DESC:Various embodiments of the invention provide novel inhibitors for BCL-2.
3-substituted indolin-2-one compounds are known to possess anticancer properties. One such chemical backbone, having the general formula (3-((2-alkyl/aralkyl)-6-arylimidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylidene)-1,3-dihydro-2H-indol-2-one, is synthesized. One embodiment of the invention provides a method for synthesizing an inhibitor for BCL-2. The method includes selecting a predefined concentration of indolin-2-one dissolved in alcohol. The indolin-2-one dissolved in alcohol is then treated with a predefined concentration of aldehyde derivative dissolved in 1ml of piperidine to obtain a reaction mixture. The aldehyde derivative is a compound of general formula I

(Formula I)
Wherein, R is selected from a list comprising of CH3, benzyl and 4-Cl-benzyl; and R’ is selected from a list comprising of phenyl, 4-bromophenyl, 4-chlorophenyl, 4-fluorophenyl, 4-methoxyphenyl, 4-methylphenyl, 4-nitrophenyl, 2H-chromen-2-one-3-yl. The reaction mixture is refluxed and then cooled under reduced pressure to obtain a precipitate. The precipitate thus obtained is then subjected to purification to obtain a compound having general formula (3-((2-alkyl/aralkyl)-6-arylimidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylidene)-1,3-dihydro-2H-indol-2-one, hereinafter referred to as compound II. The method described briefly herein shall be explained in detail, as exemplary embodiments of the invention.
In one example of the invention, 10 mmol of 2-indolinone is dissolved in alcohol. The alcohol is selected from the group including but not limited to methanol or ethanol. In one example of the invention 100 ml methanol is used and treated with 10 mmol 2-(4-chlorobenzyl)-6-(4-bromophenyl)imidazo[2,1-b] [1,3,4]-thiadiazole-5-carbaldehyde dissolved in 1ml piperidine to obtain the reaction mixture. The reaction mixture is refluxed for a time duration in the range of about 1h to about 5 h. The reflux heating of the reaction mixture is achieved at a temperature range of about 50oC to about 80oC. Subsequent to heating, the reaction mixture is cooled at a temperature range of about 20oC to about 35oC. The cooled reaction mixture is then concentrated under reduced pressure to obtain a precipitate. The precipitate obtained is filtered. The yield of the precipitate obtained is about 70-85% .The precipitate obtained is further purified by recrystallisation from ethanol.
In one example of the invention, the recrystallised precipitate obtained is a compound having the IUPAC name 3-((2-(4-chlorobenzyl)-6-(4-bromophenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylidene)-1,3-dihydro-2H-indol-2-one, hereinafter referred to as compound 5m. FIG.1 shows chemical structure of 5m, according to an embodiment of the invention.The structure of 5m includes a central imidazothiadiazole ring A, flanked by benzyl and phenyl rings (B, C) and an indole ring (D) placed in parallel and antiparallel directions.
Another embodiment of the invention provides a compound of formula II, for inhibiting BCL-2 function, the structure of which is given below:

Wherein, R is selected from a list including but not limited to CH3, benzyl and 4-Cl-benzyl. R’ is selected from a list including but not limited to phenyl, 4-bromophenyl, 4-chlorophenyl, 4-fluorophenyl, 4-methoxyphenyl, 4-methylphenyl, 4-nitrophenyl, 2H-chromen-2-one-3-yl. In alternative embodiments of the invention, derivatives, tautomers, isomers, polymorphs, solvates or intermediate of compound II are obtained. The compound 5m is chemically and biologically characterized.
In silico docking programs is used for studying the interaction of the synthesized compound II with the crystal structure of human BCL2 (PDB ID: 1GJH) and other anti-apoptotic BCL2 family members. In one example of the invention open source tool AutoDock Vina is used to study in-silico docking of 5m with the crystal structure of human BCL2 (PDB ID: 1GJH). FIG.2a shows the NMR and LC-MS spectra of the 5m. The specificity of binding of 5m is determined by binding studies and in-silico docking simulations. FIG. 2b shows the binding of 5m along the hydrophobic cleft of BCL2. 5m exhibits preferential binding towards BH-1 domain, that involves interaction with several conserved residues including but not limited to N143, W144, G145, R146,all belonging to the well conserved NWGR motif and R139 than only the two residues D103 and R107 of the BH3 domain...
