Claims:We Claim,
1. A novel cancer cell specific prooxidant drug compound, which differentiates the mode of cellular biochemistry between normal and cancer cells and exhibits renowned anticancer activity against hepatocellular carcinoma by sparing normal cell, the claimed drug compound comprises of (2E,4E)-1-hydroxy-2,2,6,6-tetramethyl piperidin-4-yl 5-(benzo[d][1,3]dioxol-5-yl)penta -2,4-dienoate) (HTMPP) of molecular formula 1

Formula 1
2. A pharmaceutical composition having novel cancer cell specific prooxidant drug, which differentiates the mode of cellular biochemistry between normal and cancer cells and exhibiting renowned anticancer activity against hepatocellular carcinoma by sparing normal cell, comprises of therapeutically effective amount of (2E,4E)-1-hydroxy-2,2,6,6-tetramethyl piperidin-4-yl 5-(benzo[d][1,3]dioxol-5-yl)penta -2,4-dienoate) (HTMPP) of molecular formula 1

Formula 1
and pharmaceutically acceptable carrier, diluents or excipient.

3. The composition as claimed in claim 2 is formulated into at least one member of the group comprising of tablet, a pill, a capsule, oral syrup, a caplet, a troche, a pouch, or sprinkles.
4. A method of preparing novel cancer cell specific prooxidant drug compound HTMPP, which differentiates the mode of cellular biochemistry between normal and cancer cells and exhibits renowned anticancer activity against hepatocellular carcinoma by sparing normal cell, the claimed preparation process comprises of covalently conjugating piperine and 4- hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) through esterification reaction.
5. A method of preparing novel cancer cell specific prooxidant drug compound HTMPP from piperine, which drug compound differentiates the mode of cellular biochemistry between normal and cancer cells and exhibits renowned anticancer activity against hepatocellular carcinoma by sparing normal cell, the claimed preparation process comprises of following steps:
a. alkaline hydrolysis of piperine to form piperic acid,

b. esterification of equimolar amount of piperic acid and TEMPOL at 0oC in dichloromethane and stirring under Nitrogen, DCC and DMAP medium for predetermined time at room temperature to form a solid material,
c. filtering the obtained solid material and washing the filtrate with 1MHCl, NaHCO3 and brime to form an organic phase and aqueous phase,
d. drying the obtained organic phase over MgSO4 and evaporated in vacuum to obtain a piperine –TEMPOL adduct

e. dissolving the obtained Piperine –TEMPOL adduct in ethanol saturated with HCl gas , and adding ascorbic acid followed by refluxing for predetermined time for the evaporation of the solvent to form HTMPP of formula 1

Formula 1
6. A novel cancer cell specific prooxidant drug compound, which differentiates the mode of cellular biochemistry between normal and cancer cells and exhibits renowned anticancer activity against hepatocellular carcinoma by sparing normal cell, the claimed drug compound comprises of (2E,4E)-1-hydroxy-2,2,6,6-tetramethyl piperidin-4-yl 5-(benzo[d][1,3]dioxol-5-yl)penta -2,4-dienoate) (HTMPP) of molecular formula 1 prepared according to the process as claimed in claim 4&5

Formula 1

Dated this 21st day of March 2016 For UNIVERSITY OF MADRAS
By its Patent Agent

Dr.B.Deepa
, Description:Form 2

THE PATENT ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)

“HTMPP ((1-HYDROXY-2,2,6,6- TETRAMETHYL PIPERIDIN-4-YL) PIPERATE} – A NOVEL DRUG AGAINST HEPATOCELLULAR CARCINOMA AND METHOD OF PREPARATION THEREOF”

in the name of UNIVERSITY OF MADRAS an Indian National having address at UNIVERSITY OF MADRAS, GUINDY CAMPUS, CHENNAI- 600 025, Tamil Nadu, India.

