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Title:
AG-08 MOLECULE THAT IS A SAPOGENOL DERIVATIVE EXHIBITNG CYTOTOXIC EFFECTS BY ACTIVATING THE NECROSIS PATHWAY AND SYNTHESIS METHOD THEREOF
Document Type and Number:
WIPO Patent Application WO/2020/222717
Kind Code:
A1
Abstract:
The invention is related to an AG-08 coded novel structured molecule having cytotoxic features against cancer cells. The aim of the invention is to primarily synthesize the astragenol (AG) molecule from cycloastragenol (CA) and then from here, the cytotoxic molecule coded AG-08 which enables to kill cancer cells via a pathway that is non-apoptotic as a difference from the compounds that exhibit traditional anticancer activity.

Inventors:
BEDİR ERDAL (TR)
BALLAR KIRMIZIBAYRAK PETEK (TR)
TAĞ ÖZGÜR (TR)
ERZURUMLU YALÇIN (TR)
ÜNER GÖKLEM (TR)
Application Number:
PCT/TR2020/050351
Publication Date:
November 05, 2020
Filing Date:
April 28, 2020
Export Citation:
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Assignee:
IZMIR YUEKSEK TEKNOLOJI ENSTITUESUE (TR)
EGE UENIVERSITESI REKTOERLUEGUE (TR)
International Classes:
A61K31/56; A61P35/00; C07J51/00; C12P33/20
Other References:
DEBELEÇ-BÜTÜNER, B. ET AL.: "Cycloartane-type sapogenol derivatives inhibit NFKB activation as chemopreventive strategy for inflammation-induced prostate carcinogenesis", STEROIDS, vol. 135, pages 9 - 20, XP025269662
Attorney, Agent or Firm:
YALCINER, Ugur G. (YALCINER PATENT & CONSULTING LTD.) (TR)
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Claims:
CLAIMS

1. An AG-08 molecule that is a saponin derivative synthesized via the exit molecules, cycloastragenol (CA) and astragenol (AG) used to induce cytotoxicity in cancer cells via regulated necrosis pathway.

2. AG-08 molecule according to claim 1, that is used to provide necrotic impact on cancer cells in human breast cancers, human bone metastasis prostate cancers and human cervical cancers where the IC50 values are respectively 3,80+0,143372; 5,73+0,46124 and 5,45+0,40893 mM for HCC1937 (human breast cancer cell line), PC3 (human prostate cancer cell line with bone metastasis) and HeLa (human cervical cancer cell line) cells.

3. AG-08 molecule according to claim 1, used to reduce the full-length protein amounts and to cut proteins that are in charge at the autophagy pathway named Beclinl, ATG5, ATG7, ATG12 and ATG16L1.

4. A method of synthesizing AG-08 molecule according to any of the preceding claims, comprising the steps of,

- dissolving cycloastragenol (CA) in methanol,

- establishing a reaction mixture by adding sulphuric acid on the solution,

- heating the reaction mixture and carrying out a reaction,

- adding the products obtained as a result of reaction into Na2C03 solution,

- applying a liquid-liquid extraction process with EtOAc to the obtained mixture,

- washing with Brine solution and treating with anhydrous Na2S04,

- drying the mixture under vacuum,

- purifying the dried astragenol (AG) mixture,

- obtaining astragenol (AG) as the intermediate product,

- dissolving astragenol (AG) in pyridine,

- adding / -TsCl (p-Toluenesulfonyl Chloride) reactive on the solution and dissolving by mixing,

- carrying out a reaction at room temperature,

- adding the products obtained as a result of reaction into HC1 solution,

- applying a liquid-liquid extraction process with EtOAc to the obtained mixture,

- washing with Brine solution and treating with anhydrous Na2S04, - drying the mixture under vacuum,

- purifying the dried AG-08 mixture,

- and obtaining the end product AG-08.

5. An AG-08 molecule synthesis method according to claim 4, wherein in the step of dissolving cycloastragenol (CA) in methanol, 6.11 mmol CA is dissolved in 20 mL methanol.

6. An AG-08 molecule synthesis method according to claim 4, wherein, in the step of forming a reaction mixture with the addition of sulphuric acid on a solvent, 1 mL concentrated sulphuric acid is added onto the solution.

