Title of Invention	NOVEL ANTICANCER HYDRAZINO DERIVATIVE OF CURCUMIN AND PROCESS FOR PREPARATION THEREOF
Abstract	The present invention describes novel hydrazino derivatives of curcumin with enhanced stability and process for preparation thereof. The said process comprises reacting curcumin or curcumin analogues with azo compounds in presence of a base to yield the final compound of formula 5 and formula 5". The compounds of the present invention function as highly potent chemotherapeutic agents.

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FORM2 THE PATENTS ACT, 1970 (39 of 1970) & The Patents Rules, 2003 COMPLETE SPECIFICATION (See section 10; rule 13) 1. Title of the invention; NOVEL ANTICANCER HYDRAZINO DEREVATIVES OF CURCUMIN AND PROCESS FOR PREPARATION THEREOF 2. Applicant(s) (a) NAME : (b) NATIONALITY (c) ADDRESS: INDIAN INSTITUTE OF TECHNOLOGY Created by an act of Parliament, Institute of Technologies Act, 1961 Indian Institute of Technology, Bombay, India 3. PREAMBLE TO THE DESCRIPTION The following specification particularly describes the invention and the manner in which it is to be performed: FIELD OF INVENTION The present invention relates to synthesis of novel hydrazino derivatives of curcumin, its analogs and the biological activity thereof. More particularly, the present invention relates to the synthesis of novel hydrazino derivatives of curcumin of formula 5 and 5'. R5 R6 O O R6 R5 R5 R6 O OH R6 R5 Wherein R1 is H or OMe, R2 is H, OH, OMe, N02, PhCH20, R3isH, OMe,N02 R4isH, OMeorN02, R5 is H, OH, OMe, NO2 CH3, R6H R7isH E is C02R, COR, CONR2, CN, CF3, RS02 wherein R is alkyl, aryl, alkaryl BACKGROUND OF THE INVENTION The natural product curcumin with structural formula as given under is well documented in the literature. 2 OMe curcumin OMe Curcumin is the non-nutritive, non-toxic compound in turmeric that has been used for centuries in India and all over the world as a dietary spice and as an herbal medicine for treatment of wounds, jaundice, and rheumatoid arthritis. In addition, curcumin inhibits the proliferation of a variety of tumour cells and has anti-metastatic activity. Curcumin has numerous biological properties including anti¬oxidant, anti-cancer, anti-HIV-I integrase, chemopreventive, anti-inflammatory, and anti-angiogenesis activities. Various groups worldwide have used curcumin as a lead compound to develop numerous analogs with different bioactivities. Zambre A. P. et al ("Novel curcumin analogs targeting TNF-induced NF-KB activation and proliferation in human leukemic KBM-5 cells" Bioorganic & Medicinal Chemistry 14 (2006) 7196 - 7204) describes preparation and characterization of curcumin Knoevenagel condensates and their Schiff bases and corresponding copper conjugates. These compounds were evaluated for their potential of inhibiting TNF-induced NF- KB activation and proliferation in human leukemic KBM-5 cells at equimolar concentrations. However, the concentrations used by Zambre, A. P et al are in higher range. Moreover, the Knoevenagel condensates reported 80% inhibition at a very high concentration of 50 uM. Also the cell lines used to evaluate anti¬proliferative activity are different. 3 0Me Curcumi H2yssVH2 R = 2-hydroxy, 2,3-dihydroxy, 3,4-dihydroxy N. Cu ,N N N Anto R. J. et al ("Anti-tumour and free radical scavenging activity of synthetic curcuminoids" International journal of Pharmaceutics 131 (1996) 1 - 7) mentions the screening of synthetic derivatives of curcumin for cytotoxic and tumoricidal activities. The anti-proliferative action of synthetic curcumins was studied on Ehrlich ascites carcinoma cells and Dalton's lymphoma ascites cells and for tissue culture, L929 Cells. However, the cell lines used to evaluate anti-tumor activity are different. An article by Joong Sup Shim et al ("Hydrazino curcumin, a novel synthetic curcumin derivative, is a potent inhibitor of endothelial cell proliferation" Bioorganic & Medicinal Chemistry