The compound 5m, obtained is used for inhibiting BCL-2 function in a cell. The cell is a mammaliam cell which includes but is not limited to a bone cancer cell, a bladder cancer cell, a brain cancer cell, a breast adenocarcinoma cell, a cervical cancer cell, a colon cancer cell, a gastrointestinal cancer cell, a leukemia cell, a lung cancer cell, a lymphoma cell, a melanoma cell, a neuroblastoma cell, an ovarian cancer cell, a pancreatic cancer cell, a prostate cancer cell, a renal cancer cell, a retinoblastoma cell, a small-cell lung cancer cell, a non-small cell lung cancer cell, a stomach cancer cell, a testicular cancer cell, a thymus cancer cell, a thyroid cancer cell, an astrocytoma cell, a glioblastoma cancer cell, a head cancer cell, and a hepatocarcinoma cell.
In another embodiment of the invention, a pharmaceutical composition comprising the compound II is provided. The compound II is formulated as a pharmaceutical composition in excipients. The excipients include but not limited to an adjuvant, a diluent, a carrier, a granulating agent, a binding agent, a lubricating agent, a disintegrating agent, a sweetening agent, a glidant, ananti-adherent, an anti-static agent, a surfactant, an anti-oxidant, a gum, a coating agent, a coloring agent, a flavoring agent, a coating agent, a plasticizer, a preservative, a suspending agent, an emulsifying agent, a plant cellulosic material, a spheronization agent and/or other conventionally known pharmaceutically acceptable excipient or any combination of excipients thereof.
In yet another embodiment of the invention a method of managing a condition associated with increased expression of BCL-2 is provided. The method includes administering the pharmaceutical composition comprising compound II to a cell afflicted with increased expression of BCL-2 to reduce or inhibit BCL-2 function. Examples of a cell described herein include but are not limited to a bone cancer cell, a bladder cancer cell, a brain cancer cell, a breast adenocarcinoma cell, a cervical cancer cell, a colon cancer cell, a gastrointestinal cancer cell, a leukemia cell, a lung cancer cell, a lymphoma cell, a melanoma cell, a neuroblastoma cell, an ovarian cancer cell, a pancreatic cancer cell, a prostate cancer cell, a renal cancer cell, a retinoblastoma cell, a small-cell lung cancer cell, a non-small cell lung cancer cell, a stomach cancer cell, a testicular cancer cell, a thymus cancer cell, a thyroid cancer cell, an astrocytoma cell, a glioblastoma cancer cell, a head cancer cell, and a hepatocarcinoma cell. The pharmaceutical composition is administered through routes which include but not limited to intravenous administration, intramuscular administration, intraperitoneal administration, hepatoportal administration, intra articular administration and pancreatic duodenal artery administration, or any combination thereof.
In one embodiment of the invention, method for managing at least one condition associated with increased expression of BCL-2 is provided. The method includes reducing the function of BCL-2 in at least one cell by administering the compound II of the invention. The cell is a mammaliam cell which includes but is not limited to a bone cancer cell, a bladder cancer cell, a brain cancer cell, a breast adenocarcinoma cell, a cervical cancer cell, a colon cancer cell, a gastrointestinal cancer cell, a leukemia cell, a lung cancer cell, a lymphoma cell, a melanoma cell, a neuroblastoma cell, an ovarian cancer cell, a pancreatic cancer cell, a prostate cancer cell, a renal cancer cell, a retinoblastoma cell, a small-cell lung cancer cell, a non-small cell lung cancer cell, a stomach cancer cell, a testicular cancer cell, a thymus cancer cell, a thyroid cancer cell, an astrocytoma cell, a glioblastoma cancer cell, a head cancer cell, and a hepatocarcinoma cell.
In one specific example of the invention, the derivative 5m is administered and is tested for efficacy. Efficacy of 5m is assayed using breast adeno carcinoma models obtained from mice models. Tumor is developed in Swiss albino mice, by injecting EAC cells following which animals are treated with 5m. Six doses each of 10 mg/kg b. wt is administered and the rate of growth of tumor is monitored over a period of 24 days. FIG.3a shows significant reduction in tumor growth in cancer induced mice models, according to an example of the invention The graph shows significant reduction in tumor growth, when administered with 5m and when compared with the tumor in the control group that remained proliferative. FIG. 3b shows histological analysis of tumor in the thigh tissue of control and 5m treated mice, according to an embodiment of the invention. The histological samples obtained on the 25th day are HE stained. The HE stained samples show reduced number of infiltrated cells in the thigh tissue of the 5m treated animals. FIG. 3c shows histological analysis of tumor in the liver tissue of control and 5m treated mice, according to an embodiment of the invention. The histological samples obtained on the 25th day are HE stained. The HE stained samples show reduced number of infiltrated cells in the thigh tissue of the 5m treated animals.
