A compound with antiproliferative activity against breast cancer and the synthesis method of this compound

Abstract

The present invention relates to a compound with antiproliferative activity against aggressive breast cancer cells and a synthesis method of this compound for use in patients with breast cancer. Said compound is a mono(thio)substituted-1,4-benzoquinone compound. The compound synthesized in the invention is a new molecule has potential use in the treatment of triple negative breast cancer (TRNC), which represents the most aggressive type of breast cancer, especially in the treatment of MDA-MB-231 coded breast cancer cell line due to its antiproliferative activity and the effects of the electrochemical / electronic properties of the compound on biological activity. In order to confirm the molecular structure of the compound of the present invention, spectroscopic analysis, electrochemical analysis and cell viability and proliferation experiments were performed to determine the anticancer effect of the compound. From the analysis, it was concluded that the compound has the ability to suppress the proliferation of breast cancer cells.

Description

[0001] A COMPOUND WITH ANTIPROLIFERATIVE ACTIVITY AGAINST BREAST CANCER AND THE SYNTHESIS METHOD OF THIS COMPOUND

[0002] Field of the Invention

[0003] The present invention relates to a compound with antiproliferative activity against aggressive breast cancer cells and a synthesis method of this compound for use in patients with breast cancer. Said compound is a mono(thio)substituted-1 ,4- benzoquinone compound.

[0004] The compound synthesized in the present invention has potential use in the treatment of triple negative breast cancer (TNBC), which represents the most aggressive type of breast cancer, especially in the treatment of MDA-MB-231 coded breast cancer cell line due to its antiproliferative activity and the effects of the electrochemical / electronic properties of the compound on biological activity.

[0005] State of the Art

[0006] Cancer is a disease caused by the uncontrolled proliferation and growth of cells in any organ or tissue of the body. Cancer is named according to the tissue in which it occurs. The most common and fatal cancer types are lung, colon and breast cancers. Breast cancers are malignant tumors that begin in breast cells and grow uncontrollably. Breast cancer is the most common type of cancer, accounting for 30% of cancers detected in women, and 15% of cancer-related deaths in women are due to breast cancer

[0001] ,

[0007] The most aggressive type of breast cancer, which is highly resistant to most drugs, is called triple negative breast cancer (TNBC). TNBC is a highly aggressive type of breast cancer that lacks the estrogen hormone receptor, progesterone hormone receptor and human epidermal growth factor receptor. TNBC is characterized by extremely high drug resistance, rapid progression, poor prognosis, and lack of clear therapeutic targets. TNBC constitutes 15-20% of breast cancers, and this type of breast cancer is mostly encountered in young pre-menopausal women under the age of 40 [2], Since options for the treatment of TNBC are quite limited, understanding the molecular basis of the disease is important for the development of effective new drugs. One of the most commonly used breast cancer cell lines in breast cancer research is the MDA-MB-231 cell line. MDA-MB-231 cells are a highly aggressive, poorly differentiated triplenegative breast cancer cell line [3].

[0008] Quinones are compounds found in nature in plants, fungi, insects and microorganisms. Apart from natural quinones, synthetically obtained quinones are also used in materials, medicine and chemistry fields. Especially the diversity of biological activities shown by quinones; their antifungal, antiviral, antibacterial, anticancer, antiproliferative, anti-inflammatory, antiplatelet, anti-allergic and cytotoxic effects make their synthesis up to date. The use of quinone and hydroquinone structures as anticancers and electron transfer reactions are of great importance in biological systems.