7. An AG-08 molecule synthesis method according to claim 4, wherein, in the step of carrying out the reaction at room temperature, the reaction mixture is mixed at room temperature for 4 hours.

8. An AG-08 molecule synthesis method according to claim 4, wherein, in the step of adding the products that are obtained as a result of the reaction to the Na2C03 solution, the ended reaction products are added into a 100 mL 9% Na2C03 solution.

9. An AG-08 molecule synthesis method according to claim 4, wherein in the step of applying a liquid-liquid extraction process with EtOAc to the obtained mixture, liquid-liquid extraction process is carried out 3 times with 50 mL EtOAc.

10. An AG-08 molecule synthesis method according to claim 4, wherein in the step of washing with Brine solution and treating with anhydrous Na2S04, the molecule is treated with anhydrous Na2S04 after being washed with Brine solution.

11. An AG-08 molecule synthesis method according to claim 4, wherein, in the step of drying the mixture under vacuum, the mixture is dried under vacuum 60°C in a rotary evaporator.

12. An AG-08 molecule synthesis method according to claim 4, wherein, in the step of purifying the dried astragenol (AG) mixture, the dried reaction mixture is mixed into 5g silica and is conditioned with 7:3 Hexane:EtOAc and is purified with 60 g silica.

13. An AG-08 molecule synthesis method according to claim 4, wherein in the step of dissolving astragenol (AG) in pyridine, 400 mg (0.408 mmol) AG is dissolved in 6 mL pyridine. 14. An AG-08 molecule synthesis method according to claim 4, wherein, in the step of adding

/ -TsCl reactive onto the solution and performing dissolution by mixing, 600 mg / -TsCl reactive is added onto the solution the mixture is dissolved with the aid of a magnetic mixer.

15. An AG-08 molecule synthesis method according to claim 4, wherein, in the step of adding the products that are obtained as a result of the reaction to the HC1 solution, the ended reaction is added into a 100 mL 5% HC1 solution.

16. An AG-08 molecule synthesis method according to claim 4, wherein, in the step of purifying the dried AG-08 mixture, the dried reaction mixture is mixed into silica and is conditioned with 80:20 Hexane:EtOAc and is purified with 15 g silica.

Description:
AG-08 MOLECULE THAT IS A SAPOGENOL DERIVATIVE EXHIBITNG CYTOTOXIC EFFECTS BY ACTIVATING THE NECROSIS PATHWAY AND

SYNTHESIS METHOD THEREOF

Technical Field

The invention is related to an AG-08 [(10S,13R,14S,16S, 17R)-16-hydroxy-17-((2R,5S)- 5-(2-hydroxypropan-2-yl)-2-methyltetrahydrofuran-2-yl)-4,4, 10,13, 14-pentamethyl-2,3,4,5,8, 10, 12,13, 14,15, 16,17-dodecahy dro- 1 H-cy clopenta[a]phenanthren-3 -yl 4-methylbenzene sulfonate] coded molecule with a novel structure having cytotoxic effects directed to cancer cells.

Prior Art

Saponins (Sapogenol); are secondary metabolites that have glycosidic groups on the triterpenic or steroidal structures that are high in molecular weight and are found commonly in plant organisms. While it is known as amphiphilic glycosides that form permanent bubbles such as in soap bubbles when mixed with aqueous solutions, structurally it is grouped as one or more hydrophilic core glycosides having combined lipophilic triterpene derivatives. The compounds in this group have been noted provide activities such as antimutagenic, molluscicidal, antihelminitic, cardiovascular, analgesic, antipyretic, adaptogenic, antiviral, and antitussive, hypoglysemic, hypocholesterolemia, antiviral, antibacterial, immunostimulant, anti-HIV, cytotoxic and antitumor.

