Compounds with unique liphagane meroterpenoid scaffold having boronic acid functionality in the skeleton are described (formula 1) together with pharmacological potential of these compounds as anticancer agents. A method of preparation and inhibiting the activity of phosphoinositide-3-kinase (PI3K-alpha and beta) has been presented. In particular, the invention describes a method of inhibiting PI3K isoforms, wherein the compounds are novel structures based on liphagane scaffold with unique boronic acid functionality. The methods and uses thereof are described herein this invention.

FIELD OF THE INVENTION

The present invention relates to boronic acid bearing liphagane compounds. The present invention particularly relates to boronic acid bearing meroterpenoid liphagane scaffold based compounds. The compounds have been designed, synthesized and their biological evaluation results for anticancer activity by inhibiting PI3K pathway are presented in this invention. The field of invention for this work relates and covers the development of novel PI3K-α/β inhibitors based on meroterpenoid liphagane scaffold for anticancer activity.

BACKGROUND OF THE INVENTION

PI3Ks are a family of related intracellular signal transducer capable of phosphorylating the 3 position hydroxyl group of the inositol ring of Phosphatidylinositol (PtdIns). They are also known as phosphatidylinositol-3-kinases. The pathway, with oncogene PIK3 and tumor suppressor (PTEN) gene is implicated in insensitivity of cancer tumors to insulin and IGF1, in calorie restriction. 3-kinase (PI3K) signaling pathway is a newly identified strategy for the discovery and development of certain therapeutic agents. Among the various subtypes of PI3K, class IA PI3K-alpha has gained increasing attention as a promising drug target for the treatment of cancer due to its frequent mutations and amplifications in various human cancers. In contrast with cytotoxic agents that do not differentiate between normal proliferating and tumour cells, targeted therapies primarily exert their action in cancer cells. Initiation and maintenance of tumours are due to genetic alterations in specific loci. The identification of the genes in these alterations occurs has opened new opportunities for cancer treatment. The PI3K (phosphoinositide 3-kinase) pathway is often overactive in human cancers and various genetic alteration have been found to cause this. In all cases, PI3K inhibition is considered to be one of the most promising targeted therapies for cancer treatment.

Owing to its widespread activation in inflammation and cancer, a growing appreciation of the therapeutic potential of inhibitors of the phosphoinositide 3-kinase (PI3K) pathway has stimulated intense interest in compounds with suitable pharmacological profiles. These are primarily directed toward PI3K itself. However, as class I PI3Ks are also essential for a range of normal physiological processes, broad spectrum PI3K inhibition could be poorly tolerated.

In recent years, patents describing a new generation of PI3K inhibitors have started to appear, with a particular focus on the development of compounds with enhanced isoform selectivity for use as anti-cancer and anti-inflammatory therapies. However, challenges remain for the efforts to pharmacologically target this enzyme family in a successful manner.

Rationale for the Selection of Phosphoinositide 3-Kinase-α (PI3K-α/β) Inhibitors:—