In another example of the invention the derivative 5m is tested for its efficacy in a Dalton’s lymphoma mice models. FIG. 4a shows effect of 5m on tumor cell proliferation in a BCL2-driven mouse tumor model, according to an embodiment of the invention. Swiss albino mice are injected with Dalton lymphoma ascetic cells for inducing Dalton’s Lymphoma following which the mice are treated with 5m. Six doses of 5m, each of 10 mg/kg b. wt is administered to the mice via an intraperitoneal route. The rate of growth of tumor is monitored over a period of 24 days. Tumor progression was monitored by measuring the changes in body weight, normalized to the weight fluctuations in normal mice. A significant reduction in tumor progression is observed in 5m treated mice as compared to the untreated control mice. Body weight fluctuation is one of the important parameters commonly employed for assessing the adverse effects of a drug on normal physiology. The effect of 5m on the overall health of normal animals is monitored. The mice is treated with 5m and is monitored for change in body weight over a period of 16 days FIG. 4b shows a graph comparing the bodyweights of control and 5m treated mice, according to an embodiment of the invention. No significant change in body weight is observed in 5m-treated mice as compared to that of untreated control group. This indicates nontoxic nature of 5m. FIG. 4c shows a graph of comparative analysis of tumour progression with known BCL-2 inhibitors, according to an embodiment of the invention. The known inhibitors used are HA 14-1, Gossypol, Epigallocatechin. Six doses of 10 mg/kg b. wt of each inhibitor is administered and the rate of growth of tumor is monitored over a period of 24 days. 5m exhibits highest potential against tumor regression, followed by Gossypol. HA 14-1 is effective to a limited extent, whereas no significant tumor reduction is observed in case of Epigallocatechin.
FIG. 4d shows a graph of in vivo progression of EAC tumor in 5m and ABT199 inhibitor treated and untreated mouse models, according to an embodiment of the invention. Six doses of 10 mg/kg b. wt of 5m and ABT199 inhibitor is administered in EAC injected mouse model. 5m treated mice exhibited a significant reduction in tumor volume, as compared to the untreated tumor control. Values are depicted as mean ±SEM. ABT199 treated group shows a reduction in the tumor volume. However, the tumor regression in ABT199 is observed to be lower than that of 5m.
FIG. 5a through d generally shows the mechanism of cell death induced by 5m, according to an embodiment of the invention. The mechanism of cell death is investigated using various assays. 5m treated cells are subjected to Annexin FITC/PI double-staining in order to assess the cell death pathway activated. Results show predomimant activation of the apoptotic pathway as shown in FIG.5a, after 48 h of 5m treatment, with no significant increase in the necrotic cell population as shown in FIG.5a and 5b. 5m treated cells stained with hoechst dye show significant increase in cells with nuclear condensation, a distinct indicator of apoptosis, as shown inFIG.5c. Further, tumor cells obtained from mice bearing DLA tumor, post 5m treatment are assayed for distinct characteristics of apoptosis, as shown in FIG.5d. Activated Caspase 3 and Caspase 9 show increased expression in 5m treated cells, as compared to untreated controls. Interestingly, Caspase 8 cleavage is not observed in treated samples, as shown in FIG.5d. Taken together, 5m activates the intrinsic pathway of apoptosis, leading to cell death.
FIG.6a show DNA fragmentation in the 5m treated EAC tumor tissues from mice, according to an embodiment of the invention. In order to delineate the mechanism of 5m-induced cell death, a TUNEL assay is performed on 5m treated EAC tumor tissues from mice. A comparative analysis is performed with the untreated control.The results show extensive DNA fragmentation following 5m treatment as compared to the control, when analysed on day 25 post treatment.