[0009] In the state of the art, hormone therapy is a frequently preferred treatment method in breast cancer patients. Hormone therapy for breast cancer works by stopping these hormones from reaching breast cancer cells. Tamoxifen, a selective estrogen receptor modulator that can be used orally, is preferred in this treatment method. Tamoxifen is the most sold and used drug in the treatment of breast cancer worldwide and works by blocking estrogen receptors [4], However, during this treatment, a number of cellular mechanisms that regulate cellular replication are disrupted. Although the overall response rate to said drug is high, in many patients the disease recurs after treatment or the patient develops resistance to said drug. Therefore, the damage caused to the patient as a result of cell disruptions caused by hormone therapy, which is frequently used in breast cancer, cannot be prevented and the risk of recurrence of the disease after treatment cannot be prevented because the patient may develop resistance to the drug.

[0010] In addition to Tamoxifen, the most commonly used classes of chemotherapeutic agents according to the routine treatment protocol are the anthracycline group (doxorubicin) and the taxane group (paclitaxel and docetaxel). The most important side effect of the anthracycline-based chemotherapy method commonly used in breast cancer patients is cardiotoxicity. Anthracycline-related cardiotoxicity often occurs late, 10-15 years after treatment, and is usually irreversible once symptomatic [5]. Paclitaxel, one of the taxane group chemotherapeutic drugs, has a very low water solubility value, and this makes it difficult to provide a suitable dosage form during treatment. Various serious side reactions such as hypertensive reactions, nephrotoxicity and neurotoxicity also occur in patients using paclitaxel during their treatment. In this case, drug-related side effects such as cardiotoxicity, hypertensive reactions, nephrotoxicity and neurotoxicity that may occur in patients undergoing breast cancer treatment cannot be prevented and difficulties in adjusting the appropriate dosage form of the drug used in treatment cannot be prevented.

[0011] In cancer treatment methods used in another known state of the art, where treatment is not restricted to a specific cell for each type of cancer, a treatment that kills a cell in one condition may actually help the cell in another condition to grow better. Because the tumor can adopt states in changing microenvironments, each of which may be sensitive to different anticancer treatments. Therefore, this method may show no or limited activity against target cells. This situation leads to the fact that the risk of cancer recurrence is high even after successful treatment of cancer.

[0012] The limitations and inadequacies of the solutions in the state of the art, the permanent damage to the patient's health as a result of cell disruption caused by hormone therapies frequently used in breast cancer treatment, the risk of the patient developing breast cancer again in the future due to the patient's resistance to said chemotherapeutic drugs, side effects such as cardiotoxicity, hypertensive reactions, nephrotoxicity and neurotoxicity that occur in the patient after treatment, and the risk of sensitization to anticancer therapies that occur when targeted treatment is not performed, have made it necessary to develop a development in this field.

[0013] Brief Description and Objects of the Invention

[0014] The present invention relates to a compound with antiproliferative activity against aggressive breast cancer cells and a synthesis method of this compound for use in patients with breast cancer. Said compound is a mono(thio)substituted-1 ,4- benzoquinone compound with the structure shown in Formula 1 . (Formula 1)

[0015] This compound synthesized in the invention is a new molecule and the effect of the electrochemical / electronic properties of the compound on biological activity is being determined. There are Chlorine (-CI) and Thio (-SR) groups on the compound synthesized in the invention. As a result of the effect of these groups on the benzoquinone structure on cancer cells, the structure was found to have suppressive properties against MDA-MB-231 cells (triple receptor negative breast cancer cells).

[0016] One object of the invention is to develop better treatment strategies and a target cell- targeted method to improve patient survival in the treatment of TNBC-type breast cancer than is currently available. The compound synthesized in the invention provides targeted therapy in the treatment of TNBC, which represents the most aggressive type of breast cancer, especially against the MDA-MB-231 coded breast cancer cell line, with its antiproliferative activity.

[0017] Another object of the present invention is to make new and original developments for compounds used in the medical field. The present invention realizes an improvement in the technical field regarding quinone compounds and / or their derivatives. The mono(thio)substituted-1 ,4-benzoquinone compound synthesized in the invention is a new molecule. The effect of electrochemical / electronic properties of this compound on biological activity and anti-proliferative activity against aggressive breast cancer cells is investigated for the first time and a unique synthesis is realized.