At cellular level, phosphoinositide-3-kinase signaling contributes to many processes, including cell cycle progression, cell growth, survival and migration and intracellular vesicular transport. The PI3K represents the family of lipid kinases that can be classified into three subfamilies according to structure and substrate specificity viz., class I, class II and class III. The class I PI3Ks are the most extensively studied among lipid kinases, are heterodimeric proteins; each containing a smaller regulatory domain and a larger 110 kDa catalytic domain, which occur in four isoforms differentiated as p110α, p110β, p110γ, and p110δ. Although, there are natural product based small molecules reported in the literature which inhibit the PI3-kinases having the IC50value in nano-gram range (viz., Wortmannin isolated fromPenicillium wortmanni, LY294002 a synthetic analogue of the flavonoid quercetin, etc) but these molecules did not reach to market because of low potency, poor isoform or kinase selectivity, limited stability and unacceptable pharmacological and pharmacokinetic properties. However, PI3 kinase inhibitors having isoform selectivity and promising drug-like properties have now begun to emerge that show promise for the treatment of cancer and other disease indications. In cancer, evidence suggests that inhibition of the class 1A PI3 kinases p110α and p110β appear to be the most appropriate to target. Recently, Andersen et al., in 2006 reported the potential isoform selective PI3K-alpha inhibitor from marine spongeAka coralliphagaunder the collaborative program to screen marine invertabrates against human PI3K-alpha keeping in mind that natural products from marine resources have emerged as a copious repository of molecular diversity and hold considerable promise as a rich source of lead structures in drug discovery. Liphagal (Joshua J. Day, Ryan M. McFadden; The catalytic enantioselective total synthesis of (+)-Liphagal;Angew. Chem. Int. Ed.2011, 50, 6814-6818; Enrique Alvarez-Manzaneda, RachidChahboun; Enantioselectivetotal synthesis of the selective PI3-kinase inhibitor Liphagal;Org. Lett.,2010, 12 (20), pp 4450-4453; Jonathan H. George, Jack E. Baldwin; Enantiospecific biosynthetically inspired formal total synthesis of (+)-Liphagal,Org. Lett.,2010, 12 (10), pp 2394-2397; Alban R. Pereira, Wendy K. Strangman, Synthesis of phosphatidylinositol 3-kinase (PI3K) inhibitory analogues of the sponge meroterpenoid Liphagal;J. Med Chem.,2010, 53 (24), pp 8523-8533; Dima A. Sabbah, Jonathan L. Vennerstrom; Docking studies on isoform-specific inhibition of phosphoinositide-3-kinases;J. Chem. Inf. Model.,2010, 50 (10), pp 1887-1898; Ram Vishwakarma and Sanjay Kumar; Efficient Synthesis of key intermediate toward Liphagal synthesis;Synthetic Communications;2010, 41(2), pp 177-183; Frederic Marion, David E. Williams, Liphagal, a selective inhibitor of PI3 kinase-α isolated from the spongeAka coralliphaga: Structure elucidation and biomimetic synthesis;Org. Lett.,2006, 8 (2), pp 321-324; Goverdhan Mehta, Nachiket S. Likhite, C. S. Ananda Kumar A concise synthesis of the bioactive meroterpenoid natural product (±)-liphagal, a potent PI3K inhibitor,Tet. Lett,2009, vol. 50, no. 37, pp 321-324) was ˜10-fold more potent against PI3K-α than against PI3K-γ. We have synthesized boron containing analog of liphagal by rational modification on this molecule following diversity oriented synthesis approach for the discovery of lead molecules.

OBJECTS OF THE PRESENT INVENTION

The main object of the present invention is to provide boronic acid bearing liphagane compounds. Another object of the invention provides a process for preparation of boronic acid functional group containing liphagane compounds.

Yet another object of the present invention is to provide process for the preparation for step A6 to A7 and A7 to A by the synthetic route mentioned in the claims of this invention document.

Still another object of the present invention is to evaluate biological activity of the boronic acid based liphagal compounds as anticancer agents.

Yet another object of the present invention is to identify isoform selectivity of these compounds for PI3K inhibition as alpha or beta specific when studied for enzyme specificity.

Yet another object of the invention is to explore the mechanism of action and growth inhibition of the liphagal boronic acid bearing compound by Annexin-V or immunofluroscent assay and by cell cycle analysis.

SUMMARY OF THE INVENTION

Accordingly the present invention provides a compound of general formula 1, and pharmaceutically acceptable salts thereof,

In another embodiment of the invention, the compound is useful as specific inhibitor of PI3K-α or β isoform in cancer treatment.

Yet another embodiment of the invention provides a process for preparation of compounds of general formula 1 and pharmaceutically acceptable salts thereof

ii) adding triethyl or trimethyl borate to the above mixture obtained in step (i) and stirring;iii) quenching the reaction of step (ii) with saturated ammonium chloride solution followed by extraction with water immiscible solvent to obtain compound of general formula 10

iv) reacting the compound 10 with BI3or DMS or AlCl3/thiourea in a proportion in the range of 1:1 to 3:4 by moles in an ether solvent;v) quenching the reaction of step (iv) by addition of hypo solution followed by extraction with a water immiscible solvent to obtain compound of general formula 1.