The following specification particularly describes the invention and the manner in which it is to be performed
FIELD OF THE INVENTION:
The present invention relates to the field of medicine, and in particular to oncology. More specifically the present invention relates to a drug for treating cancer, in particular hepatocellular carcinoma.
BACKGROUND OF THE INVENTION AND PRIOR ART:
Despite being the fifth most leading cause of cancer , Hepatocellular carcinoma is the third most cause of internal malignancy deaths , every year worldwide . Although HCC has historically been more common in the developing world, its incidence in developed countries has almost doubled in the last two decades, largely as a result of liver cirrhosis .The diversity of the etiologic factors implicated in the development of HCC , such as disease with underlying cirrhosis , viral hepatitis, alcoholism and metabolic diseases such as diabetes and hemachromatosis makes the disease critical to be resolved. Unfortunately, most HCC patients are not eligible for curative therapies such as surgical resection or liver transplantation due to the late stage diagnosis and heterogenesity of the tumour . Only 15% of patients are eligible for surgical management involving hepatic resection or transplantation . Five-year survival rates of > 70% can be achieved in these patients, but recurrences are inevitable . Many patients who are not candidates for surgical intervention but who have early-stage disease gain benefit from loco-regional therapies such as radiofrequency ablation or transarterial chemoembolization ( TACE ) ; however , their efficacy is limited by recurrence as well .
There are reports available in the literature about the existence of various Hepatocellular carcinoma treating drugs.
US 20010018445 A1 mainly discloses a pharmaceutical composition for use in the treatment of hepatocellular carcinoma, which comprises thalidomide and a pharmaceutically acceptable carrier.
EP 2508207 A1 relates to therapeutic approaches for treating cancer, in particular hepatocellular carcinoma, with nanoparticles loaded with a chemotherapeutic antitumoral agent. In particular, it relates to the treatment of cancer by administration of said nanoparticles by intravenous infusion for at least 2 hours in order to prevent toxicological side effects and increase the benefit/risk ratio of the treatment.
US 8821880 relates to methods of diagnosing, and methods of treating, hepatocellular carcinoma in a subject. The invention also relates to polypeptide antagonists of PLVAP proteins, including humanized and chimeric antibodies that specifically bind PLVAP proteins.
US 8,853,273 relates to provision of a pharmaceutical agent useful for the prevention and treatment of hepatocellular carcinoma, and the pharmaceutical agent for the prevention and/or treatment of hepatocellular carcinoma contains an acyclic retinoid, a salt thereof, or a solvate of any of these, in combination with a branched-chain amino acid, a salt thereof, or a solvate of any of these.
From the discussed prior art it is inferred about the existence various pharmaceutical agents for the treatment of hepatocellular carcinoma. However such drugs suffers various drawbacks which includes noxious side effects on normal cells because of non specific ROS mediated injury.
Further the magnitude of benefit gained from the systemic chemotherapy till now , including, sorafenib is limited to manage this neoplasm in advanced cases . Multiple etiological factors, complex molecular pathogenesis with genomic instability needs a new paradoxical approach along with the molecular targeted therapy against hepatocellular carcinoma.
OBJECT OF THE INVENTION.
The main object of the present invention relates to a novel cancer cell specific prooxidant drug compound comprises of (2E,4E)-1-hydroxy-2,2,6,6-tetramethyl piperidin-4-yl 5-(benzo[d][1,3]dioxol-5-yl)penta -2,4-dienoate) (HTMPP)
Another object of the present invention is to develop a process for preparing novel cancer cell specific prooxidant drug HTMPP compound comprises of covalently conjugating piperine and 4- hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) through esterification process
Yet another object of the present invention relates to a novel cancer cell specific prooxidant drug HTMPP which could differentiates the mode of cellular biochemistry between normal and cancer cells
Yet another object of the present invention relates to a novel cancer cell specific prooxidant drug HTMPP which exhibits renowned anticancer activity against hepatocellular carcinoma by sparing normal cell.
Yet another object of the present invention is to formulate a pharmaceutical formulation comprising of HTMPP along with pharmaceutically acceptable carrier, diluents or excipient for treating hepatocellular carcinoma
Further object of the present invention is to administer the formulated composition for treating subjects suffering from hepatocellular carcinoma.
SUMMARY OF THE INVENTION.
The present invention discloses a novel cancer cell specific prooxidant drug compound for treating hepatocellular carcinoma and method of preparation thereof. The novel drug compound of the present invention comprises of (2E,4E)-1-hydroxy-2,2,6,6-tetramethyl piperidin-4-yl 5-(benzo[d][1,3]dioxol-5-yl)penta -2,4-dienoate) (HTMPP) of molecular formula 1. The process of preparing novel cancer cell specific prooxidant drug compound HTMPP comprises of covalently conjugating piperine and 4- hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) through esterification process.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 depicts the synthetic scheme of HTMPP
Figure 2 depicts the NMR characterization of HTMPP
Figure 3 depicts the Molecular weight determination of HTMPP by GC-MS
Figure 4. depicts the chemical structure of HTMPP
