N-benzyl-alpha-aminoamides as inhibitors of the anaphase-promoting complex / cyclosome (APC / C)

Abstract

The present invention relates to compounds of formula (I), which are inhibitors of anaphase-promoting complex / cyclosome (APC / C) function, wherein R and R are as disclosed herein. These compounds are useful in the treatment of cancer, particularly breast cancer. [Formula 1] JPEG2023554404000047.jpg24169

Description

Detailed Description of the Invention

[0001] The present invention relates to compounds of formula I, which are inhibitors of the anaphase-promoting complex / cyclosome (APC / C), and pharmaceutical compositions thereof for use in the treatment of cancer, particularly breast cancer. Additionally, the present invention relates to compositions of compounds of formula I administered in combination with proTAME.

[0002] [Background technology] Cancer is a disease that affects many people and is a leading cause of death in humans. Cancer is characterized, in part, by uncontrolled cell proliferation (see Golias, CH., Charalabopoulos, A., Charalabopoulos, K. Cell proliferation and cell cycle control: a mini review. Int J Clin Pract, 2004, 58, 12, 1134-1141). Therefore, compounds that disrupt cell division (e.g., mitosis) may be part of the cancer chemotherapy armament. For example, some current mitotic disruptors in clinical use, such as paclitaxel, are thought to target microtubules and thus disrupt mitotic spindle function (see Wang, TH., Hsin-Shih Wang, MD., Soong, YK. Paclitaxel-Induced Cell Death. Cancer 1, 2000, 88 (11)). Indeed, prolonged disruption of mitosis can cause cells to undergo apoptosis. However, some tumors develop resistance to microtubule-disrupting drugs by inactivating the spindle assembly checkpoint (SAC), a highly complex signaling network orchestrated by several proteins, including Cdc20, that ensures accurate and timely segregation of chromosomes during cell division. Recruitment of SAC proteins to kinetochores, the sites for attachment of chromosomes to the microtubule polymers that separate sister chromatids during cell division, is essential for full activity and optimal function of the SAC.Binding of Cdc20 to BubR1 mediates its recruitment to kinetochores, while binding of Cdc20 to the anaphase-promoting complex / cyclosome (APC / C) regulates the interaction of APC / C with specific ubiquitin substrates for subsequent degradation by the proteasome during cell cycle progression, thus governing the cell cycle forward in a unidirectional manner (Meadows JC, Millar JB. Sharpening the anaphase switch. Biochem Soc Trans 2015, 43:19-22; Izawa D, Pines J. The mitotic checkpoint complex binds a second CDC20 to inhibit active APC / C. Nature 2015, 517: 631-34; Di Fiore B. et al. The ABBA motif binds APC / C activators and is shared by APC / C substrates and regulators. Dev Cell 2015, 32:358-72; Zich J, Hardwick KG. Getting down to the phosphorylated 'nuts and bolts' of spindle checkpoint signaling. Trends Biochem Sci. 2010, 35:18-27; and WO 2012 / 149266). To enable the development of more effective therapeutic approaches for breast tumors, it is necessary to develop novel chemical inhibitors that affect Cdc20 protein-protein interactions that are important for SAC function, including APC / C regulation, in cancer cells in which Cdc20 is abnormally overproduced and in tumors associated with abnormal SAC signaling and chromosome segregation defects. Cdc20 protein may function as an oncoprotein to promote breast cancer progression.To date, the compound apsin (Apcin) in combination with ProTAME is the only cancer therapeutic targeting strategy (see Lixia Wanga, Jinfang Zhangb, Lixin Wanb, Xiuxia Zhoua, Zhiwei Wanga, Wenyi Wei. Targeting Cdc20 as a novel cancer therapeutic strategy. Pharmacol Ther. 2015; 151: 141-151; PCTUS2011050203; and US 2013 / 0230458). Apsin (an APC / C inhibitor) binds to Cdc20 and prevents APC / C substrate recognition, thereby inhibiting ubiquitination of APC / C substrates.

[0003] Therefore, there is a need to dispose of novel inhibitors of APC / C for the treatment of cancer, particularly for the treatment of breast cancer.

[0004] [Summary of the Invention] A first aspect of the present invention relates to a compound of formula I or a pharmaceutical salt thereof:

[0005] [ka]

[0006] During the ceremony, R1 is H, aryl, C1-C 20 alkyl, -CF3, CCl3 or -CBr3; R2 represents C1-C6 alkyl optionally substituted with -NH2 or Cy1; Cy1 represents a phenyl group (-Ph), optionally substituted with -OH.

[0007] Thus, the compounds of formula I may be in the free or salt form. Examples of anions of salts of compounds of formula I include, in particular, the anion chloride (Cl - ) and anionic TFA(CF3CO2 - ) is included.

[0008] Some of the compounds of formula I may have chiral centers which can give rise to various stereoisomers, and the present invention relates to each of these stereoisomers as well as mixtures thereof.

[0009] The R1 group of the compound of formula I can be in any available ortho, meta, or para position.

[0010] In another embodiment, the invention relates to compounds of formula I as defined above, wherein Cy1 represents a phenyl group (-Ph) substituted in the para position by -OH.

[0011] In another embodiment, the present invention relates to compounds of formula I as defined above, wherein R1 is -CF3, CCl3 or -CBr3, preferably R1 is -CF3.

[0012] In another embodiment, the present invention relates to compounds of formula I as defined above, wherein R2 is C1-C4 alkyl substituted with -NH2.

[0013] In another embodiment, the present invention relates to compounds of formula I as defined above, wherein R2 is a group of formula R2-a:

[0014] [ka]

[0015] .

[0016] In another embodiment, the present invention relates to compounds of formula I as defined above, wherein R2 is a group of formula R2-b:

[0017] [ka]

[0018] .

[0019] In another embodiment, the present invention relates to a compound of formula I,

[0020] [ka]

[0021] The present invention relates to compounds of formula I as defined above, which are selected from:

[0022] In another embodiment, the present invention relates to a compound of formula I,

[0023] [ka]

[0024] The present invention relates to compounds of formula I as defined above, which are selected from:

[0025] Another embodiment of the present invention relates to a pharmaceutical composition comprising a compound of formula I as defined above or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0026] The compound of formula I or a pharmaceutically acceptable salt thereof can be administered alone or in combination with a prodrug, which is preferably pro-N-4-tosyl-L-arginine methyl ester (proTame).

