of the aforementioned acids, oxo or imino] A [3-(2-hydroxyethyl)morpholino] residue is preferred, for example. R3 is either a hydrogen residue (unsubstituted) or is selected from a ring from the list: aromatic and non-aromatic (hetero-)cycles with three to seven ring members with up to two fused cycles, e.g. B. Benzene, furan, thiophene, pyrrole, pyrazole, imidazole, pyran, thiopyran, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrrolidine, pyrazolidine, imidazolidine, tetrazole, piperidine, piperazine, morpholine, thiomorpholine, quinoline, isoquinoline, quinazoline, quinoxaline, chromene, isochromene, indole, indole, 2-oxa-5-aza-bicyclo[4.3.0]nonane, 2-oxa-5-aza-bicyclo[4.4.0]decane or indazole, which is bonded to an acylamine (C1-3), aminoalkyl (C1-3), alkoxy (C1-3), oxyalkyl (C1-3), alkyl (C1-4), alkenyl (C2-4), alkynyl (C2-4), amino, oxy or directly to the core or Halogens, haloalkyl (CM) or hydrophilic groups such as [hydroxyl, sulfhydryl, unbranched and branched (thio-)ether (CM), amino, azido, hydroxyamino, hydrazino, nitro, nitroso, cyan, isocyan, isocyanato,Isothiocyanato, Thiocyano, Fulminato, Semicarbazono, carboxylic acids, as well as their sulfur, selenium and tellurium analogues, imide, sulfonic, sulfinic, selenone, phosphonic and phosphinic acids, as well as the esters and amides of the aforementioned acids substituted with linear or branched (CM) alkyl, linear or branched (CM) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, oxo or imino; These residues can be unsubstituted or (multiple times) substituted in any position with linear or branched (CO) alkyl, linear or branched (CO) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, alkoxyalkyl (Ci-io), alkylaminoalkyl (CMO), alkyliminoalkyl (CO), rings from the list: aromatic and non-aromatic (hetero-)cycles with three to seven ring members with up to two fused cycles, e.g., benzene, furan, thiophene, pyrrole, pyrazole, imidazole, pyran, thiopyran, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrrolidine, pyrazolidine, imidazolidine, tetrazole.Piperidine, piperazine, morpholine, thiomorpholine, quinoline, isoquinoline, quinazoline, quinoxaline, chromene, isochromene, indole, 2-oxa-5-aza-bicyclo[4.3.0]nonane, 2-oxa-5-aza-bicyclo[4.4.0]decane or indazole, which are bonded to an acylamine (C1-3), aminoalkyl (C1-3), alkoxy (C1-3), oxyalkyl (C1-3), alkyl (CM), alkenyl (C2-4), alkynyl (C2-4), amino, oxy or directly bonded to halogens, haloalkyl (Ci-e) or hydrophilic groups such as [hydroxyl, sulfhydryl, unbranched and branched (thio-)ether (CM), amino, azido, hydroxyamino, hydrazino, nitro, nitroso, cyano, isocyano, isocyanato, isothiocyanato, Thiocyano, fulminato, semicarbazono, carboxylic acids, and their sulfur, selenium and tellurium analogues, imide, sulfonic, sulfinic, selenone, phosphonic and phosphinic acids, as well as those with linear or, branched (Ci-e) alkyl, linear or branched (Ci-e) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl substituted esters and amides of the aforementioned acids, oxo or imino]; these residues can in turn be unsubstituted at any position or (also multiple times) substituted with linear or branched (C1-10) alkyl, linear or branched (C1-10) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, alkoxyalkyl(Ci-io), alkylaminoalkyl (C1-10), alkyliminoalkyl (C1-10), rings from the list: aromatic and non-aromatic (hetero-)cycles with three to seven ring members with up to two fused cycles, e.g. B. Benzene, furan, thiophene, pyrrole, pyrazole, imidazole, pyran, thiopyran, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrrolidine, pyrazolidine, imidazolidine, tetrazole, piperidine, piperazine, morpholine, thiomorpholine, quinoline, isoquinoline, quinazoline, quinoxaline, chromene, isochromene, indole, 2-Oxa-5-aza-bicyclo[4.3.0]nonane,2-Oxa-5-aza-bicyclo[4.4.0]decane or indazole], which are bonded to an acylamine (C1-3), aminoalkyl (C1-3), alkoxy (C1-3), oxyalkyl (C1-3), alkyl (C1-4), alkenyl (C2-4), alkynyl (C2-4), amino, oxy or directly, halogens, haloalkyl (Ci-e) or hydrophilic groups such as [hydroxyl, sulfhydryl, unbranched and branched (thio)ether (Ci-e), amino, azido, hydroxyamino, hydrazino, nitro, nitroso, cyano, isocyano, isocyanato, isothiocyanato, thiocyano, fulminato, semicarbazono, carboxylic acids, as well as their sulfur, selenium and tellurium analogues, imide, sulfone, sulfine, selenone, phosphone, and Phosphinic acids, as well as the esters and amides of the aforementioned acids substituted with linear or branched (Ci-e) alkyl, linear or branched (Ci-e) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, oxo or imino]; these residues can in turn be unsubstituted at any position or (also multiple times) substituted with linear or branched (Ci-10) alkyl,linear or branched (C1-10) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, al-koxyalkyl(Ci-io), alkylaminoalkyl (C1-10), alkyliminoalkyl (C1-10), rings from the list: aromatic and non-aromatic (hetero-)cycles with three to seven ring members with up to two fused cycles, e.g. B. Benzene, furan, thiophene, pyrrole, pyrazole, imidazole, pyran, thiopyran, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrrolidine, pyrazolidine, imidazolidine, tetrazole, piperidine, piperazine, morpholine, thiomorpholine, quinoline, isoquinoline, quinazoline, quinoxaline, chromene, isochromene, indole, 2-Oxa-5-aza-bicyclo[4.3.0]nonane, 2-oxa-5-aza-bicyclo[4.4.0]decane or indazole] containing an akylamino (Ci-3), aminoalkyl (C1-3), alkoxy (C1-3), oxyalkyl (C1-3), alkyl (C1.4), alkenyl (C2-4), alkynyl (C2-4), amino, oxy or directly bonded, halogens, haloalkyl (Ci-e) or hydrophilic groups such as [hydroxyl, sulfhydryl, unbranched and branched (thio-)ether (Ci-e), amino, azide,Hydroxyamino, hydrazino, nitro, nitroso, cyano, isocyano, isocyanato, isothiocyanato, thiocyano, fulminato, semicarbazono, carboxylic acids, and their sulfur, selenium, and tellurium analogues, imide, sulfonic, sulfinic, selenone, phosphonic, and phosphinic acids, as well as esters and amides of the aforementioned acids substituted with linear or branched (Ci-e) alkyl, linear or branched (Ci-e) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, oxo or imino]. Preferably, R3 is equal to R2.

[0040] This results, by way of example, without representing the full range of all possible combinations, in the following list of expressly protected substances in claim 14.