Figure 5 depicts the antiproliferative property of HTMPP on Hep G2 cell line . Hep G2 cells were cultured in the presence or absence of the indicated concentrations of HTMPP for the indicated periods of time. Mitochondrial succinate dehydrogenase activity of Hep G2 cells was assessed by MTT assay as a measure of cell growth. IC50 value was calculated for both 24 and 48 h of HTMPP treatment. Data represent the mean of 3 independent experiments.
Fig 6 depicts the JC-1 staining of Mitochondrial transmembrane potential changes of HTMPP- treated HepG2 cells at the concentrations of 25 µM and 10 µM for 24 and 48 h of treatment . Images shown are representative of three independent experiments.
Figure 7 depicts the Hep G2 cells were treated with HTMPP for 24 and 48 h (A): Immunostaining of annexin V-FITC (green) in control and treated cells. (B): control and treated cells were immunostained with annexin V-FITC, co- stained with PI and analyzed by flow cytometry. Apoptotic cells were localized in the lower right (early apoptosis) and upper right (late apoptosis) quadrants of the dot-plot graph using annexin V vs PI
Figure 8 depicts the DCF-DA analysis of HTMPP (25 µM) induced cancer cell specific ROS generation after 24 h of treatment . A: Representative fluorescent images of ROS production in control and HTMPP treated chang and Hep G2 cells.
DETAILED DESCRIPTION OF THE INVENTION:
The present invention discloses a novel cancer cell specific prooxidant drug compound for treating hepatocellular carcinoma and method of preparation thereof.
Prooxidant mediated therapeutics were always underappreciated due to their noxious side effects on normal cells because of non specific ROS mediated injury . Since liver is the central organ of xenobiotic clearance , it is susceptable to drug induced hepatic injury . In this study, a drug called HTMPP was synthesized by covalently conjugating piperine and 4- hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) through esterification process to treat hepatocellular carcinoma without injuring normal liver cells based on the differences in their cellular biochemistry . HTMPP showed renowned anticancer activity against hepatocellular carcinoma by sparing normal cell in vitro.
Oxidative therapy against cancer cells by targeting redox signature has gained momentum in recent years. Cancer cells conceive increased ROS generation than their normal counterparts to promote cell prolliferation . Moderate static ROS accumulation may maintain malignancy , but in excess amounts causes DNA damage - mediated apoptosis in cancer cells. Many exogenous pro-oxidants like as Motexafin gadolinium , ß-Lapachone (ARQ 501) , Buthionine sulphoximine, Imexon , Phenylethyl isothiocyanate, 2-methoxyestradiol and arsenic trioxide showed promising anticancer activity in preclinical models by promoting ROS accumulation in multitude of cancer cells , however they failed to be a better alternatives due to their off targeted side effects . Hence a prooxidant drug which shows differential cytotoxicity, specifically to cancer cells, by sparing normal cells, can meet the urgent needs of cancer chemotherapeutics .
Hepatocellular carcinoma in advanced stages needs multimodal therapy, including chemotherapy . Unfortunately , most drugs that are investigeted are not only toxic to cancer cells but also to normal liver cells leading to disease recurrence after treatment regimens. Hence, according to hepatocellular carcinoma , a cancer with compromised liver function needs a safe and targeted molecular therapy .
Piperine is an amide alkaloid present in black and long pepper. Piperine has anti- inflammatory, antianalgesic, antipyretic and bioavailablity enhancing property. TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl) is a piperidine nitroxide, serves as a redox sensitive MRI contrast agent and EPR spin trap . Under reducing conditions, such as in cancer cells , the nitroxide (antioxidant) moiety of TEMPOL is reduced intohydroxylamine (non antioxidant) whereas in normal cells nitroxide is maintained in its stable form due to redox homeostasis. Based on this , it was hypothesised that TEMPOL , by acting as an antioxidant , may protect the normal liver cells from piperine- induced ROS- mediated injury by specifically revealing the cancer cells to piperine induced ROS mediated apoptosis.
Figure 1 illustrates the synthesis of HTMPP Piperine(1) is alkali-hydrolysed to give piperic acid (2). Carboxyl group of piperic acid is coupled with hydroxyl group of TEMPOL through esterification process using Dicyclohexylcarbodiamide (DCC) coupling reaction . Briefly, Equimolar amount of TEM`POL (1mmole) and piperic acid (1mmole) were stirred at 0ºc in Dichloromethane. Under nitrogen, DCC (1 mmole ), DMAP (0.25 mmole) were added and stirred for 12 h at room temperature. The solid material formed was filtered and filtrate was washed with 1M HCl (1 ml) and NaHCO3 (2ml) and brime (2ml). The organic phase was dried over MgSo4 and evaporated in vacuum to give a piperine –TEMPOL adduct (3) , ( Bright orange solid. (Yield – 92%) ). Further HTMPP (4) was synthesised by dissolving Piperine –TEMPOL adduct (3mmole) in ethanol (10 ml, saturated with HCl gas previously) , to which 4 mmole of ascorbic acid in 1ml of water was added and refluxed for 30 minutes. Then, the solvent was evaporated off and the procedure was repeated till the loss of EPR sensitive hyperfine splits.
Figure 2 illustrates the NMR characterization of HTMPP Yield:93% . 1,H NMR (300 MHz, CDCl3): d 7.36 (s, 1H), 6.92-6.72 (m, 6H), 5.9 (s, 2H), 3.95-3.6 (m, 1H), 1.91-0.8 (m, 18H); 13C NMR (75 MHz, CDCl3): d164.8, 147.7, 147.5, 144.4, 143.1, 139.7, 129.6, 123.4, 122.1, 107.6, 104.9, 100.7. Form this data it is inferred about the formation of ester bond between TEMPOL and piperine.