[0018] This compound is aimed to be used therapeutically in TNBC breast cancer treatments with its antiproliferative activity against triple receptor negative breast cell line MDA- MB-231 cells, an aggressive breast cancer cell line. Description of the Figures

[0019] Figure 1. Cyclic voltammogram of the compound in ACN / TBAPFe solution medium at a scan rate of 100 mV / s.

[0020] Figure 2. Effect of the compound on cell viability / proliferation in MDA-MB-231 breast cancer cells

[0021] Figure 3. Effect of the compound on clone formation in MDA-MB-231 breast cancer cells

[0022] Detailed Description of the Invention

[0023] The present invention relates to a compound with antiproliferative activity against aggressive breast cancer cells and a synthesis method of this compound for use in patients with breast cancer. Said compound is a mono(thio)substituted-1 ,4- benzoquinone compound with the structure shown in Formula 1 . (Formula 1)

[0024] The compound of the present invention shows antiproliferative activity, especially against the MDA-MB-231 coded breast cancer cell line, and the damage caused by cell degradation that may occur in the patient's cells other than cancerous cells due to treatment is prevented. The effect of electrochemical / electronic properties of this compound on biological activity and anti-proliferative activity against aggressive breast cancer cells is investigated for the first time and a unique synthesis is realized.

[0025] The compound synthesized in the invention provides targeted therapy in the treatment of TNBC, which represents the most aggressive type of breast cancer, with its antiproliferative activity, especially against the triple receptor negative breast cell MDA- MB-231 cell line. There are Chlorine (-CI) and Thio (-SR) groups on the compound synthesized in the invention. As a result of the effect of these groups on the benzoquinone structure on cancer cells, the structure was found to have suppressive properties against MDA-MB-231 cells (triple receptor negative breast cancer cells).

[0026] Synthesis method of the compound of the present invention comprises the process steps of; i) Carrying out the reaction with an equivalent ratio of p-chloronil and sodium-2- methyl-2-propanthiolate in a chloroform + dichloromethane solvent mixture,

[0027] (Reaction 1) ii) extracting the reaction mixture with water and chloroform, iii) drying and filtering the organic phase, iv) removing the organic phase by vacuum, v) purifying the crude product by column chromatography

[0028] In one embodiment of the invention, the synthesis method of the compound comprises the process steps of; i) Reacting 8.14 mmol of p-chloranil compound with 8.14 mmol of sodium-2- methyl-2-propanethiolate in 50 mL chloroform+dichloromethane solvent mixture at 55°C, (Reaction 1) ii) extract the reaction mixture with water and chloroform, (3 x 25 mL) iii) filtering the organic phase by drying with anhydrous sodium sulfate (Na2SC ) iv) removing the organic phase by vacuum, v) purifying the crude product by column chromatography in a silica gel-filled glass column, petroleum ether-chloroform solvent

[0029] The reaction of the compound synthesized according to this method is explained in

[0030] Reaction 1 .

[0031] (Reaction 1)

[0032] 1HNMR,13CNMR and Mass Spectroscopy (MS) results for the compound are given below.

[0033] Spectroscopic analyzes of the compound obtained in the invention were performed. The results of the spectroscopic analysis performed to confirm the molecular structure of the compound in question are shown in Table 1 .

[0034] Table 1. Spectroscopic analysis results

[0035] Rf (1 PET / I CHCh): 0.7 Yield: 8%; Kermis solid; UV (CHCI3), Amax (log E): 296 (4.23), 506 (2.91 ).

[0036] 1H-NMR (500 Mz, CDCh): 5: 1.38 (s, 9H, methyl group protons).13C-NMR (125 Mz, CDCh): 6:173.33, 170.28 (carbonyl group, C=O carbons); 148.67, 144.27, 142.02, 140.48 (quinone carbons) 53.92 (tertiary carbon, Cter); 32.38 (methyl carbons, CH3).

