Gypsogenin-amine semisynthesis derivative compounds with anticancer activity and synthesis methods of these compounds

Abstract

The invention relates to the gypsogenin-amine semisynthesis derivative compounds with anticancer activity and synthesis methods of these compounds. The gypsogenin-amine semisynthesis derivative compounds of the invention are derivatized from gypsogenin-amine aglycone.

Description

[0001] GYPSOGENIN-AMINE SEMISYNTHESIS DERIVATIVE COMPOUNDS WITH ANTICANCER ACTIVITY AND SYNTHESIS METHODS OF THESE COMPOUNDS

[0002] Technical Field of the Invention

[0003] The invention relates to the gypsogenin-amine semisynthesis derivative compounds with anticancer activity and synthesis methods of these compounds. The gypsogenin- amine semisynthesis derivative compounds of the invention are derivatized from gypsogenin aglycone.

[0004] State of the Art

[0005] Today, plants are actively used in the treatment of a plurality of diseases. For example, sage, mint, linden, and ginger are used to treat colds, while poppy is used as a source of morphine to relieve severe pain. Currently, the use of different plants for medicinal purposes is allowed in many countries, especially in China. The treatment provided by the aforementioned plants is due to the properties of the main active ingredients in said plants. In order to benefit more from the main active ingredients of the aforementioned plants in the treatment of diseases, it is of great importance to obtain these active ingredients synthetically under laboratory conditions. The cytotoxic properties of the main active ingredient obtained at the end of this laborious and costly process are examined and its intended purpose and area of use are determined, and some companies synthesize these active ingredients in the laboratory. It is also important to isolate the compounds in a pure form so that they can be used in the treatment of diseases. One of the said active ingredients is gypsogenin, which is a saponin.

[0006] In a patent document in the state of the art, numbered TR 2019 / 22043, the gypsogenin aglycone derivative compounds of the invention have efficacy in preventing cancer cell growth on different human cancer cell lines such as HEK 293, Hela, SHSY5Y, A549, PANC-1 , and M DA- MB- 231 .

[0007] Breast cancer is a type of cancer that starts in breast cells and has the highest incidence in the world after lung cancer. It is reported that one in every 8 women will develop breast cancer at some point in her life

[0001] . Cervical cancer, which is another deadly type of cancer, is the fourth most common cancer worldwide and the leading cause of death among gynecological cancers. Despite advanced surgical and medical treatments, cervical cancer is still one of the most lethal gynecologic cancers[2]. The World Health Organization (WHO) has warned that by 2030, the annual number of cervical cancer cases will increase to 700,000 and the annual number of deaths to 400,000, especially in low- and middle-income countries[3]. Another type of cancer, human T lymphocyte cancer (lymphoma), is a type of cancer caused by lymphocytes. Lymphoma, or lymph cancer, is a serious disease that occurs as a result of the cancerization of white blood cells called lymphocytes, in other words, the body's defense cells, and constitutes a large part of blood-borne cancers, which can cause death if not intervened in time. Leukemia, which is another deadly type of cancer, is a cancer of white blood cells created in the bone marrow. There are two types of leukemia; acute and chronic. Acute leukemias are leukemias that emerge suddenly, progress rapidly and leave the patient debilitated, and consist of two main types: acute lymphoblastic and acute myeloblastic. Chronic leukemias, on the other hand, are leukemias that have a slower course and usually do not worsen the patient suddenly. Chronic leukemia includes two main types: chronic myeloid (myelocytic) leukemia (CML) and chronic lymphocytic leukemia. In acute leukemia, the number of leukocytes in the blood may be low, normal, or high, while in chronic leukemia, the number of leukocytes in the blood increases. Chronic myeloid leukemia is a cancer of granulocytes and in this disease, the number of granulocytes and along with granulocytes, platelets, the cells that enable blood clotting, are increased in the blood[4]. The 5-year survival rate for CML, a type of blood cancer that particularly affects individuals aged 25-60 years, is 67%[5].

[0008] The present art does not include any compound or derivative thereof that shows anticancer activity on human breast cancer cell line (MCF-7), human cervical cancer cell line (HeLa), human T lymphocyte cancer (Jurkat) cell line, and human chronic myeloid leukemia cancer cell line (K562).

[0009] Although the state of the art does not include any compounds or derivatives that show anticancer activity on human breast cancer cell line (MCF-7), human cervical cancer cell line (HeLa), human T lymphocyte cancer (Jurkat) cell line and human chronic myeloid leukemia cancer cell line (K562), a resistance has been developed by the cancer cells against the compounds and derivatives that show activity, albeit separately, on said cancer cell lines as a result of long-term use, and for this reason, it has become a necessity to introduce new compounds and derivatives that are not yet recognized by cancer cells and to which said cancer cells have not yet developed resistance.

[0010] Since the present art does not include any compound or derivative thereof that exhibits anticancer activity on human breast cancer cell line (MCF-7), human cervical cancer cell line (HeLa), human T lymphocyte cancer (Jurkat) cell line, and human chronic myeloid leukemia cancer cell line (K562), and resistance has been developed by the cancer cells against the compounds and derivatives that show activity, albeit separately, on said cancer cell lines as a result of long-term use, due to reasons such as the necessity of introducing new compounds and derivatives that are not yet recognized by cancer cells and to which the cancer cells have not yet developed resistance, it has been made necessary to introduce new compounds that exhibit anticancer activity on human breast cancer cell line (MCF-7), human cervical cancer cell line (HeLa), human T lymphocyte cancer (Jurkat) cell line and human chronic myeloid leukemia cancer cell line (K562) for use in eliminating all these problems.

[0011] Summary and Objects of the Invention

[0012] The invention relates to the gypsogenin-amine semisynthesis derivative compounds with anticancer activity and synthesis methods of these compounds. The gypsogenin- amine semisynthesis derivative compounds of the invention are derivatized from gypsogenin aglycone. The general structure of the compounds of the invention is denoted by Formula X.

[0013]

[0014] Formula X

[0015] An object of the invention is to introduce new compounds that exhibit anticancer activity on human breast cancer cell line (MCF-7), human cervical cancer cell line (HeLa), human T lymphocyte cancer (Jurkat) cell line, and human chronic myeloid leukemia cancer cell line (K562), and to which cancer cells will not show resistance when used. Thanks to the novelty of the compounds of the invention, the resistance of cancer cells is eliminated.

[0016] Another object of the invention is to introduce compounds that exhibit anticancer activity on the human breast cancer cell line (MCF-7), human cervical cancer cell line (HeLa), human T lymphocyte cancer (Jurkat) cell line, and human chronic myeloid leukemia cancer cell line (K562). A common compound derivative that provides activity on all of said cancer cell lines is provided with the invention. Studies show that the compounds of the invention kill cancer cells and have the potential to be used as drugs. When the anticancer effects of the drugs prepared using the compounds of the invention on some cancer cells were examined, according to the metabolic capacity MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide] test data, among these new compounds, compound 5 synthesized with 6-bromo-1 ,3-benzothiazol-2- amine was the most effective anticancer agent with an IC50 value of 0.66±0.17 pM in MCF-7 cell line, indicating the high activity of amine in human breast cancer cell line among gypsogenin derivatives. Compound 5 also exhibited significant anticancer effect against Jurkat cells with IC50 value of 1.40±0.89pM. The activity of gypsogenin derivatives against breast cancer cell line (MCF-7) was followed by Compound 3 (IC50= 1.03±0.15 pM) and Compound 1 (IC50= 2.40±0.82 pM). Moreover, Compound 3 and Compound 1 revealed the most pronounced anticancer effects against HeLa cell line with IC50 values of 0.74±0.12 pM and 1.88±0.73 pM, respectively. The anticancer effects of these agents were higher than Imatinib (IC50= 3.03±0.26 pM). Compound 2 was found to be the most effective anticancer agent against Jurkat and K562 cells with IC50 values of 1.15±0.53 pM and 2.35±0.22 pM, respectively; the anti-leukemic effects of Compound 2 were found to be even higher than gypsogenin and imatinib. This result indicates that the aniline moiety enhances the anti-leukemic effect of gypsogenin derivatives. Overall, it is concluded that almost all gypsogenin derivatives exhibited more significant anticancer activity than gypsogenin and Imatinib against all cell lines tested except K562 cells. After MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-di- phenyltetrazolium bromide] test evaluation, apoptosis was detected by considering the effective values. In this context, measurements were taken with HOPI monitoring after cell viability tests to determine whether the first applied 325 agents played a role in apoptosis or necrosis cells in the effect groups. Accordingly, the groups with the highest number of apoptotic cells in MCF-7 cells after drug cultivation were Compound 5, Compound 3, and Compound 4 with 93.38%, 88.34%, and 84.74% values, respectively. In HeLa cells, the groups with the highest number of apoptotic cells were found to be Compound 3, Compound 1 , and Compound 6 with 92.45%, 85.68%, and 83.68%, respectively. For Jurkat cells, it was Compound 5, Compound 2, and Compound 1 with 88.66%, 87.07%, and 81.10%, respectively. Lastly, Compound 2, Compound 6, and Compound 5 showed the most significant apoptotic activity against K562 cells with 82.22%, 80.79%, and 78.89%, respectively. Briefly, the most significant apoptotic effects were detected with Compound 5 and Compound 3 against MCF-7 and HeLa cells, respectively, and these results were found to be fully consistent with the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide] test data. In summary, the gypsogenin derivatives (Compounds 1 -6) of the invention with amine substituents at position C-23 were tested for their cytotoxicity activity in different human cancer cell lines and the compounds of the invention were found to exhibit cytotoxic activity. The compounds exhibited significant cytotoxic inhibition against MCF-7, HeLa, Jurkat, and K562 cell lines. The results showed that Compound 5 was the most effective derivative against MCF-7 cell line (IC50= 0.66±0.17 pM). Compound 3 shows the most effective anticancer activity against HeLa cell line with an IC50 value of 0.74±0.12 pM. In addition, the anti-leukemic effect of Compound 2 was determined to be the highest against Jurkat and K562 cell lines and IC50 values were calculated as 1.15±0.53 pM, and 2.35±0.22 pM, respectively. Apoptosis rates were found effective with 93.38% for Compound 5 in MCF-7 cell line and 92.45% for Compound 3 in HeLa cell line.

