Active compounds for use in the treatment of breast cancer (MCF-7), prostate cancer (PCS), and colon cancer (HT29)

Abstract

The invention relates to phosphazene derivative compounds, which are two active agents usable in the treatment of breast cancer (MCF-7), prostate cancer (PCS), and colon cancer (HT29).

Description

[0001] ACTIVE COMPOUNDS FOR USE IN THE TREATMENT OF BREAST CANCER (MCF-7), PROSTATE CANCER (PC3), AND COLON CANCER (HT29)

[0002] Technical Field

[0003] The invention relates to phosphazene derivative compounds, which are two active agents usable in the treatment of breast cancer (MCF-7), prostate cancer (PC3), and colon cancer (HT29).

[0004] Prior Art

[0005] Cancers (Malignant tumors) are generally continuously and rapidly growing tumors. They lack capsules, grow without limitation, and invade surrounding tissues and vessels. They frequently metastasize, and untreated cancers or cancers with delayed treatment can be fatal. Various drugs are used to treat the disease. The body's immune system plays the primary role in the treatment of diseases. The elimination of factors that weaken the immune system is the first step of the treatment. Since it is impossible to predict how much and where cancerous cells metastasize, it is a desirable situation for the immune systems of patients undergoing cancer treatment to be strengthened to destroy these disseminated cells. In this respect, the synthesis and discovery of new drug active agents are very important.

[0006] Intensive studies have been conducted for many years on the design and synthesis of effective and valuable chemical molecules that can be used especially for therapeutic purposes in order to prevent or reduce cancer-related deaths. However, due to the existence of a wide variety of cancer types, the increase in deaths caused by cancer, and the fact that a large portion of the produced molecules are unsuitable for use for various reasons, the need for new molecules with anticancer potential isincreasing day by day. For this reason, new chemotherapeutic compounds are being synthesized and their biological activities are being investigated. Today, scientists are not content with only synthesizing new chemical compounds, but also strive for the production of compounds that have economic value, are targeted, and can be beneficial to humanity. Therefore, cancer chemotherapy continues to be an important field of research for chemists today, as it was in the past. Based on this thought, new chemotherapeutic compounds are being synthesized day by day and their biological activities are being investigated. As a result of various researches, it has been determined that a cell mass with a high division capacity is influential in the cancerization of tissues and in the acquisition of the ability to "metastasis", that is, the spreading of cancer cells in cancerous tissues in any organ to another part of the body, and these cells have been named as cancer stem cells (CSC). All different CSCs have unlimited proliferation potential and the ability to spread to other organs "metastasis". It is also known that said cells are resistant to chemotherapy. Therefore, for an effective combat against cancer, it is necessary to develop new drugs and methods having the potential to neutralize cancer stem cells.

[0007] In research conducted in 2001, organic, organometallic, or inorganic groups can be bonded to the halide atoms on the phosphazene ring via nucleophilic substitution reactions. The most important feature of phosphazene chemistry is the ability of phosphazenes to undergo substitution reactions with very different groups and that the compounds exhibit very different properties depending on the attached groups. For example, it has been observed that aziridine-substituted phosphazenes inhibit the growth of cancer cells by cleaving DNA.

[0008] In a study conducted in 2016, partially substituted tetraspirocyclotetraphosphazene derivatives were obtained from the reaction of octachlorocyclotetraphosphazene with N, N-dibenzylethylenediamine, and subsequently, fully substituted spirocyclotetraphosphazenes were obtained from the reaction of these compounds with an excess of pyrrolidine and morpholine. The structures of the compounds were elucidated by spectroscopic and crystallographic methods. The cytotoxicactivities of all compounds against HeLa cervical cancer cells were investigated. As a result of the studies, it was observed that the compounds exhibited limited cytotoxic, apoptotic, and necrotic effects on HeLa cancer cells.

[0009] In the 2018 document by Fatih CEI. IK titled " Click Reaksiyonu Uygulamalan lie Hibrit 1,2,3-Triazollerin Sentezi Ve Biyokimyasal Ozelliklerinin Incelenmesi", a method directed to the synthesis of various compounds with the thiophenecarboxaldehyde compound is mentioned.

[0010] Upon examination of the studies present in the prior art, a need has arisen to develop phosphazene derivative compounds, which are two active agents usable in the treatment of breast cancer (MCF-7), prostate cancer (PC3), and colon cancer (HT29).