Figure 3 illustrates the molecular weight determination by GC-MS analysis. Gas chromatography (GC) analysis was carried out using Agilent 6890N gas chromatography equipped with photon multiplier tube as detector coupled to front injector type 1079. The chromatograph was fitted with HP 5 MS capillary column (30 m ×0.25 mm i.d.,). The injector temperature was set at 250°C, and the oven temperature was initially at 70 °C hold for 4 mins then programmed to 200°C at the rate of 10°C/min and finally held at 200 °C for 13 min. Helium was used as a carrier gas with the flow rate of 1.5 ml/min. 0.2 microlitre of the sample (diluted with acetone 1:10) was injected in the splitless mode. The percentage of composition of the CPE was calculated by the GC peak areas. GC–mass spectrometry (GC– MS) analysis of sample was performed using Agilent gas chromatography equipped with JEOL GC MATE-II HR Mass Spectrometer. GC conditions were the same as reported for GC analysis and the same column was used. The mass spectrometer was operated in the electron impact mode at 70 eV. Ion source and transfer line temperature was kept at 250°C. The mass spectra were obtained by centroid scan of the mass range from 50 to 600 amu. The compounds were identified based on the comparison of their retention indices (RI), retention time (RT), mass spectra of WILEY, NIST library data of the GC-MS system and literature data (Adams, 2009). [M] + = 373.3.