[0017] Description of the Drawings

[0018] Fig. 1. Comparison of the apoptosis rates of the compounds (Compounds 1 -6) of the invention in HeLa Cell line with the apoptosis rates of Imatinib and gypsogenin in HeLa Cell line

[0019] Fig. 2. Comparison of the apoptosis rates of the compounds (Compounds 1-6) of the invention in Jurkat Cell line with the apoptosis rates of Imatinib and gypsogenin in Jurkat Cell line

[0020] Fig. 3. Comparison of the apoptosis rates of the compounds (Compounds 1 -6) of the invention in K562 Cell line with the apoptosis rates of Imatinib and gypsogenin in K562 Cell line

[0021] Fig. 4. Comparison of the apoptosis rates of the compounds (Compounds 1 -6) of the invention in MCF-7 Cell line with the apoptosis rates of Imatinib and gypsogenin in MCF-7 Cell line

[0022] Fig. 5.1H-NMR spectrum of compound 1 .

[0023] Fig. 6.13C-NMR spectrum of compound 1 .

[0024] Fig. 7. MSQ spectrum of compound 1 .

[0025] Fig. 8. UV spectrum of compound 1 .

[0026] Fig. 9.1H-NMR spectrum of compound 2.

[0027] Fig. 10.13C-NMR spectrum of compound 2.

[0028] Fig. 11. MSQ spectrum of compound 2. Fig. 12. UV spectrum of compound 2.

[0029] Fig. 13.1H-NMR spectrum of compound 3.

[0030] Fig. 14.13C-NMR spectrum of compound 3.

[0031] Fig. 15. MSQ spectrum of compound 3.

[0032] Fig. 16. UV spectrum of compound 3.

[0033] Fig. 17.1H-NMR spectrum of compound 4.

[0034] Fig. 18.13C-NMR spectrum of compound 4.

[0035] Fig. 19. MSQ spectrum of compound 4.

[0036] Fig. 20. UV spectrum of compound 4.

[0037] Fig. 21.1H-NMR spectrum of compound 5.

[0038] Fig. 22.13C-NMR spectrum of compound 5.

[0039] Fig. 23. MSQ spectrum of compound 5.

[0040] Fig. 24.1H-NMR spectrum of compound 6.

[0041] Fig. 25.13C-NMR spectrum of compound 6.

[0042] Fig. 26. MSQ spectrum of compound 6.

[0043] Detailed Description of the Invention

[0044] The invention relates to the gypsogenin-amine semisynthesis derivative compounds with anticancer activity and synthesis methods of these compounds. The gypsogenin- amine semisynthesis derivative compounds of the invention are derivatized from gypsogenin aglycone. The invention introduces new compounds that exhibit anticancer activity on human breast cancer cell line (MCF-7), human cervical cancer cell line (HeLa), human T lymphocyte cancer (Jurkat) cell line, and human chronic myeloid leukemia cancer cell line (K562), and to which cancer cells will not show resistance when used. The compound of the invention is denoted by Formula X.

[0045] Formula X

[0046] The compounds of the invention are the following compounds;

[0047] • (3 / 3)-3-Hydroxy-23-(diethylamine) olean-12-en-28-oic acid (Compound 1 , Formula 1),

[0048] • (3 / 3)-3-Hydroxy-23-(aniline)olean-12-en-28-oic acid (Compound 2, Formula 2),

[0049] • (3 / 3)-3-Hydroxy-23-[(2-mercaptoethyl)amine]olean-12-en-28-oic acid (Compound 3, Formula 3),

[0050] • (3 / 3)-3-Hydroxy-23-(dimethylamine) olean-12-en-28-oic acid (Compound 4, Formula 4),

[0051] • (3 / 3)-3-Hydroxy-23-[(6-bromo-1 ,3-benzothiazol-2-amine] olean-12-en-28-oic acid (Compound 5, Formula 5),

[0052] • (3 / 3)-3-Hydroxy-23-(2-amine anthracene) olean-12-en-28-oic acid (Compound 6, Formula 6).

[0053] Formula X wherein R is selected from the following formulas,

[0054]

[0055] • (3 / 3)-3-Hydroxy-23-(diethylamine) olean-12-en-28-oic acid compound (Compound 1 ) obtained when the R group in said Formula X is R=Ri is denoted by Formula 1 . Formula 1

[0056] Synthesis Yield: 87%

[0057] Molecular Weight (g / mol): 527.77g / mol

[0058] Melting temperature: 237.3-238.6°C

[0059] FT-IR (cm1): 3443 cm1, 2924 cm1, 2848 cm1, 1642 cm1, 1456 cm1, 1380 cm1, 1262 cm’1, 1027 cm’1, 749 cm’1

[0060] 1H NMR (600 MHz, pyridine-d5): 6= 0.92 (H-24), 0.96 (H-25), 0.99 (H-26), 1.03 (H-30), 1.04 (t, 6H, H-2a), 1.23 (H-27), 1.77 (H-29), 3.28 (dd, 1 H, H-18), 3.71 (q, H-1 a), 4.19 (m, 1 H, H-23), 4.31 (d, 1 H,H-3), 5.48 (t, 1 H, H-12).

[0061] 13C NMR (150 MHz, pyridine -d5): 14.5 (C-2a), 16.5 (C-24), 17.8 (C-25),19.1 (C-26), 24.3 (C-30), 26.6 (C-27), 31.5 (C-29), 42.2 (C-18), 46.9 (C-1 a), 61.8 (C-23), 73.9 (C-3), 123.1 (C-12), 145.3 (C-13), 180.9 (C-28)

[0062] UV: 290, 252 nm

[0063] MS: LC / MS (ESI-MS) m / z C34H57O3 527.77 [M]_.

[0064] (3 / 3)-3- Hydroxy-23-(aniline)olean-12-en-28-oic acid compound (Compound 2) obtained when the R group in said Formula X is R=R2is denoted by Formula 2.