[0027] Therefore, another aspect of the present invention relates to a pharmaceutical composition comprising a compound of formula I as defined above in combination with a further compound selected from pro-N-4-tosyl-L-arginine methyl ester (proTame).

[0028] Another aspect of the present invention relates to a compound of Formula I, or a pharmaceutically acceptable salt thereof, for use in therapy.

[0029] Another aspect of the present invention relates to a compound of formula I for use in the treatment of cancer:

[0030] [ka]

[0031] During the ceremony, R1 is H, aryl, C1-C 20 alkyl, -CF3, CCl3, -CBr3, or -Cl3; R2 represents C1-C6 alkyl optionally substituted with -NH2 or Cy1; Cy1 represents a phenyl group (-Ph), optionally substituted with -OH.

[0032] In another embodiment, the present invention relates to a compound of formula I for the use as defined above, said compound of formula I being

[0033] [ka]

[0034] is selected from.

[0035] In another embodiment, the present invention relates to a compound of formula I for the use as defined above, said compound of formula I being

[0036] [ka]

[0037] is selected from.

[0038] In another embodiment, the invention relates to a compound of formula I for use as defined above for the treatment of breast cancer.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. Throughout the specification and claims, the term "comprises" and variations thereof are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon examination of the description or can be learned by practicing the present invention. The following examples and figures are provided for illustrative purposes and are not intended to limit the present invention.

[0040] BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the 3D structure of the Cdc20-o-TFB-Tyr complex, the contact of o-TFB-Tyr Cdc20 and apsin (Apcin) complex, and o-TFB-Tyr-Cdc20.

[0041] Figure 2 shows the cytotoxicity analysis by MTT assay of compounds tested against triple-negative breast cancer cells, using the Cdc20 inhibitor apsin for comparison. Cytotoxicity analysis of asynchronous HCC-38 triple-negative breast cancer cells after 24 hours of treatment with the selected third-generation compound o-TFB-Tyr. HCC-38 cells were exposed to 25 μM and 5 μM concentrations of compound o-TFB-Tyr (Panel A) and 1 μM concentration of compound o-TFB-Tyr (Panel B), alone and in combination with proTAME. The negative control used was untreated cells (medium), while the positive controls used were 25 μM apsin alone, 10 μM proTAME alone, and 25 μM apsin in combination with 10 μM proTAME. Data were analyzed by one-way ANOVA and Dunnett's test using all columns compared to the negative control column (medium), p<0.001. Experiments were performed in duplicate.

[0042] Figure 3 shows the cytotoxicity analysis of compound o-TFB-Tyr at concentrations of 5 μM, 1 μM, 0.5 μM, and 100 nM. HCC-38 cancer cells were exposed to compound o-TFB-Tyr alone and to compound o-TFB-Tyr in combination with proTAME. The negative control used was untreated cells (medium), while the positive control used was 25 μM apsin in combination with 10 μM proTAME. Data were analyzed by one-way ANOVA and Dunnett's test, p<0.001, using all columns compared to the negative control column (medium). Replicate experiments were performed.

[0043] Figure 4 shows the principle of the cell membrane permeability test, which is part of the ADME test.

[0044] Figure 5 is a graph showing the relative cytotoxicity of compound o-TFB-Tyr at various concentrations in HeLa cells compared with reversine, a small compound inhibitor of Mps1 kinase and an upstream regulator of SAC, and apsin, a small binder of Cdc20 and an inhibitor of APC / C activation by Cdc20. This study confirmed that compound o-TFB-Tyr exhibited higher cytotoxic effects against different types of cancers originating from various tissues.

[0045] Figure 6 compares the relative cytotoxicity of compound o-TFB-Tyr in HeLa cells with the compounds apsin, m-TFB-Tyr, p-TFB-Tyr, and o-TFB-Lys at the same concentrations. Compounds o-TFB-Tyr, m-TFB-Tyr, and p-TFB-Tyr are closely related in terms of chemical structure. The comparative study confirmed that compound o-TFB-Tyr exhibited a higher cytotoxic effect against cancer cells in culture. Thus, we highlight key stereochemical features of o-TFB-Tyr and its isomers m-TFB-Tyr and p-TFB-Tyr that explain the anticancer activity of o-TFB-Tyr and structurally related molecules. The comparative analysis also demonstrated that the chemical nature of the R2 residue in the claimed compounds o-TFB-Tyr, m-TFB-Tyr, p-TFB-Tyr, and o-TFB-Lys explains the cytotoxicity of these molecules against cancer cells.

[0046] Figure 7 shows a clonogenic assay of HCC-38 cells treated with DMSO (1.2% v / v), the Mps1 kinase inhibitor reversine, the Cdc20 binder apsin, and the compound o-TFB-Tyr. After 12 days of incubation at 37°C, clones were stained. HCC38 cells were treated with the compounds for 24 hours, after which the medium was changed every 48 hours. In this assay, fewer triple-negative breast cancer cell clones were consistently observed in cells treated with reversine and o-TFB-Tyr, confirming the desired cytotoxicity of the latter compound in cancer cells.

[0047] In Figure 8, each well of the clonogenic assay was then scanned using an Axiozoom Zeiss Axioplan fluorescence microscope equipped for DIC imaging and fluorescence imaging and analyzed using ImageJ2 image processing software (Fiji). Here, a representative image generated by the image processing software is shown.

[0048] Figure 9 is a Western blot showing that the compound o-TFB-Tyr causes inhibition of cyclin B ubiquitination by APC / C. As a result, non-ubiquitinated cyclin B escapes degradation by the proteasome. This analysis also shows that pepsin is relatively less effective than the compound o-TFB-Tyr as an antagonist of Cdc20-mediated APC / C activation. In this experiment, HCC-38 cells with a density of 200,000 cells per well were used.

[0049] 〔Example〕 Computing Present a technique (Maestro Suite, Schrodinger) for docking a flexible ligand having chemical structure I within the binding site of Cdc20 protein (rigid) (Figure 1). This method is based on a series of pre-generated structures for the compounds (o-, m-, p-TFB-aa, ligands), and optimization based on the final flexibility gradient of the ligands within the binding site of the protein. The binding site of the receptor is defined as a cubic box, and the compound is placed at the center of the binding pocket. In all cases, the box is large enough to ensure the independence of the docking results from the definition of the binding site. The docking parameters (score docking kcal / mol) give an idea of the best compound-protein complex.