Volume 10, Issue 9, September 2002, 2987-2992) describes new synthetic curcumin derivatives with enhanced anti-angiogenic activity. Also the cell lines used for evaluation of antiproliferative activity are different. OMe H N—N OMe WO03105751 titled "Novel curcumin derivatives" relates to novel curcumin derivatives and their pharmaceutical composition for treating diseases associated 4 with unregulated angiogenesis. WO03105751 mentions the use of hydrazino derivatives of curcumin of the below structural formula H N—N R R An article by Ohori H. et al. entitled "Synthesis and biological analysis of new curcumin analogues bearing an enhanced potential for the medicinal treatment of cancer" Molecular Cancer Therapeutics 2006; 5:2563-2571 describes symmetrical 1,5-diarylpentadienone whose aromatic rings possess an alkoxy substitution at each of the positions 3 and 5. The antiproliferative activity of these analogues were screened in a variety of cancer cell lines including lines derived from stomach (GCIY, SHIOTC), lung (LK87), breast (MCF7), ovary (OVK18), prostate (PC3), pancreas (PK9), bile duct (HuCCTl), thyroid gland (8505c), skin (A431), kidney (ACHN), liver cancers (HepG2) and melanoma. This article however does not provide a detailed stability data of the analogues and other synthetic derivatives reported therein. There is no description about substitution at the active methylene position. From a biological point of view the therapeutic potential of any compound lies in its potency as well as its inherent stability of the compound, thereby allowing it to have a longer shelf life. The stability of any given compound depends on its structure. Curcumin, despite emerging as a promising anti cancer agent, is severely restricted by its unstable nature, particularly in the solution form. The need of the hour is to come up with a variety of second generation compounds having enhanced stability without compromising its potency as a chemotherapeutic agent. 5 The, present inventors have surprisingly found that the novel derivatives of curcumin of formula 5 and formula 5' address the above shortcomings of the known prior art curcumin derivatives. The present inventors have also developed a simple and efficient methodology for the synthesis of highly bioactive derivatives of curcumin of formula 5 and formula 5'. OBJECT OF INVENTION It is an object of the present invention to provide novel hydrazino derivatives of curcumin and its analogs. It is another object of the present invention to provide novel hydrazino derivatives of curcumin in high chemical yield. It is yet another object of the present invention to provide a commercially viable one-pot process for the synthesis of hydrazino derivatives of curcumin and its analogs. It is yet another object of the present invention to provide novel curcumin analogues that have greater stability and hence longer shelf life. It is further an object of the present invention to provide novel analogues of curcumin having anticancer properties. SUMMARY OF INVENTION According to one aspect of the present invention there is provided a novel curcumin derivative of the formula 5 and formula 5' represented as 6 Wherein R1 is H or OMe; R2 is H, OH, OMe, N02, PhCH20; R3 is H, OMe, N02; R4 is H, OMe or N02, R5 = H, OH, OMe, N02, CH3, R6 = R7 = H; E is C02R, COR, CONR2, CN, CF3, RS02; R = alkyl, aryl, alkaryl. According to another aspect of the present invention there is provided a one-pot process for the synthesis of novel hydrazino curcumin derivatives of formula 5 and formula 5' that comprises the steps of:, a. Treatment of curcumin or curcumin analogues with azo compound in an organic solvent in presence of base; b. Treatment of the reaction mixture of (a) with cold aqueous acid; c. Solvent extraction of the reaction mixture of (b); d. Concentration of the organic layer of (c) to yield compounds of formula 5 and formula 5'. BRIEF DESCRIPTION OF ACCOMPANYING FIGURES Figure la: Comparison of IC50 values of curcumin in HeLa cells and HCT- 116 cell lines. Figure lb: Comparison of IC50 values of AM-26 in HCT-116 and HeLa cells Figure lc: Comparison of IC50 values of AM-27 in HCT-116 and HeLa cells Figure Id: Comparison of Antiproliferative activities of Curcumin, AM-27 and AM-26 in HeLa and HCT-116. Figure 2: Comparison of % inhibition of CHO cells with Curcumin, AM-26 and AM-27 at same concentrations. Figure 3: , Percent change in the average OD values of Curcumin, AM-26 and AM-27 in 24 hours. 