[0065]

[0066] Formula 2 Synthesis Yield: 56%

[0067] Molecular Weight (g / mol): 526.72g / mol

[0068] Melting temperature: 237.6-239.0°C

[0069] FT-IR (cm1): 3444 cm1, 2924 cm1, 2853 cm1, 1633 cm1, 1462 cm1, 1376 cm1, 1264 cm’1, 742 cm’1

[0070] 1H NMR : (600 MHz, pyridine -d5): 5= 0.91 (H-29), 0.95 (H-25), 0.97 (H-30), 1.03 (H- 26), 1.04 (H-24), 1.20 (H-27), 3.29 (dd, 1H, H-18), 3.60 (1H, H-23), 3.72 (d, 1H, H-3),

[0071] 5.48 (br s, 1H, H-12), 6.87 (-NH), 7.20 (1H, H-2a / H-6a), 7.57 (1H, H-3a / H-5a), 7.90 (1H, H-4a).13C NMR: (150 MHz, pyridine -d5): 12.7 (C-24), 15.6 (C-25), 17.1 (C-26), 23.3 (C-30), 25.7 (C-27), 32.8 (C-29), 41.8 (C-18), 56.4 (C-4), 73.1 (C-3), 122.6 (C-12), 123.6 (C- 2a / C-6a), 129.0 (C-4a), 135.5 (C-3a / C-5a), 144.5 (C-13), 151.0 (C-1a).

[0072] UV: 275 nm

[0073] MS: LC / MS (ESI-MS) m / z C36H53NO3 526.70 [M-COOH+Na+1] +.

[0074] (3 / 3)-3-Hydroxy-23-[(2-mercaptoethyl)amine]olean-12-en-28-oic acid compound (Compound 3) obtained when the R group in said Formula X is R=Rs is denoted by Formula 3.

[0075] Formula 3

[0076] Synthesis Yield: 79%

[0077] Molecular Weight (g / mol): 526.69g / mol

[0078] Melting temperature: 218.5-219.5°C

[0079] FT- 1 R (cm1): 3434 cm1, 2648 cm1, 1650 cm1, 1457 cm1, 1265 cm1, 1209 cm1, 1031 , 749 cm’1.1H NMR (600 MHz, pyridine -d5): 5= 0.90 (H-29), 0.95 (H-25), 0.98 (H-30), 1.03 (H-26), 1.04 (H-24),1.22 (H-27), 1.96 (H-2a), 3.29 (dd, 1 H, H-18), 3.73 - 4.20 (m, 1 H, H-23), 4.22 (d, 1 H, H-3), 4.75 (H-1 a), 5.48 (br s, 1 H, H-12), 5.87 (NH-).

[0080] 13C NMR (150 MHz, pyridine -d5): 12.9 (C-24), 15.7 (C-25),17.1 (C-26), 23.3 (C-30), 23.4 (C-2a), 25.7 (C-27), 32.7 (C-29), 41.8 (C-18), 43.1 (C-1 a), 67.4 (C-23),73.0 (C-3), 122.5 (C-12), 144.5 (C-13), 180.0 (C-28).

[0081] UV: 284 nm

[0082] MS: LC / MS (ESI-MS) m / z C32H55NOS 526.67 [M+Na+2]+.

[0083] (3 / 3)-3-Hydroxy-23-(dimethylamine) olean-12-en-28-oic acid compound (Compound 4) obtained when the R group in said Formula X is R=R4 is denoted by Formula 4.

[0084] Synthesis Yield: 63%

[0085] Molecular Weight (g / mol): 453.22g / mol

[0086] Melting temperature: 251 .0-252.6°C FT-IR (cm1): 3434 cm1, 2924 cm1, 2853 cm1, 1644 cm1, 1415 cm1, 1382 cm1, 1275 cm’1, 749 cm’1.

[0087] 1H NMR (600 MHz, pyridine -d5): 5= 0.90 (H-29), 0.95 (H-25), 0.98 (H-30), 1.01 (H-26), 1.04 (H-24), 1.22 (H-27), 3.30 (dd, 1 H, H-18), 3.59 (H-1a / H-2a), 3.72-4.07 (m, 1 H, H-

[0088] 23), 4.19 (d, 1 H, H-3), 5.48 (br s, 1 H, H-12).

[0089] 13C NMR (150 MHz, pyridine -d5): 12.5 (C-24), 15.5 (C-25), 17.1 (C-26), 23.4 (C-30), 25.7 (C-27), 32.9 (C-29), 43.1 (C-18), 48.7 (C-1a / C-2a), 67.7 (C-23), 73.3 (C-3), 122.3 (C-12), 144.6 (C-13), 184.3 (C-28).

[0090] UV: 275 nm

[0091] MS: LC / MS (ESI-MS) m / z 453.20 [M-CH3+1]+

[0092] (3 / 3)-3-Hydroxy-23-[(6-bromo-1 ,3-benzothiazol-2-amine] olean-12-en-28-oic acid compound (Compound 5) obtained when the R group in said Formula X is R=R5is denoted by Formula 5.

[0093]

[0094] Formula 5 Yield: 87%

[0095] Molecular Weight (g / mol): 694.13g / mol

[0096] Melting temperature: 116.2-117.2°C

[0097] FT-IR (cm1): 3440 cm1, 2920 cm1, 2844 cm1, 1640 cm1, 1455 cm1, 1290 cm1, 1255 cm1, 1027 cm1, 749 cnr1.1H NMR (600 MHz, pyridine -d5): 6= 0.50 (s, H-24), 0.68 (s, H-26), 0.85 (s, H-30), 0.85 (s, H-25), 0.85 (s, H-29), 1.04 (s, H-27), 2.72 (dd, J= 6.8, 13.4 Hz, H-18), 3.04-3.31 (m, H-23), 3.43 (d, H-3), 5.13 (brs, H-12), 7.23 (1H, m, H- 2a), 7.32 (H-3a), 7.61 (-NH), 7.86 (2H, m, H-5a).13C NMR (150 MHz, pyridine -d5): 13.1 (C-24), 15.9 (C-25), 17.3 (C-26), 23.9 (C-30), 26.0 (C-27), 33.3 (C-29), 41.2 (C-18), 64.8 (C-23), 70.7 (C-3), 112.5 (C-4a), 119.5 (C- 2a), 121.9 (C-12), 123.7 (C-5a), 128.7 (C-3a), 133.5 (C-2b), 144.2 (C-13), 152.4 (C- 1 b), 167.9 (C-1 a), 179.1 (C-28).

[0098] MS: LC / MS (ESI-MS) m / z CsyHssBr^OS [M+Na]+: 694.11 (100).

[0099] (3 / 3)-3-Hydroxy-23-(2-amine anthracene) olean-12-en-28-oic acid compound (Compound 6) obtained when the R group in said Formula X is R=Re is denoted by Formula 6.

[0100] Formula 6 Yield: 50%

[0101] Molecular Weight (g / mol): 683.87g / mol

[0102] Melting temperature: 122.6-123.2°C

[0103] FT-IR (cm1): 3466 cm1, 2925 cm1, 2850 cm1, 2059 cm1, 1634 cm1, 1462 cm1, 1265 cm’1, 1250 cm’1, 761 cm’1

[0104] 1H NMR (600 MHz, DMSO-d6): 5= 0.51 (s, H-24), 0.69 (s, H-26), 0.84 (s, H-30), 0.85 (s, H-25), 0.85 (s, H-29), 1.08 (s, H-27), 2.70 (dd, J= 6.8, 13.4 Hz, H-18), 3.03-3.41 (H- 23), 4.13 (m, H-3), 5.13 (brs,H-12), 7.04 (H-1a), 7.06 (H-3a), 7.66 (H-6a / H-7a), 7.69 (H- 4a), 7.93 (H-8a), 7.99 (H-5a), 8.07 (H-9a / H-10a).

[0105] 13C NMR (150 MHz, DMSO-d6): 11.2 (C-24), 13.1 (C-25), 14.4 (C-26), 15.9 (C-27), 23.8 (C-30), 26.01 (C-29), 41.8 (C-18), 64.6 (C-23), 70.6 (C-3), 120.5 (C-9a), 121.9 (C- 12), 123.0 (C-2b), 124.3 (C-4b), 127.7 (C-3a), 127.8 (C-6a / C-7a), 129.1 (C-1 a / C-8a), 132.0 (C-3b), 132.3 (C-4a), 137.3 (C-1b), 139.3 (C-2a), 144.3 (C-13), 179.3 (C-28).

[0106] MS: LC / MS (ESI-MS) m / z C44H57NO3 [M+2K]+ : 683.84.