In yet another embodiment of the invention, the ether solvent used in step (i) and (v) is selected from a group consisting of tetrahydrofuran, dichloromethane, diethyl ether, diisopropyl ether and isopropyl ether.

In yet another embodiment of the invention, the base in step (i) is selected from a group consisting of tetramethyl ethylene diamine, triethyl amine, trimethyl amine and diisopropyl ethyl amine.

In yet another embodiment of the invention, reaction in step (i) is carried out at a temperature in the range of −78° C. to 35° C. for a period ranging between 5 to 10 min.

In yet another embodiment of the invention, reaction in step (ii) is carried out at a temperature in the range of 0-5° C., for a period ranging between 1 to 2 h.

In yet another embodiment of the invention, the water immiscible solvent in step (iii) and (v) is selected from a group consisting of ethylacetate, dichloromethane, ether or chloroform.

In still another embodiment of the invention, reaction in step (iv) is carried out at a temperature ranging between −78° C. to 35° C. for a period ranging between 1 to 3 h,

In still another embodiment of the invention, the compound of general formula 1 obtained in step (v) is converted into a pharmaceutically acceptable salt.

In still another embodiment of the invention, the compound of general formula 1 is converted into a pharmaceutically acceptable salt by a process comprising the steps of mixing the compound of general formula 1 with a base in a ratio 1:1 proportion, wherein the base is selected from a group consisting of sodium hydroxide, potassium hydroxide and ammonium hydroxide in water, stirring the reaction mixture for 1-2 h followed by drying to obtain the pharmaceutically acceptable salt of the compound of general formula 1.

Yet another embodiment of the invention provides a pharmaceutical composition comprising an effective amount of the compound of formula 1, optionally along with a pharmaceutically acceptable carrier, salt, excipients or diluents.

In still another embodiment of the invention, the pharmaceutically acceptable carrier is selected from a group consisting of water, buffered saline, glycols, glycerols, olive oil and liposomes.

Still another embodiment of the invention provides a method of treatment of cancer by specific inhibition of PI3K-α or β isoform in a human cancer cell line using a compound of general formula 1,

In another embodiment of the invention, dosage of compound of general formula 1 is in the range of 20 mg/kg to 100 mg/kg.

In another embodiment of the invention, the representative compound A has a GI50 concentration in the range of 2.4 μM-2.6 μM when used for in vitro activity against colon and breast cancer cell lines.

In another embodiment of the invention, the representative compound A demonstrates >74% optimal growth inhibition in human cancer cell lines at a concentration of 10 μM.

In another embodiment of the invention, the representative compound E when used for in vitro activity against colon cancer cell lines increases sub-G1/G0 population and shows concentration dependent growth arrest in G1/G0 population and late apoptosis in colon cancer cell lines.

ABBREVIATIONS

13CNMR: carbon nuclear magnetic resonance

h or hr: hour

1HNMR: proton nuclear magnetic resonance

MTT: mitochondrial membrane potential

TLC: thin layer chromatography

DETAILED DESCRIPTION OF THE INVENTION

In yet another embodiment, ‘Y’ is O, S, NH, and NR, wherein, R-may be substituted with alkyl, aryl, heteroaryl moiety or any cyclic aliphatic or aromatic system.

In an embodiment of the present invention, n and n1 are selected carbon chain length from 0, 1 and 2.

In an embodiment, wherein, the substituent R4is also selected from a group consisting of hydrogen, alkyl substituents viz., methyl, ethyl, propyl and the higher homologues either linear of branched, including alicyclic such as cyclopentane, cyclohexane or higher membered rings, fused rings, aryl/heteroaryl substituted alkyl groups including benzlic or its higher homologues that might include unsaturated alkyl groups such as cinnamul, crotyl and prenyl substituents.

In embodiment of the present invention, R is independently selected from H or one to ten carbon chain either linear or branched, saturated or unsaturated, alkyl group optionally substituted with OH, H, OH, ═O, ═S, OR, COR, CHO, CO2R, OCOR, NH2, NHR, NRR′, NO2, F, Cl, Br, I, OSO3H, SO2R, CN, SiRR′R″ and R. Here R, R′, R″ may be alkyl, aryl, heteroaryl or any cyclic aliphatic ring with different substitutions.