From the spectroscopic evidences it is ascertained the structure of HTMPP as depicted in Figure 4.

FIG 5 illustrates the Cytotoxicity analysis using MTT assay. Cytotoxicity of HTMPP on Hep G2 (hepatoma) and Chang (normal) cells were studied using MTT assay by treating cells with different concentrations of HTMPP (5 -100µM) for 24 -48 h . HTMPP did not show any toxicity to normal liver cells till 250 µM after 24 h of incubation . In contrast , HTMPP treated Hep G2 cells showed loss of viability with an IC 50 value of 25 and 10 µM after 24 and 48 h ,respectively, These results showed the cancer cell-specific cytotoxicity of HTMPP in Hep G 2 cells.

Figure 6 illustrates the Cancer cell - specific ROS generation by HTMPP. To determine whether HTMPP induced differential cytotoxicity in Hep G2 cells is mediated by ROS , both Hep G2 and Chang liver cells were treated with HTMPP for 24 h and intracellular concentration of ROS generated were observed by DCF fluorescence . Fluorescent microscopic analysis revealed that HTMPP induced significant ROS accumulation specifically in Hep G2 cells compared with the normal cells.

Figure 7 illustrates the mitochondrial membrane potential. Reactive oxygen species are main activators of mitochondrial membrane permeablisation in executing cancer cell apoptosis. JC-1 stained HTMPP treated Hep G2 cells showed overall significant increase in the red to green fluorescent intensity as a determinant of increase in mitochondrial permeabilisation .

Figure 8 illustrates the HTMPP induced apoptosis in Hep G2 Cells. Recently , many redox mediated cancer therapeutics have been appreciated for their ROS- mediated apoptosis inducing property in many cancer cell lines In this sequel , Annexin V/ PI stained HTMPP treated Hep G2 cells also showed dose and time dependent ROS mediated apoptosis in Hep G2 cells.

The chemicals used in the present invention were Piperine, Superoxide dismutase (SOD), 6-carboxy-2',7' dichlorodihydrofluorescein diacetate, diacetoxymethyl ester (DCF-DA), 3-(4,5- dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT), and 5-Diethoxyphosphoryl-5-methyl-1-pyrroline-N- oxide (DEPMPO) was obtained from sigma . Cell culture medium (RPMI 1640), fetal bovine serum (FBS), antibiotics, sodium pyruvate, trypsin, and phosphate-buffered saline (PBS) were purchased from Gibco (Grand Island, NY, USA).

The present study used Chang liver cells (normal liver hepatocytes) and Hep G2 cells (human hepatocellular carcinoma cells ) . Cells were grown in DMEM medium supplemented with 10% FBS, 2% sodium pyruvate, 1% penicillin, and 1% streptomycin. Cells were grown in a 25-mm flask to 70% confluence at 37°C in 5% CO2 .

Cell viability was determined by MTT assay. In the mitochondria of living cells, yellow MTT undergoes a reductive conversion to formazan , giving a purple color. Both Hep G2 and Chang liver cells , grown to ~80% confluence in 25-mm flasks, were trypsinized, counted, seeded in 96- well plates with an average population of 7000 cells/well, incubated overnight, and then treated with different concentrations of HTMPP (5-100 µM ) for 24 and 48 h . Whereas , particularly Chang liver cells were only treated with different concentrations of piperine (5-100 µM ) for 24 h .All experiments were done using three replicates and IC 50 values were determined.

The ROS levels in Chang and Hep G2 cells treated with HTMPP were determined using DCF-DA, a membrane-permeative fluorogenic probe. The acetate and acetoxymethyl ester groups of this probe are enzymatically cleaved inside living cells. The probe can then be oxidized by intracellular oxidants (ROS) to give a product, DCF, which emits a strong, green fluorescence (?ex=504 nm; ?em=529 nm). The fluorescence intensity is proportional to the level of cellular oxidants. Cells, grown to 80% confluence on 6-mm glass coverslips, were treated with 25 µM of HTMPP for 24 h, followed by incubation with DCF-DA. The cells were further incubated in the dark for 20 min and washed with protein-free medium, and then fluorescence images were immediately captured with a Nikon Eclipse TE2000-U camera system using excitation/emission at 495/520 nm. The captured images were then analyzed using Meta Morph image analysis software.