Saponin based semi-synthetic anticancer drug discoveries have been made with triterpenoids such as oleanolic acid, ursolic acid for some time, and limited number of studies have been carried out with structures such as cycloartanes, lanostanes and hopanes that are rarely found in nature. Cycloartanes are produced by living beings that only perform photosynthesis among low molecular weighted bioregulators and the plants that are the richest in terms of containing these classes of compounds are Astragalus species. The anticancer potentials of saponins are partially weak. Due to this reason, bioactivity guided drug design and synthesis is carried out in order to find new structures having high potential. Several triterpenic compounds derived from betulinic acid have been determined as potential anticancer drug candidates in cellular screening [1] Betulinic acid and some derivatives thereof, have also been determined as potential anti -HIV agents [2]

2-cyano-3,12-dioxoolean-l,9-dien-28-oic acid or CDDO, has exhibited extremely high anti-inflammatory effects at low doses (nitric oxide production inhibitor), and has been determined to be a multi-functional molecule that is promising in treating cancer at higher doses [3] It has been reported by in vitro studies that CDDO, induced differentiation in human leukemia, osteosarcoma [4], breast cancer [5] cells and inhibited growth and induced apoptosis

[6] [7] In advanced studies that have been conducted, it has been reported that several genes (LAP -2, BCL-2, C-MYC, VEGF, MMP-9) related to NF-KB (nuclear factor kappa B) activity were suppressed. In another study, it has been determined that CDDO and its derivatives exhibited cytotoxic activity dependent on NF-KB in glioblastoma and neuroblastomas [8] It has been determined that CDDO and its derivatives have carried out NF-KB inhibition via IKK (Inhibitor KappaB Kinase) inhibition. This compound has been subject to several patents and it has been primarily used in clinical studies in chronic renal patients due to its anti-inflammatory effects (US7435755 B2, US20050288363 Al, US20090048205 Al, US20090093447 Al, WO2008136838 Al, AND W02009023232 Al). CDDO-Me has been included in Phase 3 clinical studies of chronic kidney disease, however studies have been ceased due to cardiotoxicity. This compound has been included in phase 1 studies in lymphoma and pancreas cancers as combination therapy due to its anticancer effects, but successful results were not obtained.

Nowadays CDDO and some derivatives thereof, are still being used in advanced clinical research for type-2 diabetes and arterial hypertension due to their anti-inflammatory and potent antioxidant effects via the Nrf-2 pathway exhibited particularly at low doses.

As a result, there is no saponin or derivative thereof that has been used in completed clinical studies due to their anticancer effects. None of the mechanisms of action of any of these derivatives included in clinical studies have been reported as necrosis. In the United States patent numbered US20150093455 of the prior art, compositions and methods for increasing telomerase activity have been disclosed. These methods and compositions are related to being used in the treatment process of various diseases by increasing telomerase activity in the cells or tissues of a patient. As shown in the formula depicted in Figure 1, an isolated compound is protected within the scope of the application and a combined cyclopropyl ring is formed together with X 1 hydroxy or b-D-xylopyranoside, X 2 hydroxy or b-D-glucopyranoside, X 3 hydroxy; OR 1 hydroxy; and carbon numbered R 2 9 of this formula and it represents the single link between the carbons numbered 9 and 11. In some cases, this treatment application is performed in ex vivo cell therapies. One of these is natural aging, cancer, cancer treatment, acute or chronic infections, and tissue degeneration arising from genetic disorders.

In the patent application numbered WO2015179983 of the prior art, di- and tri- cationic glycosylated antitumor ether lipids (GAELs), L-glycosylated GAEL’S and rhamnose-linked GAEL’S as cytotoxic agents against epithelial cancer cells and cancer stem cells is disclosed. GAEL’S kill cancer cells via a non-apoptotic pathway as an effective strategy to avoid resistance.

In the study carried out by Feng LM et ak, of the prior art, titled“Smith degradation, an efficient method for the preparation of cycloastragenol from astragaloside IV” the preparation of cycloastragenol (CA) from astragaloside IV (ASI) is described. The optimization of Smith degradation has been carried out as follows; ASI is dissolved in 60% methanol -water solution, then oxidized inside NalCE for 12 hours and has been reduced for 4 hours with NaBH and is finally acidified in 1 M sulphuric acid (H 2 SO 4 ) at pH2 for 24 hours. Under optimal conditions CA has been obtained for ASI with a yield of 84.4%.