[0037] MS m / z: [M]’: (298.0, 100 %), (299.9, 96 %). The mass / charge ratio of the compound in the mass spectrum (in negative ion mode in MS analysis) was found to be [M]’ m / z: 298.0 (at 100%) and m / z: 299.9 (96%). The expected molecular weight of the compound is 299.6 g.mol’1on average. Electrochemical analyzes of the compound mentioned in the invention were performed. The electronic perturbation of the compound, a new benzoquinone derivative, was investigated at different scan rates (50-1000 mV / s), in the potential range of 0,3 - (-2) V and using an Ag / AgCI reference electrode. Figure 1 shows the cyclic voltammogram of the benzoquinone derivative at a scan rate of 100 mV / s.

[0038] According to the data obtained from the cyclic voltammogram, it can be seen in Figure 1 that both redox processes for the compound are reversible. The difference between the cathodic and anodic peak potentials for two redox couples was calculated as 70- 100 mV and the peak ratios were found to be approximately 1. Additionally, it was observed that there was no significant change in AEPand ipa / v1 / 2ratios with the change in scan rate. With the presence of thiolate and chlorine groups on the quinone structure, the electrochemical property of the quinone structure is greatly improved. The presence of the thiolate group reduces the electron density on the benzoquinone structure. It is seen that the E1 / 2 potential values for the thio substituted benzoquinone compound are quite negative for both redox processes. For the quinone derivative, the anodic and cathodic peak potentials of the 1stand 2ndredox couple were found to be - 195 mV and -870 mV, respectively, at a scan rate of 100 mV / s (Table 2). This result is an indication that the benzoquinone derivative is easily reduced.

[0039] Table 2. Electrochemical parameters of the quinone derivative of the invention

[0040] In Table 2, Epcand Eparefer to cathodic and anodic peak potentials, respectively, lpcand lparefer to cathodic and anodic peak currents, respectively, and AEPrefers to the anodic and cathodic peak difference (AEP= Epa- Epc).

[0041] Geometric optimization of said compound was performed using the DFT method and theoretical HOMO-LUMO energies were calculated. Using the 6-31 G (d,p) basis set and the B3LYP hybrid functional, HOMO-LUMO energies were calculated for all atoms of the benzoquinone derivative and molecular orbital analysis was performed [6]. The results obtained from the theoretical calculation are shown in Table 3.

[0042] Table 3. Quantum chemical descriptors of the benzoquinone derivative

[0043] The HOMO and LUMO energies calculated for the compound of the present invention by TD-DFT method, B3LYP functional and 6-31 g (d,p) basis set were found to be relatively small, -6.83 eV and -4.14 eV, respectively. The AEgapvalue for this compound was calculated as 2,69 eV from the HOMO-LUMO values. The small HOMO-LUMO energy range of this molecule makes it soft for chemical reactions and may underlie its bioactivity. However, the automatically corresponding hardness value was also found to be low at 1 ,345, suggesting the high reactivity of the investigated compound.

[0044] In the synthesis of the compound of the present invention, the progress of the reaction was controlled by Thin Paper Chromatography (TLC) under UV light. In order to test the effect of benzoquinone synthesized in aggressive triple receptor negative breast cancer cells (MDA-MB-231 ) on cell viability / proliferation, 1000 cells were added to 100 pl medium and incubated in an oven for proliferation. Then, different concentrations of benzoquinone were applied to the cells at the 72ndhour of incubation. 24 hours after application, cell viability / proliferation was measured by MTS analysis.

[0045] In order to test the effect of benzoquinone synthesized in aggressive triple receptor negative breast cancer cells (MDA-MB-231 ) on clone formation, an important feature of cancer cells, 1500 cells were added to 2000 pl medium and incubated in an oven for proliferation. Different concentrations of benzoquinone were applied to the cells at the 48thhour of incubation. 12 days after the application, incubation of the cells was terminated and the number of colonies formed was counted.