[0050] <Synthesis of o-, m- or p-trifluorobenzyl-L-amino acid derivatives (o-TFB-Tyr, o-TFB-Lys and m-TFB-Tyr, p-TFB-Tyr)> 1. Protection of the amino group of L-amino acid using the t-butoxycarbonyl group:

[0051]

Chemical formula

[0052] L-amino acids were suspended in a 1:1 mixture of water and dioxane (Procedure A) or water and 2-propanol (Procedure B) under argon. Sodium hydroxide (Procedure A) or potassium hydroxide (Procedure B) in water was then added with constant stirring. After the addition of di-tert-butyl carbonate, the reaction mixture was stirred at room temperature. Upon completion of the reaction, the solvent was removed under reduced pressure to half its volume, and potassium hydrogen sulfate was then added until the solution reached pH 2. The reaction mixture was extracted with ethyl acetate, and the organic phase was washed with saturated sodium chloride solution and water. The solution was dried over sodium sulfate and then filtered. The filtrate was concentrated to dryness. We used the product in the next reaction without further purification.

[0053] [Table 1]

[0054] 2. Coupling reaction of Boc-L-amino acid with trifluorobenzylamine 2.1 Use of 2-trifluorobenzylamine as a coupling reagent

[0055] [ka]

[0056] Boc-L-amino acids were dissolved in dry DMF under argon. Then, diisopropylethylamine (Procedure A) or 2,4,6-collidine (Procedure B) and HBTU were added sequentially at room temperature (rt) and stirred for 30 minutes. Trifluorobenzylamine was then added at room temperature, and the reaction was stirred overnight at room temperature. Upon completion of the reaction, the solvent was removed under reduced pressure. The crude product was then purified by silica gel chromatography.

[0057] [Table 2]

[0058] (2S)-2,6-bis[(tert-butoxycarbonyl)amino]-N-[2-(trifluoromethyl)benzyl]hexanamide (3)

[0059] [ka]

[0060] 1 H-NMR (500 MHz, CDCl3): δ 7.63 (1H, d, J = 7.5 Hz), 7.54-4.47 (2H, m), 7.36 (1H, t, J = 7.5 Hz), 6.65 (1H, bs), 5.17 (1H, bs), 4.65-4.56 (2H, m), 4.07 (1H, bs), 3.09 (2H, m), 1.89-1.81 (1H, m), 1.69-1.59 (1H, m), 1.53-1.44 (2H, m), 1.42 (9H, s), 1.40 (9H, s), 1.40-1.39 (2H, m) ppm. 13 C-NMR (125 MHz, CDCl3): δ 172.1, 156.2, 155.8, 136.4, 132.3, 130.1, 128.0 (q, J = 30.9 Hz), 127.5, 125.9 (q, J = 5.8 Hz), 124.4 (q, J = 273.9 Hz), 80.2, 79.2, 54.6, 39.9 (q, J = 2.5 Hz), 39.7, 31.5, 29.7, 28.4, 28.2, 22.6 ppm. + ): m / z 504 (M+H) + , 526 (M+Na) + IR (KBr): ν 3318, 3080, 2978, 2934, 2867, 1693, 1610, 1525, 1457, 1392, 1367, 1315, 1250, 1166, 1121, 1059, 1039, 867, 769, 655cm -1 .

[0061] (2S)-2-[(tert-butoxycarbonyl)amino]-3-{4-[(tert-butoxycarbonyl)hydroxy]phenyl}-N-[2-(trifluoromethyl)benzyl]propanamide (4)

[0062] [ka]

[0063] 1 H-NMR (500 MHz, CDCl3): δ 7.60 (1H, d, J = 7.6 Hz), 7.47 (1H, t, J = 7.8 Hz), 7.37-7.30 (2H, m), 7.14 (2H, d, J = 8.4 Hz), 7.03 (2H, d, J = 8.4 Hz), 6.34 (1H, bs), 5.03 (1H, bs), 4.57 (1H, dd, J = 15.6, 6.4 Hz), 4.52 (1H, dd, J = 15.6, 6.4 Hz), 4.34 (1H, bs), 3.11-3.00 (2H, m), 1.55 (9H, s), 1.38 (9H, s) ppm. 13 C-NMR (125 MHz, CDCl3): δ 171.0, 155.4, 151.8, 150.0, 136.1, 133.9, 132.2, 130.2, 130.1, 128.0 (q, J = 31.3 Hz), 127.5, 125.9 (q, LRMS (EI): m / z 538 (M + , 0.1), 321 (100), 231 (6), 159 (22), 136 (21).

[0064] (2S)-2-[(tert-butoxycarbonyl)amino]-3-[4-(hydroxy)phenyl]-N-[2-(trifluoromethyl)benzyl]propanamide (5)

[0065] [ka]

[0066] 1 H-NMR (400 MHz, CDCl3): δ 7.58 (1H, d, J = 7.6 Hz), 7.43 (1H, t, J = 7.6 Hz), 7.32 (1H, t, J = 7.6 Hz), 7.22 (1H, bs), 6.93 (2H, d, J = 8.1 Hz), 6.66 (2H, d, J = 8.1 Hz), 6.41 (1H, t, J = 6.2 Hz), 5.19 (1H, bs), 4.59 (1H, dd, J = 15.4, 6.1 Hz), 4.46 (1H, dd, J = 15.5, 5.6 Hz), 4.31 (1H, bs), 2.97 (1H, J = 14.3, 6.5 Hz), 2.92 (1H, dd, J = 14.3, 7.8 Hz), 1.39 (9H, s) ppm. 13 C-NMR (100 MHz, CDCl3): δ 171.5, 155.6, 155.1, 135.9, 132.2, 130.3, 130.0 (q, J = 3.3 Hz), 127.9 (q, J = 29.7 Hz), 127.8, 127.5, LRMS (EI): m / z 438 (M + , 0.5), 321 (100), 231 (5).