[0041] Particularly potent derivatives with a particularly strong anti-tumor effect were obtained from the common core shown in Figure 23, where Y is preferably N or C, most preferably N, and X is preferably C, N, O, P or S, most preferably N, where RI is selected from a ring from the list: [benzene, furan, thiophene, pyrrole, pyrazole, imidazole, pyran, thiopyran, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrrolidine, pyrazolidine, imidazolidine, tetrazole, piperidine, piperazine, morpholine, thiomorpholine, quinoline, isoquinoline, quinazoline, quinoxaline, chromene, isochromene, indole or indazole], which is bonded to the core with an alkyl (C2-4), alkenyl (C2-4) or alkynyl (C2-4) ring;These residues can be unsubstituted or (multiple times) substituted in any position with linear or branched (CI-6) alkyl, linear or branched (CI-6) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, rings from the list: [furan, thiophene, pyrrole, pyrazole, imidazole, pyrrolidine, pyrazolidine, imidazolidine or tetrazole], halogens, haloalkyl (CI-6) or hydrophilic groups such as [hydroxyl, sulfhydryl, unbranched and branched (thio)ether (CI-6), amino, azido, hydroxyamino, hydrazino, nitro, nitroso, cyano, isocyano, isocyanato, isothiocyanates, thiocyano, fulminato, semicarbazono, carboxylic acids, as well as their sulfur, selenium and tellurium analogues, imide, sulfone, sulfine, selenone, Phosphonic and phosphinic acids, as well as esters and amides of the aforementioned acids substituted with linear or branched (CI-6) alkyl, linear or branched (CI-6) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, oxo or imino;These residues can in turn be unsubstituted or (multiple times) substituted in any position with linear or branched (Cl-6) alkyl, linear or branched (CI-6) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, halogens or hydrophilic groups such as [hydroxyl, sulfhydryl, unbranched and branched (thio-)ether (CI-6), prim, sec. and tert. Amines, azido, hydroxyamino, hydrazino, nitro, nitroso, cyano, isocyano, isocyanato, isothiocyanates, thiocyano, fulminato, carboxylic acids, and their sulfur, selenium and tellurium analogues, imide, sulfonic, sulfinic, selenone, phosphonic and phosphinic acids, and esters and amides of the aforementioned acids substituted with linear or branched (Cl-6) alkyl, linear or branched (Cl-6) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, oxo or imino]. R2 is either unsubstituted or (multiple) substituted with linear or branched (Cl-10) alkyl, linear or branched (Cl-10) alkenyl or alkynyl, (C3-7) cycloalkyl or alkenyl, hydrophilic groups such as [hydroxyl, sulfhydryl], unbranched and branched (thio) ethers (Cl-6), amino, azido, hydroxyamino, hydrazino, nitro, nitroso, cyano, isocyano, isocyanato, isothiocyanates, thiocyano, fulminato, semicarbazono, carboxylic acids, as well as their sulfur, selenium and tellurium analogues, imide, sulfonic, sulfinic, selenone, phosphonic and phosphinic acids, as well as those with linear or branched (Cl-6) alkyl, linear or branched (Cl-6) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl substituted esters and amides of the aforementioned acids.]. R3 is either a hydrogen residue (unsubstituted) or is selected from a ring from the list: [Benzene, Furan, Thiophene, Pyrrole, Pyrazole, Imidazole, Pyran, Thiopyran, Pyridine, Pyridazine, Pyrimidine, Pyrazine, triazine, pyrrolidine, pyrazolidine, imidazolidine, tetrazole, piperidine, piperazine, morpholine, thiomorpholine, quinoline, isoquinoline, quinazoline, quinoxaline, chromene, isochromene, indole, indole, 2-0xa-5-aza-bicyclo[4.3.0]nonane, 2-oxa-5-aza-bicyclo[4.4.0]decane or indazole], which is bonded to an acylamine (CI-3), aminoalkyl (CI-3), alkoxy (CI-3), oxyalkyl (CI-3), alkyl (CI-4), alkenyl (C2-4), alkynyl (C2-4), amino, oxy or directly to the core or halogens, haloalkyl (C1-6) or hydrophilic groups such as [hydroxyl, sulfhydryl, unbranched and branched (thio)ether (CI-6), Amino, azido, hydroxyamino, hydrazino, nitro, nitroso, cyan, isocyan, isocyanato, isothiocyanate, thiocyano, fulminato, semicarbazono, carboxylic acids, and their sulfur, selenium and tellurium analogues, imide, sulfonic, sulfinic, selenone, phosphonic and phosphinic acids, as well as those with linear or branched (Cl-6) alkyl, linear or branched (Cl-6) alkenyl or alkynyl,(C3-7) Cycloalkyl or -alkenyl substituted esters and amides of the aforementioned acids, oxo or imino]; These residues can be unsubstituted or (multiple times) substituted at any position with linear or branched (C 1-10) alkyl, linear or branched (Cl -10) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, alkoxyalkyl(Cl-1O), alkylaminoalkyl (Cl-10), alkyliminoalkyl (Cl-10), rings from the list: [benzene, furan, thiophene, pyrrole, pyrazole, imidazole, pyran, thiopyran, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrrolidine, pyrazolidine, imidazolidine, tetrazole, piperidine, piperazine, morpholine, thiomorpholine, quinoline, isoquinoline, quinazoline, quinoxaline, chromene, isochromene, indole, 2-Oxa-5-aza-bicyclo[4.3.0]nonane, 2-Oxa-5-aza-bicyclo[4.4.0]decane or indazole], which are bonded to an acylamine (CI-3), aminoalkyl (CI-3), alkoxy (CI-3), oxyalkyl (CI-3), alkyl (CI-4), alkenyl (C2-4), alkynyl (C2-4), amino, oxy or directly bonded to halogens,Haloalkyl (CI-6) or hydrophilic groups such as [hydroxyl, sulfhydryl, unbranched and branched (thio)ethers (CI-6), amino, azido, hydroxyamino, hydrazino, nitro, nitroso, cyano, isocyano, isocyanato, isothiocyanates, thiocyano, fulminato, semicarbazono, carboxylic acids, and their sulfur, selenium, and tellurium analogues, imide, sulfonic, sulfinous, selenone, phosphonic, and phosphinic acids, as well as esters and amides of the aforementioned acids substituted with linear or branched (CI-6) alkyl, linear or branched (CI-6) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, oxo or imino]; these residues can in turn be unsubstituted or (multiple times) substituted at any position with linear or branched (Cl-10) Alkyl, linear or branched (Cl-10) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, alkoxyalkyl(Cl-1O), alkylaminoalkyl (Cl-10), alkyliminoalkyl (Cl-10), rings from the list: [benzene, furan, thiophene, pyrrole, pyrazole, imidazole, pyran, thiopyran,Pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrrolidine, pyrazolidine, imidazolidine, tetrazole, piperidine, piperazine, morpholine, thiomorpholine, quinoline, isoquinoline, quinazoline, quinoxaline, chromene, isochromene, indole, 2-oxa-5-aza-bicyclo[4.3.0]nonane, 2-oxa-5-aza-bicyclo[4.4.0]decane or indazole], which are bonded to an acylamine (CI-3), aminoalkyl (CI-3), alkoxy (CI-3), oxyalkyl (CI-3), alkyl (CI-4), alkenyl (C2-4), alkynyl (C2-4), amino, oxy or directly bonded to halogens, haloalkyl (CI-6) or hydrophilic groups such as [hydroxyl, sulfhydryl, unbranched and branched (thio)ethers]. (CI -6), Amino, Azido, Hydroxyamino, Hydrazino, Nitro, Nitroso, Cyan, Isocyan, Isocyanato, Isothiocyanates, Thiocyano, Fulminato, Semicarbazono, carboxylic acids, as well as their sulfur, selenium and tellurium analogues, imide, sulfonic, sulfinic, selenone, phosphonic and phosphinic acids, as well as the esters and amides of the aforementioned acids substituted with linear or branched (CI -6) alkyl, linear or branched (CI -6) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, oxo or imino; These residues can in turn be unsubstituted or (multiple times) substituted at any position with linear or branched (C 1-10) alkyl, linear or branched (C 1-10) alkenyl or alkynyl, (C 3-7) cycloalkyl or -alkenyl, alkoxyalkyl(Cl-1O), alkylaminoalkyl (C 1-10), alkyliminoalkyl (C 1-10), rings from the list: [benzene, furan, thiophene, pyrrole, pyrazole, imidazole, pyran, thiopyran, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrrolidine, pyrazolidine, imidazolidine, tetrazole, piperidine, piperazine, morpholine, thiomorpholine, quinoline, isoquinoline, quinazoline, quinoxaline, chromene, isochromene,Indole, 2-oxa-5-aza-bicyclo[4.3.0]nonane, 2-oxa-5-aza-bicyclo[4.4.0]decane or indazole], which are bonded to an acylamine (Cl-3), aminoalkyl (Cl-3), alkoxy (Cl-3), oxyalkyl (Cl-3), alkyl (Cl-4), alkenyl (C2-4), alkynyl (C2-4), amino, oxy or directly, halogens, haloalkyl (Cl-6) or hydrophilic groups such as [hydroxyl, sulfhydryl, unbranched and branched (thio)ether (Cl-6), amino, azido, hydroxyamino, hydrazino, nitro, nitroso, cyano, isocyano, isocyanato, isothiocyanates, thiocyano, fulminato, semicarbazono, carboxylic acids, as well as their sulfur, selenium and tellurium analogues, imide, sulfone, Sulfinic, selenone, phosphonic, and phosphinic acids, as well as esters and amides of the aforementioned acids substituted with linear or branched (CI-6) alkyl, linear or branched (CI-6) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, oxo or imino]. R4, R5, R6, R7 and / or R8 are each either unsubstituted or selected from linear or branched (C1-10) alkyl, linear or branched (C1-10) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, alkoxyalkyl(Cl-1O), alkylaminoalkyl (C1-10), alkyliminoalkyl (C1-10), rings from the list: [benzene, furan, thiophene, pyrrole, pyrazole, imidazole, pyran, thiopyran, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrrolidine, pyrazolidine, imidazolidine, tetrazole, piperidine, piperazine, morpholine, thiomorpholine, quinoline, isoquinoline, quinazoline, quinoxaline, chromene, isochromene, indole, 2-Oxa-5-aza-bicyclo[4.3.0]nonane, 2-Oxa-5-aza-bicyclo[4.4.0]decane or indazole], which are bonded to an acylamine (Cl-3), aminoalkyl (Cl-3), alkoxy (Cl-3), oxyalkyl (Cl-3), alkyl (Cl-4), alkenyl (C2-4), alkynyl (C2-4), amino, oxy or directly bonded, halogens, haloalkyl (Cl-6) or hydrophilic groups such as [hydroxyl, sulfhydryl, unbranched and branched (thio)ether (Cl-6), amino, azide,Hydroxyamino, hydrazino, nitro, nitroso, cyano, isocyano, isocyanato, isothiocyanates, thiocyano, fulminato, semicarbazono, carboxylic acids, and their sulfur, selenium, and tellurium analogues, imide, sulfonic, sulfinic, selenone, phosphonic, and phosphinic acids, as well as esters and amides of the aforementioned acids substituted with linear or branched (C1-6) alkyl, linear or branched (C1-6) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, oxo or imino]; these residues may in turn be unsubstituted at any position or (multiple times) substituted with linear or branched (C1-10) alkyl, linear or branched (C1-10) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, Alkoxyalkyl(Cl-1O), alkylaminoalkyl (C 1-10), alkyliminoalkyl (C 1-10), rings, from the list: [Benzene, furan, thiophene, pyrrole, pyrazole, imidazole, pyran, thiopyran, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrrolidine, pyrazolidine, imidazolidine, tetrazole, piperidine, piperazine, morpholine, thiomorpholine, quinoline, isoquinoline, quinazoline, quinoxaline, chromene, isochromene, indole, 2-oxa-5-aza-bicyclo[4.3.0]nonane, 2-oxa-5-aza-bicyclo[4.4.0]decane or indazole], which are bonded to an acylamine (Cl-3), aminoalkyl (Cl-3), alkoxy (Cl-3), oxyalkyl (Cl-3), alkyl (Cl-4), alkenyl (C2-4), alkynyl (C2-4), amino, oxy or directly, halogens, haloalkyl (CI -6) or hydrophilic groups such as [hydroxyl, sulfhydryl, unbranched and branched (thio-)ethers (CI -6), amino, azido, hydroxyamino, hydrazino, nitro, nitroso, cyanocyanin, isocyanin, isocyanate, isothiocyanate, thiocyanate, fulminate, semicarbazonate, carboxylic acids, as well as their sulfur, selenium and tellurium analogues, imide, sulfonic, sulfinic, selenone, phosphonic and phosphinic acids,as well as the esters and amides of the aforementioned acids substituted with linear or branched (CI-6) alkyl, linear or branched (CI-6) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl, oxo or imino; These residues can in turn be unsubstituted or (multiple times) substituted at any position with linear or branched (C 1-10) alkyl, linear or branched (C 1-10) alkenyl or alkynyl, (C 3-7) cycloalkyl or -alkenyl, alkoxyalkyl(Cl-1O), alkylaminoalkyl (C 1-10), alkyliminoalkyl (C 1-10), rings from the list: [benzene, furan, thiophene, pyrrole, pyrazole, imidazole, pyran, thiopyran, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrrolidine, pyrazolidine, imidazolidine, tetrazole, piperidine, piperazine, morpholine, thiomorpholine, quinoline, isoquinoline, quinazoline, quinoxaline, chromene, isochromene, indole, 2-Oxa-5-aza-bicyclo[4.3.0]nonane, 2-oxa-5-aza-bicyclo[4.4.0]decane or indazole] containing an akylamino (Cl-3), aminoalkyl (Cl-3), alkoxy (Cl-3),Oxyalkyl (Cl-3), alkyl (Cl-4), alkenyl (C2-4), alkynyl (C2-4), amino, oxy or are directly bonded, halogens, haloalkyl (Cl-6) or hydrophilic groups such as [hydroxyl, sulfhydryl, unbranched and branched (thio-)ethers (Cl-6), amino, azido, hydroxyamino, hydrazino, nitro, nitroso, cyano, isocyano, isocyanato, isothiocyanato, thiocyano, fulminato, semicarbazono, carboxylic acids, as well as their sulfur, selenium and tellurium analogues, imide, sulfonic, sulfinous, selenone, phosphonic and phosphinic acids, as well as the esters and amides of the previously mentioned substituted with linear or branched (Cl-6) alkyl, linear or branched (Cl-6) alkenyl or alkynyl, (C3-7) cycloalkyl or -alkenyl [mentioned acids, oxo or imino], where R4, R5, R6, R7 and R8 can be different. R4 and R5 or R5 and R6 can also be cyclized together in the first substitution plane to form a ring with a maximum of seven ring members.

[0042] This results, by way of example, without representing the range of all possible combinations, in the following list of expressly protected substances in claim 15 for which a particularly strong anti-tumor effect could be demonstrated.

[0043] Figures 24 and 25 show the results of incubating fibroblasts with 3-BDO and MHY1485, respectively, and illustrate the reduction of TGF-beta-induced expression of TGF-beta-dependent factors such as collagen 1 by MHY1485. Fibroblasts were incubated for 24 h (qPCR) or 48 h (in-cell ELISA) either alone with 3-BDO or MHY1485, or with simultaneous administration of [missing information]. incubated with TGF-β (1 µL / ml). Shown are the results of the protein and RNA expression of collagen 1 (COL1A1) determined by in-cell ELISA and qPCR.

[0044] Figure 26 illustrates the individual pharmacological characteristics of potential inhibitors with respect to their effects on the signaling pathway activities of mTORC1 and TGFBR1 based on PPI inhibition of FKBP1A with mTORC1 and of FKBP1A with TGFBR1. Drug characteristic 1 shows strong inhibition of the FKBP1A-mTORC1 interaction, but only slight or no inhibition or even enhancement of the FKBP1A-TGFBRI interaction, resulting in increased mTORC1 and unchanged or decreased TGFBR1 signaling pathway activity. Drug characteristic 2 shows strong inhibition of the FKBP1A-mTORC1 and FKBP1A-TGFBR1 interactions, resulting in increased mTORC1 and TGFBR1 signaling pathway activity. Drug characteristic 3 shows little or no inhibition or even enhancement of the FKBP1A-mTORC1 interaction and an enhancement of the FKBP1A-TGFBR1 interaction, resulting in reduced or unchanged mTORC1 and reduced TGFBR1 pathway activity.The following is a summary of the various diseases that can be therapeutically addressed with a corresponding active ingredient.