Wherein, further the substituent R at various positions is also selected from a group consisting of hydrogen, alkyl substituents viz., methyl, ethyl, propyl and the higher homologues either linear or branched, including alicyclic such as cyclopentane, cyclohexane or higher membered rings, fused ringsm aryl/heteroaryl substituted alkyl groups including benzlic or its higher homologues that might include unsaturated alkyl groups such as cinnamul, crotyl and prenyl substituents.

In an embodiment in the present invention, the routine method was used for the in silico bioinformatics study of liphagal and its boronic acid based compounds, it is as mentioned below: all the computational studies were carried out in the Schrodinger suite 2010 molecular modeling software. The 2D structures of all the molecules were built in the maestro window. All the molecules were then converted to their respective 3D structure, with various conformers, tautomers and ionization states using the Ligprep and Confgen modules. The molecules were then minimized using the OPLS—2005 force field. The 3D crystal structure of PI3Kα reported in Protein Data Bank (PDB) was used as receptor for docking studies (PDB ID: 3HHM). The protein was downloaded from the PDB and was prepared for docking using the Protein Preparation wizard. Hydrogen's were added to the protein and the missing loops were built. Bond length and bond order correction was also carried out for preparing the protein for docking studies. The active site grid was generated based on the already co-crystallised ligand of the receptor using receptor grid generation module. The ligands were docked on to the receptor through this grid using Glide module and flexible docking was carried out for all the conformers in order to find out the binding mode of these ligands. The extra precision (XP) scoring function of Glide was used for carrying out these studies. In yet another embodiment of the present invention, wherein, the results obtained in the in silico studies of liphagal and its boronic acids based compounds are as: based on the docking studies, it was found that the boronic acid analogues of liphagal bind with better affinity to PI3Kα than liphagal. The interaction studies show that boronic acid (OH) are involved in strong H-bond interactions with Val851 and Gln859, whereas liphagal is involved in H-bond interaction at one place only with Gln859. Also the dock score of boronic acid based compound was about −10 and that of liphagal was about −8.5, which shows a stronger affinity of boronic acid analogues towards PI3Kα.

EXAMPLES

The invention is further described by reference to following examples which are intended to illustrate and should not be construed to limit the scope of the present invention.

Materials and Method:

General: Solvents were purified according to the standard procedures, and reagents used were of highest purity available. All reactions were performed in flame-dried glass apparatus under argon/nitrogen atmosphere unless mentioned otherwise. Anhydrous solvents like CH2Cl2, Et2O, THF, CH3OH, CH3CN, DMF, pyridine, Et3N were freshly dried using standard methods. NMR measurements (1H and13C) were recorded on either 400 or 500 MHz spectrometer (Bruker) fitted with pulse-field gradient probe, and trimethylsilane (TMS) or residual resonance of deuterated solvent were used as internal reference. Chemical shifts are expressed in (δ) parts per million and coupling constants J in hertz. Mass spectra were recorded on ESI MS or MALDI-TOF/TOF MS/MS-MS spectrophotometer using 2,5-Dihydroxy benzoic acid/α-Cyano-4-hydroxy benzoic acid/Sinapinic acid (Sigma-Aldrich) as matrix in acetonitrile:water containing 0.01% TFA. Optical rotations were measured on a digital PerkinElmer-241 polarimeter. Analytical TLC was performed on Merck 60 F254plates, and compounds were visualized by spraying and charring with phosphomolybdic acid or 20% H2SO4in MeOH as developing reagent. Preparative TLC was performed on pre-coated silica gel 60 F254plates (20×20 cm) purchased from Merck. Silica column chromatography was carried out with silica gel (100-200 mesh) or flash silica gel (230-400 mesh) purchased from Merck.

Synthesis of Compound A

Step 6: Synthesis of Compounds (A6)

Step 7: Synthesis of Compound (A7)

For the purpose of this application, compound A7 has been interchangeably referred to as compound E.