In the study carried out by WANG QZ et ak, of the prior art, titled,“Effects of Couplet Medicines (Astragalus Membranaceus and Jiaozhen) on Intestinal Barrier in Postoperative Colorectal Cancer Patients”, the effects of a binary drug obtained from Astragalus membranaceus and Jiaozhen on patients with postoperative colon cancer is disclosed.

In the study carried out by Auyeung KK et ak, of the prior art, titled, Astragalus saponins modulate mTOR and ERK signaling to promote apoptosis through the extrinsic pathway in HT-29 colon cancer cells”, the modulation of mTOR and ERK signaling by Astragalus saponins to promote apoptosis through the extrinsic pathway in HT-29 colon cancer cells is disclosed. Brief Description of the Invention

The aim of the invention is to primarily synthesize the astragenol (AG) molecule from cycloastragenol (CA) and then from here, the cytotoxic molecule coded AG-08 which enables to kill cancer cells via a pathway that is non-apoptotic as a difference from the compounds that exhibit traditional anticancer activity. Detailed Description of the Invention

“The AG-08 molecule that is a sapogenol derivative exhibiting cytotoxic effects by activating the necrosis pathway and synthesis method thereof’ carried out to reach the aims of this invention has been illustrated in the Figures, and according to these figures;

Figure 1 - Schematic view of the synthesis of the AG-08 coded compound via the chemical structures of cycloastragenol (CA), astragenol (AG) and AG-08.

Figure 2 - Immunoblotting view by which the effect of the AG-08 coded compound on

PARPl protein is examined.

Figure 3 - Immunoblotting view by which the effect of the AG-08 coded compound on caspase 8, 9 and 3 proteins is examined.

Figure 4 - Immunoblotting view by which the effect of the AG-08 coded compound on proteins related with autophagy is examined.

Figure 5 - View of the effect of AG-08 coded compound with acridine orange/Ethidium bromide cell dye.

Figure 6 - Shows the flow cytometry results of the effects of the AG-08 coded compound on apoptotic/necrotic cell death examined with Annexin-V and 7-AAD staining.

Figure 7 - View of sapogenol structures similar to AG-08 that can have effect via the same pathway.

The invention is related to the usage of the synthesized AG-08 molecule that is a saponin derivative, for providing cytotoxic effect on cancer cells by activating the necrosis pathway via the cycloastragenol (CA) and astragenol (AG) exit molecules.

The IC50 values of the AG-08 molecule is respectively 3, 80±0, 143372; 5,73±0,46124 and 5,45±0,40893 mM (micromolar) for HCC1937 (human breast cancer cell line), PC3 (human prostate cancer cell line with bone metastasis) and HeLa (human cervical cancer cell line) cells.

Proteins in charge at the autophagy pathway named Beclinl, ATG5, ATG7, ATG12 and ATG16L1 are cut off with AG-08 and the amounts of full-length proteins are reduced.

Method for synthesizing the AG-08 coded compound that exhibits necrotic effect on cancer cells comprising the steps of,

dissolving cycloastragenol (CA) in methanol,

establishing a reaction mixture by adding sulphuric acid on the solution,

- heating the reaction mixture and carrying out a reaction,

adding the products obtained as a result of reaction into NaiCCh solution,

applying a liquid-liquid extraction process with EtOAc to the obtained mixture,

- washing with Brine solution and treating with anhydrous NaiSCri,

drying the mixture under vacuum,

- purifying the dried astragenol (AG) mixture,

obtaining astragenol (AG) as the intermediate product,

dissolving astragenol (AG) in pyridine,

adding -TsCl (p-Toluenesulfonyl Chloride) reactive on the solution and dissolving by mixing,

carrying out a reaction at room temperature,

adding the products obtained as a result of reaction into HC1 solution,

applying a liquid-liquid extraction process with EtOAc to the obtained mixture,

- washing with Brine solution and treating with anhydrous Na2S04,

drying the mixture under vacuum,

- purifying the dried AG-08 mixture,

and obtaining the end product AG-08.

The invention is related to the drug research-development field and particularly to the subject of discovering, from natural products (triterpenic saponin group), molecules that can be used as anti-cancer drugs that have effect via novel impact mechanisms, and presenting them to the pharmaceutical/health sector to be used in preclinical and clinical studies.