[0045] The invention makes a significant contribution to the targeted treatment of TNBC, particularly addressing the high tendency of the disease to metastasize, which is crucial for prognosis. The implementation of this targeted treatment approach is essential for effective future treatment of TNBC.

[0046] According to the invention, this therapeutic goal is achieved by significantly reducing the migration of tumor cells in TNBC through pharmacological activation of the "Mammalian Target of Rapamycin" (also "mechanistic Target of Rapamycin", mTOR), in particular mTORC1.

[0047] mTOR is a key kinase that regulates various cellular processes such as cell differentiation and proliferation. By synergistically using it with therapies that directly target tumor cell viability (chemotherapy or immunotherapy), the efficacy of TNBC treatment can be significantly increased and the prognosis for patients substantially improved.

[0048] To date, no drugs are used to specifically target and reduce the high tendency of triple-negative breast cancer cells to metastasize. The pathophysiology of this early and extensive spread of the disease to other organs remains poorly understood. One theoretical approach is the targeted intervention of cellular signaling pathways.

[0049] While targeted pharmacological inhibition of mTOR, especially mTROCl, shows a significant anti-tumor effect in other breast cancer subtypes (E. Paplomata et al., Ther. Adv. Med. Oncol. 6, 154–166 (2014)), clinical studies have shown that Studies with mTORC1 inhibitors such as everolimus in TNBC have so far not led to any improvement in prognosis (NCT00499603; NCT00930930).

[0050] In vitro experiments surprisingly revealed that the migration of TNBC cells is significantly inhibited by mTOR activation. This inhibition is achieved primarily through activation of mTOR, particularly isolated activation of mTOR complex 1 (mTORC1). This inhibition is especially effective under hypoxic conditions, i.e., nutrient- and oxygen-deficient conditions such as those found in the extracellular matrix of the tumor microenvironment, which tumor cells must penetrate during metastasis. Specifically, the migration of TNBC cells of the mesenchymal stem-like subtype, as well as those of the BRCA1-mutated subtype (BReast CAncer gene 1) and the BRCA2-mutated subtype (BReast CAncer gene 2), is significantly inhibited (Figure 1).

[0051] Under these conditions, no significant reduction in cell viability could be demonstrated (Figure 3). A reduction in tumor cell migration due to decreased cell viability could therefore be ruled out.

[0052] Interestingly, in comparative experiments, mTOR inhibition did not show a comparable reducing effect on TNBC cell migration, as is known to occur in many tumors, including other breast cancer subtypes (E. Paplomata et al., Ther. Adv. Med. Oncol. 6, 154–166 (2014)). Instead, surprisingly, inhibition of mTOR, particularly mTORC1, actually increased migration under hypoxia (Figure 1).

[0053] These findings—that activation of mTOR, particularly mTORC1, especially under conditions such as those found in the extracellular matrix of the tumor microenvironment (which tumor cells must penetrate during metastasis), leads to a significant reduction in the migration of TNBC cells, and that mTOR inhibition, especially mTORC1 inhibition, leads to an increase in this migration—are novel and have not been previously described. Targeting the early stages of metastasis, i.e., migration within the tumor microenvironment, via mTOR activation (especially isolated mTORC1 activation) thus represents a new approach for future personalized therapy in TNBC.

[0054] The invention addresses a central cellular mechanism which, surprisingly, is only weakly activated in TNBC even under nutrient- and oxygen-rich conditions (Figure 6). This evidently plays a major role in the high migratory capacity of TNBC cells, particularly under nutrient- and oxygen-depleted conditions. It can therefore be assumed that, unlike many other tumors, pharmacological activation of mTOR activity (especially isolated mTORC1 activity) in TNBC results in strong anti-tumor activity with high tumor specificity.

[0055] In addition to the surprisingly discovered anti-tumor effect on cell migration in TNBC, it was also surprisingly found that activation of mTOR, especially mTORC1, activates the immune system and probably also intratumorally against the tumor. A directed immune response is activated. This is particularly crucial in TNBC, as the intra-tumoral immune response is significantly suppressed by various cellular processes.

[0056] In TNBC, mTOR activation inhibits the secretion of immunosuppressive cytokines. Experimental findings indicate that mTOR activation, particularly of mTORC1, in TNBC cells, especially under hypoxic conditions, leads to reduced secretion of the immunosuppressive cytokine TGF-beta. This was demonstrated by an ELISA test (Figure 10). TGF-beta inhibits the immune activity of T cells and other immune cells. Activators of mTOR, especially mTORC1, thus exert an immunostimulatory effect by inhibiting the secretion of immunosuppressive cytokines from TNBC cells within the tumor. The secretion induced by the cell interaction of TNBC cells with the extracellular matrix, particularly collagen 1, is crucial in this process. Furthermore, TGF-beta has a pro-carcinogenic effect on other cells in the tumor microenvironment, especially tumor-associated fibroblasts.

[0057] Activation of mTOR, particularly mTORC1, has further anti-tumor effects in TNBC. Surprisingly, activation of mTOR, especially mTORC1, reduces cell viability during prolonged cultivation under oxygen-deprived and nutrient-reduced conditions (hypoxia). Specifically, a reduction in cell viability was observed when cells were cultured under nutrient- and oxygen-deprived conditions for an extended period (Figure 7). Surprisingly, a reduction in TNBC cell proliferation was also observed over a longer period when cultured under nutrient-deprived and oxygen-rich conditions. Newly developed, more potent mTORC1 activators even showed a significant reduction in the cell viability of TNBC cells under nutrient-rich culture conditions with normoxia (Figure 8).

[0058] Activation of mTOR, particularly mTORC1, also inhibits the secretion of procarcinogenic molecules. Experimental results indicate that mTORC1 activation specifically inhibits the secretion of procarcinogenic molecules, particularly signaling molecules including cytokines and chemokines such as II-1B, -6, CCL2, -5, CXCL1, -2, -8, -12, CCL20, CX3CL1, CCL18, and matrix metalloproteinases (MMPs; including MMP-2, -7, -9). Bioinformatic correlations can be established for these proteins, analogous to those for pS6K and FKBP1A (Fig. 9).

[0059] Activation of mTOR, particularly mTORC1, may also inhibit the expression of plakophilin 2 (PKP2). It is already known that PKP2 knockdown leads to reduced EGFR activation and decreased proliferation and, in particular, migration of TNBC cells (K. Arimoto et al., Mol Cell Biol. 2014 Oct;34(20):3843-54. doi: 10.1128 / MCB.00758-14.). The experimental results obtained now indicate that, via mTOR activation, especially isolated activation of mTORC1, there is a cellular downregulation of plakophilin 2. This represents another novel mechanism of inhibition of cell proliferation and, in particular, migration in TNBC.

[0060] A particularly preferred activator of mTOR, especially of mTORC1, according to the invention is MHY1485 (4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazine-2-amine; CAS No.: 326914-06-1):

[0061] Another particularly preferred activator of mTOR according to the invention, especially of mTORC1, is 3-BDO (3-benzyl-5-[(2-nitrophenoxy)methyl]-2,3,4,5-tetrahydrofuran-2-one; CAS No. 890405-51-3):

[0062] 3-BDO is particularly advantageous because it activates mTORC1 via an interaction with FKBP1A (peptidyl prolyl cis-trans isomerase), which is essential for its efficacy (http: / / dx.doi.org / 10.4161 / auto.28363), thus exhibiting high TNBC specificity. 3-BDO acts as an activator of mTOR, especially mTORC1, by binding to FKBP1A, which is highly expressed in TNBC, particularly in metastatic TNBC cells within the tumor microenvironment (WO2021 / 180840 Al). This further enhances the tumor specificity of 3-BDO. Previous studies show that 3-BDO binds to the rapamycin binding site on FKBPla (D. Ge et al., Autophagy, 10(6), 957–971. https: / / doi.org / 10.4161 / auto.28363). Interestingly, the inventors were the first to demonstrate that, unlike rapamycin, the binding of 3-BDO in docking studies largely covers domain C of FKBP1A (see Fig. 11).Since this domain binds to both the FRB domain (FKBP12-rapamycin binding domain) of mTOR and the central mTORC1 activator Rheb ("Ras homolog enriched in brain"; NCBI Gene: 6009; UniProtKB / Swiss-Prot: Q15382), 3-BDO apparently addresses a previously undescribed dual mechanism via FKBPla for the activation of mTOR. 1. The inhibitory effect of FKBP1A on mTORC1 is prevented because the binding of FKBP1A (or the C domain of FKBP1A) to the FRB domain of mTORC1 is inhibited by 3-BDO. 2. By additionally preventing Rheb from binding to the C domain of FKBP1A, Rheb remains free in the cell and can efficiently bind to and activate mTORC1 (X. Bai et al., Cell Mol Life Sci. 2010 Jan;67(2):239-53. doi: 10.1007 / s00018-009-0163-7).

[0063] Since FKBP1A has so far only been described as an inhibitory component of the mTORC1 complex (not mTORC2), mTORC1 activation is specifically achieved by 3-BDO (L. Hu et al., iScience. 2021 Sep 27;24(11): 103177. doi: 10.1016 / j.isci.2021.103177).

[0064] Regarding the pathophysiology of TNBC, the inventors' data indicate that FKBP1A is highly expressed even under nutrient-rich and normoxic conditions, and that mTORC1 activation, as normally induced by nutrients, is apparently prevented. A similar effect can be assumed for FKBP38 or other members of the FKBP family that possess a comparable domain C.

[0065] Interestingly, the inventors were the first to discover that MHY1485 also binds to domain C of FKBP1A (see Fig. 12). In contrast to 3-BDO, MHY1485 binds more completely to the region Tyr82 to Pro88 of domain C of FKBP1A and barely overlaps with the rapamycin binding site.

[0066] Since other members of the FKBP protein family, especially FKBP38, also possess the described domain C or homologous domains or domain elements (see: [0026, 0027]) and for other members of the FKBP protein family, especially for FKBP38 (Y. Yan et al., Arch Biochem Biophys. 2024 Feb:752: 109891. doi: 10.1016 / j.abb.2024.109891), where a comparable mTORC1 inhibition could be demonstrated, the effect of the domain C-specific active substances described in this invention is to be applied to all members of the FKBP family with a comparable protein domain C, in particular FKBP38 (structurally similar to FKBP1A).

[0067] According to the invention, a specific activation of mTOR, in particular of mTORC1, can thus be used specifically for the TNBC, whereby other cells with low FKBP1A expression, or low expression of other mTORC1 regulatory FKBPs with a domain C or a homologous domain, in particular FKBP38, are addressed less. In this therapeutic approach, a specific active ingredient binding the FKBP C domain, in particular of FKBP1A and FKBP38, preferably MHY1485- or 3-BDO or one of their derivatives (see Figures 16, 17 and claims 14, 15), preferably an FKBP1A-3-BDO-, FKBP38-3-BDO-, FKBP1A-MHY1485-, FKBP38-MHY1485-, FKBPIA-derivative or FKBP38-derivative complex, which inhibits the binding of domain C to both mTORC1 and Rheb. Of particular importance is deacetylated FKBP 1 A, which has a high affinity for the FRB domain of mTORC1 and efficiently inhibits it (L. Hu et al., iScience 24(11): 103177, DOI:10.1016 / j.isci.2021.103177).

[0068] In addition to tumor cells, this specificity of such activators of mTOR, especially mTORC1, also implies non-tumor cells of the tumor microenvironment in TNBC, such as tumor-associated macrophages, tumor-associated fibroblasts, adipocytes, endothelial cells, pericytes, other immune cells including microglia, especially tumor-infiltrating leukocytes, particularly cytotoxic T-cell lymphocytes, as well as dendritic cells. It has been found that TNBC cells strongly secrete FKBP1A, preferably deacetylated FKBP1A, and other (preferably deacetylated) FKBPs including FKBP38, among others, in vesicles, whereby the FKBPs, especially FKBP1A and / or FKBP38, are taken up by surrounding cells of the tumor microenvironment and can have an immunosuppressive effect in leukocytes, including in the cytosol, especially in T lymphocytes, by inhibiting mTORC1.