Step 8: Synthesis of Compound A

The solution of BI3in DCM was added slowly and drop wise in the round bottom flask containing solution of compound A7 in DCM at −78° C. The mixture of this was stirred at same temperature for half an hour the slowly raised to rt. The progress of reaction was monitored by TLC. The reaction mixture was neutralized using potassium thiosulphate solution and extracted with DCM solution and separated the organic layer, dried over sodium sulphate, concentrated in vaccuo. The crude was purified by column chromatography using hexane/EtOAc as eluent.1H NMR (500 MHz, CDCl3) δ 6.83 (s, 1H), 2.56-2.49 (m, 1H), 1.56-1.52 (m, 4H), 1.48 (s, 3H), 1.37 (s, 3H), 0.99 (s, 3H), 0.96 (d, 3H) ppm. Mass: ESI [M+Na]+: 372.211; Elemental anal. calcd. for C21H29BO5; C, 67.75; H, 7.85; B, 2.90. found C, 67.65; H, 7.61; B, 2.30.

All the compounds disclosed in formula 1, are prepared by employing the similar method containing different substitutions at R1, R2, R3 and R4 positions, as described for the preparation of compound A. The details of reaction conditions are depicted in the table given below—

Step 2: Synthesis of compound 13 (4,5-dimethoxy-2-(methoxymethoxy)benzaldehyde)

Step 5: Synthesis of compound 18 a starting material 3-(2,6,6-trimethylcyclohex-1-enyl)propanoic acid: 17 g (425.001 mmol) of NaOH was dissolved in water to make a 70 ml solution in a 250 ml conical flask with a magnetic stirrer. The alkali solution was then cooled in an ice bath and 17 g (106.25 mmol) of bromine was added to the solution after stirring for 1 h, 4.5 g (23.19 mmol) of dihydro-β-ionone 17 in 10 ml of dioxane was dropped into the solution, the stirring was continued at rt for 4 h. The excess of hypobromite was neutralized with 10% sodium bisulfite and solution was extracted with diethylether to remove remaining impurities. Acidification of the alkaline solution with conc. hydrochloric acid was done under usual conditions and workup gave 18 (4.1 g, 90.1%) as a colorless liquid.1H NMR (CDCl3, 400 MHz): δ 2.44-2.39 (m, 2H), 2.37-2.31 (m, 2H), 1.93-1.89 (t, J=8 Hz, 2H), 1.61 (s, 3H), 1.58-1.54 (s, 2H), 1.44-1.41 (m, 2H), 1.00 (s, 6H).13CNMR (CDCl3, 100 MHz): 180.3, 135.4, 128.5, 39.7, 34.9, 34.7 (multiple merged peaks), 28.4, 23.5, 19.6, 19.4. HRMS (ESI) m/z: [M+H]+calcd for C12H20O2+H+197.1541. Found 197.1530.

The MTT assay (MTT Assay (Legrier M E, Yang C P, Yan H G et al. Targeting protein translation in human non small lung cancer via combined MEK and mammalian target of rapamycin suppression.Cancer Res67:11300-8(2007).) is useful for measuring the effect of a wide range of compounds on the in vitro growth of either normal or cancer cell lines. The assay was set up in a 96-well, flat-bottomed polystyrene microtiter plate. 3-5000 cells were suspended per well in appropriate growth medium, and the cells were added to replicate wells (triplicates were preferred). It was preferable to add the cells to the required number of wells in the plate prior to adding the drugs or the test agents. After the cells were added to the plate, it was placed on the incubator for overnight incubation, while the agents to be tested were being prepared. After overnight incubation drugs or test compounds were added at defined concentrations to each set of replicate wells and incubated for 48 hrs in CO2incubator. Most of these compounds were dissolved in dimethyl sulfoxide (DMSO) for the final addition. After 48 hr incubation, diluted the MTT stock solution (2.5 μg/ml) with an equal volume of tissue culture medium and added 20 μl of this solution directly to each well with a multichannel pipette. As with the adherent-cell method, return the plates to the incubator for a period of at least 4 h. After 4 hr incubation centrifuge the plates at 1000 g for 10 min at ambient temperature, followed by inversion of the plates and blotting of excess medium. Add 150 μl of working DMSO to solubilize the MTT formazan product. A standard micro plate reader with adjustable wavelength across the visible spectrum was used. The OD values at 570 nm obtained for each set of triplicates corresponding to a specific concentration of a test agent was then transferred into a spreadsheet program.