Mitochondrial membrane permeablisation (MMP) was determined using JC-1 probe as described by manufacturer’s protocol (Sigma Aldrich) . JC-1 is widely used to monitor mitochondrial membrane depolarization. In healthy cells with high mitochondrial membrane potential, JC-1 spontaneously forms complexes known as J-aggregates with intense red fluorescence. Whereas, in apoptotic or unhealthy cells with low mitochondrial membrane potential, JC-1 remains in the monomeric form, which shows only green fluorescence. Thus , mitochondrial depolarization is indicated by a decrease in red/green fluorescence intensity ratio. Briefly , after treating the Hep G2 cells with 25 and 10 µM of HTMPP for 24 and 48 h , cells were stained with 10 µM of JC-1 for 25 min at 37°C. After washing, cells were analysed with Carl Zeiss fluorescence microscope.
Hep G2 cells were treated with 25 and 10 µM of HTMPP for 24 and 48 h and immunostained with annexin V-FITC/PI, analyzed by flow cytometry (a FACS Calibur flow cytometer) according to manufacturer's protocol (Sigma Aldrich). Only green fluorescein- positive cells without PI staining were regarded as apoptotic cells.

Thus the inventiveness of the present invention lies in the following aspects.
? HTMPP is a targeted anticancer drug against hepatocellular carcinoma
? HTMPP specificity is based on the differences in the cellular uptake and reducing environment between normal and cancer cells
? HTMPP potentially induced apoptosis in Hep G2 cells while protectiing normal Chang liver cells
? Anticancer activity of HTMPP is based on the reactive oxygen species mediated mitochondrial membrane permeablisation.

In one of the preferred embodiment the present invention shall disclose a novel cancer cell specific prooxidant drug compound, which differentiates the mode of cellular biochemistry between normal and cancer cells and exhibits renowned anticancer activity against hepatocellular carcinoma by sparing normal cell. The novel cancer cell specific prooxidant drug compound of the present invention comprises of (2E,4E)-1-hydroxy-2,2,6,6-tetramethyl piperidin-4-yl 5-(benzo[d][1,3]dioxol-5-yl)penta -2,4-dienoate) (HTMPP) of molecular formula 1

Formula 1
In another preferred embodiment the present invention shall disclose a pharmaceutical composition having novel cancer cell specific prooxidant drug, which differentiates the mode of cellular biochemistry between normal and cancer cells and exhibits renowned anticancer activity against hepatocellular carcinoma by sparing normal cell. The composition of the present invention comprises of therapeutically effective amount of (2E,4E)-1-hydroxy-2,2,6,6-tetramethyl piperidin-4-yl 5-(benzo[d][1,3]dioxol-5-yl)penta -2,4-dienoate) (HTMPP) of molecular formula 1

Formula 1
and pharmaceutically acceptable carrier, diluents or excipient.
As per the invention the composition can be formulated into at least one member of the group comprising of tablet, a pill, a capsule, oral syrup, a caplet, a troche, a pouch, or sprinkles.
In yet another preferred embodiment the present invention shall disclose a method of preparing novel cancer cell specific prooxidant drug compound HTMPP, which differentiates the mode of cellular biochemistry between normal and cancer cells and exhibits renowned anticancer activity against hepatocellular carcinoma by sparing normal cell. The preparation process comprises of covalently conjugating piperine and 4- hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) through esterification reaction.
In yet another preferred embodiment the present invention shall disclose a method of preparing novel cancer cell specific prooxidant drug compound HTMPP from piperine, which drug compound differentiates the mode of cellular biochemistry between normal and cancer cells and exhibits renowned anticancer activity against hepatocellular carcinoma by sparing normal cell. The preparation process comprises of following steps:
a. alkaline hydrolysis of piperine to form piperic acid,

b. esterification of equimolar amount of piperic acid and TEMPOL at 0oC in dichloromethane and stirring under Nitrogen, DCC and DMAP medium for predetermined time at room temperature to form a solid material,
c. filtering the obtained solid material and washing the filtrate with 1MHCl, NaHCO3 and brime to form an organic phase and aqueous phase,
d. drying the obtained organic phase over MgSO4 and evaporated in vacuum to obtain a piperine –TEMPOL adduct

e. dissolving the obtained Piperine –TEMPOL adduct in ethanol saturated with HCl gas , and adding ascorbic acid followed by refluxing for predetermined time for the evaporation of the solvent to form HTMPP of formula 1

Formula 1
In yet another preferred embodiment the present invention discloses a novel cancer cell specific prooxidant drug compound comprises of (2E,4E)-1-hydroxy-2,2,6,6-tetramethyl piperidin-4-yl 5-(benzo[d][1,3]dioxol-5-yl)penta -2,4-dienoate) (HTMPP) of molecular formula 1 prepared according to the procedures discussed above.