[0067] 2.2 Use of 3- or 4-trifluorobenzylamine as a coupling reagent

[0068] [ka]

[0069] Boc-L-tyrosine was dissolved in dry DMF under argon. Then, 2,4,6-collidine and HBTU were added sequentially at room temperature and stirred for 30 minutes. Then, 3-trifluorobenzylamine (Procedure A) or 4-trifluorobenzylamine (Procedure B) was added at room temperature, and the reaction mixture was stirred overnight at room temperature. Upon completion of the reaction, the solvent was removed under reduced pressure. The crude product was purified by silica gel chromatography.

[0070] [Table 3]

[0071] (2S)-2-[(tert-butoxycarbonyl)amino]-3-{4-[(tert-butoxycarbonyl)hydroxy]phenyl}-N-[3-(trifluoromethyl)benzyl]propanamide (6)

[0072] [ka]

[0073] 1 H-NMR (500 MHz, CDCl3): δ 7.48 (1H, d, J = 7.5 Hz), 7.43 (1H, s), 7.61 (1H, t, J = 7.5 Hz), 7.27 (1H, bs), 7.15 (2H, d, J = 8.3 Hz), 7.04 (2H, d, J = 8.3 Hz), 6.58 (1H, bs), 5.16 (1H, bs), 4.36 (3H, s), 3.05 (2H, s), 1.54 (9H, s), 1.36 (9H,s) ppm. 13C-NMR (125 MHz, CDCl3): δ 171.3, 155.5, 151.8, 150.0, 138.8, 134.0, 130.9, 130.7 (q, J = 32.9 Hz), 130.2, 129.1, 124.3, 124.2, LRMS (EI): m / z 321 (100), 231 (6), 159 (34), 136 (22).

[0074] (2S)-2-[(tert-butoxycarbonyl)amino]-3-[4-(hydroxy)phenyl]-N-[3-(trifluoromethyl)benzyl]propanamide (7)

[0075] [ka]

[0076] 1 H-NMR (500 MHz, CDCl3): δ 7.92 (1H, bs), 7.47 (1H, d, J = 7.7 Hz), 7.41 (1H, s), 7.36 (1H, t, J = 7.7 Hz), 7.20 (1H, bs), 6.97 (1H, bs), 6.95 (2H, d, J = 8.4 Hz), 6.65 (2H, d, J = 8.4 Hz), 5.38 (1H, bs), 4.38 (1H, dd, J = 15.5, 5.2 Hz), 4.31 (1H, dd, J = 15.5, 5.6 Hz), 4.24 (1H, q, J = 7.1 Hz), 2.91 (2H, d, J = 7.1 Hz), 1.36 (9H, s) ppm. 13C-NMR (125 MHz, CDCl3): δ 171.9, 155.7, 155.4, 138.7, 130.9 (×2C), 130.3, 129.0, 127.4, 124.2, 124.1 (q, J = 3.1 Hz), 123.9 (q, J = 272.3 Hz), 115.4, 80.4, 56.1, 42.8, 37.7, 28.1 ppm. + , 0.3), 321 (100), 231 (3), 159 (54), 136 (24).

[0077] (2S)-2-[(tert-butoxycarbonyl)amino]-3-{4-[(tert-butoxycarbonyl)hydroxy]phenyl}-N-[4-(trifluoromethyl)benzyl]propanamide (8)

[0078] [ka]

[0079] 1 H-NMR (300 MHz, CDCl3): δ 7.55 (2H, d, J = 8.1 Hz), 7.20 (2H, d, J = 8.5 Hz), 7.18 (2H, d, J =8.1 Hz), 7.07 (2H, d, J = 8.5 Hz), 6.23 (1H, bs), 5.00 (1H, bs), 4.41 (2H, d, J = 6.3 Hz), 4.32 (1H, q, J = 7.1 Hz), 3.14 (1H, dd J = 13.7, 7.1 Hz), 3.02 (1H, dd, J = 13.7, 7.1 Hz), 1.57 (9H, s), 1.41 (9H, s) ppm. 13C-NMR (125 MHz, CDCl3): δ 171.1, 155.4, 151.9, 150.1, 141.7, 133.9, 130.2, 129.6 (q, J = 33.8 Hz), 127.7, 125.6 (q, J = 3.7 Hz), LRMS (EI): m / z 538 (M + , 0.1), 321 (100), 231 (4), 159 (20), 136 (15).

[0080] (2S)-2-[(tert-butoxycarbonyl)amino]-3-[4-(hydroxy)phenyl]-N-[4-(trifluoromethyl)benzyl]propanamide (9)

[0081] [ka]

[0082] 1 H-NMR (500 MHz, CDCl3): δ 7.45 (2H, d, J = 7.0 Hz), 7.35 (1H, bs), 7.11-7.04 (3H, m), 6.93 (2H, d, J = 8.3 Hz), 6.65 (2H, d, J = 8.3 Hz), 5.56 (1H, bs), 4.37 (1H, dd, J = 15.5, 6.0 Hz), 4.27-4.14 (2H, m), 2.85 (2H, d, J = 7.0 Hz), 1.33 (9H, s) ppm. 13C-NMR (125 MHz, CDCl3): δ 172.0, 155.6, 152.0, 141.8, 130.2, 129.3 (q, J = 34.5 Hz), 127.6, 127.1, 125.2, (q, J = 4.2 Hz), 124.0 (q, J = 272.0 Hz), 115.3, 80.2, 56.0, 42.6, 37.7, 28.0 ppm. + , 0.5), 321 (100), 231 (5), 159 (25), 136 (25).

[0083] 3. Deprotection reaction of Boc-trifluorobenzyl amino acid derivatives

[0084] [ka]

[0085] The Boc-trifluorobenzylamide derivative was dissolved in a mixture of CHCl:TFA [2:1] under argon at room temperature, and the solution was stirred at this temperature. Upon completion of the reaction, the solvent was removed under reduced pressure. The crude reaction product was purified by two procedures: 1) Procedure A: reversed-phase chromatography using a reveleris cartridge SRC C18; 2) Procedure B: anion-exchange chromatography using Dowex 50WX4 resin, followed by silica gel chromatography.