Within the scope of the invention, the derivatives that have been obtained by carrying out semi-synthesizing on cycloartanes within the group of triterpene saponins have been screened in terms of their cytotoxic effects. The AG-08 coded compound that is obtained after the treatment of -TsCl (p-Toluenesulfonyl chloride) reactive with our exit molecule, has been reported to have cytotoxic effect against cancer cells. When advanced studies were carried out in relation to the impact mechanism of this compound, it has been noted that it eliminates cancer cells via a necrosis pathway. The fact that the exit molecules (CA and AG) do not have such an effect, this shows that by means of the reaction mentioned above, triterpeni c/steroidal saponins can be turned into anti-cancer drug candidate molecules that have an impact on the necrosis pathway.

The scope of the invention is based on synthesizing the AG-08 coded compound. The synthesis of the AG-08 coded compound is carried out in steps and first of all astragenol (AG) is obtained as an intermediate product from cycloastragenol (CA) and following this the AG-08 coded compound that has necrotic effect on cancer cells as a difference from (CA and AG) exit molecules is obtained from astragenol. The synthesis of AG-08 has been schematically illustrated in Figure 1, and the synthesis is described below, according to the study carried out within the scope of the invention.

Synthesis of AG-08:

6.11 mmol CA is dissolved in 20 mL methanol. 1 mL concentrated sulphuric acid is added, and the reaction mixture is heated in a condenser for 6 hours. The progress of reaction is tracked by a thin layer chromatography. The ended reaction is added into a 100 mL 9% NaiCCL solution. Liquid-liquid extraction process with 50 mL EtOAc is carried out 3 times. After being washed with brine solution and treated with anhydrous NaiSCL, it is dried under vacuum at 60°C in a rotary evaporator. The reaction mixture that is dried is mixed into 5g silica and is conditioned with 7:3 Hexane:EtOAc and is purified with 60 g silica.

Astragenol (AG) is obtained with 60.6% yield. 400 mg AG (0.408 mmol) is dissolved in 6 mL pyridine. 600 mg p-TsCl reactive is added thereon and is dissolved with the aid of a magnetic mixer. The progress of the reaction that continues for 4 hours at room temperature is tracked with thin layer chromatography. The ended reaction is added into a 100 mL 5% HC1 solution. Liquid-liquid extraction process with 50 mL EtOAc is carried out 3 times. After EtOAc extract is washed with brine solution and treated with anhydrous NaiSCL, it is dried under vacuum at 60°C in a rotary evaporator. The reaction mixture that is dried is mixed into silica and is conditioned with 80:20 Hexane:EtOAc and is purified with 15 g silica. AG-08 is obtained with a yield of 34.7%.

Empirical study

Biologic Activity Screening Studies:

The cytotoxicity of the AG-08 and AG molecules in HeLa (human cervical cancer cell line), PC3 (human prostate cancer cell line) and HCC1937 (human breast cancer cell line) cells have been evaluated in terms of usage, according to the protocol of the company producing the WST1 reactive (ROCHE). While the AG molecule did not exhibit cytotoxicity even at 30 mM dose, the IC 50 values of the AG-08 molecule have been determined to be 3, 80±0.143372; 5,73±0.46124 and 5,45±0.40893 pM respectively for HCC1937, PC3 and HeLa cells.