[0011] Object of the Invention

[0012] The object of this invention is to develop phosphazene derivative compounds, which are two active agents usable in the treatment of breast cancer (MCF-7), prostate cancer (PC3), and colon cancer (HT29).

[0013] Detailed Description of the Invention

[0014] A compound of Formula A for the use of treatment of breast cancer (MCF-7), prostate cancer (PC3), and colon cancer (HT29),

[0015] (Formula A)

[0016]

[0017] wherein;

[0018] X: N (nitrogen);

[0019] Y: SC4H3CH2 (thiophenylmethyl),

[0020] R: CH3and C2H5(methyl and ethyl)

[0021] R: / / -OHxN (^-propylamine).

[0022] The chemical structure of the compound A-l subject to the invention, synthesized from the derivative of the main compound;

[0023] H3C(H2C)2HN I II NH(CH2)2CH3

[0024] p\Y''

[0025]

[0026] H3C(H2C)HN^ ^NH(CH2)CH3

[0027] (Formula A-l)

[0028] The IUPAC name of the molecule subject to the invention is stated below:

[0029] Tetrapropylamino-(N-methyl-N'-2-thiophenylmethyl)-ethane-l,2-diamino-(N / N) spirocyclotriphosphazene (A-l)

[0030] The chemical structure of the compound A-2 subject to the invention, synthesized from the derivative of the main compound;H3C(H2C)2HN I II NH(CH2)2CH3

[0031]

[0032] H3C(H2C)HN^ ^NH(CH2)CH3

[0033] (Formula A-2)

[0034] The IUPAC name of the molecule subject to the invention is stated below:

[0035] T etrapropy 1 amino-(N -ethyl -N1-2 -thi opheny Imethy 1 )-ethane- 1, 2 -di amino-(N / N)spirocyclotriphosphazene (A-2)

[0036] These 2 compounds synthesized from the main compound group are organic cyclic compounds. In the synthesis of the compounds, 2-Thiophenylspirotetrachlorophosphazene compounds (3 and 4) were obtained from the reaction of diamine compounds (1 and 2), prepared using thiophenecarboxaldehyde, with trimeric phosphazene, which is an inorganic cyclic compound. Subsequently, 2-Thiophenylspirotetraaminophosphazene compounds (A-l and A-2) were obtained from the reaction of these compounds with propylamine.

[0037] The development of phosphazene derivative compounds carried out to achieve the objectives of this invention is illustrated in the attached figures.

[0038] Figure 1 A view of the table showing the time-dependent variation of the measurable change occurring in MCF-7 (CD24 / CD44*) micro-tissues according to the invention.

[0039] [Results were analyzed using the Two-way ANOVA test.]

[0040] ****P<0.0001, *** P<0.0005, ** P<0.01, * P<0.05 vs. Control)Figure 2 A view of the table showing the time-dependent variation of the measurable change occurring in HT29 (CD133* / CD44*) micro-tissues according to the invention.

[0041] [Results were analyzed using the Two-way ANOVA test.]

[0042] ****P< O. OOOl, *** P<0.0005, ** P<0.01, * P<0.05 vs. Control)

[0043] The synthesis stages of the 2 active agent molecules subject to the invention are described below.

[0044] \ I. EtOH R Comptumd CHO JfeN NHR - < n3(I)

[0045] 2 -tMopfeenytaiethyMisiH! ees CHJCHJ (2J

[0046] 2-SlsiopheHylspirotetra^hlorophospbazcne

[0047] i>-propylaisutie NR R R' CoKipsmnrf EtjN ’" N BryTHFR HNi|. NHR' < H' n-CIIJfl- M Ar zP% ''FX

[0048]

[0049] RUN NUR' CH2CH3nCHyet (A-2)

[0050] 2*tbio pheaykpirotetraaaiiBop hasp haze sse

[0051] The method steps in the synthesis of the Formula A-l active agent from the Formula A compound are respectively as follows:

[0052] First, a Schiff base was synthesized as a result of the condensation reactions of the starting compound, 2-thiophenecarboxaldehyde, with N-m ethyl- 1,2-diaminoethane in an ethanol medium; and from the reduction of this Schiff base with NaBH, the N / N donor atom unsymmetrical N-m ethyl-N'-(2 -thiophenylmethyl)- 1,2-diaminoethane (1) ligand was synthesized.The partially substituted 2-thiophenylmethylspirocyclotriphosphazene derivative (3) was synthesized from the reaction of Hexachlorocyclotriphosphazene (HCCP) with the N / N donor atom unsymmetrical N-methyl-N'-(2-thiophenylmethyl)-l,2-ethanediamine (1) compound in a 1:1 molar ratio at room temperature, in the presence of EtaN, in THF solvent, and under an argon atmosphere. In the studies conducted, only the spiro product was isolated regioselectively. In the reaction, NEh is used to remove the released HC1 from the medium by forming NEtvHCl salt.

[0053] The fully amine-substituted cyclotriphosphazene compound (A-l) was synthesized as a result of the reaction of the synthesized partially substituted 2-thiophenylmethylspirocyclotriphosphazene derivative (3) with an excess of n-propylamine in a dry THF medium, under an argon atmosphere and under reflux, in the presence of triethylamine. The phosphazene derivative was purified by column chromatography.

[0054] The method steps in the synthesis of the Formula A-2 active agent from the Formula A compound are respectively as follows:

[0055] First, a Schiff base was synthesized as a result of the condensation reactions of the starting compound, 2-thiophenecarboxaldehyde, with N-ethyl-l,2-diaminoethane in an ethanol medium; and from the reduction of this Schiff base with NaBH, the N / N donor atom unsymmetrical N-ethyl-N'-(2-thiophenylmethyl)-l,2-diaminoethane (2) ligand was synthesized.

[0056] The partially substituted 2-thiophenylmethylspirocyclotriphosphazene derivative (4) was synthesized from the reaction of Hexachlorocyclotriphosphazene (HCCP) with the N / N donor atom unsymmetrical N-ethyl-N'-(2-thiophenylmethyl)-l,2-ethanediamine (2) compound in a 1:1 molar ratio at room temperature, in the presence of EtaN, in THF solvent, and under an argon atmosphere. In the studies conducted, only the spiro product was isolated regioselectively. In the reaction,NEta is used to remove the released HC1 from the medium by forming NEh. HCl salt.

[0057] The fully amine-substituted cyclotriphosphazene compound (A-2) was synthesized as a result of the reactions of the synthesized partially substituted 2-thiophenylmethylspirocyclotriphosphazene derivative (4) with an excess of n-propylamine in a dry THF medium, under an argon atmosphere and under reflux, in the presence of triethylamine. The phosphazene derivative was purified by column chromatography.

[0058] The process steps applied to determine the antiproliferative effects of the compounds, which are 2 active agents subject to the invention, on cancer cells and CSCs are stated below.

[0059] In the first step of the isolation, cultivation, and characterization of cancer stem cells from cancer cell lines, cancer stem cell isolation studies were performed from MCF7 breast cancer, PC3 prostate cancer, and HT29 colon cancer cell lines. Cancer stem cell separation was carried out for isolation via a magnetic isolation kit (MACS-Miltenyi Biotec), which was also supplied within the scope of the invention. The separation was performed according to the recommended protocol. During this separation, CD133, CD24, and CD44 loaded magnetic nanoparticles, which are also included in the kit content, were utilized.

[0060] The MACS technique enables the separation of desired cells in the presence of a magnetic field using magnetic micro-particles labeled to bind to the target surface protein. It has been emphasized that the size of the used magnetic nanoparticles is approximately 100 nm and they do not cause any toxic effect on the cells. The separation method via MACS according to the recommended protocol was used in all studies requiring stem cell isolation within the scope of the work. Modifications made in the protocol were adjusted according to the type of magnetic marker nanoparticles (CD133+, CD44+, and CD24-) and the amount of cells used. The MiniMACS separator was used when the cell count was below 2xl07, and theMidiMACS separator was used when it exceeded 2xl07. Furthermore, in addition to the separator change, as stated above, the amounts of buffer, micro-bead, and FcR blocking solutions to be used were also increased proportionally.

[0061] The breast cancer (MCF7), prostate cancer (PC3), and colon cancer (HT29) stem cells, isolated as described above, were characterized by the qRT-PCR method in the subsequent steps of the invention.