7 DETAILED DESCRIPTION OF THE INVENTION The natural product curcumin and its derivatives possess numerous biological properties including anti-oxidant, anti-cancer, anti-HIV-1 integrase, chemopreventive, anti-inflammatory, and anti-angiogenesis activities. Although curcumin has been known as an anti-cancer agent against a wide range of cancer cells, its inherent instability and poor potency has been the limiting factors from its use as a potent chemotherapeutic agent. The present invention provides a one-pot process for the synthesis of a novel curcumin derivative of the formula 5, which possesses enhanced stability and anticancer properties. The compound of present invention is prepared by treating curcumin or its analogs in a solvent with azo compound in the presence of a base, typpically an amine. The reaction proceeds to completion in an hour. On completion of the reaction, the reaction mixture is treated with cold aqueous acid and extracted with a suitable solvent. The combined organic layer is concentrated to afford substantially pure amino/hydrazino curcumin. The typical curcumin analogs, besides parent curcumin [diferuloylmethane or 1, 7-bis-(4-hydroxy-3-methoxyphenyl)-l, 6-heptadiene-3, 5-Dione], include those with substituents on the aromatic ring or on the linear chain. Further, the substituent could be cyclic or open chain, branched or straight chain, could contain or devoid of hetero atoms. Following are representative examples of curcumins suitable for the present invention: • (\E, 6E)-l,7-bis-(4-hydroxy-3-methoxyphenyl)-l,6-heptadiene-3,5-dione, • (1E, 6E0-l,7-bis-(2,5-dimethoxyphenyl)-l,6-hepta.diene-3,5-dione, • (IE, 6£)-l,7-bis-(4-nitrophenyl)-l,6-heptadiene-3j5-dione, • (1E, 6E)-l,7-bis-(2-nitrophenyl)-I,6-heptadiene-3i5-dione, 8 • (1E, 6E)-1,7-bis-(4-methoxyphenyI)-1,6-heptadiene-3,5-dione, • (\E, 6iT)-l)7-bis-(3,4-dimethoxyphenyl)-l,6-heptadiene-3,5-dione, • (lE",6E)-l,7-bis-(2,4-dimethoxyphenyl)-l,6-heptadiene-3,5-dione, • \\E, 6£)-I, 7-bis-(4-benzyloxy-3-methoxyphenyl)-l, 6-heptadiene-3, 5-Dione. The azo compounds include those with two of the groups such as carboxylic acid (CO2H), esters of carboxylic acid (CO2R, where R = ethyl, isopropyl, benzyloxycarbonyl), COR, CONR2, CN, CF3, RS02 attached to -N=N-. The ratio of curcumin to the azo compound may be 1:0.5 to 0.5:2, but the preferred ratio is 1:2 Typical bases include amines such as secondary or tertiary amines, cyclic or acyclic, containing one or more N atoms. Representative examples are 4-N, N-dimethylaminopyridine (DMAP), its isomers and substituted analogs, imidazole and its substituted analogs, amines in which these moieties are fused to other rings, e.g. a benzene ring, l,4-diazabicyclo[2.2.2]octane (DABCO), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), l,5-diazabicyclo[4.3.0]non-5-ene (DBN). However, DMAP is preferred. The base may vary from 0.1 to 2.0 equivalents, most preferred being 1.2 equivalents. The reaction can be carried out in a range of solvents or mixtures thereof selected from alcohols such as methanol, ethanol, cyclohexanol, open chain and cyclic ether solvents such as diethyl ether, tetrahydrofuran (THF), 1, 4-dioxan, halogenated solvents such as dichloromethane, chloroform, hydrocarbon solvents such as toluene, benzene. Other suitable solvents include acetonitrile, dimethyl formamide, dimethyl sulfoxide, acetone and ethyl acetate. These solvents do not react with either of the reactants, viz. curcumin or azo compound. 