[0107] A synthesis method of gypsogenin-amine semisynthesis derivative compound of the invention with anticancer activity comprises the process steps of i. adding diethyl amine (R1), aniline (R2), 2-mercaptoethylamine (R3), dimethylamine (R4), 6-bromo-1 ,3-benzothiazol-2-amine (R5) or 2-amine anthracene (R6) compounds dissolved in dichloromethane to the solution of gypsogenin dissolved in dichloromethane in the same way in inert medium (N2), ii. initiating the reduction reaction by conversion of the carbon-nitrogen imine (C=N) bond to an amine (C-NH) bond with the addition of sodium triacetoxyborohydride (NaBH(OAc)3) to the resulting mixture, iii. continuing to stir the reaction mixture under reflux condenser, iv. eliminating borohydride remaining in the reaction after completion of the reaction by adding aqueous sodium bicarbonate solution, v. extracting the resulting reaction mixture with dichloromethane, vi. drying the collected organic phase with the addition of anhydrous sodium sulfate (Na2SC>4) and evaporating the solvent collected in the organic phase after extraction, vii. introducing the resulting organic phase with solvent into the column with silica gel filler, viii. obtaining the compounds (3 / 3)-3-Hydroxy-23-(diethylamine) olean-12-en-28-oic acid (Compound 1 ), (3 / 3)-3-Hydroxy-23-(aniline)olean-12-en-28-oic acid (Compound 2), (3 / 3)-3-Hydroxy-23-[(2-mercaptoethyl)amine]olean-12-en-28-oic acid(Compound 3), (3 / 3)-3-Hydroxy-23-(dimethylamine) olean-12-en-28-oic acid (Compound 4), (3 / 3)-3-Hydroxy-23-[(6-bromo-1 ,3-benzothiazol-2-amine] olean- 12-en-28-oic acid (Compound 5) or (3 / 3)-3-Hydroxy-23-(2-amine anthracene) olean-12-en-28-oic acid (Compound 6) after the purification of the organic phase by column chromatography.

[0108] In an embodiment of the invention, a synthesis method of gypsogenin-amine semisynthesis derivative compound of the invention with anticancer activity comprises the process steps of i. adding diethyl amine (Ri), aniline (R2), 2-mercaptoethylamine (R3), dimethylamine (R4), 6-bromo-1 ,3-benzothiazol-2-amine (R5) or 2-amine anthracene (R6) compounds dissolved in dichloromethane to the solution of gypsogenin dissolved in dichloromethane in the same way in inert medium (N2) at a temperature of 20-25°C, ii. initiating the reduction reaction by conversion of the carbon-nitrogen imine (C=N) bond to an amine (C-NH) bond with the addition of sodium triacetoxyborohydride (NaBH(OAc)3) to the resulting mixture, iii. continuing to stir the reaction mixture for 2 hours under reflux condenser, iv. eliminating borohydride remaining in the reaction by adding aqueous sodium bicarbonate solution 1 hour after the reaction is complete, v. extracting the resulting reaction mixture 3 times (10 mL x 3) with 10 mL of dichlormethane, vi. drying the collected organic phase with the addition of anhydrous sodium sulfate (Na2SO4) and evaporating the solvent collected in the organic phase after extraction by an evaporator, vii. introducing the resulting organic phase with hexane / ethyl acetate (6 / 4; 8 / 2) or dichloromethane / ethyl acetate (1 / 1 ) as solvent into a column with silica gel filler, viii. obtaining the compounds (3 / 3)-3-Hydroxy-23-(diethylamine) olean-12-en-28-oic acid (Compound 1 ), (3 / 3)-3-Hydroxy-23-(aniline)olean-12-en-28-oic acid (Compound 2), (3 / 3)-3-Hydroxy-23-[(2-mercaptoethyl)amine]olean-12-en-28-oic acid(Compound 3), (3 / 3)-3-Hydroxy-23-(dimethylamine) olean-12-en-28-oic acid (Compound 4), (3 / 3)-3-Hydroxy-23-[(6-bromo-1 ,3-benzothiazol-2-amine] olean- 12-en-28-oic acid (Compound 5) or (3 / 3)-3-Hydroxy-23-(2-amine anthracene) olean-12-en-28-oic acid (Compound 6) after the purification of the organic phase by column chromatography.

[0109] The cytotoxic properties of the synthesized new gypsogenin-amine semi-synthesis derivative compounds (Compounds 1 -6) of the invention were examined with reference to the substance "Imatinib". Further, after the result of metabolic capacity MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide] test evaluation, apoptosis determination studies were carried out by HOPI staining, taking into account the effective values (Table 1 , 2 and Fig. 1 ).

[0110] Table 2. IC50 values of compounds 1 - 6. Table 3. Apoptosis values for compounds 1-6.

[0111] The anticancer effect of the prepared drugs on some cancer cells is shown in Fig. 1 . For this purpose, prostate cancer cells were plated in 48 wells with 100,000 cells in each well, and only drug treatment was performed at different concentrations and compared with the control groups. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-di- phenyltetrazolium bromide] test protocols were applied 24 hours after the application. The tests were repeated 3 times and the absorbance values of each group were normalized with the control group values and plotted. Accordingly, according to the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide] test data obtained, it was determined that Compound 5 was the most effective drug for the MCF-7 cell line, and it was observed that Compound 3 and Compound 1 were also effective, respectively. Compound 6 was observed to have a similar effect compared to Imatinib. In Hela cells, Compound 3 was found to be effective, followed by Compound 1 as the most effective agent. Similarly, when compared with Imatinib, the effect of these agents was more effective than Imatinib. It was also observed that Compound 2 was the most effective agent in Jurkat cells, followed by Compound 5 and Compound 6. Almost all compounds (Compounds 1 -6) of the invention were found to be more effective than Imatinib in the Jurkat cell line. Finally, in the K562 cell line, Compound 2 was found to be the most effective agent, while gypsogenin, Compound 5 and Compound 6 were also found to be more effective than imatinib. After MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide] test evaluation, apoptosis was determined by HOPI staining considering the effective values. In this context, firstly, measurements were taken with Hoechst and Propidium Iodide (HOPI) staining after cell viability tests to determine whether apoptosis or necrosis activity plays a role in the mechanism of action of the applied agents. Cells stained blue by HOPI staining are considered viable cells, while cells containing blue and particles indicate early apoptotic cells. Similarly, round cells stained with pink color indicate necrotic cells, while cells with pink color and particles indicate late apoptotic cells. Accordingly, the agents with the highest number of apoptotic cells after drug treatment in MCF-7 cells was determined as Compound 5, Compound 3, and Compound 2, respectively. Similarly, the groups with the highest number of apoptotic cells observed in Hela cells were Compound 3, Compound 1 , and Compound 6, respectively. In Jurkat cells, this order was Compound 5, Compound 2, and Compound 1 . Lastly, in K562 cells, the groups with the highest number of apoptotic cells were Compound 2, Compound 6, and Compound 5, respectively. It is an important point that these data obtained are similar to the results of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- di-phenyltetrazolium bromide] test.

[0112] NMR spectra of the synthesized compounds (Compounds 1 -6) of the invention were obtained using Bruker brand 600 MHz NMR device, UV spectra were obtained using Thermo / Evolution Array-EA 1002017 spectrophotometer, IR spectra were obtained using Perkin-Elmer 1600 FT-IR (4000-400 cm1) spectrophotometer, and mass spectra (MS) were obtained using Thermo Scientific / Surveyor MSQ spectrophotometer.

[0113] In the purifications used in the process step of the synthesis method of the invention, column chromatography (CC) and thin layer chromatography (TLC) as well as crystallization and extraction techniques were used. The melting points of the compounds (Compounds 1 -6) of the invention were determined using a Gallenkamp brand melting point analyzer.

[0114] NMR spectra were adjusted to the TMS peak and 13C and / or APT spectra were adjusted to the solvent peaks of CDCI3 (5 77.0 ppm), pyridine-d5 (5 150.4, 135.9, 123.8 ppm), CD3OD (5 49.1 ppm), and DMSO-d6 (5 39.7 ppm). Mass spectra were obtained using the electron spray (ES) method. In column chromatography (CC), normal phase 70-230 mesh silica gel was used, while in thin layer chromatography (TLC) normal phase silica gel 60 F254 was used. A 254 nm UV lamp in the chamber was used to control thin layer separations. For the control of UV inactive compounds, acid spraying on the TLC plate followed by burning on the hot plate were applied.