[0086] [Table 4]

[0087] (2S)-2,6-Diamino-N-[2-(trifluoromethyl)benzyl]hexanamide (o-TFB-Lys-TFA) (Procedure A) (10)

[0088] [ka]

[0089] 1 H-NMR (500 MHz, D2O): δ 7.79 (1H, d, J = 7.8 Hz), 7.65 (1H, t, J = 7.5 Hz), 7.54 (1H, d, J = 7.5 Hz), 7.52 (1H, t, J = 7.8 Hz), 4.70 (1H, d, J = 15.4 Hz), 4.57 (1H, d, J = 15.4 Hz), 4.04 ( 1H, t, J = 6.6 Hz), 2.94 (2H, t, J = 7.8 Hz), 1.98 (2H, m), 1.72-1.63 (2H, m), 1.43-1.34 (2H, m) ppm. 13 C-NMR (125 MHz, D2O): δ 170.1, 135.4 (q, J = 1.7 Hz), 133.2, 130.7, 128.8, 128.1 (q, J = 30.7 Hz), 127.0 (q, J = 6.0 Hz), 125.0 (q, J = 274.4 Hz), 53.6, 41.2 (q, J = 2.8 Hz), 39.6, 31.0, 26.9, 21.8 ppm. LRMS (ESI-ES + ): m / z 304 (M+H) + , 326 (M+Na) + IR (KBr): ν 3080, 2882, 2824, 1673, 1433, 1316, 1203, 1128, 1061, 1040, 840, 800, 770, 723 cm -1 .

[0090] (2S)-2-amino-N-[2-(trifluoromethyl)benzyl]-3-[4-(hydroxy)phenyl]propanamide (o-TFB-Tyr-TFA) (Procedure A) (11)

[0091] [ka]

[0092] 1H-NMR (500 MHz, CD3OD): δ 7.72 (1H, d, J = 7.7 Hz), 7.56 (1H, t, J = 7.5 Hz), 7.50 (1H, t, J = 7.7 Hz), 7.13 (1H, d, J = 7.5 Hz), 6.97 (2H, m), 6.66 (2H, m), 4.64 (1H, d, J =15.3 Hz), 4.28 (1H, d, J = 15.3 Hz), 4.14 (1H, dd, J = 10.0, 5.9 Hz), 3.17 (1H, dd, J = 13.6, 5.9 Hz), 2.94 (1H, dd, J = 13.6, 10.0 Hz) ppm. 13 C-NMR (125 MHz, CD3OD): δ 167.9, 154.0, 133.6, 131.6, 129.7, 129.5, 127.2, 126.5 (q, J = 30.1 Hz), 125.3 (q, J = 5.1 Hz), 124.4, 123.4 (q, J = 273.6 Hz), 114.8, 53.7, 39.2, 35.2 ppm. LRMS (ESI-ES + ): m / z 339 (M+H) + , 361(M+Na) + , 699 (2M+Na) + IR (KBr): ν 3416, 3089, 2928, 1677, 1615, 1518, 1439, 1370, 1317, 1204, 1122, 1061, 1041, 840, 801, 770, 723 cm -1 .

[0093] (2S)-2-amino-N-[2-(trifluoromethyl)benzyl]-3-[4-(hydroxy)phenyl]propanamide (o-TFB-Tyr) (Procedure B) (12)

[0094] [ka]

[0095] 1H-NMR (500 MHz, CDCl3): δ 7.69 (1H, t, J = 6.1 Hz), 7.63 (1H, d, J = 7.6 Hz), 7.50 (1H, dd, J = 7.7, 7.4 Hz), 7.43 (1H, d, J = 7.7 Hz), 7.37 (1H, dd, J = 7.6, 7.4 Hz), 7.02 (2H, m), 6.77-6.74 (2H, m), 4.62 (2H, d, J = 6.1 Hz), 3.61 (1H, dd, J = 8.8, 4.3 Hz), 3.13 (1H, dd, J = 13.8, 4.3 Hz), 2.68 (1H, dd, J = 13.8, 8.8 Hz), 3.05 (3H, bs) ppm. 13 C-NMR (125 MHz, CDCl3): δ 147.7, 155.2, 136.4, 132.3, 130.6, 130.4, 128.7, 128.2 (q, J = 30.6 Hz),127.6, 126.0 (q, J = 6.3 Hz), 124.4 (q, J = 274.2 Hz), 115.7, 56.4, 40.0, 39.8 (q, J = 2.1 Hz) ppm. LRMS (EI): m / z 321 (34), 231 (25), 159 (56), 136 (100).

[0096] (2S)-2-amino-N-[3-(trifluoromethyl)benzyl]-3-[4-(hydroxy)phenyl]propanamide (m-TFB-Tyr) (Procedure B) (13)

[0097] [ka]

[0098] 1H-NMR (500 MHz, CDCl3): δ 7.75 (1H, t, J = 5.8 Hz), 7.50-7.44 (2H, m), 7.39 (1H, t, J = 7.5 Hz), 7.33 (1H, d, J = 7.5 Hz), 6.97 (2H, d, J = 8.2 Hz), 6.71 (2H, d, J = 8.2 Hz), 4.41 (2H, s), 3.58-3.50 (1H, m), 3.04 (1H, dd, J = 13.4, 4.2 Hz), 2.86 (3H, bs), 2.65 (1H, dd, J = 13.4, 9.0 Hz) ppm. 13 C-NMR (125 MHz, CDCl3): δ 174.9, 155.6, 139.0, 130.9 (q, J = 1.4 Hz), 130.8 (q, J = 32.3 Hz), 130.2, 129.0, 128.0, 124.2 (q, J = 3.7 Hz), 124.1 (q, J = 3.9 Hz), 123.9 (q, J = 272.1 Hz), 115.5, 56.3, 42.5, 40.0 ppm. LRMS (EI): m / z 338 (M + , 0.2), 321 (50), 231 (26), 159 (95), 136 (100).