FIG.6b shows immunohistochemistry analysis on sections of mouse tumor tissues following 5m treatment, according to an embodiment of the invention. Six doses of 10 mg/kg of 5m is injected into mouse tumor tissues and an immunohistochemical analysis is done on the 25th day. The results show robust expression of Ki67, a cell proliferation marker in the control tumors, which is negligible in the treated sections. Significantly, 53BP1, a DNA damage sensing protein, is found to be expressed more in treated tissues, as compared to untreated ones. Also the apoptotic marker cleaved CASPASE 3 is found to be overexpressed in the treated sections confirming the activation of apoptosis in treated tumors. The aforementioned experiments distinctly and specifically demonstrate 5m induced apoptosis in tumor cells, both ex vivo and in vivo.
FIG.7 shows a model for the mechanism of 5m induced cytotoxicity, according to an embodiment of the invention. Upon diffusing into the cells, 5m binds to hydrophobic binding pocket of BCL-2 at the outer mitochondrial membrane, inhibiting its physiological function. Reduction in overall BCL-2 function inside cells leads to the loss of MMP and release of the pro-apoptotic factors, CYTOCHROME C and SMAC/DIABLO. These can cause the downstream activation of CASPASE 3, CASPASE 9 and PARP1 resulting in DNA fragmentation and cell death via intrinsic pathway of apoptosis.
FIG. 8a. shows relative expression of BCL-2 in different cell lines, according to an embodiment of the invention. The Levels of BCL-2 expression is shown in six independent cancer cell lines HeLa, LNCaP, P388D1, A2780, NALM6 and Molt4. In a specific example of the invention, the efficacy of 5m and ABT199 is measured. FIG. 8b shows efficacy of 5m and ABT199 in different cell lines, according to an example of the invention. Six independent cancer cell lines HeLa, LNCaP, P388D1, A2780, NALM6 and Molt4 is subjected to 5m and ABT199 treatment at concentrations of 0µM, 1 µM, 2 µM, 5 µM and 10 µM respectively. The cell lines are analyzed for cell death using the MTT assay. 5m exhibits higher cytotoxic potential, as compared to ABT199, in all the cancer cell lines tested.
FIG.8c shows the cytotoxic effects of 5m in various cancer cell lines and a non-cancerouscell line, 293T, along with the endogenous BCL-2 levels in these cell lines, according to an embodiment of the invention. Results showed that viability is significantly reduced at 48 h of treatment in cells expressing higher levels of BCL-2 (REH, NALM6, CEM P388D1, A2780, Molt4), while cells with low BCL-2 (K562, T47D, 293T, HeLa, LNCaP) exhibited reduced cytotoxicity. The effect is marginal in the non-cancerous cell line, 293T. This shows that 5m-induced cytotoxicity is dependent on the levels of BCL2 expression.
FIG.9 shows Pharmacokinetic studies of plasma from 5m treated animals, according to an embodiment of the invention. 10 mg/kg of 5m is administered to mice and the plasma is collected after time intervals of 5 min, 10 min, 30 min, 1 hour, 2 hours and 6 hours. A HPLC analysis shows that the maximum circulating concentration, Cmax of 5m is obtained in 30 min following its treatment indicating that it is amenable to absorption in vivo. The plasma levels of 5m exhibit a bi-exponential decline with a t1/2 of 2.3 hours and a clearance rate, CL of 6.3 ml/min/kg. Thus, 5m administration in mice did not show any significant side effects, as analyzed using various parameters reiterating its specificity, and rendering it suitable for clinical studies.
Another embodiment of the invention provides a kit comprising the compound II for inducing apoptosis or managing at least one condition with over expressionof BCL-2. . The method includes administering to a cell afflicted with such a condition an effective amount of the pharmaceutical composition to reduce or inhibit BCL-2 function. Examples of cell include but is not limited to a bone cancer cell, a bladder cancer cell, a brain cancer cell, a breast adenocarcinoma cell, a cervical cancer cell, a colon cancer cell, a gastrointestinal cancer cell, a leukemia cell, a lung cancer cell, a lymphoma cell, a melanoma cell, a neuroblastoma cell, an ovarian cancer cell, a pancreatic cancer cell, a prostate cancer cell, a renal cancer cell, a retinoblastoma cell, a small-cell lung cancer cell, a non-small cell lung cancer cell, a stomach cancer cell, a testicular cancer cell, a thymus cancer cell, a thyroid cancer cell, an astrocytoma cell, a glioblastoma cancer cell, a head cancer cell, and a hepatocarcinoma cell.
The present invention provides a BCL-2-specific small molecule inhibitor that acts selectively in cancer cells with high BCL-2 expression, while sparing normal cells. In vivo studies show that the administration of the inhibitor in mice causes minimal side effects, as opposed to several other BCL-2 inhibitors. Further, the inhibitor does not affect platelets, deeming it fit for clinical cancer therapy.