Results: Cytotoxicity assay based on MTT was performed on the panel of cancer cell lines using compound A and compound E as a test material. In order to determine the effect of compound A and compound E on cell proliferation and in relative IC50values, MCF-7, caco-2 & HCT-115 were treated with compound A and compound E at indicated concentrations (0.01, 0.1, 1, 10 μM) for 48 h. In the present study, compound A and compound E produced concentration dependent inhibition of cell proliferation. From the MTT based inhibition in cell proliferation, the calculated cell based IC50value of 2.6 μM and 2.4 μM in breast (MCF-7) and colon (caco-2) cell line were observed for compound A and 5.6, 3.7 and 3.1 μM in colon (caco-2, HCT-115) and breast (MCF-7) for compound E was calculated (FIG. 3). These results depicted that both compound A and compound E showed more effectiveness against colon cell proliferation as reflected by relative IC50values and therefore towards the colon cancer in general.

PI3K inhibition assay (PI3K Assay (Emmanuelle M, Huang Y, Yan H G et al. Targeting Protein Translation in Human Non-Small Cell Lung Cancer via Combined MEK and Mammalian Target of Rapamycin Suppression.Cancer Res67:(23). (2007).) was carried out by PI3 Kinase activity/inhibitor assay kit, where PI3 kinase reaction was set up in Glutathione-coated strips/plate for inhibitor reaction. Kinase and inhibitors were pre-incubated for 10 minutes prior to the addition of PIP2 substrate. 5 μL of 5× kinase reaction buffer were added in each well followed by the further addition of 5 μL/well of PIP2 substrate. Then distilled H2O was added to each well so as to make up a final volume of 25 μL/well. Incubation was done at rt for 1 hour which was followed by washing the wells 3 times with 200 μL of 1×TBST per well and then 2 times with 200 μL of IX TBS per well. Then 100 μL of the Substrate TMB per well was added and then to keep for colour development in the dark for 5-20 minutes. However, appearance of the blue color to avoid over-development were monitored. 100 μL of the stop solution per well was used to stop the reaction. Readings were recorded at 450 nm.

The IC50value of a drug measures the effectiveness of a compound in inhibiting biological or biochemical function. Drug molecules can be categorized as low, active or highly active based on IC50values. The determination of enzyme based IC50values helps in early analysis and estimation of the drug activities in order to narrow down drug candidates for further experimental purpose. The liphagal, compound A and compound E used in the present study inhibited PI3Kα enzyme activity in dose dependent pattern with varying concentration i.e 20, 40, 80, 160, 320 and 640 nM respectively. Moreover, an IC50of 108, 140 and 102 nM for liphagal, compound A and compound E against PI3Kα was observed and 100 nM for compound A against PI3Kβ was also determined (FIGS. 4 and 5). This approach will not only enhance origin specific cancer drug discovery process, but will also save time and resources committed.

Analysis (Cell cycle (Waxman D J, Schwartz P S, Harnessing apoptosis for improved anti-cancer gene therapy,Cancer Res.63:8563-8572(2003).) of a population of cells replication state can be achieved by fluorescence labeling of the nuclei of cells in suspension and then analyzing the fluorescence properties of each cell in the population. The experiment was performed using caco-2, colon human cancer cell line. Cells were seeded in 6 well plates at the concentration of 3×105cells/ml/well. Plate was incubated in CO2incubator for overnight. After overnight incubation test sample(s) were added at desired concentration, sparing wells for negative and positive control and incubated for 24 hrs. After 24 hr incubation, cell were trypsinized along with test sample from each well was extracted using a micropippete and separately transferred into 15 ml centrifuge tubes. Tubes were centrifuged at 3000 rpm for 5 min. The supernatant was discarded and pellet was resuspended in 1 ml filtered PBS and centrifuged at 2000 rpm for 5 min. After 5 mins supernatant was discarded and pellet was resuspended in 70% ethanol. Cells were fixed for at least 1 hour at 4° C. (cells may be stored in 70% ethanol at −20° C. for several weeks prior to PI staining and flow cytometric analysis). Cells were again centrifuged at 2000 rpm for 5 minutes and washed twice in filtered PBS by centrifuging at 2000 rpm for 5 min. Supernatant was discarded and tubes were placed in inverted position over tissue paper till all the supernatant drained over the paper. 1 ml of cell cycle reagent (CCR) was added in each acquisition tube in dark. Reading was taken on flow cytometer (BD Biosciences).