[0069] The finding that TNBC cells strongly secrete FKBP1A, preferably mTORC1-inhibiting deacetylated FKBP1A, and other (preferably deacetylated) FKBPs, possibly especially FKBP38, can be used to advantage according to the invention. In particular, T cells hardly express FKBP1A, probably because (specifically deacetylated) FKBP1A can inhibit mTOR, preferentially mTORC1 (L. Hu et al., iScience 24(11): 103177, DOI: 10.1016 / j.isci.2021.103177). Analyses of biobanks show that FKBP1A has the strongest anti-proportional correlation with the expression of p70 S6 kinase of all FKBPs (Figure 9), a direct effector of mTORC1.

[0070] However, immune cells require mTOR, and especially mTORC1, activity for their function. Therefore, everolimus also has an immunosuppressive effect via the inhibition of mTORC1. For example, high expression of FKBP1A has been observed in leukocytes, such as B cells, CD8 T cells, CD4 T cells, and T cells in the pancreas or hepatocellular carcinoma (P. Watcharanurak et al., Sci Rep 14, 7888 (2024). https: / / doi.org / 10.1038 / s41598-024-58324-z; Zhonggu-ang Li et al., Int. J. Mol. Sci. 2022, 23(21), 12797; https: / / doi.org / 10.3390 / ijms232112797).

[0071] The finding that TNBC cells secrete FKBP1A, preferentially the mTORC1-inhibiting deacetylated FKBP1A, which is taken up by immune cells in the tumor microenvironment, suggests that TNBC likely promotes an immunosuppressive environment in this way. This also applies to other secreted FKBPs, possibly especially FKBP38.

[0072] TNBC cells inhibit mTOR, preferably mTORC1, in surrounding immune cells, and according to the invention, these immune cells can be reactivated in a TNBC-specific manner via mTOR activators, preferably mTORC1 activators, since normal T cells do not have high FKBPIA expression, especially of deacetylated FKBP1A, or high FKBP38 expression.

[0073] This intracellular FKBP1A or, if applicable, FKBP38, which originates from TNBC cells and is taken up by (surrounding) immune cells, can be bound via a specific FKBP1A- or FKBP38-binding agent, preferably 3-BDO or MHY1485 or one of its derivatives presented here. The resulting FKBP1A-drug complex or FKBP38-drug complex (preferably FKBP1A-3-BDO-, MHY1485- or derivative complex or FKBP38-3-BDO-, MHY1485- or The derivative complex can then specifically activate mTOR, particularly mTORC1, in these cells. The dual interaction of the active ingredient—both the binding to domain C of FKBP1A or FKBP38, which is essential for its effect, and the interaction with the FRB domain of mTORC1 or with Rheb—causes the activation of mTOR, particularly mTORC1 (see [0060 - 0063]). This activation is particularly important therapeutically in TNBC, since intratumoral immune cells are inactivated by the tumor cells, and targeted activation of immunosuppressed leukocytes rich in FKBP1A, particularly deacetylated FKBP1A, or FKBP38, can be achieved through the specific activation of FKBP1A- or FKBP38-binding mTOR activators, particularly mTORC1 activators. This can be combined particularly efficiently with immunotherapeutic approaches.

[0074] Furthermore, in the aforementioned cells of the tumor microenvironment, the expression of FKBP1A, especially deacetylated FKBP1A, or of FKBP38, which is increased by cellular (signaling) molecules of the TNBC cells, can be specifically targeted via a specific FKBP1A- or FKBP38-binding agent, preferably 3-BDO or MHY1485 or one of its derivatives, and thus mTOR, especially mTORC1, can be specifically activated in these cells.

[0075] Another way to pharmacologically activate mTOR, particularly mTORC1, is through SIRT2 inhibitors. Inhibition of SIRT2 blocks the deacetylation of FKBP1A, thus preventing the inhibition of mTOR activity by deacetylated FKBP1A. SIRT2 is overexpressed in TNBC and generally in tumors with high mesenchymal stem cell properties (W. Zhou et al., Cell Rep. 2016 Oct 25;17(5): 1302-1317. doi: 10.1016 / j.celrep.2016.10.006.).

[0076] The experimental data indicate that preventing the expression of FKBP1A via siRNA through a resulting depletion of deacetylated FKBP1A can also activate mTORC1, i.e., it represents another mechanism of activation.

[0077] Regarding systemic activation of mTOR, particularly mTORC1, in humans in vivo, good tolerability can generally be expected, especially when using 3-BDO as an mTORC1 activator (presumably due to specific activation primarily in FKBPlA-high-expressing cells). In vivo experiments with 3-BDO in mice have shown good tolerability (Ge Di et al., Autophagy. 2014 Jun; 10(6): 957-71. doi: 10.4161 / auto.28363; Peng Nan etal., Sei Rep. 2014 Jul 1:4:5519. doi: 10.1038 / srep05519).

[0078] In further structural analyses and docking studies, the inventors surprisingly discovered that MHY1485, 3-BDO, and the developed active substances not only inhibit the binding of domain C of FKBP1A to FRB of mTORC1, but also, to varying degrees, inhibit the binding of domain C of FKBP1A to the TGF-beta receptor 1 (TGFBR1) (see Fig. 15). It is known that domain C of FKBP1A also binds to TGFBR1 (Huse M, Chen YG, Massague J, Kuriyan J. Crystal structure of the cytoplasmic domain of the type I TGF beta receptor in complex with FKBP12. Cell. 1999 Feb 5;96(3):425-36). At physiological concentrations FKBP1A thus prevents the activation of the receptor in the absence of its ligand; at higher concentrations, FKBP1A can also inhibit TGFBR1 in the presence of the ligand (Chen YG, Liu F, Massague J. Mechanism of TGFbeta receptor inhibition by FKBP12. EMBO J. 1997 Jul 1; 16(13): 3866-76). In the inventors' work, the differences between binding to domain C of FKBP1A to the FRB of mTORC1 and binding to TGFBR1 were elucidated and taken into account in the characterization and development of mTORC1 activators via the targeted inhibition of the FKBP1A-mTORC1 interaction.The structural differences between the two binding motifs lie in the fact that while TGFBR1 utilizes the rapamycin binding site in FKBP1A, it interacts little with it except for an interaction with the 80's loop of domain C (Tyr82, Thr85, and His87 to Ile97). Consequently, a large portion of the domain C area, with which many of our derivatives interact in docking studies, is not occupied by TGFBR1. It was found that MHY1485 interacts only very weakly, while 3-BDO interacts significantly with TGFBR1 binding (see Fig. 15). For FKBPlA-bound MHY1485, binding studies at the interaction site with TGFBR1 even indicate an enhancing binding to TGFBR1, which in this context acts as a protein-protein interaction stabilizer (“molecular glue”) on both proteins (FKBP1A and TGFBR1) (see .

[0031] ). MHY1485 binds within a cavity formed by both proteins, both on the FKBPIA side and with the nitrophenyl group above FKBPlAs, thus apparently enhancing the physiological binding of both proteins (see Fig. 15). In TGF-beta activation studies in fibroblasts, no inhibition of TGFBR1 activation by its ligand TGF-beta could be demonstrated for 3-BDO, whereas for MHY1485 this was very pronounced, and fibroblast activation was even below the value without the ligand TGF-beta – evidently due to the enhanced inhibition of TGFBR1 mediated by the increased binding of FKBPlAs to TGFBR1 by MHY1485 (see Figs. 24, 25).

[0079] The TGF-beta signaling pathway plays a crucial role in oncology, particularly in triple-negative breast cancer (TNBC) and other tumors with high mesenchymal stem cell properties. While an anti-cancer effect is frequently described in early-stage epithelial tumors, a pro-cancer effect has generally been demonstrated in later stages of the disease and in tumors with high mesenchymal properties (Principe DR, Doll JA, Bauer J, Jung B, Munshi HG, Bartholin L, Pasche B, Lee C, Grippo PJ. TGF-β: duality of function between tumor prevention and carcinogenesis. J Natl Cancer Inst. 2014 Feb; 106(2):djt369). Therefore, considering the influence on the TGFBR1 signaling pathway is important for the development of an mTORC1 activator that aims to inhibit the FKBPlA-mTORC1 interaction.While additional activation of the TGFBR1 signaling pathway appears therapeutically beneficial in early epithelial tumors, activation should generally be avoided, or even inhibited, in later tumor stages and tumors with high mesenchymal stem cell properties. These considerations were taken into account in this patent application, and in addition to the aforementioned derivatives that inhibit the interaction of FKBP1A and TGFBR1, further derivatives were developed that inhibit this interaction. The goal is to avoid disrupting or even enhancing the interaction of FKBP1A with mTORC1 as much as possible, while maintaining the most efficient inhibition of this interaction. This is achieved by these drug candidates covering larger portions of domain C, but not those parts crucial for the interaction of FKBP1A with TGFBR1. This includes, as described in paragraph

[0066] described, the rapamycin binding site and part of the 80' loop of domain C. This disrupts the interaction between mTORC1 and FKBP1A, while TGFBR1 can still bind to FKBP1A. This particularly affects drug candidates that interact with Gln53 and Glu60 of FKBP1A during docking and therefore do not cover the loop of domain C with which TGFBR1 interacts as much as those that disrupt the interaction between FKBP1A and TGFBR1. In the docking studies, some of these ligands even interacted in such a way that they filled a pocket formed between FKBP1A and TGFBR1, further enhancing the interaction between FKBP1A and TGFBR1. This then leads to strong mTORC1 activation with simultaneous unchanged or even inhibited TGFBR1 activity.In this way, particularly for advanced tumor stages and tumors with high mesenchymal stem cell properties, an efficient anti-tumor effect is generally achieved through a simultaneous, pathophysiologically meaningful addressing of both signaling pathways. Furthermore, it has been found that FKBP1A functions as a novel central regulatory element of both signaling pathways and could essentially centrally control the (patho-)physiologically opposing activities of both pathways, i.e., high TGFBR1 activity and low mTORC1 activity (or vice versa). A functional relationship between the mTORC1 and TGFBR1 signaling pathways has been little investigated to date, particularly in tumors with high mesenchymal properties. Studies in immune cells show that high TGFBR1 activity in CD44-positive cells inhibits mTORC1 (Gabriel SS, et al., Transforming growth factor-β-regulated mTOR activity preserves cellular metabolism to maintain long-term T-cell responses in chronic infection. Immunity. 2021 Aug 10;54(8): 1698-1714. e5.).Since CD44 is an important tumor stem cell marker in TNBC, this finding suggests a potential role for both opposing signaling pathways in TNBC, as well as the possible role of FKBP1A in the targeted, direct regulation of both pathways. This may be determined by the acetylation status of FKBP1A: When strongly deacetylated, FKBP1A binds strongly to mTORC1 and efficiently inhibits it (L. Hu et al., iScience 24(11): 103177). This results in less regulation and increased activity of the TGFBR1 signaling pathway. In the acetylated state, FKBP1A binds only weakly or not at all to mTORC1 and can bind efficiently to TGFBR1 (possibly with increased affinity) and inhibit it. Since SIRT2, which deacetylates FKBP1A, is overexpressed in TNBC (and other tumors with high mesenchymal stem cell properties), the deacetylated FKBP1A centrally downregulates the mTORC1 signaling pathway and simultaneously activates the TGFBR1 signaling pathway.

