Two drug molecules for use in the treatment of MCF-7 breast cancer, PC3 prostate cancer, and HT29 colon cancer

Abstract

The present invention relates to two drug molecules having the structure of Formula I and Formula II, which have an antiproliferative effect against cancer, and to the synthesis of these molecules.

Description

[0001] TWO DRUG MOLECULES FOR USE IN THE TREATMENT OF MCF-7 BREAST CANCER, PC3 PROSTATE CANCER, AND HT29 COLON CANCER Technical Field

[0002] The present invention relates to two active agents for use in the treatment of breast cancer (MCF-7), prostate cancer (PC3), and colon cancer (HT29), and to the development of synthesis methods thereof.

[0003] Prior Art

[0004] Intensive studies have been carried out for many years on the design and synthesis of effective and valuable chemical molecules that can be used especially for therapeutic purposes with the aim of preventing or reducing cancer-related deaths. However, the need for new molecules having anticancer potential is increasing day by day due to the existence of very diverse types of cancer, the increase in deaths caused by cancer, and the unsuitability of the use of a large portion of the produced molecules for various reasons. Therefore, 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 possess economic value, are targeted, and can be beneficial to humanity. Therefore, as in the past, cancer chemotherapy continues to be an important research area for chemists today as well. Based on this consideration, new chemotherapeutic compounds are being synthesized day by day and their biological activities are being investigated. As a result of various studies, it has been determined that a cell mass with high proliferative capacity plays a role in the cancerization of tissues and in the acquisition of the ability of cancer cells in any organ of the cancerous tissues to spread to another part of the body, i.e., "metastasis", and these cells have been termed Cancer Stem Cells (CSCs). All different CSCs possess unlimited proliferation potential and the ability to spread to other organs ("metastasis"). It is also known that the 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 render cancer stem cells ineffective.Organic, organometallic, or inorganic groups can be attached to the halide atoms on the phosphazene ring via nucleophilic substitution reactions. The most important feature of phosphazene chemistry is that phosphazenes can undergo substitution reactions with very different groups and the compounds can exhibit very different properties depending on the attached groups.

[0005] In a study conducted on the syntheses of new di spiro, bino, and dispiro-ansa spermine cyclotriphosphazene derivatives used as anti-cancer agents and the investigation of the cytotoxic and apoptotic properties of the structures of these compounds, first, dispirobino and dispiro-ansa spermine cyclotriphosphazene derivatives were synthesized as a result of the reactions of phosphazenes with spermine. Subsequently, the structures of these compounds were investigated by spectroscopic methods. Furthermore, it was observed that the obtained compounds possess apoptotic, cytotoxic, and anti-cancer activity for HT-29 and Hep2 cells (Yildinm et al., 2012, Eur. J. Med. Chem. 52, 213-20).

[0006] In the early periods of cancer research, it was thought that genetic differentiations were effective during the transformation of healthy cells into cancerous cells, and that cancerous cell masses (tumors) were formed by mutations occurring consecutively on a cell population. However, in later periods, it was suggested that the occurrence of the same type of genetic mutation millions of times in the same tissue and on the same cell mass is a very low probability; therefore, genetic mutation alone would not be sufficient to initiate carcinogenesis (Ghiaur et al., 2012, Stem Cells, 30, 89-93). Based on this consideration, as a result of various researches carried out by some study groups, it was noted that a cell mass with high proliferative capacity is effective in the cancerization of tissues and in the acquisition of metastasis (spread of cancer cells in any organ to another part of the body) ability of cancerous tissues, and these cells were termed Cancer Stem Cells (CSCs). CSCs are cells defined as a sub-population of tumor cells that can trigger tumor initiation and cause recurrences.

[0007] In the document titled “Bicyclic phosphazenes derived from (amino) cyclotetraphosphazenes” (Krishnamurthy, S. S. (1989). Phosphorus, Sulfur, and Silicon and the Related Elements, 41(3-4), 375-391), the derivation of bicyclic molecules from amino cyclotetraphosphazenes is described.In the document titled “Mechanism of formation of bicyclic phosphazenes. Isolation of a versatile tetrakis (amino) intermediate, N4P4C14(NMe2)2(NHEt)2” (Narayanaswamy, P. Y., Dhathathreyan, K. S., & Krishnamurthy, S. S. (1985). Inorganic Chemistry, 24(5), 640-642.), the formation of bicyclic tetraphosphazenes is described.