[0062] As the next step, the method of determining the gene expressions of CD44+, CD133+, and CD24- cells obtained via the MACS technique by qRT-PCR was applied.

[0063] Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed to determine the expressions of CD 133, CD24, and CD44 proteins, which are cancer stem cell (CSC) markers, and OCT3 / 4, Sox2, Nestin, and Nanog proteins, defined as sternness genes, in CD133+, CD44+, and CD24- cells separated from MCF7, PC3, and HT29 cells via the MACS method. The "comparative AACT" method was used for the quantification of amplification products obtained by Realtime PCR. The AACT value refers to the first cycle where a significant increase in the amount of PCR products occurs. A reference gene was utilized as a calibrator, and a control group was utilized to enable comparison. The relative expression of the target gene with respect to a control gene (GAPDH) was examined. This method is a very suitable method for expression studies of genes synthesized in low abundance. Since PCR is an exponential process, the variation is expressed as an exponential function of 2. Since the AACT values obtained for the control are approximately "0", the values for 2AACTare "1". The result obtained is the relative amount of the expression of the target gene of interest in the sample group with respect to the control. Since it is a ratio expression, it is unitless. It indicates how many fold the expression of the target gene in the sample has increased or decreased compared to the control.During the determination of the antiproliferative effects of the compounds on cancer cell lines;

[0064] The following operating procedure was applied to determine the anti-proliferative effects on cancer cell lines of the compounds subject to the invention, which were synthesized and for which information regarding synthesis and characterization was provided in the previous steps. The anti-proliferative effects of the compounds on cancer cell lines were determined by the MTT method. In this application, breast cancer (MCF-7), prostate cancer (PC3), and colon cancer (HT29) cell lines, and as controls, healthy colon (CCD-18Co), healthy breast (MCF12A), and healthy prostate (RWPE-1) were used; and the tested compounds were tested at six different concentrations (200 pM, 100 pM, 50 pM, 25 pM, 12.5 pM, 6.25 pM, 3.13 pM, 1.56 pM). The solvent used for solubilizing the relevant compounds is DMSO, and care was taken to ensure that the maximum DMSO concentration in the main stock was 0.1% (v / v) during calculation. Thus, any cytotoxic effect originating from DMSO was prevented. The initial working concentration was set to 200 pM, then serial dilutions were performed to ensure the dilution of the substance in eight different concentrations. Prepared solutions were applied to the wells 24 hours after the initial cell numbers were seeded into the plates (while the cells were in the logarithmic phase). Said application was performed in triplicate. The plates to which the substance was added were placed in an incubator with 95% humidity containing 5% CO2 at 37°C to perform viability analysis with the MTT test at 24, 48, and 72 hours. For MTT, the used medium in the plate was removed, and medium containing MTT at a concentration of 0.5 mg / ml was added onto the cells. After the cells were incubated in the dark in an incubator with 5% CO2 at 37°C for 3 hours, DMSO was added onto the wells to ensure the dissolution of the formed formazan crystals. It was read in a UV spectrophotometer at a wavelength of 570 nm and absorbance values were recorded. The absorbance value of each sample was proportioned to the absorbance value of the cell control belonging to that experiment set; thus, % viability rates were determined and graphed. In the graphs, the mean of three repetitions ± standard deviations (SD) are given as a bar graph.GraphPad Prism program (version 8, GraphPad Software, Inc., San Diego California, USA) was used for the statistical evaluation of the significance levels of the data obtained from the applied test.

[0065] The IC50values of the compounds in cancer cell lines and at different times are included in the table below.

[0066] MCF-7 PC-3 HT-29 24 48 72 24 48 72 24 48 72 hours hours hours hours hours hours hours hours hours A-l 189,6 96,79 33,35 210,2 60,75 42,69 185,5 51,91 38,79 A-2 80,96 35,18 16,16 132,9 51,37 12,6 53,09 22,82 11,52

[0067]

[0068] In the stage of determining the effects of the compounds on cancer stem cells;

[0069] The effects of the active compounds (A-l and A-2), determined by the method mentioned above, on cancer stem cells were determined using the MTT method again. 5-Fu was used as a positive control agent in this step as well. As a result of the test, the anti-proliferative effects of the relevant compounds on cancer stem cells were determined, and the IC50values were calculated using the GraphPad Prism 8 program.