[0115] CDCI3, CD3OD, pyridine-d5, and DMSO-d6 were used as solvents when obtaining the NMR spectra of the compounds (Compounds 1 -6) of the invention. Samples were placed in quartz NMR tubes and measurements were made. FT-IR spectra were taken on solid samples and measured in the 400-4000 cm-1region.

[0116] The cytotoxic effects and IC50 values of the new Compounds 1 -6 of the invention, the efficacy of which tested on cancer cell lines (MCF-7, Hela, Jurkat, and K562), were screened by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide] test. The test is based on the conversion of the tetrazolium salt MTT [3-(4,5-dimethylthiazol- 2-yl)-2,5-di-phenyltetrazolium bromide] to dissolved formazan by mitochondrial reductase in living cells. For this purpose, cell lines were incubated in 96-well cell culture plates at an initial concentration of 1 *105 cells / ml for 24 hours at 37 °C in a humidified oven containing 5% CO2. Then, different concentrations of the substance were added to the cultured cells and the cells were incubated for 24 hours. Imatinib was used as a positive control agent. After incubation, MTT [3-(4,5-dimethylthiazol-2- yl)-2,5-di-phenyltetrazolium bromide] solution was added to 96 wells and cells were incubated at 37 °C for 4 hours, and then the incubated cells were dissolved with dimethylsulfoxide (DMSO). Lastly, cytotoxicity measurements were performed at 550 nm wavelength using a spectrometer.

[0117] Cell viability % : (Optical density of control group - optical density of sample) / (optical density of control group - optical density of empty well)

[0118] The results of the cytotoxicity measurements of the synthesized drugs were determined as percentage increase relative to the control ± SD not exposed to any agent. The control value was taken as 0% cytotoxic. Cytotoxicity data were fitted to a sigmoidal curve and a four-parameter logistic model was used to calculate the IC50 value, which is the substance concentration value that causes 50% inhibition compared to the control group without any substance treatment. IC50 is the concentration value that reduces cell growth by 50% under experimental conditions. This value is the average of at least three independent reproducible and statistically significant calculations. IC50 value was determined within ±95% confidence interval. This analysis was performed with GraphPad Prism (San Diego, CA).

[0119] The effects of the synthesized compounds (Compounds 1 -6) of the invention on cancer cells were examined. In this context, MCF-7 breast cancer cell line, Jurkat T lymphocyte cell line, HeLa cervical cancer cell line, and K562 chronic myeloid leukemia cell line were used. These lines were obtained commercially from ATCC company.

[0120] RPMI-1640 medium was used to maintain the viability of PC3 and LNCaP cells used for medium preparation and cell proliferation. The medium used contained 10% FBS (Fetal Bovine Serum), 1% Penicillin-streptomycin, and 1% L-glutamine. The prepared medium was filtered into 50 ml falcon tubes and stored at +4 °C. After the medium was prepared, the frozen-provided cell lines were shaken gently for 1 -2 minutes in a 37°C water bath to thaw the cells. The cell suspension tube contents were then transferred to a 15 ml centrifuge tube, 3-4 mL of warm fresh medium was added and centrifuged at 800 rpm for 5 minutes. The supernatant was removed, the pellet was resuspended in fresh medium, and the cells were transferred to culture flasks. The proliferation, passaging and follow-up processes of the cells were monitored with an inverted microscope and the cells were incubated in an incubator at 37 °C with 95% humidity and 5% carbon dioxide (CO2), which is a suitable culture medium for them.

[0121] Incubated cells were checked every day and passaging process was performed for confluent cells. Since the cell lines used are cells that reproduce by adhesion, 0.75 ml of trypsin-ethylene diamine tetra acetic acid (EDTA) was added and incubated for 5 minutes before passaging, thereby lifting the cells. After incubation, 5 ml of fresh medium was added to the cell suspension and the cells were resuspended using a micropipette and then centrifuged at 800 rpm for 5 minutes. After centrifugation, the supernatant was removed and the pellet was gently pipetted into flasks with 5 ml of fresh medium.

[0122] For freezing and thawing of cells, cells were lifted with trypsin during passaging process, transferred to a 15 ml falcon and centrifuged at 800 rpm for 5 minutes at 4 °C. After removing the supernatant, fresh medium, DMSO and FBS were added to the falcon, the pellet was homogenized, and the cell contents were transferred to freezing tubes. The tubes were stored overnight at -80 °C and then stored at -196 °C in a liquid nitrogen tank.

[0123] After the cells were removed from -196 °C and thawed in a water bath (37 °C), the cell suspension was taken into a 15 ml falcon. The cells were diluted by adding medium on the cell suspension and the cells were centrifuged at 800 rpm for 5 minutes and washed. After centrifugation, the supernatant was removed, fresh medium was added to the obtained pellet, and the pellet was homogenized and taken to the flasks for the continuation of the culture.

[0124] For cell counting and installing the experimental setup, cells that were considered to be sufficient in number for the experimental setup were again treated with trypsin-EDTA, centrifuged, and the supernatant was removed, fresh medium was added to the pellet and cells were gently pipetted using a micropipette. Cells were then transferred into 48- well cell culture plates with 100,000 cells per ml for cell viability tests and 24-well cell culture plates with 1 ,000,000 cells per ml for other tests. While preparing the well cell culture plate, the cell density of the cell line to be used first was determined by counting with Thoma slide. Considering the amount of culture to be prepared, the final concentration was adjusted to 100,000 or 1 ,000,000 cells per ml and the required amount was taken from the stock vial according to these calculations and added into the well cell culture plate.

[0125] Cell viability tests were performed 24 hours after treatment with MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide] stain. Accordingly, 5mg / ml MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide] stain was first weighed and dissolved in phosphate buffered saline (PBS). Then, the 48-well cell culture plate containing the cells was removed from the incubator and 40 pl MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide] stain was added to each well and incubated for 4 hours. After incubation, the cell suspension was removed with a micropipette, 100 pl DMSO was added to the wells and measured at 560 and 620 nm in a spectrophotometer. From the results obtained, cell viabilities after the treatments were calculated using "formula a". Cell viability = (OD value for treatment group / OD value for control group) X 100

[0126] (Formula a)

[0127] In other words, the viability of cells without agent application (0 pM) was assumed to be 100%, and the ratio of viable cells at all concentrations was expressed in % form. The calculated cell viabilities were plotted against the concentration of the agents in GraphPad Prism XY table and IC50 values were calculated using the necessary functions of the software.

[0128] Whether the apoptotic or necrotic effects of the applied drugs occur in the cells was determined using HOPI staining technique. In this context, firstly, the stains to be used for HOPI applications (hoechst and propidium iodide) were prepared by mixing them in a 1 :1 ratio in an Eppendorf tube using a micropipette. The prepared stain was stored in the refrigerator at +4 °C. 24 hours after the agents were applied, all the medium containing the cells in the wells was removed with a micropipette. Then 150 pl trypsin was added to each well in the 24-well cell culture plate and incubated for 5 minutes. After incubation, 750 pl of fresh medium was added to each well in the cell culture well plate in the sterile chamber and the cells were lifted with a micropipette and transferred to Eppendorf tubes. The cells in Eppendorfs were then centrifuged at 800 rpm for 5 minutes. After centrifugation, the supernatant was removed from the medium with a micropipette. 50 pl of cell suspension was left in the tubes and 5 pl of HOPI stain was added. After adding the stain to all tubes, the Eppendorf tubes were sealed and lined up in an Eppendorf container, the container was covered with aluminum foil and kept at 37 °C with 5% CO2 and 96% humidity for 30 minutes. Lastly, the cells inside the Eppendorf tubes were examined under a fluorescence microscope. From the images obtained, the ratio of normal cells and apoptotic / necrotic cells was determined using the imaged program.

[0129] As a result of all these analyzes, it has been proven that the compounds (Compounds 1 -6) of the invention are more effective compared to imatinib, which is considered as the reference drug. The new semi-synthesis derivative compounds of the invention have been synthesized with naturally sourced starting material, which represents a significant step in terms of economic feasibility. It is also noteworthy that the compounds of the invention are more effective than Imatinib, which is considered to be the reference drug.

[0130] References

[0131] [1] Wikimedia Foundation. (2024, January 7). Meme Kanseri. Wikipedia. https: / / tr.wikipedia.org / wiki / Meme_kanseri

[0132] [2] §ahin, G., Duran , T., Kuggukturk, S., Kogak, N., Bayramoglu, D., Kebapgilar, A., &

[0133] Qelik, . (n.d.). Neferinin Servikal Kanser Hiicreleri (HeLa) Uzerinde Apoptotik Etkisi.