[0080] The above paragraphs suggest a further development possibility for drug candidates that specifically target the FKBP1A-mTORC1 or -TGFBR1 interaction: substances that specifically enhance the interaction of FKBP1A with TGFBR1 while simultaneously increasing the mTORC1- The interaction may remain unaffected or, in the sense of mTORC1 inhibition, may also be enhanced. The background for such drug candidates is their use in diseases involving a pathological increase in the TGFBR1 signaling pathway. The novel approach of pharmacologically enhancing FKBP1A binding to TGFBR1 to inhibit the broad TGFBR family via the endogenous ligand FKBP1A is previously undescribed and represents a novel approach. Since the seven different type I TGFBR receptors (ALKI-7) can be activated by five different type II TGFBR receptors (TGFBR2, BMPR2, ACVR2A, ACVR2B, and AMHR2), efficient inhibition of the TGFBR1 signaling pathway can be achieved via the described enhanced FKBP1A-TGFBR1 interaction (Heldin CH, Moustakas A. Signaling Receptors for TGF-β Family Members. Cold Spring Harb Perspect Biol. 2016 Aug 1;8(8):a022053). In particular, MHY1485, or...Derivatives of MHY1485 with little or no inhibition of the FKBP1A-mTORC1 interaction are particularly interesting drug candidates and have not yet been described as enhancing the FKBP1A-TGFBR1 interaction. Pathologically increased activity of the TGFBR1 signaling pathway is observed in fibrotic diseases such as systemic sclerosis (SSc), which affects not only the skin but also the lungs and other internal organs. Attenuation of the TGFBR1 signal reduces the differentiation of fibroblasts into myofibroblasts, leading to a reduction in extracellular matrix deposits. Furthermore, vascular changes associated with chronic ischemia and ulceration are reduced (PMID: 29355590). In addition to SSc, other chronic inflammatory diseases also exhibit an overactive TGFBR1 signaling pathway. For example, in the late stages of Crohn's disease and ulcerative colitis, chronic inflammatory processes often lead to fibrosis in the intestine, resulting in stenoses and intestinal narrowing, frequently necessitating surgical interventions (PMID: 40967215, PMID: 38475672).Furthermore, increased TGF-β signaling has been observed in asthma and allergic diseases, which is associated with airway remodeling (PMID: 38514615). Finally, patients with myopathy (e.g., Marfan syndrome, sarcopenia) benefit from TGFBR1 inhibition, as overactivation of this signaling pathway promotes muscle degeneration and wasting (PMID: 21798096). In summary, overactivation of the TGFBR1 signaling pathway has been described particularly for the following diseases: 1. Fibrotic diseases such as idiopathic pulmonary fibrosis, post-COVID pulmonary fibrosis, liver fibrosis / cirrhosis (e.g., in non-alcoholic steatohepatitis, NASH), renal fibrosis, cardiac fibrosis, systemic sclerosis, keloids and hypertrophic scars, Dupuytren's contracture, proliferative vitreoretinopathy, endometriosis 2. Autoimmune and chronic inflammatory diseases such as rheumatoid arthritis, psoriatic arthritis, Crohn's disease, ulcerative colitis 3. Vascular and cardiovascular diseases such as atherosclerosis, pulmonary arterial hypertension, Marfan syndrome, Loeys-Dietz syndrome, cardiac remodeling after myocardial infarction 4. Metabolic and chronic organ diseases such as diabetic nephropathy, diabetic retinopathy, non-alcoholic fatty liver disease (NASH) 5. Neurological and neuromuscular diseases such as multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, gliosis following central nervous system injury 6. Allergic and immunological diseases The extent to which downregulation or increased deacetylation of FKBP1A (especially acetylated FKBP1A) is pathophysiologically involved in or even causative of the overactivity in these diseases has not yet been conclusively investigated scientifically. However, given the central role of FKBP1A in the regulation of the TGF-beta receptor, it seems plausible that reduced endogenous inhibition by FKBP1A is pathophysiologically relevant. For such diseases, an active substance like the one described above, which enhances the PPI between FKBP1A and TGF-β1, would also possess high disease specificity and would be characterized not only by high efficacy but also by a low side effect profile.

[0081] Therapeutically, the enhancement of the FKBP 1A-TGFBR1 interaction can be used to specifically inhibit the TGFBIR signaling pathway, particularly in fibrotic diseases where current inhibitors of the TGF-beta-1 signaling pathway are nonspecific and inefficient. Although both antibody-based and small molecule inhibitors against the TGF-β signaling pathway have been developed, none of the drug candidates tested in clinical trials have yet been approved (https: / / clinicaltrials.gov / study / NCT03834662?tab=results). This was partly due to low efficacy, but also to potential side effects (PMID: 29355590). TGF-β plays an important role in regulating the immune system, and dysregulation promotes autoimmune processes (PMID: 38514615). MHY1485 and corresponding

[0080] Developed active substances that enhance the FKBP1A-TGFBR1 interaction have a decisive advantage over direct TGFBR1 inhibitors because they attenuate the TGFBR signaling pathway but do not completely block it. This still allows signal transduction but prevents overactive activation of the system at high extracellular TGF-β levels. MHY1485 or corresponding active substances (see

[0038] to

[0042] ) could be implemented in a corresponding therapy in a timely manner, since the tolerability of the active ingredient MHY1485 has already been extensively investigated in preclinical studies. In particular, topical application appears to be efficiently feasible.

[0082] Figure 26 summarizes the different possibilities of pharmacologically influencing the FKBPlA-mTORC1 or FKBP 1A-TGFBR1 interactions and illustrates the respective therapeutic use.

[0083] According to the invention, the activator of mTOR, in particular of mTORC1, is preferably chemically coupled to tumor-specific peptides / antibodies. In this way, the tumor specificity of the activator of mTOR, in particular of mTORC1, can be further increased, and potential side effects of systemic activation of mTOR, in particular of mTORC1, can be reduced.

[0084] Preferably, the specific activators of mTOR, in particular mTORC1, preferably MHY1485 or 3-BDO, are conjugated to tumor-specific peptides or antibodies (e.g., CD44- or TROP-2-specific) via chemical linker constructs (Y. Liu et al., Mol. Cancer 22, 145 (2023)). In this way, a local effect is induced in the tumor tissue in TNBC.

[0085] In preferred embodiments, enzymatically cleavable linkers are used with antibodies / peptides, e.g. lysosomal cathepsin B cleavable linkers.

[0086] Compounds of general formula (I) are particularly preferred where R1 stands for a peptide or antibody; and R2 and R3 each independently represent a side chain of a naturally occurring alpha-amino acid.

[0087] A preferred example of such a linker cleaved via lysosomal cathepsin B is valine-alanine p-aminobenzyloxycarbonyl (PABC), i.e., R2 stands for -CH2CH2CH2-NH(C=O)NH2; and R3 stands for -CH3:

[0088] Another favored example is valine-citrulline-p-aminobenzyloxycarbonyl (PABC), i.e. R2 stands for -CH2CH2CH2-NH(C=O)NH2; and R3 stands for -CH(CH3)2:

[0089] Following cellular uptake of the antibody or peptide-MHY1485 conjugate, chemically unchanged MHY1485 is preferentially released endosomally locally as a result of enzymatic cleavage by lysosomal cathepsin B:

[0090] Analogously, other chemical linker constructs with other chemically reactive terminal groups can also be used according to the invention (Y. Liu et al., Mol. Cancer 22, 145 (2023)).

[0091] The same applies to other activators of mTOR, such as 3-BDO derivatives containing a primary or secondary amine (see Figure 16).

[0092] Preferred peptides for conjugation with the activators of mTOR according to the invention are selected from CD44, in particular CD44v6, and TROP-2.

[0093] The antibody or peptide conjugates can also contain couplings to other anti-tumor agents, so-called dual-drug antibody-drug conjugates, in which the mTOR- The activating component then has a direct adjuvant effect (CM Yamazaki, et al., Nat. Commun. 12, 1-13 (2021)).

[0094] According to the invention, the activators of mTOR, in particular of mTORC1, are preferably used synergistically with other anti-tumor agents, especially with immunomodulating substances (synergistic immunotherapy), for the treatment of TNBC. Alternatively, monotherapy is possible.

[0095] According to the invention, the activators of mTOR, in particular of mTORC1, are preferably used synergistically together with inhibitors of the MAP kinase signaling pathway for the treatment of TNBC.

[0096] According to the invention, the activators of mTOR, in particular of mTORC1, are preferably used synergistically with PPAR-gamma modulators or PPAR-gamma activators for the treatment of TNBC.

[0097] According to the invention, FKBPlA-dependent activators of mTOR, especially mTORC1, such as 3-BDO and MHY1485, can preferably be used synergistically with FKBPlA-degrading agents such as RC32 for the treatment of TNBC in the future, since the effect of these mTORC1 activators is stoichiometrically reduced at high concentrations of FKBPlA, and experiments have shown that RC32 enhances the effect of 3-BDO and MHY1485. A comparable synergism can be assumed with FKBP38-degrading agents.

[0098] Another aspect of the invention relates to a method for finding activators of mTOR, in particular mTORC1, which are suitable for the treatment of TNBC, preferably for adjuvant or neoadjuvant (preferably synergistic with another TNBC therapy) or possibly also monotherapeutic treatment, wherein the method comprises the following steps: (a) Providing an activator of mTOR; this includes, in particular, FKBP1A- or FKBP38- or other FKBP-dependent mTORC1 activators that bind specifically to the C domain of FKBP1A or FKBP38 or other FKBPs and prevent interaction with the FRB domain of mTOR and / or Rheb, such as 3-BDO or MHY1485, or one of the derivatives described in this invention disclosure (see Figures 16, 17 and claims 14, 15). It is also preferable to consider a possible inhibitory effect of the mTORC1 activators on FKBP1A-TGFBR1 binding, which can be specifically excluded. (b) Incubating cancer cells of a TNBC cancer cell line with the activator of mTOR, preferably mTORC1, and determining a property of the cancer cells; (c) Incubating cancer cells of the same TNBC cancer cell line as in step (b) in the absence of the activator of mTOR, preferably mTORC1, and determining the same property of the cancer cells as in step (b) under the same conditions as step (b); (d) Comparing the determined property of the cancer cells according to steps (b) and (c).

[0099] The suitability of mTOR activators, preferably mTORC1, can relate to the monotherapeutic treatment of TNBC, so that the inventive method serves to discover activators which, on their own, i.e., without interaction with other substances or cells, enable therapy. According to this embodiment, the absence of the mTOR activator is preferably the only difference between step (c) and step (b). According to this embodiment, steps (b) and (c) are each preferably carried out in the absence of (i) cytotoxic substances, (ii) substances with anti-tumor activity, and (iii) immune cells directed against cancer cells.

[0100] Unless expressly defined otherwise, the term "substance" shall below refer to both cytotoxic substances and substances with anti-tumor activity for the purpose of describing the invention.

[0101] In a preferred embodiment, the method according to the invention serves to discover activators of mTOR, preferably mTORC1, which are suitable for the adjuvant treatment of TNBC, wherein step (b) is carried out in the presence of (i) a cytotoxic substance, (ii) a substance with anti-tumor activity, or (iii) immune cells directed against cancer cells; and wherein step (c) is carried out in the presence of the same substance or the same immune cells as in step (b).

[0102] The order of steps (a) to (d) of the inventive procedure is not fixed, although step (a) will naturally occur at the beginning and step (d) naturally towards the end. Steps (b) and (c) can be performed consecutively in any order, simultaneously, or partially simultaneously.

[0103] In step (a) of the inventive process, an activator of mTOR, preferably mTORC 1, is provided. Activators of mTOR, preferably mTORC 1, are known and, among other things, commercially available. Two particularly preferred activators of mTOR, especially mTORC1, according to the invention are MHY1485 and 3-BDO.

[0104] In steps (b) and (c) of the inventive process, cancer cells from a TNBC cell line are used. Suitable TNBC cell lines are commercially available. Preferably, the TNBC cell line is selected from MDA-MB-231 and MDA-MB-436 cancer cells.