[0046] In the synthesis of the compound of the present invention, MDA-MB-231 cell line was purchased from ATCC for cell viability and proliferation experiments (MTS analysis) in order to determine the anticancer effect of the compound. Cells were cultured in medium comprising 10% fetal bovine serum at 37°C in a 5% CO2 incubator. The compound 1 -(1 -hydroxyhexane-3-ylthio)anthracene-9, 10-dione was dissolved in dimethylsulfoxide.

[0047] Cell viability and proliferation were measured by MTS (3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4 sulfophenyl)-2H-tetrazolium) assay (Promega, Madison, Wl). Cells grown in culture were counted under an inverted microscope using a toma slide. Then, the cells were seeded in 96-well plates with 1.20 x 103 Z100 pl medium in each plate. Cells were treated with benzoquinone synthesized at different concentrations (5, 10, 20, 50, 100, 200 and 500 pM) for 24 hours at the 72ndhour of the cultures. After 24 h of treatment with the compound of interest, a solution comprising MTS and phenazine methosulfate (20:1 v / v) was added to the cells. It was kept at 37°C for 2-3 hours. Afterwards, the cells' viability / proliferation rate was calculated by measuring the absorbance at 490 nm using Elisa Reader (Promega Glomax Multiplex Detection System).

[0048] According to the results of MTS analysis, there was a significant decrease in the number of viable cells in cells treated with the compound of interest at the indicated concentrations compared to cells treated with no treatment (NT) and cells treated with DMSO, the solvent of the substance of interest (Figure 2: p<0.05, p<0.01 , p<0.001 ). Clone formation analysis was performed to investigate the effect of the compound of the present invention on the proliferation and colony formation of MDA-MB-231 cells. With this clonogenic analysis, the growth / proliferation and neoplastic (colony-forming) tendency of cancer cells are measured. While normal cells stop multiplying after multiplying at a certain rate and do not form colonies, cancer cells continue to multiply continuously and form colonies. With this analysis, the growth / proliferation and neoplastic (colony-forming) tendency of cancer cells are measured. For this purpose, it was planted in 6-well plates with 1.5 x 103 / 2 ml medium in each plate. At the 48thhour of incubation, the cells were treated with the relevant compound at 5, 10, 15, 20, 25 pM concentrations, and some cells were not treated for control purposes. During the incubation, untreated cells were monitored and the incubation was terminated when these cells completely filled the bottom of the container in which they were planted (12 days later). At the end of 12 days, the medium (the medium in which the cells grow) was removed and stained with crystal violet comprising methanol (the substance used to dye the cells) for 5 minutes. After staining, the cells were washed with distilled water, images were taken and colonies were counted.

[0049] The results of clone analysis show that treatment with the respective compound at concentrations of 10, 15, 20 and 25 pM significantly suppressed colony formation of MDA-MB-231 cells compared to untreated (NT) and DMSO-treated cells. (Figure 3: p<0,05, p<0,01 , p<0,001 ). The analysis showed that the compound has the ability to suppress the proliferation of breast cancer cells.

[0050] REFERENCES

[0051] [1] Siegel, R. L., Miller, K. D., Jemal, A. (2020). Cancer statistics, CA: a cancer journal for clinicians, 70(1 ), 7-30.

[0052] [2] Yin L., Duan J. J., Bian X.W., Yu S.C. (2020). Triple-negative breast cancer molecular subtyping and treatment progress. Breast Cancer Res, Jun 9;22(1 ):61 .

[0053] [3] Brian, L., Joshua, B., Xi, C. (201 1 ). Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies Clinical Inv, 121 : 2750-67.

[0054] [4] Cuzick J., Decensi A., Arun B., Brown P.H., Castiglione M., Dunn B. (201 1 ). Preventive therapy for breast cancer: a consensus statement. Lancet Oncol, 12:496- 503.