[0134] [3] DSO: Rahim Agzi Kanserinden yilda ortalama 311 bin Ki§i Hayatini Kaybediyor. Anadolu Ajansi. (n.d.). https: / / www.aa.com.tr / tr / saglik / dso-rahim-agzi-kanserinden- yilda-ortalama-311 -bin-kisi-hayatini-kaybediyor / 2046685

[0135] [4] Kronik Miyeloid Lbsemi. Turk Hematoloji Dernegi. (n.d.). https: / / www.thd. org.tr / thd_hal k / ?sayfa=kronik_miyeloid

[0136] [5] Lbsemi (Kan Kanseri) nedir? Belirti ve Tedavi Ybntemleri Nelerdir?. Medical Park, (n.d.). https: / / www.medicalpark.com.tr / kan-kanseri-nedir-belirti-ve-tedavi-yontemleri- nelerdir / hg-

[0137] 1817#:~:text=%C3%96zellikle%2025%2D60%20ya%C5%9F%20aras%C4%B1 ndaki,o ran%C4%B1 %20ise%20%67’dir.

Claims

CLAIMS1 . A gypsogenin-amine semisynthesis derivative compound with anticancer activity, denoted by Formula X,wherein R is selected from the following formulas;2. A compound according to claim 1 , having any one of the following formulas, characterized in that it is:• (3p)-3-Hydroxy-23-(diethylamine) olean-12-en-28-oic acid (Compound 1 , Formula 1),• (3p)-3-Hydroxy-23-(aniline)olean-12-en-28-oic acid (Compound 2, Formula 2),• (3p)-3-Hydroxy-23-[(2-mercaptoethyl)amine]olean-12-en-28-oic acid (Compound 3, Formula 3), • (3p)-3-Hydroxy-23-(dimethylamine) olean-12-en-28-oic acid (Compound 4,Formula 4),• (3p)-3-Hydroxy-23-[(6-bromo-1 ,3-benzothiazol-2-amine] olean-12-en-28-oic acid (Compound 5, Formula 5),• (3p)-3-Hydroxy-23-(2-amine anthracene) olean-12-en-28-oic acid (Compound 6, Formula 6).

3. A compound according to claim 1 or 2, indicated by Formula 1 , wherein R=Ri4. A compound according to claim 3, characterized in that the synthesis yield of (3 / 3)-3-Hydroxy-23-(diethylamine) olean-12-en-28-oic acid compound (Formula 1 ) is 87%.

5. A compound according to claim 3, characterized in that the molecular weight of (3 / 3)-3-Hydroxy-23-(diethylamine) olean-12-en-28-oic acid compound (Formula 1 ) is 527.80 g / mol.

6. A compound according to claim 3, characterized in that the melting temperature of (3 / 3)-3-Hydroxy-23-(diethylamine) olean-12-en-28-oic acid compound (Formula 1 ) is 237.3-238.6°C.

7. A compound according to claim 3, characterized in that the FT-IR spectrum of (3 / 3)-3-Hydroxy-23-(diethylamine) olean-12-en-28-oic acid compound (Formula 1 ) comprises peaks at 3443, 2924, 2848, 1642, 1456, 1380, 1262, 1027, 749 cm1.

8. A compound according to claim 3, characterized in that1H NMR spectrum of (3 / 3)-3-Hydroxy-23-(diethylamine) olean-12-en-28-oic acid compound (Formula 1 ) comprises peaks at 0.92 (H-24), 0.96 (H-25), 0.99 (H-26), 1.03 (H-30), 1.04 (t, 6H, H-2a), 1.23 (H-27), 1.77 (H-29), 3.28 (dd, 1 H, H-18), 3.71 (q, H-1 a), 4.19 (m, 1 H, H-23), 4.31 (d, 1 H,H-3), 5.48 (t, 1 H, H-12).

9. A compound according to claim 3, characterized in that13C NMR spectrum of (3 / 3)-3-Hydroxy-23-(diethylamine) olean-12-en-28-oic acid compound (Formula 1 ) comprises peaks at 14.5 (C-2a), 16.5 (C-24), 17.8 (C-25),19.1 (C-26), 24.3 (C- 30), 26.6 (C-27), 31.5 (C-29), 42.2 (C-18), 46.9 (C-1 a), 61.8 (C-23), 73.9 (C-3), 123.1 (C-12), 145.3 (C-13), 180.9 (C-28).

10. A compound according to claim 3, characterized in that the UV spectrum of (3 / 3)- 3-Hydroxy-23-(diethylamine) olean-12-en-28-oic acid compound (Formula 1 ) comprises peaks at 290, 252 nm.11 . A compound according to claim 3, characterized in that the MS spectrum of (3 / 3)- 3-Hydroxy-23-(diethylamine) olean-12-en-28-oic acid compound (Formula 1 ) comprises LC / MS (ESI-MS) m / z Calc for C34H57O3 527.77 [M]_peaks.

12. A compound according to claim 1 or 2, indicated by Formula 2, wherein R=R2Formula 213. A compound according to claim 12, characterized in that the synthesis yield of (3 / 3)-3-Hydroxy-23-(aniline)olean-12-en-28-oic acid compound (Formula 2) is 56%.

14. A compound according to claim 12, characterized in that the molecular weight of (3 / 3)-3-Hydroxy-23-(aniline)olean-12-en-28-oic acid compound (Formula 2) is 526.72 g / mol.

15. A compound according to claim 12, characterized in that the melting temperature of (3 / 3)-3-Hydroxy-23-(aniline)olean-12-en-28-oic acid compound (Formula 2) is 237.6-239.0°C.

16. A compound according to claim 12, characterized in that the FT-IR spectrum of (3 / 3)-3-Hydroxy-23-(aniline)olean-12-en-28-oic acid compound (Formula 2) comprises peaks at 3444, 2924, 2853, 1633, 1462, 1376, 1264, 742 cm1.

17. A compound according to claim 12, characterized in that1H NMR spectrum of (3 / 3)-3-Hydroxy-23-(aniline)olean-12-en-28-oic acid compound (Formula 2) comprises peaks at 0.91 (H-29), 0.95 (H-25), 0.97 (H-30), 1.03 (H-26), 1.04 (H- 24), 1.20 (H-27), 3.29 (dd, 1 H, H-18), 3.60 (1 H, H-23), 3.72 (d, 1 H, H-3), 5.48 (br s, 1 H, H-12), 6.87 (-NH), 7.20 (1 H, H-2a / H-6a), 7.57 (1 H, H-3a / H-5a), 7.90 (1 H, H-4a).

18. A compound according to claim 12, characterized in that13C NMR spectrum of (3 / 3)-3-Hydroxy-23-(aniline)olean-12-en-28-oic acid compound (Formula 2) comprises peaks at 12.7 (C-24), 15.6 (C-25), 17.1 (C-26), 23.3 (C-30), 25.7 (C- 27), 32.8 (C-29), 41.8 (C-18), 56.4 (C-4), 73.1 (C-3), 122.6 (C-12), 123.6 (C- 2a / C-6a), 129.0 (C-4a), 135.5 (C-3a / C-5a), 144.5 (C-13), 151.0 (C-1 a).

19. A compound according to claim 12, characterized in that the UV spectrum of (3 / 3)-3-Hydroxy-23-(aniline)olean-12-en-28-oic acid compound (Formula 2) comprises a peak at 275 nm.

20. A compound according to claim 12, characterized in that the MS spectrum of (3 / 3)-3-Hydroxy-23-(aniline)olean-12-en-28-oic acid compound (Formula 2) comprises LC / MS (ESI-MS) m / z C36H53NO3 526.70 [M-COOH+Na+1]+peaks.21 . A compound according to claim 1 or 2, indicated by Formula 3, wherein R=R3Formula 322. A compound according to claim 21 , characterized in that the synthesis yield of (3 / 3)-3-Hydroxy-23-[(2-mercaptoethyl)amine]olean-12-en-28-oic acid compound (Formula 3) is 79%.

23. A compound according to claim 21 , characterized in that the molecular weight of (3 / 3)-3-Hydroxy-23-[(2-mercaptoethyl)amine]olean-12-en-28-oic acid compound (Formula 3) is 526.69 g / mol.