[0008] In the document titled “Syntheses and spectral characterizations of tetraspirocyclotetraphosphazenes containing bis(4-fluorobenzyl) pendant arms” (Okumu§, A., Elmas, G., & KIIIQ, Z. (2021). Ata-Kimya Dergisi, 1(1), 1-19.), the synthesis of 4, 4,8,8-tetrachloro-2-trans-6-bis-N-(4-fluorobenzyl)-N'-ethylethane-l,2-diaminocyclotetraphosphazene is disclosed. It has also been suggested that the compound can be used against cancer.

[0009] When current medical developments are examined, it is known that a large number of cancer types are highly likely to be treatable with traditional treatments such as surgery, chemotherapy, and radiotherapy when diagnosed at an early stage. However, despite developing methods, many cancer types can still be diagnosed at late stages, during the metastasis process. This situation perpetuates the quests in the related field in the scientific world and even causes them to continue with increasing momentum. Studies conducted in this context are generally directed towards cancer stem cells. It has been clearly demonstrated that even if cancer is diagnosed and treated at an earlier stage, the said cells can cause the tumor to recur after a while, the related cells are resistant to traditional treatment methods (chemotherapy, radiotherapy, surgery, etc.), and they can trigger the development of more aggressive metastatic processes. Therefore, the primary objective is now to render cancer stem cells ineffective alongside cancerous cells. It has been determined that traditional cancer treatment methods applied to date are not effective against CSCs having high proliferation and differentiation capacities, and that the said cells show resistance to known therapeutic agents. When various studies conducted are examined carefully, the necessity of developing new drugs and methods having the potential to render CSCs ineffective, which can self-renew by dividing within the tumor and cause the formation of many cell types capable of forming tumors, has emerged (Tuna, 2009, Turk Onkoloji Dergisi, 24 (1), 42-47).

[0010] At the basis of the stated R&D process lies, primarily, the carrying out of the isolation of CSCs from the tumor population in a reliable manner and the determination of the responsesgiven by the isolated cells to molecules that are used as drugs or have the potential to be used.

[0011] Objectives and Brief Desciption of the Invention

[0012] The object of the present invention is to develop two molecules having an antiproliferative effect against cancer and the synthesis method of these molecules.

[0013] With this invention, a new dimension has been introduced to cancer research through partially and fully substituted mono(4-fluorobenzyl)spiro cyclotriphosphazenes targeting CSCs. The fact that the relevant compounds have also been studied in three-dimensional tumor models has enabled the acquisition of comprehensive preclinical data prior to transitioning to in vivo studies. Thus, a perspective distinct from the applications existing in the prior art has been presented. It has been observed that the two compounds developed with the invention exhibit superior antiproliferative effects at high concentrations when compared to the positive control agent 5-Fu, which is currently used clinically in existing treatment methods. This reinforces the prediction that these compounds could be strong candidates for chemotherapeutic agents. In reaching this conclusion, care was taken to ensure that the doses inhibiting cancer cell proliferation were also doses that did not exhibit cytotoxic effects on healthy cells. Thus, the potential of the studied compounds to be chemotherapeutic agents with a significantly low side-effect profile has been demonstrated more clearly and accurately.

[0014] Detailed Desciption of the Invention

[0015] The molecules of the present invention are explained with reference to the attached figures.

[0016] Figure 1 Percent cell viability graphs generated following the interaction of the compound of Formula I with HT29 (a), the compound of Formula II with HT29 (b), the compound of Formula I with PC3 (c), the compound of Formula II with PC3 (d), the compound of Formula I with MCF-7 (e), and the compound of Formula II with MCF-7 (f) cancer cells for a determined period. [Results were analyzed using the Two-way ANOVA test (* * * *P<0.0001, * * *P<0.0005, * *P<0.01, *P<0.05 vs Control) ]