9 Although the reaction can be carried out over a wide range of temperatures from -50 to 100 °C depending on the solvent used, the preferred temperature range is -10 to 30 °C. In an embodiment of the invention the reaction is preferably carried out in the dark, since the azo compounds of the present invention are photosensitive in nature. In another embodiment of the invention the reaction is carried out under inert atmosphere. In yet another embodiment the stability of curcumin derivatives of formula 5 is assessed by spectrometric methods based on Lambert-Beer's law. To assess the stability of any compound spectrometric methods are used based on Lambert-Beer's law which states that: The Absorbance of any compound is - Directly proportional to its concentration, and - Distance traveled by the light in the solution, i.e., Aac Aal Hence, Amax = eel, Where, £ = Molar Extinction Coefficient. Based on this law, the concentration of the compound at which its Absorbance value is maximum, is referred to as AmaXjand its corresponding wavelength is X^ax. Ideally, the Amax value obtained at a specific concentration must remain constant. Hence, any variation in Amax of a compound might be regarded as an indicator of any variation in its concentration either due to reaction or due to its inherent instability in the solution. 10 Thus by incubating a compound at fixed concentration, and observing its Absorbance in a time dependent manner for 24 h, we get an idea about the stability of the compound. The invention is now illustrated with a few representative but non-limiting examples. EXAMPLES Example 1 Preparation of Diisopropyl 1-((1E, 6E)-1, 7-bis (4-nitrophenyl)-3, 5- dioxohepta-1, 6-dien-4-yI) hydrazine-!, 2-dicarboxylate (3c) 3c To a solution of curcumin lc (91.5 mg, 0.25 mmol) in CH3CN (2 ml) was added diisopropyl azodicarboxylate 2a (101 mg, 0.5 mmol, 2 equiv) and the reaction mixture was stirred at 0 C for 5 min under N2 and protected from light. DMAP (41 mg, 0.3 mmol, 1.2 equiv) was added very slowly and the reaction mixture was stirred for 15-20 min at room temperature. After the completion of the reaction, 20% of cold aqueous HC1 solution was added and the product was extracted with CH2CI2 (4 x 25 ml) and the combined organic layer was washed with brine (10 ml) and dried over anhydrous sodium sulphate. Then the solvent was evaporated in vacuum and the product was purified by silica gel column chromatography by eluting with (EtOAc /n-hexane 30:70) and further recrystallised from ethanol. Yellow crystalline solid; Yield 119 mg, 84%; Mp 225 °C. 11 Example 2 Preparation of Diisopropyl 1-((1E, 6E)-1, 7-bis (4-methoxyphenyl)-3, 5-dioxohepta-1, 6-dien-4-yI) hydrazine-!, 2-dicar boxy late (3e) CO2P12 3e To a solution of curcumin le (84 mg, 0.25 mmol) in CH3CN (2 ml) was added diisopropyl azodicarboxylate 2a (101 mg, 0.5 mmol, 2 equiv) and the reaction mixture was stirred at 0°C for 5 min under N2 and protected from light. DMAP (41 mg, 0.3 mmol, 1.2 equiv) was added very slowly and the reaction mixture was stirred for 15-20 min at room temperature. After the completion of the reaction., 20% of cold aqueous HCI solution was added and the product was extracted with CH2C12 (4 x 25 ml) and the combined organic layer was washed with brine (10 ml) and dried over anhydrous sodium sulphate. Then the solvent was evaporated in vacuum and the product was purified by silica gel column chromatography by eluting with (EtOAc/n-hexane 30:70) and fiirther recrystallised from ethanol. Yellow crystalline solid; Yield 108 mg, 80%; Mp 223 °C. Example 3 Preparation of Diethyl 1-((1£, 6E)-1, 7-bis (4-methoxyphenyl)-3, 5-dioxohepta-1, 6-dien-4-yl) hydrazine-1, 2-dicarboxylate (4e) O O MeCT^^ Et02C'N~NH ^^\)Me C02Et 4e To a solution of curcumin le (84 mg, 0.25 mmol) in CH3CN (2 ml) was added diethyl azodicarboxylate 2b (87 mg, 0.5 mmol, 2 equivalents) and the