[0055] [5] Lotrionte, M., Biondi-Zoccai, G., Abbate, A., Lanzetta, G., et al. (2013) Review and meta-analysis of incidence and clinical predictors of anthracycline cardiotoxicity. The American journal of cardiology, 1 12(12):1980-1984.

[0056] [6] The Becke three parameter hybrid exchange functional and the Lee-Yang-Parr correlation functional) (Becke, J. Chem. Phys. 1993, 98, 5648; Lee vd., Phys. Rev. B. 1988, 37, 785.

Claims

CLAIMS1. A compound with antiproliferative activity against aggressive breast cancer cells having the following chemical formula (Formula 1 )(Formula 1)2. A compound according to claim 1 for use in the treatment of breast cancer.

3. A compound according to claim 1 for use in the treatment of Triple Negative Breast Cancer (TNBC).

4. A compound according to claim 3 with antiproliferative activity against the breast cancer cell line MDA-MB-231 .

5. A compound according to any one of claims 1 -4, wherein said compound is a mono(thio)substituted-1 ,4-benzoquinone compound.

6. A compound according to any one of claims 1 -4, wherein said compound comprises a mass / charge ratio [M]- m / z in the mass spectrum (negative ion mode in MS analysis): 298.0 (at 100%) and m / z: 299.9 (at 96%).

7. A compound according to any one of claims 1 -4, wherein said compound has an average molecular weight of (299.6 g.mol’1).

8. A compound according to any one of claims 1 -4, wherein the1H-NMR spectrum of the compound comprises 5:1.38 ppm (3 methyl protons: 3CH3) peaks.

9. A compound according to any one of claims 1 -4, wherein the13C-NMR spectrum of the compound comprises 5: 173.33, 170.28 ppm (carbonyl group carbons), 148.67, 144.27, 142.02, 140.48 ppm (four carbons belonging to quinone structure), 53.92 ppm (tertiary carbon), 32.38 ppm (methyl carbons) peaks.

10. Synthesis method of mono(thio)-substituted benzocinoid compound with antiproliferative activity against Triple Negative Breast Cancer (TNBC) breast cancer type, comprising the process steps of;i) Carrying out the reaction with an equivalent ratio of p-chloronil and sodium-2-methyl-2-propanthiolate in a chloroform + dichloromethane solvent mixture,ii) extracting the reaction mixture with water and chloroform, iii) drying and filtering the organic phase, iv) removing the organic phase by vacuum, v) purifying the crude product by column chromatography.

11. Synthesis method of mono(thio)-substituted benzocinoid compound with antiproliferative activity against Triple Negative Breast Cancer (TNBC) breast cancer type, comprising the process steps of; i) Reacting 8.14 mmol of p-chloranil compound with 8.14 mmol of sodium- 2-methyl-2-propanethiolate in 50 mL chloroform+dichloromethane solvent mixture at 55°C,ii) extracting the reaction mixture with water and chloroform, (3 x 25 mL) iii) filtering the organic phase by drying with anhydrous sodium sulfate (Na2SO4) iv) removing the organic phase by vacuum, v) purifying the crude product by column chromatography in a silica gel- filled glass column, petroleum ether-chloroform solvent.

12. A compound according to any one of claims 1 -11 for use in the treatment of breast cancer.

13. A compound according to claim 12 for use against the breast cancer cell line MDA-MB-231 for the treatment of triple negative breast cancer (TNBC).

14. Use of a pharmaceutical composition of the compound according to claim 13 in the production of a medicament for the treatment of cancer.

15. Use of a pharmaceutical composition of the compound according to claim 13 for the production of a medicament for the treatment of breast cancer against the breast cancer cell line MDA-MB-231 for the treatment of triple negative breast cancer (TNBC).

16. A medicament with anti-cancer activity comprising a pharmaceutical composition according to claim 13.

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