[0017] Figure 2 Percent cell viability graphs generated following the interaction of the compound of Formula I with CCD-18CO (a), the compound of Formula II with CCD-18CO (b), thecompound of Formula I with RWPE-1 (c), the compound of Formula II with RWPE-1 (d), the compound of Formula I with MCF-12A (e), and the compound of Formula II with MCF-12A (f) healthy cell lines for a determined period. [Results were analyzed using the Two-way ANO VA test ( * * * *P<0.0001, * * *P<0.0005, * *P<0.01, *P<0.05 vs Control) ]

[0018] Figure 3 Percent cell viability graphs generated following the interaction of the compound of Formula I with HT29 CD44+(a), HT29 CD133+(b), PC3 CD44+(c), PC3 CD133+(d), MCF-7 CD44+(e), and MCF-7 CD24' (f) cancer stem cell lines for a determined period.

[0019] [Results were analyzed using the Two-way ANOVA test (****P<().0001, ***P<0.0005, * *P<0.01, *P<0.05 vs Control) ]

[0020] Figure 4 Percent cell viability graphs generated following the interaction of the compound of Formula II with HT29 CD44+(a), HT29 CD133+(b), PC3 CD44+(c), PC3 CD133+(d), MCF-7 CD44+(e), and MCF-7 CD24' (f) cancer stem cell lines for a determined period.

[0021] [Results were analyzed using the Two-way ANOVA test (****P<().0001, ***P<0.0005, * *P<0.01, *P<0.05 vs Control) ]

[0022] Figure 5 Graphs showing the time-dependent variation of the dimensional change occurring in HT29 CD133+(a) and CD44+(b) micro-tissues. [Results were analyzed using the Two-way ANOVA test (*** *P<Q.0001, * * *P<Q.0005, * *P<Q.01, *P<Q.05 vs Control) ]

[0023] Figure 6 Graphs showing the time-dependent variation of the dimensional change occurring in PC3 CD133+(a) and CD44+(b) micro-tissues. [Results were analyzed using the Two-way ANOVA test (*** *P<Q.0001, * * *P<Q.0005, * *P<0.01, *P<Q.05 vs Control) ]

[0024] Figure 7 Graphs showing the time-dependent variation of the dimensional change occurring in MCF-7 CD24' (a) and CD44+(b) micro-tissues. [Results were analyzed using the Two-way ANOVA test (****P<0.0001, ***P<0.0005, **P<0.01, *P<0.05 vs Control)]

[0025] With the invention, 2-c7.s-6-bis(V-ethyl-V'-4-fluorobenzyl)cyclotetraphosphazene (Formula I) and di / .wpropylamino bicyclic 2- / ra / / .s-6-bis(4-fluorobenzylspiro)cyclotetraphosphazene (compounds of Formula II), which exhibit antiproliferative activity against cancer, have been developed.FPhCH, NHPr' I

[0026]

[0027] Et NHPr'

[0028] (Formula I) (Formula II)

[0029] The method for the synthesis of the molecules according to the invention is explained hereinafter.

[0030] First, the starting compound, the bidentate N / N donor atom unsymmetrical 7V-ethyl-7V'-(4-fluorobenzyl)-l,2-ethanediamine compound, was obtained by the reduction of the imine compound, formed by reacting 4-fluorobenzaldehyde with 7V-ethyl-l,2-ethanediamine in an ethyl alcohol medium, with NaBFU.

[0031] NHC2H5

[0032] EtOH

[0033] NaBH4

[0034] CH,— Ml NHC2H5

[0035]