[0099] (2S)-2-amino-N-[4-(trifluoromethyl)benzyl]-3-[4-(hydroxy)phenyl]propanamide (p-TFB-Tyr) (Procedure B) (14)

[0100] [ka]

[0101] 1H-NMR (500 MHz, CDCl3): δ 7.75 (2H, J = 5.6 Hz), 7.50 (2H, d, J = 8.0 Hz), 7.22 (2H, d, J = 8.0 Hz), 6.96 (2H, d, J = 8.4 Hz), 6.70 (1H, d, J = 8.4 Hz), 4.39 (2H, s), 3.51 (1H, dd, J = 8.3, 5.0 Hz), 3.10 (3H, s), 3.00 (1H, dd, J = 13.8, 5.0 Hz), 2.66 (1H, dd, J = 13.8, 8.3 Hz) ppm. 13 C-NMR (125 MHz, CDCl3): δ 174.9, 155.7, 142.0 (q, J = 1.4 Hz), 130.2, 129.4 (q, J = 33.6 Hz), 127.9, 127.6, 125.4 (q, J = 5.4 Hz), 124.0 (q, J = 272.1 Hz), 115.4, 56.3, 42.4, 40.0 ppm. LRMS (EI): m / z 338 (M + , 0.2), 321 (44), 231 (25), 159 (85), 136 (100) 107 (33).

[0102] Functional and Pharmacological (ADME) Assays (Effect of interaction with target molecules) (Functional (biological) testing) In vitro cytotoxicity analysis based on the MTT assay was performed to confirm the desired biological effects of the new low-molecular-weight compounds on cancer cells. A total of 45 unique molecules were tested using a triple-negative breast cancer cell line (HCC38), which is known to have Cdc20 amplification in the cell line.

[0103] Results for Set 1. The lead compound (o-TFB-Tyr) was tested at 25 μM and 5 μM, alone and in combination with the APC / C antagonist proTAME. Apsin, a reported Cdc20 inhibitor, was used for comparison (see Figure 2).

[0104] Results for Set 2. The lead compound (o-TFB-Tyr) was tested at concentrations ranging from 5 μM to 100 nM, alone and in combination with the APC / C antagonist proTAME. Apsin, a reported Cdc20 inhibitor, was used for comparison (see Figure 3).

[0105] From the functional studies summarized in Figures 2 and 3, one compound (o-TFB-Tyr) was selected for pharmacological studies, including cell permeability (Figure 4).

[0106] (Pharmacological testing) These included determination of the ADME (Adsorption, Distribution, Metabolism, and Excretion) assays of o-TFB-Tyr. The results of these studies are summarized as follows:

[0107] (Kinetic solubility) This is a useful initial screening performed before initiating ADME studies to identify potential problems and determine appropriate concentration ranges. Kinetic solubility was measured using turbidimetry. The results of this study are shown in Table 1 below.

[0108] [Table 5]

[0109] This data demonstrated that o-TFB-Tyr is readily soluble in aqueous solution.

[0110] (absorption) This was determined using an intestinal permeability assay in Caco-2 cells, a human colorectal adenocarcinoma cell line (see Figure 4). The results of this study are shown in Table 2 below.

[0111] [Table 6]

[0112] They also show that the compounds diffuse freely in both directions across the semipermeable membrane, which in turn indicates that they are not actively transported by membrane proteins such as ABC transporters, which may limit their use as drugs.

[0113] (Distribution, Metabolism and Excretion) (Metabolic stability test) The liver is the primary drug-metabolizing organ for the majority of pharmaceuticals, and a good in vitro model for studying drug metabolism is based on the use of microsomes, a subcellular fraction of the liver.

[0114] The results of this study are shown below in Table 3. The results showed that after 45 minutes there were 8% intact molecules, demonstrating the stability of the compound.

[0115] [Table 7]

[0116] (drug clearance) Two-thirds of drugs eliminated by metabolism are metabolized, at least in part, by cytochrome P450 (CYP) enzymes and the isoform CYP3A4, which accounts for nearly 50% of all CYP activity. Therefore, we tested whether CYP3A4 is involved in the clearance of o-TFB-Tyr. Table 4 below shows the cytochrome P450 (CYP3A4 isoform) inhibition (IC) of o-TFB-Tyr. 50) measurements are shown. The potential inhibition of CYP3A4 by the lead compound (o-TFB-Tyr) was tested using midazolam and testosterone as CYP3A4 substrates.

[0117] [Table 8]

[0118] In both cases, IC 50 is the IC of a reference compound known to be metabolized by the cytochrome P450 isoform CYP3A4. 50 For comparison, data for the control compound (ketoconazole) with midazolam and testosterone are shown in Table 5 below.

[0119] [Table 9]

[0120] Taken together, the data presented in Tables 4 and 5 suggest that the cytochrome P450 isoform CYP3A4 likely plays a minor role in the clearance of o-TFB-Tyr, although further studies are needed to confirm these observations.

[0121] (Plasma protein binding assay) Non-specific plasma protein binding can have a significant effect on the extent of free drug concentration, which can affect the subsequent inhibitory potential of lead compounds (see Table 6 below).

[0122] [Table 10]

[0123] In both cases (human and mouse), total recovery of protein was observed, indicating the absence of non-specific plasma protein binding.

[0124] (Cytotoxicity results) (Major results) Cytotoxicity and clonogenicity studies performed in HeLa cells confirm the moderate cytotoxic activity (i.e., in the range of 200–10 µM) of compound o-TFB-Tyr in this cancer cell line. The cytotoxic effects observed in HeLa cells (shown in Figures 5 and 6) were comparable to those observed in the triple-negative breast cancer cell line HCC-38. Furthermore, Western blot analysis of HCC-38 cells treated with compound o-TFB-Tyr confirmed the inhibitory effect of this compound on APC / C activation by Cdc20, as monitored by the inhibition of cyclin B, a substrate of the APC / C E3 ubiquitin ligase. The cytotoxicity of a series of compounds structurally related to compound o-TFB-Tyr was also tested in both HCC-38 and HeLa cells, confirming that the specific stereochemical features of compound o-TFB-Tyr have a significant effect on the desired biological activity of this compound.

[0125] (methodology) (cell proliferation) The entire following protocol was performed under sterile conditions. HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Sigma F7524). Cells were counted and seeded at a density of 6,000 cells / well in a clear-bottom 96-well plate (Greiner Bio-One). 100 μL of cells were added to each well and placed in an incubator overnight. The following day, the medium was aspirated, and 100 μL of treatment was added to the well. Cells were treated with controls (medium alone, reversine 5 μM, apsin 25 μM). Apsin and all compound stock solutions were prepared by resuspending the solids in dimethyl sulfoxide (DSMO). The stock solutions were then diluted in medium to achieve a concentration of 200 μM, and then diluted again in medium to achieve the final concentrations tested.