Formula 1

Certain modifications and improvements will occur to those skilled in the art upon a reading of the foregoing description. The above-mentioned details are provided to serve the purpose of clarifying aspects of the invention and it will be apparent to one skilled in the art that they do not serve to limit the scope of the invention. All modifications and improvements have been deleted herein for the sake of conciseness and readability but are properly within the scope of the present invention. It is understood that the foregoing detailed description is given merely by way of illustration and that modification and variations may be made therein without departing from the spirit and scope of the invention.

We Claim,
1. A novel cancer cell specific prooxidant drug compound, which differentiates the mode of cellular biochemistry between normal and cancer cells and exhibits renowned anticancer activity against hepatocellular carcinoma by sparing normal cell, the claimed drug compound comprises of (2E,4E)-1-hydroxy-2,2,6,6-tetramethyl piperidin-4-yl 5-(benzo[d][1,3]dioxol-5-yl)penta -2,4-dienoate) (HTMPP) of molecular formula 1

Formula 1
2. A pharmaceutical composition having novel cancer cell specific prooxidant drug, which differentiates the mode of cellular biochemistry between normal and cancer cells and exhibiting renowned anticancer activity against hepatocellular carcinoma by sparing normal cell, comprises of therapeutically effective amount of (2E,4E)-1-hydroxy-2,2,6,6-tetramethyl piperidin-4-yl 5-(benzo[d][1,3]dioxol-5-yl)penta -2,4-dienoate) (HTMPP) of molecular formula 1

Formula 1
and pharmaceutically acceptable carrier, diluents or excipient.

3. The composition as claimed in claim 2 is formulated into at least one member of the group comprising of tablet, a pill, a capsule, oral syrup, a caplet, a troche, a pouch, or sprinkles.
4. A method of preparing novel cancer cell specific prooxidant drug compound HTMPP, which differentiates the mode of cellular biochemistry between normal and cancer cells and exhibits renowned anticancer activity against hepatocellular carcinoma by sparing normal cell, the claimed preparation process comprises of covalently conjugating piperine and 4- hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) through esterification reaction.
5. A method of preparing novel cancer cell specific prooxidant drug compound HTMPP from piperine, which drug compound differentiates the mode of cellular biochemistry between normal and cancer cells and exhibits renowned anticancer activity against hepatocellular carcinoma by sparing normal cell, the claimed preparation process comprises of following steps:
a. alkaline hydrolysis of piperine to form piperic acid,

b. esterification of equimolar amount of piperic acid and TEMPOL at 0oC in dichloromethane and stirring under Nitrogen, DCC and DMAP medium for predetermined time at room temperature to form a solid material,
c. filtering the obtained solid material and washing the filtrate with 1MHCl, NaHCO3 and brime to form an organic phase and aqueous phase,
d. drying the obtained organic phase over MgSO4 and evaporated in vacuum to obtain a piperine –TEMPOL adduct

e. dissolving the obtained Piperine –TEMPOL adduct in ethanol saturated with HCl gas , and adding ascorbic acid followed by refluxing for predetermined time for the evaporation of the solvent to form HTMPP of formula 1

Formula 1
6. A novel cancer cell specific prooxidant drug compound, which differentiates the mode of cellular biochemistry between normal and cancer cells and exhibits renowned anticancer activity against hepatocellular carcinoma by sparing normal cell, the claimed drug compound comprises of (2E,4E)-1-hydroxy-2,2,6,6-tetramethyl piperidin-4-yl 5-(benzo[d][1,3]dioxol-5-yl)penta -2,4-dienoate) (HTMPP) of molecular formula 1 prepared according to the process as claimed in claim 4&5

Formula 1

Dated this 21st day of March 2016 For UNIVERSITY OF MADRAS
By its Patent Agent

Dr.B.Deepa