Results: Cell cycle is the life cycle of a cell. Each stage of the cell cycle i,e. G1 (Gap1), S, G2 (Gap 2), & M (mitosis) have unique events that occur within each of them. Two of the most popular flow cytometric applications are the measurement of cellular DNA content and the analysis of the cell cycle which are fundamental processes of cell survival. In the present study, the effect of compound E on the DNA content by cell cycle phase distribution was assessed by using colon (caco-2) cell line. In addition to determining the relative cellular DNA content, flow cytometry also enables the identification of the cell distribution during the various phases of the cell cycle. Cells (2×106/ml/6-well plate), exposed to different concentrations of compound E were stained with propidium iodide (PI) to determine DNA fluorescence and cell cycle phase distribution. The percentage of compound E treated sub-G0 cells with 1, 5, 7 and 9 μM for 24 h was found to be 62.5%, 64.3%, 65.6% and 70.2% respectively. Under similar conditions, Liphagal treated cultures showed 64.9% cells in sub-G0 phase. Further, the cell cycle at G2/M phase was not affected indicating that compound E treatments does not produce any mitotic block or cause delay in cell cycle. Overall, each treatment with an increase in concentration led to an increase in sub-G0 after 24 h treatment. Thus, it is clear that compound E induced early cell cycle arrest with concentration dependent manner (FIG. 6).

The cell death status was analysed using Annexin-V (Annexin-V apoptotic assay (Yunqing Li, FadilaGuessous, SherwinKwon, Manish Kumar. PTEN Has Tumor-Promoting Properties in the Setting of Gain-of-Function p53 Mutations, 2008Cancer Res;68: (6) (2008).) Flow cytometery. The experiment was performed using caco-2 colon human cancer cell line. Cells were seeded in 6 well plates at the concentration of 2×105cells/ml/well. Plates were incubated in CO2incubator for overnight. After overnight incubation test sample(s) were added at desired concentration, sparing wells for negative and positive control and incubated for 48 hrs. After 48 hr incubation, cell were trypsinized and separately transferred into 15 ml centrifuge tubes. Tubes were centrifuged at 3000 rpm for 5 min. The supernatant was discarded and pellet was resuspended in 1 ml filtered PBS and centrifuged at 2000 rpm for 5 min. After 5 mins supernatant was discarded and pellet was resuspended in 400 ml of 1× binding buffer to make cell suspension. From this suspension, 100 μl of cells is transferred in falcon tube and then 10 μl of propidium iodide (PI) and 5 μl Annexin-V antibody were added and incubated for 30 min in dark. After 30 min incubation in dark, apoptosis were analysed by flow cytometer (BD Biosciences).

Results: In the present study, the percentage of compound E treated late apoptotic cells with 1, 5, 7 and 9 μM for 48 h was found to be 36.3%, 34.8%, 38.5% and 56.8% respectively. Under similar conditions, Liphagal treated cultures showed 42.7% cells late apoptotic phase and reverse was found in early apoptotic phase with cell population decreasing 21.9%/o, 23.4%, 21.4% and 14.7% in early apoptotic phase. Further, there were not so much population of cell in necrotic phase indicating that compound E, treatments does not produce any early apoptosis and necrosis. Overall, there was a concentration dependent net increase in late apoptotic cell population (FIG. 7).