reaction mixture was stirred at 0 C for 5 min under N2 and protected from light. DMAP 12 (0.036 g, 0.3 mmol, 1.2 equiv) was added very slowly and the reaction mixture was stirred for 15-20 min at room temperature. After the completion of the reaction, 20% of cold aqueous HC1 solution was added and the product was extracted with CH2CI2 (4 * 25 ml) and the combined organic layer was washed with brine (10 ml) and dried over anhydrous sodium sulphate. Then the solvent was evaporated in vacuum and the product was purified by silica gel column chromatography by eluting with (EtOAc/n-hexane 30:70) and further recrystallised from ethanol. Yellow crystalline solid; Yield 103 mg, 81%; Mp 230 °C. Other examples including those detailed above are listed in the following tables. Table 1 Synthesis of 4-hydrazino derivatives 3 of curcuminoids 1 o o Ri ]^ R4 1 R/ "f "R R3 + R3 PrJ0JC-N=N-C02Pri 2a o o 1 DMAP,CH3CN Ri^ Ti^^T -^Y^^\ H\ ^R rt 1 h 2 R^ R3 ^R- E E NH R4 E = co2Fy 3 R3 ^R Entry Product Ri R2 R3 R4 Yield (%) 1 3a H OH OMe H 1 3b OMe H H OMe 65 2 3c H N02 H H 84 3 3d H H H N02 34 4 3e H OMe H H 80 13 5 3f H OMe OMe H 70 6 3g H OMe H OMe 40 7 3h H PhCH20 OMe H 42 Table 2 Synthesis of 4-hydrazino derivatives 4 of curcuminoids 1 Ri^[fYRl R/ y R4 1 R4 Y^R2 *1h R/ y R4 E NH R4 V% R3 + R3 R3 E R3 EtOsC- -N-N-2b -C02Et E = C02Et 4 Entry Product */ R2 R3 R4 Yield (%) 1 4b OMe H H OMe 75 2 4c H OMe H H 81 3 4f H OMe OMe H 71 Antiproliferative action ofcurcumin and its analosues Sf and 4e on cancer cell lines Experiments conducted 1. Determination of antiproliferative activity and the IC50 values ofcurcumin and its analogues 3f and 4e in the following cell lines: a. HeLa cells, and b. HCT-116 cells. 2. Determining the stabilities of the derivative compounds and comparing them with curcumin by spectrophotometer. 14 3. Confirming that the cellular death induced by these compounds evidenced in the two cell lines is by apoptosis by annexin-V/propidium iodide staining of HeLa cells. 4. Elucidating the role of p53 as a possible mediator in their anti-proliferative actions by immunostaining of HCT-116 cells. RESULTS 1. Determination of antiproliferative activity and IQo values of curcumin and its analogues AM-26 (3f) and AM-27 (4e) in HeLa and HCT-116 cell lines: Experimental Details The assay used in the present invention is referred to as SRB assay. • Cells were seeded in 96 well plates at a concentration of 1 x 105/ml. • Cells were grown for one cell-cycle and curcumin, AM-26 (31) and AM-27 (4e) were added in different concentrations. • After another cell-cycle, medium was removed and cells were fixed with 10% TCAforlhrat4°C. • Washed the cells with tap water. • 100 ul of 0.4% SRB in 1% acetic acid was added and stained for 30 min at room temperature. • Washed the cells in 1% acetic acid. • Plates were air dried and stain was dissolved in 100 ul of 10 mM unbuffered Tris. • Absorbance was measured at 540 nm. RESULTS: The results are provided in Table 3 below and in Figures la-d Table 3 Name of compound IC50 in HeLa cells ICso in HCT-116 cells Curcumin 12.7 ± 0.3 uM 4.3 ±0.1 uM 15 AM-26 (3f) 6.3 ± 0.2 uM 4.75 ± 0.66 uM AM-27 (4e) 11.55 ±0.51 uM 3.875±0.13uM • It was found that in normal CHO (Chinese Hamster Ovary cell lines), the range of anti-proliferative activity of analogues AM-26 (3f) and AM-27 (4e) was nearly equivalent to that of standard anti cancer agent, Curcumin. Thus, the compounds were equally potent in both cancerous as well as normal cell lines The comparison of the efficacy data of formula 5 with that of curcumin in the studies conducted in normal CHO (Chinese Hamster Ovary cell lines) is shown in Table 4 and in Figure 2. Table 4 % Inhibition Compound 5uM 10 uM 20 uM 30 uM Curcumin 20 40 60 98 AM-27 (3f) 18 40 83 99 AM-26 (4e) 36 90 100 100 2. Stability Assay: To determine the relative stability of the analogues 3f and 4e with respect to curcumin. Experimental Details • The compounds were dissolved in DMSO to prepare stock concentrations 10 mM, • Working concentration of 20 uM was prepared in deionized water for all the three compounds. • They were incubated in dark at room temperature for 24 h. • Blank was set with the deionized water. • Spectrophotometer was set for baseline correction under the scanning wavelength in the range of 340 nm-500 nm. • Amax reading was taken at zero hour at the onset of experiment. The corresponding wavelength >.max was noted. 