After 24 hours following the cultivation of HCC1937 and HeLa cells into a 6 well cell culture container, they were treated at 5, 10 and 20 pM doses with AG-08 and AG molecules. In empirical studies, besides these molecules, apoptosis positive control staurosporin (Sta), autophagy inhibitors Bafilomycin (Baf) and 3-methyladenine (3-MA) and histone deacetylase inhibitor trichostatin A (TSA) were used. Following an incubation period of 24 hours, the cells were lysated after being kept for 20 minutes in a RIPA buffer solution (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 10 mg/ml phenylmethylsulfonyl fluoride) that is a cell lysate buffer. The total protein concentrations of cell lysates obtained following 10 minutes of centrifugation at 14.000 rpm have been designated using the bicinchoninic method with the BCA protein concentrations determination kit (Thermo). Each sample has been divided into protein fractions using SDS-PAGE such as to contain 40 pg protein and their transfer to PVDF membrane has been performed. The expression levels of some proteins have been examined using the immunoblotting method. Poli(ADP-ribose)polymerase (PARP) that is a nuclear enzyme, is a protein that has various cellular functions such as DNA repair, transcription, aging, and molecular signalling. PARPl enzymes, are cropped under different cellular conditions by different proteases such as caspases, calpains, cathepsins and granzymes. The well-known reaction is to bring, by caspases, the 113 kDa PARP to an 89 kDA cropped state when apoptosis is induced. Therefore the 89 kDA cropped-PARP formation is used as an apoptosis indicator. A 50 kDA cropped PARP product different from this form, is present in literature as the necrotic cropping product. As it can be seen in Figure 2, AG-08 (but not AG) has led to the cropping of PARP proteins. However, the cropping product that is formed is a 50 kDA product that is related with necrosis rather than 89 kDa as is, in staurosporine which is an apoptosis agent.

In a similar study, the transition to active caspase enzymes from procaspase state of caspase enzymes after being treated with AG-08, via caspase 8, caspase 9 and caspase 3, have been examined using immunoblotting. While caspase 8, is a starter in extrinsic apoptosis caspase 9 also functions the same in intrinsic apoptosis. Starter caspases enable other caspases to be activated such as caspase 3. As it can be seen in Figure 3, while full length caspase 8 and caspase 3 are cut into their active state with AG-08, caspase 9 is not activated.

In a similar study, the expression levels of some functional proteins that are included in the autophagy pathway have been examined. In literature it has been shown that proteases that are linked to caspases and calpains cut some proteins that are essential for autophagy. In our results, it has been observed that proteins in charge at the autophagy pathway named Beclinl, ATG5, ATG7, ATG12 and ATG16L1 are not cut off with AG but are cut off with AG-08 and the amounts of full length proteins are reduced (Figure 4). The transition of the LC3 protein from LC3I to LC3II that is used as an autophagy indicator for searching the effect of AG-08 on autophagy has been examined with immunoblotting and it has been determined that transition into LC3II has increased. The intra cellular LC3II amount can increase both when autophagy increases and when it decreases but, together with the reduction of essential autophagy proteins, this result supports the hypothesis that autophagy is inhibited. Additionally, the p62 protein that is expressed as one of the autophagy substrates and is used in evaluating the autophagy pathway flow, has also been examined. Interestingly it has been noted that the p62 protein also degrades by giving a fragment of approximately 20 kDa with the AG-08 application.

Not only is it believed that the reduction of protein levels such as p62 and ATG proteins can be carried out directly via caspase 8, it is also believed that different necrosis proteases such as cathepsins can be activated by AG-08. In another assay, HCC1937 cells are cultivated on coverslips placed into 6 well cell culture conatiners and the next day the cells subjected to staurosporine, boiled water, H2O2 and 10 mM AG-08 application. In the Acridine Orange/Ethidium bromide cell staining, while acridine orange stain stains nucleus of live and dead cells, ethidium bromide stain only the nucleus of cells that have destabilized membrane. While the nucleus of cells that are applied with staurosporine exhibit degraded nuclear morphology specific to apoptosis, cells applied with boiled water and AG-08 have exhibited proper nuclear morphology however red staining (Figure 6).

Finally, cells that have been treated with AG-08 for 24 hours have been stained with 7-AAD and Annexin V in flow cytometry. It has been noted that excessive numbers of cells in cells treated with AG-08 are located at the left top region in comparison to control cells and that this region is associated with necrosis in literature (Figure 7).

Based on the data presented above, it is foreseen that it was highly probable for the tosyl carrying saponin derivatives (Steroidal and/or Triterpenic) at the aglycone ring to exhibit cytotoxicity by way of necrotic effects. Therefore, it is possible for them to be developed as anticancer drugs.