[0036] Subsequently, partially substituted mono(4-fluorobenzyl)spiro, 2-trans-6- and 2-cis-6-bis(4-fluorobenzylspiro) cyclotetraphosphazene compounds were synthesized from the reaction of the unsymmetrical 7V-ethyl-7V-(4-fluorobenzyl)-l,2-ethanediamine compound with bidentate N / N donor atoms with the tetramer (octachlorocyclotetraphosphazene) in a 1 :2 stoichiometric ratio at -10 °C, under argon atmosphere, in tetrahydrofuran (THF) solvent and in the presence of EtaN. The solution was left to stir for 72 hours, filtered, its solvent (THF) was removed in a rotary evaporator, and the remaining solid matter was dissolved in toluene and purified by column chromatography using a silica gel packed column prepared with the same solvent. Bbylece sentezi hedeflenen birinci molekul olan, 2-cis-6-bisspirosiklotetrafosfazen tiirevi Forrmil I bi 1 e§igi sentezlenmi§tir.Isopropylamine substituted monocyclic cyclotetraphosphazene and bicyclophosphazene compounds were synthesized as a result of the reactions of the synthesized partially substituted 2- / ra / / .s-6-bisspirocyclotetraphosphazene compound with an excess amount of / .w-propylamine in the presence of triethylamine in a dry THF medium under reflux in an argon atmosphere. The reaction mixture was stirred at room temperature and under an argon atmosphere for 3 days. Subsequently, the solution was refluxed for 2 days. The solution was filtered, the solvent (THF) was removed in a rotary evaporator, and the remaining solid matter was dissolved in toluene-THF (1:2) and separated by column chromatography using a silica gel-packed column prepared with the same solvent. The white solid product was purified by crystallization in hexane. As a result of this reaction, the / .w-propylamine substituted bicyclophosphazene derivative compound of Formula II, which is the second targeted molecule for synthesis, was synthesized.

[0037] FPhCH FPbCH2

[0038] I

[0039] r~N„ Cl FPhCH2NH NHC2HS'{-a ip-sN —

[0040] N C2H5N CH,PhF Q li i N - < cu f 1 — iC1Z "XNJ 2-lnw-6-btsspwo cH,PhF-4M-fi-bisspiro target product _ THF j jw-propylamine

[0041] NHPr’ Cl — PC "'NUFr1N E*

[0042] CHjPhF

[0043] NHPr’

[0044]

[0045] target product

[0046] The experiments performed to demonstrate the antiproliferative activity of the compound of the invention on cancer cells and CSCs are described below.

[0047] Isolation, Cultivation, and Characterization of Cancer Stem Cells from Cancer Cell Lines

[0048] In the first step, cancer stem cell isolation studies were carried out from MCF7 breast cancer, PC3 prostate cancer, and HT29 colon cancer cell lines. For isolation, the separation of cancer stem cells was performed via a magnetic isolation kit (MACS-Miltenyi Biotec) supplied within the scope of the project, according to the manufacturer's application protocol. Duringthis separation, CD133, CD24, and CD44 loaded magnetic nanoparticles contained in the kit were utilized.

[0049] The MACS technique enables the separation of desired cells in the presence of a magnetic field using magnetic microparticles labeled to bind to the target surface protein. It is emphasized that the size of the magnetic nanoparticles used is approximately 100 nm and they do not produce any toxic effect on the cells. According to the protocol recommended by the manufacturer, the MACS separation method was used in all studies requiring stem cell isolation within the scope of the project. Changes made in the protocol were adjusted according to the type of magnetic labeling nanoparticles (CD133+, CD44+, and CD24 ) and the cell quantity used. The MiniMACS separator was used when the cell number was below 2xl07, and the MidiMACS separator was used when it exceeded 2xl07. Furthermore, in addition to the separator change, as also stated above, the amounts of buffer, micro-bead, and FcR blocking solutions to be used were also increased proportionally.

[0050] 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 project.

[0051] Determination of gene expressions of CD44+. CD133+. and CD24~ cells obtained by the MACS technique via qRT-PCR

[0052] 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 by the MACS method. The "comparative AACT" method was used for the quantification of amplification products obtained by Real-time PCR. The AACT value refers to the first cycle where there is a significant increase in the amount of PCR products. A reference gene was used 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 change is expressed as an exponential function of 2. Since the AACT values obtained for the control are approximately "0", the values for 2'AACTare "1". The obtained result is the relative amount of the expression of thetarget gene in the sample group compared to the control. It does not contain units as it is a ratio expression. It shows how many fold the expression of the target gene in the sample has increased or decreased compared to the control.