24. A compound according to claim 21 , characterized in that the melting temperature of (3 / 3)-3-Hydroxy-23-[(2-mercaptoethyl)amine]olean-12-en-28-oic acid compound (Formula 3) is 218.5-219.5°C.

25. A compound according to claim 21 , characterized in that the FT-IR spectrum of (3 / 3)-3-Hydroxy-23-[(2-mercaptoethyl)amine]olean-12-en-28-oic acid compound(Formula 3) comprises peaks at 3434 cm'1, 2648 cm'1, 1650 cm'1, 1457 cm'1, 1265 cm1, 1209 cm1, 1031 ,749 cm1.

26. A compound according to claim 21 , characterized in that1H NMR spectrum of (3 / 3)-3-Hydroxy-23-[(2-mercaptoethyl)amine]olean-12-en-28-oic acid compound (Formula 3) comprises peaks at 0.90 (H-29), 0.95 (H-25), 0.98 (H-30), 1.03 (H- 26), 1.04 (H-24),1.22 (H-27), 1.96 (H-2a), 3.29 (dd, 1 H, H-18), 3.73 - 4.20 (m, 1 H, H-23), 4.22 (d, 1 H, H-3), 4.75 (H-1 a), 5.48 (br s, 1 H, H-12), 5.87 (NH-).

27. A compound according to claim 21 , characterized in that13C NMR spectrum of (3 / 3)-3-Hydroxy-23-[(2-mercaptoethyl)amine]olean-12-en-28-oic acid compound (Formula 3) comprises peaks at 12.9 (C-24), 15.7 (C-25),17.1 (C-26), 23.3 (C- 30), 23.4 (C-2a), 25.7 (C-27), 32.7 (C-29), 41.8 (C-18), 43.1 (C-1a), 67.4 (C- 23), 73.0 (C-3), 122.5 (C-12), 144.5 (C-13), 180.0 (C-28).

28. A compound according to claim 21 , characterized in that the UV spectrum of (3 / 3)-3-Hydroxy-23-[(2-mercaptoethyl)amine]olean-12-en-28-oic acid compound (Formula 3) comprises a peak at 284 nm.

29. A compound according to claim 21 , characterized in that the MS spectrum of (3 / 3)-3-Hydroxy-23-[(2-mercaptoethyl)amine]olean-12-en-28-oic acid compound (Formula 3) comprises LC / MS (ESI-MS) m / z C32H55NOS 526.67 [M+Na+2]+ peaks.

30. A compound according to claim 1 or 2, indicated by Formula 4, wherein R=R431 . A compound according to claim 30, characterized in that the synthesis yield of (3 / 3)-3-Hydroxy-23-(dimethylamine) olean-12-en-28-oic acid compound (Formula 4) is 63%.

32. A compound according to claim 30, characterized in that the molecular weight of (3 / 3)-3-Hydroxy-23-(dimethylamine) olean-12-en-28-oic acid compound (Formula 4) is 453.22 g / mol.

33. A compound according to claim 30, characterized in that the melting temperature of (3 / 3)-3-Hydroxy-23-(dimethylamine) olean-12-en-28-oic acid compound (Formula 4) is 251 ,0-252.6°C.

34. A compound according to claim 30, characterized in that the FT-IR spectrum of (3 / 3)-3-Hydroxy-23-(dimethylamine) olean-12-en-28-oic acid compound (Formula 4) comprises peaks at 3434 cm-1, 2924 cm-1, 2853 cm-1, 1644 cm-1, 1415 cm-1, 1382 cm1, 1275 cm1, 749 cm1.

35. A compound according to claim 30, characterized in that1H NMR spectrum of (3 / 3)-3-Hydroxy-23-(dimethylamine) olean-12-en-28-oic acid compound (Formula 4) comprises peaks at 0.90 (H-29), 0.95 (H-25), 0.98 (H-30), 1.01 (H-26), 1.04(H-24), 1.22 (H-27), 3.30 (dd, 1 H, H-18), 3.59 (H-1 a / H-2a), 3.72-4.07 (m, 1 H, H- 23), 4.19 (d, 1 H, H-3), 5.48 (br s, 1 H, H-12).

36. A compound according to claim 30, characterized in that13C NMR spectrum of (3 / 3)-3-Hydroxy-23-(dimethylamine) olean-12-en-28-oic acid compound (Formula4) comprises peaks at 12.5 (C-24), 15.5 (C-25), 17.1 (C-26), 23.4 (C-30), 25.7 (C-27), 32.9 (C-29), 43.1 (C-18), 48.7 (C-1 a / C-2a), 67.7 (C-23), 73.3 (C-3), 122.3 (C-12), 144.6 (C-13), 184.3 (C-28).

37. A compound according to claim 30, characterized in that the UV spectrum of(3 / 3)-3-Hydroxy-23-(dimethylamine) olean-12-en-28-oic acid compound (Formula 4) comprises a peak at 275 nm.

38. A compound according to claim 30, characterized in that the MS spectrum of (3 / 3)-3-Hydroxy-23-(dimethylamine) olean-12-en-28-oic acid compound (Formula4) comprises LC / MS (ESI-MS) m / z 453.20 [M-CH3+1]+ peaks.

39. A compound according to claim 1 or 2, indicated by Formula 5, wherein R=RsFormula 540. A compound according to claim 39, characterized in that the synthesis yield of (3 / 3)-3-Hydroxy-23-[(6-bromo-1 ,3-benzothiazol-2-amine] olean-12-en-28-oic acid compound (Formula 5) is 87%.41 . A compound according to claim 39, characterized in that the molecular weight of (3 / 3)-3-Hydroxy-23-[(6-bromo-1 ,3-benzothiazol-2-amine] olean-12-en-28-oic acid compound (Formula 5) is 694.13 g / mol.

42. A compound according to claim 39, characterized in that the melting temperature of (3 / 3)-3-Hydroxy-23-[(6-bromo-1 ,3-benzothiazol-2-amine] olean-12-en-28-oic acid compound (Formula 5) is 116.2-117.2°C.

43. A compound according to claim 39, characterized in that the FT-IR spectrum of (3 / 3)-3-Hydroxy-23-[(6-bromo-1 ,3-benzothiazol-2-amine] olean-12-en-28-oic acid compound (Formula 5) comprises peaks at 3440 cm-1, 2920 cm-1, 2844 cm-1, 1640 cm1, 1455 cm1, 1290 cm1, 1255 cm1, 1027 cm1, 749 cm1.

44. A compound according to claim 39, characterized in that1H NMR spectrum of (3 / 3)-3-Hydroxy-23-[(6-bromo-1 ,3-benzothiazol-2-amine] olean-12-en-28-oic acid compound (Formula 5) comprises peaks at 0.50 (s, H-24), 0.68 (s, H-26), 0.85 (s, H-30), 0.85 (s, H-25), 0.85 (s, H-29), 1.04 (s, H-27), 2.72 (dd, J= 6.8, 13.4 Hz, H- 18), 3.04 - 3.31 (m, H-23), 3.43 (d, H-3), 5.13 (brs, H-12), 7.23 (1 H, m, H-2a), 7.32 (H-3a), 7.61 (-NH), 7.86 (2H, m, H-5a).

45. A compound according to claim 39, characterized in that13C NMR spectrum of (3 / 3)-3-Hydroxy-23-[(6-bromo-1 ,3-benzothiazol-2-amine] olean-12-en-28-oic acid compound (Formula 5) comprises peaks at 13.1 (C-24), 15.9 (C-25), 17.3 (C-26), 23.9 (C-30), 26.0 (C-27), 33.3 (C-29), 41.2 (C-18), 64.8 (C-23), 70.7 (C-3), 112.5 (C-4a), 1 19.5 (C-2a), 121.9 (C-12), 123.7 (C-5a), 128.7 (C-3a), 133.5 (C-2b), 144.2 (C-13), 152.4 (C-1 b), 167.9 (C-1 a), 179.1 (C-28).

46. A compound according to claim 39, characterized in that the MS spectrum of (3 / 3)-3-Hydroxy-23-[(6-bromo-1 ,3-benzothiazol-2-amine] olean-12-en-28-oic acid compound (Formula 5) comprises LC / MS (ESI-MS) m / z C37H53BrN2OS [M+Na]+: 694.11 (100) peaks.