The structures that are described below and shown in Figure 8, are prepared from this consideration. The X and R groups on the chemical structures numbered 1-32 presented in Figure 7, express the following:

1. Each of XI, X2, X3, X4, X5 and X6 may independently be hydrogen, hydroxy, 1-6 carbon containing alkoxy, 1-6 carbon containing acyloxy, keto and glycoside;

2. Each of XI, X2, X3, X4, X5. X6 and X7 can independently be p-toluenesulfonyl m-Toluenesulfonyl, o-Toluenesulfonyl, Methanesulfonyl N-Acetylsulfanylil, benzenesulfonyl, 4-Fluorobenzenesulfonyl 2,4-dimethylbenzenesulfonyl,

-dimethylbenzeneulfonyl, 3,4-dimethylbenzenesulfonyl, 2-mesitylenesulfonyl, 2,4,6-trifluorobenzenesulfonyl, 4-tert-Amyl Benzene Sulfonyl,

4-Fenoxybenzenesulfonyl, 2,3,5,6-Tetramethyl Benzene Sulfonyl, 4-Fluoro-3 -methyl benzene sulfonyl, 4-ethoxynaphthalene-l -sulfonyl, 3- (Trifluoromethyl) benzenesulfonyl, 4- (aminosulfonyl) benzenesulfonyl chloride 4- (methylthio) benzenesulfonyl, 4-Fluoronaophthalene-l -sulfonyl, 2-fluoro-4-methyl benzene sulfonyl, 4- (2-methylpropoxy) benzene- 1-sulfonyl chloride, 3 -Carbamoyl-4-propoxybenzene- 1-sulfonyl chloride and

2,3,5 -trifluorob enzenesulfony 1 chi ori de .

3. Each of XI, X2, X3, X4, X5 and X6 may independently have alpha and beta configuration;

4. If glycosidation is present on hydroxy groups new glycosidations can be present on the glucose derivative directly linked to the structure and the numbers of glucose on the glycosidic chain can stretch up to a total of 3;

5. Rl, R2, R3 and R4 groups can independently be methyl or alcohol, aldehyde or carboxylic acid that are formed of this methyl at different oxidation levels;

6. Each Rl group may have alpha and beta configuration;

7. If Rl, R2, R3 and R4 groups are in primary alcohol forms, alkoxy containing 1-6 carbons, acyloxy containing 1-6 carbons and glycoside may be available independently on the present hydroxy group.

8. If glycosidation is present via the primary alcohol function located on the Rl, R2, R3 and R4 groups, new glycosidations can be present on the glucose derivative directly linked to the structure and the numbers of glucose on the glycosidic chain can stretch up to a total of 3;

9. If the Rl, R2, R3 and R4 groups are in carboxylic acid form, ester or amides can be made with alcohols or amines that independently carry 1-16 carbons;

10. The R4 group located in 5-8 structures, may also contain hydrogen, hydroxy, alkyl chain comprising 2-6 carbons , haloalkyl chain comprising 2-6 carbons, aryl, heteroaryl, monocyclic cycloalkyl chain comprising 3-8 carbons, bicyclic cycloalkyl chain comprising 4-8 carbons , monocyclic structured heterocyclic chain comprising 3-8 carbons, bicyclic structured heterocyclic chain comprising 4-8 carbons, and these chains may be subjected to substitution from 1-3 different points via the carbon atoms in the chain and this substitution can be an alkyl substitution comprising 1-3 carbons.

11. Symbol ~~~ , shows that single or double bonds can be found between the carbon atoms located in the positions where this symbol is found.

The AG-08 molecule inhibits cancer cells with an impact mechanism that has not been reported for this group of compounds before. Nowadays while most of the compounds that exhibit anticancer activity exhibit effect via apoptosis, the AG-08 coded molecule has a different impact mechanism and it kills cells with a non-apoptotic pathway. The empirical studies carried out within the scope of the invention, shows that this molecule has an impact both on autophagy and necrosis. It is foreseen that transition to molecules that exhibit highly potent and reliable profiles can be carried out by performing advanced modifications both on this molecule and by deriving other saponin structures similarly. It is believed that AG-08 and/or other derivatives can be good candidates for anticancer drug research in combination with compounds that have impact directly or via apoptosis.

It is believed that within the scope of the invention, the molecular mechanism of AG-08, which has been shown to induce the death of cancer cells by in vitro experiments, can be associated with other pathways. Thereby in the future the effect may be verified by in vivo pre-clinical studies.

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