For immunofluorescence microscopic analysis, 4×104 CACO-2 cells/ml were seeded on 18-mm coverslips in 6-well plates, one day before experiment. Cells were serum starved overnight and treated with liphagal and compound E, 4 and 3 μM respectively for 24 hr. Following treatment, cells were washed in PBS, followed by fixation in absolute methanol at −20° C. for 5 min19. The fixed cells were blocked with 10% goat serum in PBS for 20 min at room temperature to eliminate non-specific binding of secondary antibody. Cells were incubated with polyclonal rabbit pAKT (serine 473) primary antibody (1:100 in 0.5% BSA in PBS; Santa Cruz Biotechnology) for 1 h at 25° C. in moist chamber, then washed and incubated with secondary antibody. The cells were washed and incubated for 45 min with a Texas red-conjugated goat antirabbit antibody (1:500 in 0.5% BSA in PBS; Santa Cruz Biotechnology) at 25° C. The coverslips were mounted on glass slides with 4′,6-diamidino-2-phenylindole-containing ProLong Gold Antifade mounting medium (Invitrogen) and visualized by fluorescence microscope (Olympus, IX81) under an Olympus 60× oil immersion objective lens. The negative controls were also used in which incubation of cells with primary antibody was omitted.

Results: Phosphorylation (activation) of Akt is associated with protection of cells from apoptosis20(K. Nicholson, N. Anderson. 2002. The protein kinase B/Akt signaling pathway in human malignancy.Cell signal14: 381-395). In the present studies it was observed that treatment of CACO-2 cells with liphagal and compound E, 4 and 3 M respectively for 24 hr caused the inhibition of pAkt (Ser 473). The inhibition of Akt consequently leads to apoptosis. The untreated cells showed the pAKT in the cytoplasm (FIG. 8).

ADVANTAGES OF THE PRESENT INVENTION

Advantages of introducing boronic acid functionality: Recent report on synthetic analog of liphagal (Alban R. Pereira, Wendy K. Strangman, Synthesis of phosphatidylinositol 3-kinase (PI3K) inhibitory analogues of the sponge meroterpenoid Liphagal; J. Med. Chem., 2010, 53 (24), pp 8523-8533) with an IC50 of 66 nM and selectivity towards PI3K-α, suggests that this analog possess greater chemical structure stability and gives opportunity for developing this skeleton into lead preclinical candidate. As a part of our ongoing program on developing isoform selective PI3K inhibitors, it occurred to us that it would be interesting to embark a program on the preparation of compounds based on this modified structure, leveraging the evidence of biological activity exhibited by this molecule. In this direction, we initiated our efforts, and planed to replace aldehyde functionality with boronic acid. Further, the 14-formyl-15,16-dihydroxy substitution pattern in the aromatic ring of liphagal is required to achieve nanomolar potency. It is also demonstrated that the absence of the C-14 formyl group appears to destabilize the liphagane heterocyclic ring system, making it more susceptible to air oxidation and skeletal rearrangements involving ring B contraction. This evidence suggests that the C-8 desmethyl analog with contracted B ring to six-membered, must be ultimately responsible for the activity, which supports our envision. Therefore, instead of formyl at the C-14, we designed a contracted B ring analog without formyl functionality having boronic acid in this place, assuming that this analog would offer more rigidity to the structure. Also, using a boronic acid instead of an aldehyde could circumvent the associated drawbacks. Moreover, boron has ability to biomimic carbon and forms the covalent adducts with the serine or histidine residues of the active site ((a) Adams, J. A.; Behnke, M.; Chen, S.; Cruichshank, A. A.; Dick, L. R.; Grenier, L.; Klunder, J. M.; Ma, Y. T.; Plamondon, L.; Stein, R. L. Bioorg. Med. Chem. Lett. 1998, 98, 333. (b) Paramore, A.; Frantz, S. Nat. Rev. Drug Discovery 2003, 2, 611).

Keeping in view the role of boron, the importance of boronic acid bearing compounds of liphagal are visualized as potential PI3K inhibitor. The evidence from the computational in silico docking of this boronic acid bearing liphagal compounds PI3K showed excellent H-bonding interactions with key amino acids, which are also previously reported as a key amino acid to be involved in inhibitory interactions in the p110α active site of PI3K-α with improved docking score of −8.08 over 1 and 2.12 The biological potential of boronic acid as PI3K inhibitor was also examined, which has shown PI3K-α isoform selectivity and excellent inhibitory activity (IC5023 nM) for one of the compound i.e. compound-AZ.