[0105] The number of cancer cells used in each of steps (b) and (c) of the inventive method is not limited. According to the invention, at least one single cancer cell is used in each step, but preferably a defined plurality of cancer cells.

[0106] In a preferred embodiment, where the inventive method serves to discover mTOR activators suitable for the adjuvant treatment of TNBC, in steps (b) and (c) of the inventive method the cancer cells are preferably incubated (i) with a cytotoxic substance, (ii) with a substance with anti-tumor activity, or (iii) with immune cells directed against cancer cells, step (b) additionally in the presence of the mTOR activator. preferably mTORC1. Step (c), however, in the absence of the mTOR activator, preferably mTORC1. For this purpose, the substance or the immune cells are preferably provided in a medium and combined with the cancer cells. Suitable concentrations of the substance or the immune cells depend on their respective specific efficacy and can be determined by simple routine experiments.

[0107] Suitable incubation conditions, such as temperature, medium, pH, etc., are known to a person skilled in the art and depend, among other things, on the chosen TNBC cancer cell line. A medium preferred according to the invention is RPMI medium (developed by the Roswell Park Memorial Institute), preferably RPMI-1640, which preferably contains 10% FCS (fetal calf serum). Such media are known to a person skilled in the art and are commercially available.

[0108] The duration of the incubation is preferably selected according to the invention such that a potential effect of (i) the cytotoxic substance, (ii) the anti-tumor substance, or (iii) the immune cells directed against cancer cells can be observed. Depending on the selected TNBC cancer cell line and the selected substance or immune cell, the appropriate duration of the incubation can range from a few minutes to several hours or days. Suitable time periods can be determined by simple routine experiments. In preferred embodiments, the duration of the incubation is 1 to 12 days, more preferably 3 to 9 days, and particularly preferably 6 days.

[0109] In a preferred embodiment, the cancer cells are first seeded and only after a certain period of time, preferably after 12 to 36 hours, particularly preferably after 24 hours, are (i) incubated with the cytotoxic substance, (ii) with the substance with anti-tumor activity or (iii) with the immune cells directed against cancer cells.

[0110] In a preferred embodiment, pre-incubation takes place for (i) the toxic substance, (ii) the anti-tumor substance or (iii) the immune cells directed against cancer cells, but after the addition of the activator of mTOR, preferably mTORC1, preferably 1 to 6, preferably 3 days.

[0111] In steps (b) and (c) of the method according to the invention, a property of the cancer cells is determined after incubation. The aim is to determine different properties of the cancer cells which are attributable to the different incubation in steps (b) and (c), in particular to the presence of the activator of mTOR, preferably mTORC1, in step (b) of the method according to the invention, either in the absence (monotherapy) or in the presence (adjuvant therapy) of (i) the cytotoxic substance, (ii) the substance with anti-tumor activity, or (iii) the immune cells directed against cancer cells.

[0112] An expert recognizes that fundamentally different properties of cancer cells are suitable for determining the effect of incubation in the presence or absence of certain substances.

[0113] The preferred property of cancer cells is their viability. This is particularly preferred if (i) the cytotoxic substance, (ii) the anti-tumor substance, or (iii) the immune cells directed against cancer cells are typically suitable for or aim to induce the death of the cancer cells, for example, by apoptosis.

[0114] Methods for determining the viability of cancer cells are known to those skilled in the art, and suitable tests are commercially available. In a preferred embodiment, the viability of the cancer cells is determined according to the invention using the CellTiter-Glo® test (Promega).

[0115] The evaluation of the properties of the cancer cells after incubation, for example the evaluation of their viability, can be carried out individually and subjectively by the experimenter, for example using a color reaction.

[0116] In a preferred embodiment of the inventive method, the determination of the property comprises a quantitative measurement, preferably of the cellular ATP concentration, via the measurand luminescence. For this purpose, suitable devices such as spectrometers are preferably used according to the invention.

[0117] In a preferred embodiment of the inventive method, the determination of the property comprises a quantitative measurement, preferably of cellular migration, preferably under hypoxic conditions with glucose-free RPMI medium and 5 wt% FCS. Suitable devices are preferably used for this purpose according to the inventive method.

[0118] In a preferred embodiment of the inventive method, the determination of the property comprises a quantitative measurement, preferably of the metabolic properties of the (tumor) cell. mTORC1 activation alters the cell metabolism of the cells from catabolic to anabolic and enables the quantification of corresponding markers, whereby catabolic markers such as lipolysis and fatty acid oxidation decrease and anabolic markers such as fatty acid synthesis increase. In this context, there is also an increase in anaerobic glycolysis and increased lactate production.

[0119] The cytotoxic substance (i) preferably used in steps (b) and (c) of the inventive process is not specified. Preferably, it is a known cytotoxic substance, preferably one that is approved for clinical applications and preferably already used for the treatment of TNBC. In preferred embodiments, the cytotoxic substance is selected from the group consisting of Taxol, docetaxel, cisplatin, carboplatin, cytocalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etopside, tenopside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine, mitoxantrone, mithramycin, actinomycin, d,l-dehydrotestosterone, glycocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin; preferably Taxol.

[0120] The substance (ii) with anti-tumor activity preferably used in steps (b) and (c) of the inventive method is not specified. Preferably, it is a known substance with anti-tumor activity, which is preferably approved for clinical applications and preferably already used for the treatment of TNBC. In preferred embodiments, the substance with anti-tumor activity is selected from the group consisting of targeted anti-tumor agents such as tyrosine kinase inhibitors, preferably selected from the group consisting of EGFR inhibitors, BRAF inhibitors, PIK3 inhibitors, PIK3 / mTOR inhibitors and HER2 inhibitors; preferred examples according to the invention are: mTORC2 inhibitors; EGFR inhibitors: e.g. B. Erlotinib, Gefrtinib, Lapatinib, Cetuximab, Neratinib, Osimertinib, Panitumumab, Vandetanib, Necitumumab, Dacomitinib; BRAF inhibitors: e.g. B. Vemurafenib, Dabrafenib, Sorafenib, Encorafenib; PIK3 inhibitors: e.g. B. Buparlisib, Idelasisib, Copanlisib, Duvelisib, Alpelisib, Taselisib, Perifosine, Umbrasilib; dual PIK3 / mTOR inhibitors: e.g. B. Dactolisib, Apitolisib, Gedatolisib, Bimiralisib, P7170, SF-1126, GDC-0084, LY3023414; HER2 inhibitors, e.g., trastuzumab, lapatinib, neratinib, ado-trastuzumab, emtansine, pertuzumab, dacomitinib; and / or Immunostimulating antibodies, such as antibodies directed against PD-Ll, against PD-1, or against CTLA-4. In this case, co-incubation with (iii) immune cells directed against the cancer cells may be preferred.

[0121] The immune cells directed against cancer cells preferably used in steps (b) and (c) of the inventive method (iii) are not specified. Preferably, they are therapeutically modified immune cells, which are preferably approved for clinical applications and preferably already used for the treatment of TNBC. In preferred embodiments, the immune cells directed against cancer cells are modified cytotoxic T cells. Modifications may include, for example, changes to the T-cell receptor for the recognition of cancer-specific antigens, or T-CAR cells may be used.

[0122] According to the invention, individual species (i), (ii) or (iii) can be used, or any combinations thereof, in particular - individual cytotoxic substances or combinations of several cytotoxic substances, - single substances with anti-tumor effects or multiple substances with anti-tumor effects, - immune cells of a single type directed against cancer cells or immune cells of different types directed against cancer cells, - a cytotoxic substance in combination with a substance with anti-tumor activity, - a cytotoxic substance in combination with immune cells directed against cancer cells, or - a substance with anti-tumor activity in combination with immune cells directed against cancer cells.

[0123] Preferably, the property of the cancer cells is determined in steps (b) and (c) of the method according to the invention at several concentrations of (i) the cytotoxic substance, (ii) the substance with anti-tumor activity, or (iii) the immune cells directed against cancer cells. Preferably, an ICso value is then determined based on the results for different concentrations as the concentration (i) of the cytotoxic substance, (ii) of the substance with anti-tumor activity, or (iii) of the immune cells directed against cancer cells at which 50% of the cancer cells exhibit the determined property. A comparison of the different ICso values ​​for different mTOR activators then allows an assessment of the potential suitability of the mTOR activators for the treatment of TNBC. The lower the ICso value, the better suited the inhibitor may be.An expert recognizes that for actual clinical application, a whole range of other factors are relevant which are not captured by the ICso value.

[0124] A key difference between step (b) and step (c) of the method according to the invention is that step (b) is carried out in the presence of the mTOR activator, while step (c) is carried out in the absence of the activator.

[0125] In a preferred embodiment of the method according to the invention, the absence of the mTOR activator is the only difference between step (c) and step (b).

[0126] In another preferred embodiment of the method according to the invention, besides the absence of the mTOR activator, preferably mTORC1, a further difference between step (c) and step (b) is that step (c) is carried out in the presence of an inhibitor. The inhibitor in step (c) is preferably an HMG-CoA reductase inhibitor; more preferably a statin; and most preferably simvastatin.

[0127] In a preferred embodiment of the method according to the invention, the property of the cancer cells is determined in step (b) at several concentrations of the activator of mTOR, preferably of mTORC1.

[0128] The method according to the invention is suitable for testing known mTOR activators, preferably mTORC1, with regard to their suitability for treating TNBC, either as monotherapy or as adjuvant therapy, optionally in combination with (i) a cytotoxic substance, (ii) an anti-tumor substance, or (iii) immune cells directed against cancer cells. The specific TNBC is determined by the choice of the TNBC cancer cell line used.

[0129] In a preferred embodiment, the inventive method for providing the activator of mTOR, preferably mTORC1, comprises in step (a) the upstream screening of a substance library or other collection of several test substances with regard to their potential activating effect on mTOR, preferably mTORC1. Preferably, this upstream screening is based on a comparatively simple in vitro test for which no TNBC cancer cell lines are required. Preferably, this upstream screening is designed as a high-throughput screening (HTS) and is optionally performed automatically or semi-automatically.

[0130] Step (a) preferably includes the sub-steps (ai) Providing multiple test substances; (a2) Screening the test substances for their activating effect on mTOR, preferably mTORC1; (as) Selecting at least one screened test substance whose activating effect is stronger than the activating effect of at least one other screened test substance, and providing this selected test substance as an activator of mTOR, preferably mTORC1.

[0131] Preferably, in step (as), a screening using phospho-specific antibodies in lysed cells is performed as a measure of mTOR activation, preferably mTORC1, in the presence of each individual test substance separately under otherwise identical conditions. Kinases encoded downstream of mTOR, preferably mTORC1 (e.g., S6 kinase; see Promega CS366176), or mTOR itself (Rewity, Inc., Part # 64TORPEG & 64TORPEH HTRF PHOSPHO-MTOR (SER2448) DETECTION KITS), are preferably used.

[0132] In addition, the protein concentration of non-phosphorylated kinase is measured using specific antibodies. Subsequently, the ratio of phosphorylated to non-phosphorylated kipase is determined.

[0133] Alternatively, for step (a2), genetic approaches using mTOR, especially mTORC1, activity reports are also suitable, particularly via FRET-based detection of changes in the phosphorylation state of mTOR, preferably mTORC1, itself or preferably of the kinases encoded downstream of mTOR, preferably mTORC1, such as 4-EBP1 or P70-S6K, as recently published (X. Zhou et al., Curr Protoc Chem Biol. 2016 Dec 7;8(4):225-233. doi: 10.1002 / cpch.l 1). Furthermore, cellular mTOR-specific, preferably mTORC1-specific, biochemical assays are also suitable for step (a2).