47. A compound according to claim 1 or 2, indicated by Formula 6, wherein R=R6.Formula 648. A compound according to claim 47, characterized in that the synthesis yield of (3 / 3)-3-Hydroxy-23-(2-amine anthracene) olean-12-en-28-oic acid compound (Formula 6) is 50%.

49. A compound according to claim 47, characterized in that the molecular weight of (3 / 3)-3-Hydroxy-23-(2-amine anthracene) olean-12-en-28-oic acid compound (Formula 6) is 683.87 g / mol.

50. A compound according to claim 47, characterized in that the melting temperature of (3 / 3)-3-Hydroxy-23-(2-amine anthracene) olean-12-en-28-oic acid compound (Formula 6) is 122.6-123.2°C.

51. A compound according to claim 47, characterized in that the FT-IR spectrum of (3 / 3)-3-Hydroxy-23-(2-amine anthracene) olean-12-en-28-oic acid compound (Formula 6) comprises peaks at 3466 cm1, 2925 cm1, 2850 cm1, 2059 cm1, 1634 cm1, 1462 cm1, 1265 cm1, 1250 cm1, 761 cm1.

52. A compound according to claim 47, characterized in that1H NMR spectrum of (3 / 3)-3-Hydroxy-23-(2-amine anthracene) olean-12-en-28-oic acid compound (Formula 6) comprises peaks at 0.51 (s, H-24), 0.69 (s, H-26), 0.84 (s, H-30), 0.85 (s, H-25), 0.85 (s, H-29), 1.08 (s, H-27), 2.70 (dd, J= 6.8, 13.4 Hz, H-18),3.03-3.41 (H-23), 4.13 (m, H-3), 5.13 (brs,H-12), 7.04 (H-1 a), 7.06 (H-3a), 7.66 (H-6a / H-7a), 7.69 (H-4a), 7.93 (H-8a), 7.99 (H-5a), 8.07 (H-9a / H-10a).

53. A compound according to claim 47, characterized in that13C NMR spectrum of (3 / 3)-3-Hydroxy-23-(2-amine anthracene) olean-12-en-28-oic acid compound (Formula 6) comprises peaks at 1 1.2 (C-24), 13.1 (C-25), 14.4 (C-26), 15.9 (C- 27), 23.8 (C-30), 26.01 (C-29), 41.8 (C-18), 64.6 (C-23), 70.6 (C-3), 120.5 (C-9a), 121.9 (C-12), 123.0 (C-2b), 124.3 (C-4b), 127.7 (C-3a), 127.8 (C-6a / C-7a), 129.1 (C-1 a / C-8a), 132.0 (C-3b), 132.3 (C-4a), 137.3 (C-1b), 139.3 (C-2a), 144.3 (C- 13), 179.3 (C-28).

54. A compound according to claim 47, characterized in that the MS spectrum of (3 / 3)-3-Hydroxy-23-(2-amine anthracene) olean-12-en-28-oic acid compound (Formula 6) comprises LC / MS (ESI-MS) m / z C44H57NO3 [M+2K]+: 683.84 peaks.

55. A synthesis method of a compound according to any one of the preceding claims, characterized in that it comprises the process steps of: i. adding diethyl amine (R1), aniline (R2), 2-mercaptoethylamine (R3), dimethylamine (R4), 6-bromo-1 ,3-benzothiazol-2-amine (R5) or 2-amine anthracene (Re) compounds dissolved in dichloromethane to the solution of gypsogenin dissolved in dichloromethane in the same way in inert medium (N2), ii. initiating the reduction reaction by conversion of the carbon-nitrogen imine (C=N) bond to an amine (C-NH) bond with the addition of sodium triacetoxyborohydride (NaBH(OAc)3) to the resulting mixture, iii. continuing to stir the reaction mixture under reflux condenser, iv. eliminating borohydride remaining in the reaction after completion of the reaction by adding aqueous sodium bicarbonate solution, v. extracting the resulting reaction mixture with dichloromethane, vi. drying the collected organic phase with the addition of anhydrous sodium sulfate (Na2SO4) and evaporating the solvent collected in the organic phase after extraction, vii. introducing the resulting organic phase with solvent into the column with silica gel filler,viii. obtaining the compounds (3 / 3)-3-Hydroxy-23-(diethylamine) olean-12-en-28- oic acid (Compound 1 ), (3 / 3)-3-Hydroxy-23-(aniline)olean-12-en-28-oic acid (Compound 2), (3 / 3)-3-Hydroxy-23-[(2-mercaptoethyl)amine]olean-12-en-28- oic acid (Compound 3), (3 / 3)-3-Hydroxy-23-(dimethylamine) olean-12-en-28- oic acid (Compound 4), (3 / 3)-3-Hydroxy-23-[(6-bromo-1 ,3-benzothiazol-2- amine] olean-12-en-28-oic acid (Compound 5) or (3 / 3)-3-Hydroxy-23-(2- amine anthracene) olean-12-en-28-oic acid (Compound 6) after the purification of the organic phase by column chromatography.

56. A synthesis method according to claim 55, characterized in that it comprises the process steps of: i. adding diethyl amine (Ri), aniline (R2), 2-mercaptoethylamine (R3), dimethylamine (R4), 6-bromo-1 ,3-benzothiazol-2-amine (R5) or 2-amine anthracene (Re) compounds dissolved in dichloromethane to the solution of gypsogenin dissolved in dichloromethane in the same way in inert medium (N2) at a temperature of 20-25°C, ii. initiating the reduction reaction by conversion of the carbon-nitrogen imine (C=N) bond to an amine (C-NH) bond with the addition of sodium triacetoxyborohydride (NaBH(OAc)3) to the resulting mixture, iii. continuing to stir the reaction mixture for 2 hours under reflux condenser, iv. eliminating borohydride remaining in the reaction by adding aqueous sodium bicarbonate solution 1 hour after the reaction is complete, v. extracting the resulting reaction mixture 3 times (10 mL x 3) with 10 mL of dichlormethane, vi. drying the collected organic phase with the addition of anhydrous sodium sulfate (Na2SO4) and evaporating the solvent collected in the organic phase after extraction by an evaporator, vii. introducing the resulting organic phase with hexane / ethyl acetate (6 / 4; 8 / 2) or dichloromethane / ethyl acetate (1 / 1 ) as solvent into a column with silica gel filler, viii. obtaining the compounds (3 / 3)-3-Hydroxy-23-(diethylamine) olean-12-en-28- oic acid (Compound 1 ), (3 / 3)-3-Hydroxy-23-(aniline)olean-12-en-28-oic acid (Compound 2), (3 / 3)-3-Hydroxy-23-[(2-mercaptoethyl)amine]olean-12-en-28- oic acid (Compound 3), (3 / 3)-3-Hydroxy-23-(dimethylamine) olean-12-en-28- oic acid (Compound 4), (3 / 3)-3-Hydroxy-23-[(6-bromo-1 ,3-benzothiazol-2-amine] olean-12-en-28-oic acid (Compound 5) or (3 / 3)-3-Hydroxy-23-(2- amine anthracene) olean-12-en-28-oic acid (Compound 6) after the purification of the organic phase by column chromatography.

57. A compound according to any one of claims 1 -54 for use in the treatment of cancer.

58. A compound according to any one of claims 1 -54 for use in the treatment of breast cancer.

59. A compound according to any one of claims 1 -54 for use in the treatment of human cervical cancer.

60. A compound according to any one of claims 1 -54 for use in the treatment of human T lymphocyte cancer.

61. A compound according to any one of claims 1 -54 for use in the treatment of human chronic myeloid leukemia cancer.

62. A pharmaceutical composition comprising a compound according to any one of claims 1 -54.

63. A pharmaceutical composition according to claim 62 for use in the treatment of cancer.

64. A pharmaceutical composition according to claim 62 for use in the treatment of breast cancer.

65. A pharmaceutical composition according to claim 62 for use in the treatment of human cervical cancer.

66. A pharmaceutical composition according to claim 62 for use i the treatment of human T lymphocyte cancer.

67. A pharmaceutical composition according to claim 62 for use in the treatment of human chronic myeloid leukemia cancer.

68. An anti-cancer drug comprising a pharmaceutical composition according to any one of claims 62-67.

69. A drug comprising a compound according to any one of claims 1 -54 as an active ingredient.

70. A compound obtained by a synthesis method according to any one of claims 55-56.

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