The foregoing description of the invention has been set for merely to illustrate the invention and is not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to person skilled in the art, the invention should be construed to include everything within the scope of the appended claims and equivalents thereof.
,CLAIMS:1. A method of synthesizing a compound for inhibiting BCL-2 function, the method comprising:
-Reacting a predefined concentration of indolin-2-one in alcohol with a predefined concentration of an aldehyde derivative in presence of piperidine to obtain a precipitate; and
- Purifying the precipitate to obtain the compound.
2. The method of claim 1, wherein the step of reaction includes at least one cycle of heating and/or cooling.
3. The reaction of claim 2, wherein the heating is achieved at a temperature in the range of about 50ºC to about 80ºC.
4. The reaction of claim 2, wherein the cooling is achieved at a temperature in the range of about 20ºC to about 35ºC.
5. The method of claim 1, wherein the alcohol is selected from the list comprising of methanol or ethanol.
6. The method of claim 1, wherein the aldehyde derivative is a compound of general formula (I),

Wherein,
R is selected from a list comprising of CH3, benzyl and 4-Cl-benzyl; and
R’ is selected from a list comprising of phenyl, 4-bromophenyl, 4-chlorophenyl, 4-fluorophenyl, 4-methoxyphenyl, 4-methylphenyl, 4-nitrophenyl, 2H-chromen-2-one-3-yl.

7. A compound represented by formula II,

Wherein,
R is selected from a list comprising of CH3, benzyl and 4-Cl-benzyl; and
R’ is selected from a list comprising of phenyl, 4-bromophenyl, 4-chlorophenyl, 4-fluorophenyl, 4-methoxyphenyl, 4-methylphenyl, 4-nitrophenyl, 2H-chromen-2-one-3-yl.
8. The compound of claim 7, wherein the compound is at least one of a derivative, a tautomer, an isomer, a polymorph, a solvate or an intermediate thereof.
9. The compound of claim 7, wherein the compound is for inhibiting BCL-2 function in a cell.
10. A pharmaceutical composition comprising a compound of claim 7-10 and at least one pharmaceutically acceptable excipient.
11. The composition of claim 10, wherein the composition is for inhibiting or reducing the function of BCL-2 in at least one cell.
12. The pharmaceutical composition as claimed in claim 10, wherein the pharmaceutically acceptable excipient is selected from a group comprising an adjuvant, a diluent, a carrier, a granulating agent, a binding agent, a lubricating agent, a disintegrating agent, a sweetening agent, a glidant, ananti-adherent, an anti-static agent, a surfactant, an anti-oxidant, a gum, a coating agent, a coloring agent, a flavoring agent, a coating agent, a plasticizer, a preservative, a suspending agent, an emulsifying agent, a plant cellulosic material, a spheronization agent, other conventionally known pharmaceutically acceptable excipient or any combination of excipients thereof.
13. The composition of claim 10, wherein the composition is administered through at least one route comprising of intravenous administration, intramuscular administration, intraperitoneal administration, hepatoportal administration, intra articular administration and pancreatic duodenal artery administration, or any combination thereof.
14. A method for managing at least one condition associated with increased expression of BCL-2, the said method comprising inhibiting or reducing the function of BCL-2 in at least one cell, by administering the compound of claim 7.
15. A kit for inducing apoptosis or managing at least one condition associated with over expression of BCL-2, comprising compound of structure II as claimed in claim 7, thereby inhibiting or reducing the function of BCL-2 in at least one cell.
16. The cell of claims 9, 11, 15 and 16, wherein the cell is a mammalian cell selected from the group comprising of a bone cancer cell, a bladder cancer cell, a brain cancer cell, a breast cancer cell, a cervical cancer cell, a colon cancer cell, a gastrointestinal cancer cell, a leukemia cell, a lung cancer cell, a lymphoma cell, a melanoma cell, a neuroblastoma cell, an ovarian cancer cell, a pancreatic cancer cell, a prostate cancer cell, a renal cancer cell, a retinoblastoma cell, a small-cell lung cancer cell, a non-small cell lung cancer cell, a stomach cancer cell, a testicular cancer cell, a thymus cancer cell, a thyroid cancer cell, an astrocytoma cell, a glioblastoma cancer cell, a head cancer cell, and a hepatocarcinoma cell.