[0053] Determination of the Anti-Proliferative Effects of the Compound on Cancer Cell Lines

[0054] The following experimental procedure was applied to determine the anti -proliferative effects on cancer cell lines of the compounds, the syntheses of which were performed and information regarding the synthesis and characterization of which was presented in the previous sections. 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 (50 pM, 25 pM, 12.5 pM, 6.25 pM, 3.13 pM, 1.56 pM). The solvent used for the dissolution of the related compounds was DMSO, and during the calculation, attention was paid to ensure that the maximum DMSO concentration was 0.1% (v / v). Thus, any cytotoxic effect originating from DMSO was prevented. The initial working concentration was set to 50 pM, and subsequently, the substance was diluted to six different concentrations by performing serial dilutions. The prepared solutions were applied to the wells 24 hours after seeding the initial cell numbers into the plates (while the cells were in the logarithmic phase). The said application was performed in triplicate. The plates to which the substance was added were placed in an incubator at 37 °C with 5% CO2 and 95% humidity to perform viability analysis via the MTT test at 24 and 48 hours. For MTT, the used culture medium in the plate was removed, and culture medium containing MTT at a concentration of 0.5 mg / ml was added onto the cells. After the cells were incubated for 3 hours in the dark at 37 °C in a 5% CO2 incubator, DMSO was added to the wells to ensure the dissolution of the formed formazan crystals. Readings were taken at a wavelength of 570 nm in a UV spectrophotometer, and absorbance values were recorded. The absorbance value of each sample was normalized to the absorbance value of the cell control belonging to that experimental set; thus, % viability rates were determined and graphed. In the graphs, the mean of three replicates and standard deviations (SD) are presented as column charts. GraphPad Prism software (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.Determination of the Effects of the Compounds on Cancer Stem Cells

[0055] The effects of the two active compounds determined by the above method on cancer stem cells were determined again using the MTT method. 5-Fu was used as a positive control agent at this stage as well. As a result of the test, the anti-proliferative effects of the related compounds on cancer stem cells were determined, and the IC50 values were calculated using the GraphPad Prism 8 program.

[0056] Determination of the Effects of the Compounds on Cancer Stem Cell-Based Three-Dimensional Micro-Tissues

[0057] This method is utilized for the evaluation of the tumorigenicity of cells in an in vitro environment and for the investigation of clonogenicity, which is a stem cell characteristic. In this context, within the scope of the project, the colony -forming efficiency in soft agar of MCF7 (CD247CD44+), PC3 (CD133+ / CD44+), and HT29 (CD133+ / CD44+) stem cells and the effects of the compounds on 3D micro-tissues were investigated. Briefly, in this technique, wells that are homogeneous in terms of depth and diameter size are created on agarose gel using molds termed "3D Petri Dish". When cells are cultured in these created wells, micro-tissues are formed eventually as a result of the process of spontaneous selfassembly and adhesion over time.

[0058] Inoculation of stem cells into three-dimensional aearose matrices

[0059] The 24-well culture plate containing agarose gels, prepared 1 day prior according to the method described above, was placed into a laminar flow cabinet, and the equilibration medium on the gels was aspirated. In this study, molds used to create a well diameter of 400 pm were employed.

[0060] The isolation and characterization of MCF7 (CD247CD44+), PC3 (CD133+ / CD44+), and HT29 (CD133+ / CD44+) cancer stem cells were performed according to the method specified in the project, and the cells were cultured and expanded for further experiments.

[0061] The cells to be inoculated into agarose matrices were detached using the trypsinization method and counted.After the cells were obtained, they were inoculated into the gels at a density of 5xl05cells / 60 pL per gel in triplicate for each cell type. Following inoculation, the cell-containing matrices were incubated for 1 hour at 37 °C in a 5% CO2environment.

[0062] At the end of the incubation, 600 pL of culture medium containing serum, L-glutamine, and gentamicin was added onto the gels. Subsequently, the gels were incubated under standard culture conditions.

[0063] The micro-tissues expected to form during incubation were photographed every 24 hours, analyzed using the ImageJ program, and tissue diameters were calculated.

[0064] When the micro-tissues reached approximately 400 pm, test compounds (active compounds) were added onto them at effective doses; they were photographed every 24 hours, and the change in the diameters of the micro-tissues was recorded.

[0065] Size analysis of the photographs taken over 4 days was performed using the ImageJ program, and the change in micro-tissue size occurring over time was plotted.

[0066] The results obtained are presented in Table 1, Table 2, and Table 3 below and in the attached figures.