[0126] (Cytotoxicity analysis) In vitro cytotoxicity assays involved quantitative measurement of cell proliferation and subsequent assessment of the relative toxicity of compounds. (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltertolazolium bromide (MTT) is a widely used cytotoxicity assay that measures cellular metabolic activity as an indicator of cell viability, proliferation, and cytotoxicity. In this cytotoxicity assay, the water-soluble yellow tetrazole MTT is reduced to insoluble purple formazan crystals by mitochondrial dehydrogenase. The insoluble purple crystalline product was dissolved in DMSO, and the resulting colored solution was quantified by measuring absorbance (570 nm). Reduction can only occur if mitochondrial reductase is active and therefore directly correlates with the number of viable cells. Comparison of the purple formazan produced by compound-treated cells with that of untreated control cells allowed confirmation of compound cytotoxicity, as calculated by the percentage of cell viability. Tested treatments were performed in triplicate.

[0127] The cells were cultured and exposed for 72 hours; 3 hours before the end of the exposure, 5 μL of MTT (5 mg / mL) (Invitrogen M6494) was added to each well, and the plate was then placed in an incubator for the remaining exposure time. The solution was aspirated from each well, and then 100 μL of DMSO was added to the well, and the plate was placed on a shaker at room temperature for 15 minutes. Once a uniform color was observed for each well, the absorbance was measured (570 nm) (Spectramax i3x). The cytotoxicity readings for the treated cells were normalized to the negative control (medium alone), and the cell viability percentage was calculated using the following formula:

[0128]

number

[0129] Data were analyzed by one-way analysis of variance (ANOVA) and post hoc Dunnett's test using GraphPad Prism 7.0, GraphPad Software, Inc. For the data obtained, all treatments were compared with the control (medium alone), p<0.001.

[0130] (Clonogenicity test) The entire procedure was performed under sterile conditions. HeLa cells were counted and seeded into clear-bottom 6-well plates (Greiner Bio-One) at a density of 500 cells / well (250 cells / mL). 2 mL of cells were added to each well and placed in an incubator (37°C, 5% CO2) overnight. The next day, the medium was aspirated, and 1.5 mL of treatment was added to the well. Cells were treated with control (medium alone) and compounds. All compound stock solutions were prepared by resuspending the solids in DMSO, and then the stock solutions were diluted in medium to the final concentration to be tested. HeLa cell viability was measured using a clonogenic assay, a cell survival-based assay that measures cell reproductive death after treatment with cytotoxic agents. The cells were cultured and exposed for 72 hours. The solution was then aspirated from each well, 2 mL of medium was added, and the plate was returned to the incubator. The plates were cultured for an additional 9 days (10 days total). Every few days, the cells were washed with 1x PBS and the 2 mL of medium in each well was replaced with 2 mL of fresh medium. After 10 days of culture following treatment, the solution was aspirated from each well and the cells were washed twice with 1x PBS. Next, 500 μL of 4% paraformaldehyde (Alfa Aesar J61899) in PBS was added to each well, and the plates were incubated at room temperature for 30 minutes. The solution was aspirated from each well, and then 4–5 drops of crystal violet (0.5% w / v in methanol) were added to each well. The plates were incubated at room temperature for 15 minutes. The solution was gently removed by washing each well with water, and clones were visualized. Representative images of the results of the clonogenic assay are shown in Figure 7.

[0131] Each well of the clonogenic assay was then scanned using an Axiozoom Zeiss Axioplan fluorescence microscope equipped for DIC and fluorescence imaging and analyzed using ImageJ2 image processing software (Fiji). A representative image generated by the image processing software is shown below (Figure 8).

[0132] (Confirmation of Cdc20-mediated inhibition of APC / C activation by measuring cyclin B1 levels) The entire procedure was performed under sterile conditions. HeLa cells were counted and seeded into clear-bottom 6-well plates (Greiner Bio-One) at a density of 200,000 cells / well in a volume of 2 mL and placed in an incubator (37°C, 5% CO2) overnight. The next day, the medium was aspirated, and 1.5 mL of treatment was added to the wells. Cells were treated with control (medium alone) and compounds. All stock solutions of small compounds were prepared by resuspending the solids in DSMO, and then the stock solutions were diluted in medium to the final concentration to be tested. The effects of these compounds on mitosis were analyzed by measuring the levels of cyclin B1, a downstream target of APC / C-Cdc20. Cells were cultured and exposed for 24 hours. The plates were then placed on ice, and the solution was aspirated from each well. The cells were washed twice with PBS, and then 300 μL of lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 5 mM ETDA, 1% Triton X-100, 5 mM βe, bovine pancreatic deoxyribonuclease I, cOmplete Mini EDTA-free protease inhibitor cocktail tablet (1 tablet / 50 mL of dissolution)) was added to each well, and the plate was incubated for 10 minutes with agitation. Each well was scraped for 2 minutes using a cell scraper, and the solution from each well was then transferred to the corresponding labeled Eppendorf tube. The tubes were then centrifuged at 14,500 rpm at 4°C for 30 minutes. The supernatant from each tube was transferred to a clean Eppendorf tube, flash-frozen, and stored at -20°C. Figure 9 shows a Western blot of HCC-38 cells treated with compound o-TFB-Tyr. Figure 9 confirms the inhibition of APC / C activation by Cdc20, as monitored by the inhibition of cyclin B. Mouse anti-cyclin B1 antibody (BD Pharmingen 554177) was used as the primary antibody. AP-conjugated anti-mouse IgG antibody was used as the secondary antibody (Sigma SAB3701107-1). Mouse anti-α-tubulin antibody (Santa Cruz Biotechnology sc-32293) was used as an internal control for protein concentration loading. [Brief explanation of the drawings]