16 • Subsequently, absorbance readings were taken after every one hour till 24 h. The spectra of each compound were obtained. The absorbance value corresponding to the ^max observed at zero time was taken. • A graph is plotted with Amax versus time (h) and the fall in the A^ax values was calculated as a % change in 24 h. RESULTS The results are provided in the form of graph as in Figure 3 and Table 5 Table 5 Avg % Change in24h Std Dev Curcumin 69 10.5 69 ±11% AM-26 (3f) 20 8.8 20 ± 8.8 AM-27 (4e) 16 1.73 16 ±1.7 The stability assay had irrevocably demonstrated that AM-27 (4e) was the most stable of the compounds under in vitro conditions. 1. Annexin- W propidium iodide staining of HeLa cells treated with curcumin and its analogues at IC50 and 2 x IC50 concentrations Experimental details • Cells were seeded ion cover slips in 24 well plate at concentration of 0.5 * 105/ml. • Cells were grown for one cell-cycle and curcumin, AM-26 (3f) and AM-26 (4e) were added in IC50 and 2 x IC50 concentrations. • Cells were washed with 1% PBS buffer followed by another wash with 1 x concentration of the assay buffer provided with the kit. • Recommended concentrations of Annexin-V and propidium iodide were added to the cells, which were then incubated in dark at room temperature for 15 minutes. • The cells were washed again in 1% PBS and were mounted on the slides. They were observed under florescence microscope under appropriate filters. 17 RESULTS a. In curcumin treated cells, there was a clear evidence of on-going apoptosis both in the early as well as late phases. b. Cells treated with AM-26 at both concentrations exclusively featured early phase of apoptosis with a few cells undergoing the late phase. c. AM-27 recapitulated the findings seen in curcumin treated cells, featuring both the early and late apoptotic cells. 1. Elucidating the role of p53 as a possible mediator in their anti-proliferative actions atlCso and 2 x IC50 concentrations by immunostainingof HCT-116 cells Experimental details • Cells were seeded ion cover slips in 24 well plate at concentration of 0.5 * 105 /ml. • Cells were grown for one cell-cycle and curcumins, AM-26 (3f) and AM-27 (4e) were added in IC50 and 2 x IC50 concentrations. • Cells were fixed with 3.75% of formaldehyde. • They were permeabilised with ice cold methanol and were incubated at -40 °C for 10 minutes and washed afterwards with 2% PBS. • The cells were then blocked by adding 2% BSA and incubated at 37°C for half an hour. • Primary antibody for p53 was added at working concentration of 1:300 and incubation was carried out for one and a half-hour in dark at room temperature. • This was followed by two washes with 1% PBS and addition of Alexa-Iabeled secondary antibody at 1:500 concentrations. They were incubated at room temperature in dark for 1 hour. • Another wash, once with 1% PBS and another with deionised water was given. 18 • To stain the nucleus Hoechst dye at concentration of l^g/ml was added to the cover-slips and incubated in dark for 7-8 minutes. • The cover-slips were mounted on the slides and observed under fluorescence microscope with appropriate filters. RESULTS a. In the cells treated with curcumin, p53 was found to be translocated to their respective nuclei at both the concentrations. b. In case of AM-26 (3f) and AM-27 (4e), the cells exhibited similar nuclear translocation in response to both their IC50 and 2 * IC50 concentrations. 