[0067] Table 1 : IC50 values of the compound in different cancer cell lines and at different time points

[0068] 24 h 48 h

[0069] MCF-7 PC3 HT-29 MCF-7 PC3 HT-29 Compound

[0070] of 36,5 22,6 43,8 19,3 15,4 21,5 Formula I

[0071] Compound

[0072] of 38,6 53 55,1 35,1 43,5 44,7 Formula II

[0073] 5-Fu 33,9 20,9 50,1 20,5 15,6 21,71

[0074]

[0075] Table 2: IC50 values of the compound in different healthy cell lines and at different time points

[0076] 24 h 48 h

[0077]

[0078] MCF-12A RWPE-1 CCD-18CO MCF-12A RWPE-1 CCD-18CO Compound

[0079] of 65,5 55,9 * 76,5 47,8 * Formula I

[0080] Compound

[0081] of 91,1 * 190 * 95,3 * Formula II

[0082] 5-Fu 107,1 96,7 62,9 36,3 49,5 37,95

[0083]

[0084] Table 3: IC50 values of the compound in cancer stem cell lines and at different time points

[0085] Compound of Formula Compound of Formula 5-Fu

[0086] I II

[0087] 24 h 48 h 24 h 48 h 24 h 48 h HT29 CD44+66 15,22 53,57 24,10 46,2 17,86 HT29 CD133+33,83 30,8 49,05 17,7 35,78 8,9 PC3 CD44+30,4 16,09 47,41 45,41 18,38 8,6 PC3 CD133+34,5 17,5 57,6 45,57 28,36 7,2 MCF-7 CD24" 55,97 28,77 55,5 40,16 52,51 12,47 MCF-7 CD44+56,6 21,24 60,77 25,16 58,28 8,29

[0088]

[0089] As can be clearly seen in the tables and figures, when compared to the antiproliferative 5-Fu known in the art, the compound according to the invention exhibits significantly higher efficacy in both cancer cell lines and cancer stem cell lines, while exhibiting limited cytotoxicity against healthy cells.

Claims

CLAIMS1. A synthesis method for a compound having the structure of Formula I,Et(Formula I)characterized in that, it comprises;reducing the imine compound, formed by reacting 4-fluorobenzaldehyde with 7V-ethyl-l,2-ethanediamine in an ethyl alcohol medium, with NaBFL, reacting the synthesized bidentate N / N donor atom unsymmetrical A-ethyl- / ' / '-(4-fluorobenzyl)-l,2-ethanedi amine compound with octachlorocyclotetraphosphazene in a 1 :2 stoichiometric ratio in the presence ofEt3N,purifying the partially substituted 2-cz -6-bisspirocyclotetraphosphazene compound from the synthesized partially substituted mono(4- fluorobenzyl)spiro, 2-trans-6- and 2-cz.s-6-bis(4-fluorobenzylspiro) cyclotetraphosphazene compounds.

2. A synthesis method according to Claim 1 for a compound having the structure of Formula II,NHPr’NHPr*(Formula II)characterized in that, it comprises;reacting the synthesized partially substituted 2-trans-6- bisspirocyclotetraphosphazene compound with an excess amount of isopropylamine in the presence of triethylamine,purifying those having the / .w-propyl amine substituted bicyclophosphazene structure from the synthesized / .w-propylamine substituted monocyclic cyclotetraphosphazene and bicyclophosphazene compounds.

3. A compound having the structure of Formula I.Et(Formula I)4. A compound according to Claim 3, having an antiproliferative effect against cancer.

5. A compound according to Claim 4, having an antiproliferative effect against breast cancer, prostate cancer, and colon cancer.

6. A compound having the structure of Formula I.NllPr'NHPr’(Formula II)7. A compound according to Claim 6, having an antiproliferative effect against cancer.

8. A compound according to Claim 7, having an antiproliferative effect against breast cancer, prostate cancer, and colon cancer.

9. A pharmaceutical composition comprising a compound having the structure of Formula I, a compound having the structure of Formula II, or a combination thereof, and at least one excipient.Et NHPr*(F ormul a I) (F ormul a II)10. A pharmaceutical composition according to Claim 9, having an antiproliferative effect against cancer.

11. A compound according to Claim 10, having an antiproliferative effect against breast cancer, prostate cancer, and colon cancer.

Images