[0133] [Figure 1] The Cdc20-o-TFB-Tyr complex, the contact between o-TFB-Tyr Cdc20 and the apsin (Apcin) complex, and the 3D structure of o-TFB-Tyr-Cdc20 are shown. [Figure 2] Figure 1 shows cytotoxicity analysis by MTT assay of compounds tested against triple-negative breast cancer cells, using the Cdc20 inhibitor apsin for comparison. Cytotoxicity analysis of asynchronous HCC-38 triple-negative breast cancer cells after 24 hours of treatment with the selected third-generation compound o-TFB-Tyr. HCC-38 cells were exposed to 25 μM and 5 μM concentrations of compound o-TFB-Tyr (Panel A) and 1 μM concentration of compound o-TFB-Tyr (Panel B), alone and in combination with proTAME. The negative control used was untreated cells (medium), while the positive controls used were 25 μM apsin alone, 10 μM proTAME alone, and 25 μM apsin in combination with 10 μM proTAME. Data were analyzed by one-way ANOVA and Dunnett's test using all columns compared to the negative control column (medium), p<0.001. Experiments were performed in duplicate. [Figure 3] Cytotoxicity analysis of compound o-TFB-Tyr at concentrations of 5 μM, 1 μM, 0.5 μM, and 100 nM is shown. HCC-38 cancer cells were exposed to compound o-TFB-Tyr alone and to compound o-TFB-Tyr in combination with proTAME. The negative control used was untreated cells (medium), while the positive control used was 25 μM apsin in combination with 10 μM proTAME. Data were analyzed by one-way ANOVA and Dunnett's test with all columns compared to the negative control column (medium), p<0.001. Replicate experiments were performed. [Figure 4] This shows the principle of cell membrane permeability testing, which is part of ADME testing. [Figure 5]Figure 1 shows the relative cytotoxicity of compound o-TFB-Tyr at various concentrations in HeLa cells compared with reversine, a small compound inhibitor of Mps1 kinase and an upstream regulator of SAC, and apsin, a small binder of Cdc20 and an inhibitor of APC / C activation by Cdc20. This study confirmed that compound o-TFB-Tyr exhibited higher cytotoxic effects against different types of cancers originating from various tissues. [Figure 6] The relative cytotoxicity of compound o-TFB-Tyr was compared with that of compounds apsin, m-TFB-Tyr, p-TFB-Tyr, and o-TFB-Lys at the same concentration in HeLa cells. Compounds o-TFB-Tyr, m-TFB-Tyr, and p-TFB-Tyr are closely related in terms of chemical structure. Comparative studies confirmed that compound o-TFB-Tyr exhibited a greater cytotoxic effect against cancer cells in culture. Thus, we identify important stereochemical features of o-TFB-Tyr and its isomers m-TFB-Tyr and p-TFB-Tyr that explain the anticancer activity of o-TFB-Tyr and structurally related molecules. Comparative analysis also demonstrated that the chemical nature of the R2 residue in the claimed compounds o-TFB-Tyr, m-TFB-Tyr, p-TFB-Tyr, and o-TFB-Lys explains the cytotoxicity of these molecules against cancer cells. [Figure 7] Clonogenic assay of HCC-38 cells treated with DMSO (1.2% v / v), the Mps1 kinase inhibitor reversine, the Cdc20 binder apsin, and the compound o-TFB-Tyr. After 12 days of incubation at 37°C, clones were stained. HCC38 cells were treated with the compounds for 24 hours, after which the medium was changed every 48 hours. In this assay, fewer triple-negative breast cancer cell clones were consistently observed in cells treated with reversine and o-TFB-Tyr, confirming the desired cytotoxicity of the latter compound in cancer cells. [Figure 8]Each well of the clonogenic assay was then scanned using an Axiozoom Zeiss Axioplan fluorescence microscope equipped for DIC imaging and fluorescence imaging and analyzed using ImageJ2 image processing software (Fiji). Representative images generated by the image processing software are shown here. [Figure 9] Figure 1 shows a Western blot showing that compound o-TFB-Tyr inhibits ubiquitination of cyclin B by the APC / C. As a result, non-ubiquitinated cyclin B escapes proteasomal degradation. This analysis also shows that apsin is relatively less effective than compound o-TFB-Tyr as an antagonist of APC / C activation by Cdc20. HCC-38 cells at a density of 200,000 cells per well were used in this study.

Claims

1. A compound of formula I or a pharmaceutical salt thereof: 【Chemistry 1】 During the ceremony, R １ represents —CF3; R ２ is -NH ２ or Cy １ C substituted by １ -C ６ represents alkyl; Cy １ represents a phenyl group (-Ph) substituted with -OH.

2. Cy １ 2. The compound of formula I according to claim 1, or a pharmaceutical salt thereof, wherein represents a phenyl group substituted at the para position with -OH (-Ph).

3. R ２ is -NH ２ C substituted by １ -C ６ 2. The compound of formula I according to claim 1, or a pharmaceutical salt thereof, wherein: R is alkyl;

4. R ２ is the formula R ２ 3. The compound of formula I according to claim 1 or 2, or a pharmaceutical salt thereof, wherein: 【Chemistry 2】 。

5. R ２ is the formula R ２ -b: The compound of formula I according to claim 1 or 3, or a pharmaceutical salt thereof: 【Transformation 3】 。

6. The compound of formula I is 【Chemistry 4】 2. The compound of formula I according to claim 1, selected from: or a pharmaceutical salt thereof.

7. The compound of formula I is 【Transformation 5】 7. The compound of formula I of claim 6, wherein: or a pharmaceutical salt thereof.

8. The compound of formula I is 【Transformation 6】 2. The compound of formula I according to claim 1, selected from: or a pharmaceutical salt thereof.

9. A pharmaceutical composition comprising a compound of formula I according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

10. A pharmaceutical composition comprising a compound of formula I according to any one of claims 1 to 8 in combination with a further compound selected from pro-N-4-tosyl-L-arginine methyl ester (proTame).

11. A compound of formula I according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof for use in therapy.

12. 1. A compound of formula I or a pharmaceutical salt thereof for use in the treatment of cancer: 【Transformation 7】 During the ceremony, R １ represents —CF3; R ２ is -NH2 or Cy １ C substituted by １ -C ６ represents alkyl; Cy １ represents a phenyl group (-Ph) substituted with -OH.

13. The compound of formula I is 【Transformation 8】 13. The compound of formula I or a pharmaceutical salt thereof for use according to claim 12, selected from:

14. The compound of formula I is 【Chemistry 9】 14. The compound of formula I or a pharmaceutical salt thereof for use according to claim 13, wherein:

15. The compound of formula I is 【Chemistry 10】 13. The compound of formula I or a pharmaceutical salt thereof for use according to claim 12, selected from:

16. A compound of formula I or a pharmaceutical salt thereof for use according to any one of claims 12 to 15 for the treatment of breast cancer.

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