[0134] Alternatively, the inhibition of protein-protein interaction substances of the domain C of FKBP1A or FKBP38, or other members of the FKBP family, with the FRB domain of mTORC1, or alternatively with Rheb, or alternatively with TGFBR1, can be specifically investigated in cell-based assays, cell-free protein-based assays, or via bioinformatics methods, using different throughputs, including high-throughput assays. The development and Performing such assays is known to those skilled in the art and includes methods such as: 1. Cell-based methods: Mammalian Two-Hybrid (M2H) assays, Bioluminescence Resonance Energy Transfer (BRET) / Förster Resonance Energy Transfer (FRET), Protein Complementation Assays (PCA), Co-Immunoprecipitation (Co-IP), Pull-Down Assays & Western Blot Proximity Ligation Assays (PLA), Knock-down or Knock-out of a binding partner, Reporter Gene Assays (indirect) and Confocal Microscopy / Live Cell Imaging, Cellular Thermal Shift Assay (CETSA), or 2. Cell-free methods: Fluorescence-luminescence-based assays (e.g.FP, FRET, HTFR, AlphaScreen), surface-based methods (SPR, BLI), attachment of two inactive fragments of a fluorescent protein (BiFC, bimolecular fluorescence complementation), calorimetric methods (ITC), thermal stability analyses (DSF / TSA), microscale thermophoresis (MST), structural elucidation of the complexes (with and without ligand, also individually with ligand) such as X-ray crystallography, small-angle X-ray scattering (SAXS), cryo-electron microscopy (cryo-EM) or NMR spectroscopy and mass spectrometric methods for complex analysis or such as 3. bioinformatic methods: virtual screening (structure-based virtual screening (docking of molecules to the PPI surface), ligand-based virtual screening (comparison with known inhibitors)), molecular docking & dynamics (simulation of ligand binding to PPI interfaces) and analysis of stability, including physics-based methods (e.g.all-atom MD simulations in explicit or implicit solvers) or statistical methods (e.g. Monte Carlo simulations)), free energy perturbation calculations (e.g.FEP), methods for protein complex prediction such as homology modeling or protein-protein docking, pharmacophore modeling (identification of common chemical features of known inhibitors), fragment-based drug design (FBDD, in silico; virtual combination of small molecular fragments to optimize binding), AI / machine learning-assisted methods (prediction of PPI inhibitors based on chemical and structural data), molecular similarity analysis (comparison of compound libraries with known PPI inhibitors), co-evolutionary analyses, binding hotspot mapping (identification of critical interaction sites ("hot spots") on the protein surface), de novo design of PPI inhibitors (algorithmic generation of new chemical structures to match the PPI surface), but also AI-based analogs of the aforementioned methods (e.g., Boltz2, AlphaFold, ColabFold, PoseX) (Wade M, Mendez J, Coussens NP, et al. Inhibition of Protein -Protein Interactions: Cell-Based Assays. 2017 Nov 20. In: Markossian S, Grossman A, Arkin M, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004; Arkin MR, Glicksman MA, Fu H, et al. Inhibition of Protein-Protein Interactions: Non-Cellular Assay Formats. 2012 Mar 18 [Updated 2012 Oct 1]. In: Markossian S, Grossman A, Arkin M, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eh Lilly & Company and the National Center for Advancing Translational Sciences; 2004).

[0135] In addition to the described protein-protein interaction of the domain C of FKBP1A or FKBP38 or other members of the FKBP family with the FRB domain of mTORC1, analogous to

[0134] also substances for their effect on the interaction of domain C of FKBP1A or FKBP38 or other FKBPs with the TGF-beta receptor 1 can be investigated. This allows for combinations with

[0134] specific inhibitors can be found that selectively inhibit one of the two PPIs (i.e., either specifically FKBPlA-mTORC1 or specifically FKBPlA-TGF-beta receptor 1), or both PPIs simultaneously.

[0136] Suitable reaction conditions such as medium, temperature, pH value, cofactors, etc., are known to a person skilled in the art and can be determined through standard routine experiments. Suitable substrates for the reaction are also known to a person skilled in the art.

[0137] The following examples serve to further illustrate the invention, but are not to be interpreted restrictively:

[0138] Example 1:

[0139] MDA-MB-231 and MDA-MB-436 cells were grown at a cell density of 4 x 0. AFive cells / ml were seeded onto collagen-coated plates in RPMI-1640 medium. After 16 h, the cells were washed three times with glucose- and serum-free solution and either (A) specifically according to the volume specifications from (H. Rinderknecht et al., Oxygen 2021, 1, 46-61. https: / / doi.org / 10.3390 / oxygenl010006) or (B) in a hypobaric chamber with glucose-free medium containing 5% FCS and 20 pM MHY1485 or an equivalent amount of DMSO for 16 h. Then, a pipette tip was used for scratching, and the cells were washed intensively four times with glucose- and serum-free solution. Microscopic images were then taken at t=0, the medium was added again as described above, and the cells were incubated with the drug or DMSO for 24 h. Microscopic images were then taken again, analyzed using ImageJ, and the cell-free areas of identical regions at t=24 were subtracted from t=0. The determined DMSO value was set as 100% migration and compared to the values ​​of the active ingredient.

[0140] The experimental results are shown in Figure 1.

[0141] Example 2:

[0142] Selection of representative microscopic images from Example 1. The experimental results are shown in Figure 2.

[0143] Example 3:

[0144] MDA-MB-231 and MDA-MB-436 cells were grown at a cell density of 4 x 0. AFive cells / ml were seeded onto collagen-coated plates in RPMI-1640 medium. After 16 h, the cells were washed once with PBS and incubated for 16 h with glucose-free medium containing 5% FCS and 20 pM MHY1485 or an equivalent amount of DMSO, according to the volume specifications from (H. Rinderknecht et al., Oxygen 2021, 1, 46-61. https: / / doi.org / 10.3390 / oxygenl010006). Then, analogous to Example 1, the cells were washed intensively four times with medium. Subsequently, medium was added again as described above, and the cells were incubated with the active ingredient or DMSO for 24 h. Afterward, the cellular ATP content was determined using CellTiter-GLO® (Promega), and cell viability was assessed.

[0145] The experimental results are shown in Figure 3.

[0146] Example 4:

[0147] MDA-MB-231 and MDA-MB-436 cells were grown at a cell density of 4 x 0. AFive cells / ml were seeded onto collagen-coated plates in RPMI-1640 medium. After 16 h, the cells were washed three times with glucose- and serum-free solution and incubated for 16 h with glucose-free medium containing 5% FCS and 60 pM 3-BDO or an equivalent amount of DMSO, specifically according to the volume specifications from (H. Rinderknecht et al., Oxygen 2021, 1, 46-61. https: / / doi.org / 10.3390 / oxygenl010006). A pipette tip was then used for scratching, and the cells were washed intensively four times with glucose- and serum-free solution. Microscopic images were then taken at t=0, the medium was added again as described above, and the cells were incubated with the drug substance or DMSO for 24 h. Microscopic images were then taken again, analyzed using ImageJ, and the cell-free areas of identical regions at t=24 were subtracted from t=0. The determined DMSO value was set as 100% migration and compared to the values ​​of the active ingredient.

[0148] The experimental results are shown in Figure 4.

[0149] Example 5:

[0150] PANCl cells were grown at a cell density of 4xO AFive cells / ml were seeded onto collagen-coated plates in RPMI-1640 medium. After 16 h, the cells were washed three times with glucose- and serum-free solution and incubated for 16 h according to the volume specifications from (H. Rinderknecht et al., Oxygen 2021, 1, 46-61. https: / / doi.org / 10.3390 / oxygenl010006) with glucose-free medium containing 5% FCS and 60 pM 3-BDO or an equivalent amount of DMSO. A pipette tip was then used for scratching, and the cells were washed intensively four times with glucose- and serum-free solution. Microscopic images were then taken at t=0, the medium was added again as described above, and the cells were incubated with the drug substance or DMSO for 24 h. Microscopic images were then taken again, analyzed using ImageJ, and the cell-free areas of identical regions at t=24 were subtracted from t=0. The determined DMSO value was set as 100% migration and compared to the values ​​of the active ingredient.

[0151] The experimental results are shown in Figure 5.

[0152] Example 6:

[0153] MDA-MB-231 cells were grown at a cell density of 4 x L0 A Five cells / ml were seeded onto collagen-coated plates in RPMI-1640 medium. After 16 h, the cells were washed with medium and incubated for 16 h with 20 pM MHY1485 or DMSO. Subsequently, the cells were lysed and the proteins separated by SDS-gel electrophoresis. Finally, the proteins were immobilized on a PVDF membrane by Western blot and the proteins phosphorylated p70-S6K, p70-S6K, and beta-actin were detected using specific primary antibodies and peroxidase-conjugated secondary antibodies.

[0154] The experimental results are shown in Figure 6.

[0155] Example 7:

[0156] MDA-MB-231 were cultured at a cell density of 4xO AFive cells / ml were seeded onto collagen-coated plates in RPMI-1640 medium. After 16 h, the cells were washed three times with glucose- and serum-free medium and incubated for 72 h according to the volume specifications (H. Rinderknecht et al., Oxygen 2021, 1, 46-61. https: / / doi.org / 10.3390 / oxygenl010006) in glucose- and serum-free medium with 20 pM MHY1485 or an equivalent amount of DMSO. The medium was then changed, and the cells were incubated for 72 h with the addition of the active ingredient or DMSO as described above. Cellular ATP levels were then determined using CellTiter-GLO® (Promega), and cell viability was assessed.

[0157] The experimental results are shown in Figure 7.

[0158] Example 8: MDA-MB-231 were produced in a cell density of lxl0 A5 cells / ml were seeded onto collagen-coated plates in RPMI-1640 medium. After 16 hours, the medium was changed and various concentrations of the medicinally optimized derivative 1 were added. The cells were incubated for 4 days, and subsequently, the cellular ATP content was determined using CellTiter-GLO® (Promega), and cell viability was assessed. The experimental results are shown in Figure 8.

[0159] Example 9:

[0160] The co-expression of PS6K and FKBP1A was analyzed using data analysis of the METABRIC database in “cBioPortal for Cancer Genomics”.

[0161] The results obtained are shown in Figure 9.

[0162] Example 10:

[0163] MDA-MB-231 cells were grown at a cell density of 4 x L0 A5 cells / ml were seeded onto collagen-coated plates in RPMI-1640 medium. After 16 h, the cells were washed four times with serum-free medium and incubated for 3 days with 60 pM 3-BDO or DMSO in serum-free Advanced DMEM / F12 medium (Gibco™, Art. No.: 12634010). Subsequently, the cell culture supernatant was removed, centrifuged for 2 minutes at 2000 g, and analyzed by TGF-beta-specific ELISA according to the manufacturer's instructions.

[0164] The experimental results are shown in Figure 10.

[0165] Example 11 MDA-MB-231 -tdTomato cells were grown at a cell density of lxl0 A 5 cells / ml were seeded in RPMI-1640 medium. After 16 hours, the medium was changed and the cells were incubated for 24 hours. Cells were incubated with 20 pM MHY or DMSO as a control. They were then washed once with PBS and detached with trypsin-free cell dissociation buffer, centrifuged with 300 g for 5 minutes, resuspended in PBS and centrifuged again with 300 g for 5 minutes, and finally resuspended in 100 µl PBS and injected into the perivitellin chamber. Fluorescence microscopy images were taken after 3 hours and after 3 days, and metastasis to the head and tail regions of the larva was quantified. The experimental results are shown in Figure 19.

[0166] Example 12: MDA-MB-231 cells were cultured in 96-well plates using a 3D Matrigel culture system. After embedding the cells in Matrigel domes, they were cultured with RPMI medium (containing 10% FCS, 1% PenStrep). After 24 hours, the cells were incubated for 4 days with 10 pM and 20 pM MHY1485 or DMSO as a control (three technical replicates per concentration). Cell viability was then determined by measuring cellular ATP content using the CellTiter-Glo® 3D assay according to the manufacturer's instructions. Relative luminescence intensities (RLU) were determined using a Luminome-ter microtiter plate analyzer and are shown in the diagram for the respective conditions. The experimental results are shown in Figure 21.