[0070] The IC50values of the compounds in cancer stem cells and at different times are included in the table below.

[0071] MCF-7 CD24 / 44* PC-3 CD133* / 44* HT-29 CD133* / 44* 24 48 72 24 48 72 24 48 72 hours hours hours hours hours hours hours hours hours A-l 199 44,15 36,23 114 60,52 24,94 89,33 33,87 25,22 A-2 145,7 26,64 23,54 101,2 49,44 9,661 39,63 13,99 12,32

[0072]

[0073] Finally, the effects of the developed compounds on cancer stem cell-based three-dimensional (3D) micro-tissues were determined.

[0074] This method is used for the evaluation of the tumorigenicity of cells in the in vitro medium and for the examination of the clonogenicity feature, which is a stem cell characteristic. In this context, within the scope of the invention, the soft agar colony formation efficiency of MCF7 (CD24- / CD44+), PC3 (CD133+ / CD44+), and HT29 (CD133+ / CD44+) stem cells and the effects of the compounds on 3D micro-tissue were examined. Briefly, in this technique, wells that are homogeneous in terms of depth and diameter size are formed on agarose gel using molds called "3D Petri Dish". When cells are cultured in these created wells, micro-tissues are formed at the end of a process of spontaneous self-assembly and attachment over time.

[0075] Stem cell inoculation into three-dimensional agarose matrices;

[0076] - The 24-well culture plate containing agarose gels prepared according to the method mentioned above 1 day in advance was taken into a laminar flow cabinet, and the conditioning medium on the gels was removed. In this study, molds used to create a 400 pm well diameter were used.

[0077] Isolation and characterization of MCF7 (CD24- / CD44+), PC3 (CD133+ / CD44+), and HT29 (CD133+ / CD44+) cancer stem cells were performed according to the method stated in the invention, and the cells were taken into culture and expanded for further trials.

[0078] - The cells to be taken into agarose matrices were detached by the trypsinization method and counted.

[0079] - After the cells were obtained, 5x105cells / 60 pL of cells per gel were inoculated into the gels in triplicate for each cell type. Following the inoculation, the matrices containing cells were left for incubation for 1 hour at 37°C and in a 5% CO2environment.- At the end of the incubation, 600 pL of culture medium containing serum, L-glutamine, and gentamicin was added onto the gels. Then, the gels were incubated under standard culture conditions.

[0080] - Micro-tissues expected to form during incubation were photographed every 24 hours and analyzed in the Image J program, and tissue diameters were calculated.

[0081] - When the micro-tissues reached approximately 400 pm, test compounds (active compounds) were added onto them at effective doses, and the change in the diameters of the micro-tissues was recorded by photographing every 24 hours.

[0082] Size analysis of the photographs taken over 4 days was performed in the " Image J" program, and the change occurring in micro-tissue size over time was graphed.

Claims

CLAIMS1. A compound of Formula A for the use of treatment of breast cancer (MCF- 7), prostate cancer (PC3), and colon cancer (HT29),(Formula A)X: N (nitrogen);Y: SC4H3CH2 (thiophenylmethyl),R: CH3and C2H5(methyl and ethyl)R: -C3H8N (-propylamine).which is compound A-l synthesized from the 2-thiophenecarboxaldehyde compound,H3C(H2C)2HN I II NH(CH2)2CH3p\Y'' H3C(H2C)HN^ ^NH(CH2)CH3(Formula A-l)characterized in that it is Tetrapropylamino-(N-methyl-N'-2- thiophenylmethyl)-ethane-l,2-diamino-(N / N)spirocyclotriphosphazene (A- 1).

2. A compound of Formula A for the use of treatment of breast cancer (MCF- 7), prostate cancer (PC3), and colon cancer (HT29),(Formula A)X: N (nitrogen);Y: SC4H3CH2 (thiophenylmethyl),R: CH3and C2H5(methyl and ethyl)R: -C3H8N (-propylamine).which is compound A-2 synthesized from the 2-thiophenecarboxaldehyde compound,H3C(H2C)2HN NH(CH2)2CH3H3C(H2C)HN^ ^NH(CH2)CH3(Formula A-2)characterized in that it is Tetrapropylamino-(N-ethyl-N'-2-thiophenylmethyl)-ethane-l,2-diamino-(N / N)spirocyclotriphosphazene (A-2).

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