[0167] Dermal or synovial fibroblasts were seeded in a 6-well plate at a cell density of 50,000 cells / well in RPMI-1640 medium (containing 10% FBS, 1% PenStrep, 1% glutamate, 1% sodium mupyruvate). After 72 h of growth, stimulation was performed with 3-BDO or MHY1485 with or without the addition of TGF-β. After 24 h, mRNA was extracted and purified according to the manufacturer's instructions (NucleoSpin RNA isolation kit). Subsequently, the cDNA was reverse transcribed and quantified by quantitative PCR.

[0168] The experimental results are shown in Figure 24.

[0169] Dermal or synovial fibroblasts were seeded in a 96-well plate at a cell density of 10,000 cells / well in RPMI-1640 medium (containing 3% BSA, 1% PenStrep, 1% glutamate, 1% sodium mupyruvate). The following day, stimulation was performed with 3-BDO or MHY1485 with or without the addition of TGF-β. After 48 hours, the cells were fixed with 4% formalin for 20 minutes at room temperature. Following a wash with wash buffer (PBS / 0.1% Triton X-100), nonspecific binding sites were blocked with block buffer (3% BSA in PBS / 0.1% Triton X-100) for 1 hour at room temperature. Subsequently, incubation with primary antibody (1:2000, 0.947 mg / mL, anti-Col 1 A, abeam, ab 138492) in block buffer was performed overnight in the dark at 4°C. After five washes, incubation with secondary antibody in block buffer was performed for 1 hour at room temperature (Dako, polyclonal goat anti-rabbit immunoglobulin / HRP, P044801, 1:5000). This was followed by 5 Wash steps in wash buffer were followed by two wash steps with pure PBS. The color reaction was initiated by adding 50 pL of TMB substrate solution and stopped after 15 min by adding 50 pL of stop solution (2 N H₂SO₄). The results were read out at 450 nM in a plate reader with a reference wavelength of 595 nM. The results are shown in Figure 25.

[0170] Synthesis of mTOR derivatives Synthesis based on EE Symanek et al. Org. Synth. 2009, 86, 141-150 DOI:10.15227 / orgsyn.086.0141 120-2 1. 2,4,6-Trichloro-1,3,5-triazine (S)-3-Ethylmorpholines 2. NaHCO 3(aq) DIPEA 1. Acetone, 0°C, 1 h THF, reflux, 15.5 h 2. 0°C - rt, 15 h 1 5 4 1. 2,4,6-Trichloro-1,3,5-triazine 2. NaHCO 3(aq) 1. Acetone, 0°C, 3 h 2. 0°C - rt, 15 h -(morpholin-3-yl)ethan-1-one hydrochloride DIPEA 1,4-Dioxane, reflux, 42 h 120-3 / 4 1 -tert-Butyl-(S)-2-ethyl- piperazine carboxylate Morpholine DIPEA DIPEA THF, reflux, 17.5 h 1,4-Dioxane, reflux, 24-48 h Phenyl acetic acid HATU DMF, rt, 21 h DIPEA 29Ge30 2,4,6-Trichloro-1,3,5-triazine 3-(Hydroxymethyl)-morpholine DIPEA DIPEA THF, 0°C, 3.5 h THF, reflux, 19 h 20 MeOH, NaOH (aq) reflux, 4 h 1. Methanesulfonyl chloride, 1. DCM, rt, 2 h Morpholine DMF, DIPEA 2. ACN, 70°C, 16 h 5 M HCI in ethyl acetate Ethyl acetate

Claims

Patent claims:

1. An activator of mTOR (Mammalian Target of Rapamyciri) for use in the treatment of the - Triple-negative breast cancer (TNBC), - Pancreatic ductal carcinoma (PDAC), - Melanoma, - Glioblastoma (GBM), - Colorectal cancer (CRC), - Hepatocellular carcinoma (HCC), - Non-small cell lung cancer, - high-grade serous ovarian carcinoma (HGSOC), and / or - Prostate cancer; preferably triple negative breast cancer (TNBC).

2. The activator for use according to claim 1, wherein the - Triple negative breast cancer (TNBC) of the “mesenchymal stem-like” or “mesenchymal” subtype, - Ductal pancreatic carcinoma (PDAC) of the “squamous / basal-like” subtype, - Melanoma is the invasive, dedifferentiated phenotype, - Glioblastoma (GBM) of the “mesenchymal subtype” according to TCGA classification is, - Colorectal carcinoma (CRC) of the mesenchymal subtype is, - Hepatocellular carcinoma (HCC) exhibits high EMT and TGF-beta signaling, - Non-small cell lung cancer is basal-like / mesenchymal adenocarcinoma or squamous cell carcinoma, - high-grade serous ovarian carcinoma (HGSOC) of the mesenchymal subtype, and / or - prostate carcinoma of the neuroendocrine, or the basal-like / EMT-high subtype, or the prostate cancer subtype 1.

3. The activator for use according to claim 1 or 2, which is an activator of mTORC1 (mTOR complex 1).

4. The activator for use according to one of the preceding claims, which binds specifically to the domain C of FKBP1A or FKBP38 or other members of the FKBP family.

5. The activator for use according to one of the preceding claims, which binds specifically to the domain C of FKBP1A or FKBP38 or other members of the FKBP family and additionally prevents interaction with the FRB domain of mTOR and / or Rheb.

6. The activator for use according to one of the preceding claims, which binds specifically to the domain C of FKBP1A or FKBP38 or other members of the FKBP family and additionally prevents interaction with the FRB domain of mTOR and additionally leaves interaction with TGFBR1 unaffected or enhances it.

7. The activator for use according to one of the preceding claims, wherein the migration of tumor cells in the tumor microenvironment is reduced.

8. The activator for use according to any of the preceding claims, wherein TNBC is characterized by cells of the mesenchymal stem-like subtype, the mesenchymal subtype, the BRCA1-mutated subtype or the BRCA2-mutated subtype.

9. The activator for use according to any of the preceding claims, wherein the activator is of mTORMHY1485 10. The activator for use according to any one of the preceding claims, wherein the mTOR activator is a conjugate of MHY1485 with a peptide or antibody; preferably a compound of general formula (I) (I) where RI stands for a peptide or antibody; and R2 and R3 each independently represent a side chain of a naturally occurring alpha-amino acid.

11. The activator for use according to any one of the preceding claims, wherein the activator of mTOR TNBC-specifically activates mTOR via FKBP 1 A; preferably wherein the activator of mTOR 3-BDO 12. The activator for use according to any one of the preceding claims, wherein the mTOR activator is a conjugate of 3-BDO with a peptide or antibody.

13. The activator for use according to any of the preceding claims, wherein the treatment is performed in conjunction with concomitant chemotherapy or immunotherapy for triple-negative breast cancer.

14. A compound, in particular an activator of mTOR (Mammalian Target of Rapamycin), especially an activator of mTORC1, which is selected from the group consisting of HH0010-WO HH0010-WO HH0010-WO HH0010-WO HH0010-WO HH0010-WO £9 HH0010-WO HH0010-WO HH0010-WO 99 HH0010-WO 89 HH0010-WO oz HH0010-WO HH0010-WO HH0010-WO HH0010-WO HH0010-WO 9L HH0010-WO HH0010-WO 8 HH0010-WO Z8 HH0010-WO £8 HH0010-WO 98 OA1-OIOOHH Z6 HH0010-WO £6 HH0010-WO 96 HH0010-WO 86 and / or a physiologically acceptable salt thereof.

15. A compound, in particular an activator of mTOR (Mammalian Target of Rapamycin), in particular an activator of mTORC1 according to claim 14, which is selected from the group consisting of F OH HH0010-WO 106 HH0010-WO 801 110 HH0010-WO 111 HH0010-WO HH0010-WO to HH0010-WO HH0010-WO HH0010-WO HH0010-WO 9ZI HH0010-WO HH0010-WO O HH0010-WO and / or a physiologically acceptable salt thereof.

16. The compound according to claim 14 or claim 15 for use as a drug.

17. The compound according to claim 14, 15 or 16 for use in the treatment of triple-negative breast cancer (TNBC), - Pancreatic ductal carcinoma (PDAC), - Melanoma, - Glioblastoma (GBM), - Colorectal cancer (CRC), - Hepatocellular carcinoma (HCC), - Non-small cell lung cancer, high-grade serous ovarian carcinoma (HGSOC), and / or Prostate cancer; preferably triple negative breast cancer (TNBC).

18. The compound for the application according to claim 17, wherein the - Triple negative breast cancer (TNBC) of the “mesenchymal stem-like” or “mesenchymal” subtype, - Ductal pancreatic carcinoma (PDAC) of the “squamous / basal-like” subtype, - Melanoma is the invasive, dedifferentiated phenotype, - Glioblastoma (GBM) of the “mesenchymal subtype” according to TCGA classification is, - Colorectal carcinoma (CRC) of the mesenchymal subtype is, - Hepatocellular carcinoma (HCC) exhibits high EMT and TGF-beta signaling, - Non-small cell lung cancer is basal-like / mesenchymal adenocarcinoma or squamous cell carcinoma, - high-grade serous ovarian carcinoma (HGSOC) of the mesenchymal subtype, and / or - prostate carcinoma of the neuroendocrine, or the basal-like / EMT-high subtype, or the prostate cancer subtype 1.

19. A therapeutic agent comprising at least one compound according to claim 14 or claim 15, in particular MHY1485, and especially for fibrotic diseases.

20. A method for finding activators of mTOR, in particular mTORC1, suitable for the treatment of TNBC, comprising the steps (a) Provide an mTOR activator; (b) Incubating cancer cells of a TNBC cancer cell line with the activator of mTOR, and determining a property of the cancer cells; (c) Incubating cancer cells of the same TNBC cancer cell line as in step (b) in the absence of the mTOR activator and determining the same property of the cancer cells as in step (b) under the same conditions as step (b); (d) Comparing the determined property of the cancer cells according to steps (b) and (c).

21. The method of claim 20 for finding mTOR activators suitable for the adjuvant treatment of TNBC, wherein step (b) is performed in the presence of (i) a cytotoxic substance, (ii) an anti-tumor substance, or (iii) anti-cancer immune cells; and wherein step (c) is performed in the presence of the same substance or immune cells as in step (b).

22. The method according to claim 20 or 21, wherein the determined property of the cancer cells is their viability.

23. The method according to claim 21 or 22, wherein - the (i) cytotoxic substance is selected from the group consisting of Taxol, Docetaxel, Cisplatin, Carboplatin, Cytocalasin B, Gramicidin D, Ethidium bromide, Emetine, Mitomycin, Etopside, Tenopside, Vincristine, Vinblastine, Colchicine, Doxorubicin, Daunorubicin, Dihydroxyantracindione, Mitoxantrone, Mithramycin, Actinomycin, d,l-Dehydrotestosterone, Glycocorticoids, Procaine, Tetracaine, Lidoceine, Propranolol and Puromycin; preferably Taxol; - the (ii) anti-tumor substance is selected from the group consisting of targeted anti-tumor agents, anti-hormone therapy agents and immunostimulatory antibodies; or - the (iii) immune cells directed against cancer cells are modified cytotoxic T cells.

24. The method according to any one of claims 20 to 23, wherein step (a) comprises the sub-steps (ai) providing several test substances; (a2) Screening the test substances for their activating effect on mTOR; (as) Selecting at least one screened test substance whose activating effect is stronger than the activating effect of at least one other screened test substance and providing this selected test substance as an activator of mTOR.

25. The method according to any one of claims 20 to 24, comprising at least one cell-based method, cell-free method, bioinformatic method, and / or combinations thereof, which may optionally further comprise.

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