19 WE CLAIM 1. Hydrazino derivatives of curcumin of formula 5 and formula 5' as given Wherein R1 is H or OMe; R2 is H, OH, OMe, N02, PhCH20; R3 is H, Ome or N02; R4 is H, OMe or N02; R5 = H, OH, OMe, N02, CH3; R6 = R7 = H; E is C02R, COR, CONR2, CN, CF3j RS02; R = alkyl, aryl, aralkyl 2. Hydrazino derivatives of curcumin as claimed in claim 1 wherein the representative compounds are: i. (l£,6£)-l,7-bis-(4-hydroxy-3-methoxyphenyl)-l,6-heptadiene-3,5- dione; ii. (\E, 6£)-l)7-bis-(2,5-dimethoxyphenyl)-l,6-heptadiene-3,5-dione; ■jii. (\E, 6£)-l,7-bis-(4-nitrophenyl)-l,6-heptadiene-3,5-dione; iv. (\E, 6£)-l,7-bis-(2-nitrophenyl)-l,6-heptadiene-3,5-dione; v. (\E, 6£}-l,7-bis-(4-methoxyphenyl)-l,6-heptadiene-3,5-dione; vi. (\E, 6£)-l,7-bis-(3,4-dimethoxyphenyl)-l,6-heptadiene-3,5-dione; vii. (l£,6£)-l,7-bis-(2,4-dimethoxyphenyl)-l,6-heptadiene-3,5-dione; viii. (1£, 6E)-1, 7-bis-(4-benzyloxy-3-methoxypVieny\)-l, 6-heptadiene-3, 5-Dione. 3. A process for synthesis of hydrazino derivatives of curcumin of formula 5 and formula 5' that comprises the steps of: a. Treatment of curcumin or curcumin analogues with azo compound in an organic solvent in presence of base. b. Treatment of the reaction mixture of (a) with cold aqueous acid. c. Solvent extraction of the reaction mixture of (b). 20 d. Concentration of the organic layer of (c) to yield compounds of formula 5 and formula 5'. 4. The process for synthesis of hydrazino derivatives of curcumin of formula 5 and formula 5' as claimed in claim 3 wherein the azo compound is selected from diisopropyl azodicarboxylate, diethyl azodicarboxylate, diphenyl azodicarboxylate, dibenzyl azodicarboxylate, other azodicarbonyl compounds and azo groups possessing electron withdrawing groups such as CN, CF3 and S02R, 5. The process for synthesis of hydrazino derivatives of curcumin of formula 5 and formula 5' as claimed in claim 4 wherein the most preferred azo compound is diethyl azodicarboxylate or diisopropyl azodicarboxylate. 6. The process for synthesis of hydrazino derivatives of curcumin of formula 5 and formula 5' as claimed in claim 3 and claim 4 wherein the ratio of curcumin to the azo compound is 1:0.5 to 0.5:2 7. The process for synthesis of hydrazino derivatives of curcumin of formula 5 and formula 5' as claimed in claim 6 wherein the most preferred ratio of curcumin to the azo compound is 1:2. 8. The process for synthesis of hydrazino derivatives of curcum'm of formula 5 and formula 5' as claimed in claim 3 wherein the base of step (a) is selected from 4-N, N-dimethylaminopyridine (DMAP), its isomers and substituted analogs, imidazole and its substituted analogs, amines in which these moieties are fused to other rings, e.g. a benzene ring, 1,4-diazabicyclo[2.2.2]octane (DABCO), l,8-diazabicyclo[5.4.0]undec-7-ene (DBU), l,5-diazabicyclo[4.3.0]non-5-ene (DBN). 21 9. The process for synthesis of hydrazino derivatives of curcumin of formula 5 and formula 5* as claimed in claim 8 wherein the most preferred base is dimethylamino pyrimidine. 10. The process for synthesis of hydrazino derivatives of curcumin of formula 5 and formula 5' as claimed in claims 8 and 9 wherein the quantity of base is in the range of 0.1 equivalents to 2 equivalents. 11. The process for synthesis of hydrazino derivatives of curcumin of formula 5 and formula 5' as claimed in claim 10 wherein the most preferred quantity of base is 1.2 equivalents. 12. The process for synthesis of hydrazino derivatives of curcumin of formula 5 and formula 5' as claimed in claim 3 wherein the organic solvent in step (a) is selected from methanol, ethanol, cyclohexanol, diethyl ether, tetrahydrofuran, 1,4-dioxan, dichloromethane, chloroform, toluene, benzene, acetonitrile, dimethyl formamide, dimethyl sulfoxide, acetone or ethyl acetate. 13. The process for synthesis of hydrazino derivatives of curcumin of formula 5 and formula 5' as claimed in claim 12 wherein the most preferred solvent is acetonitrile. 14. The process for synthesis of hydrazino derivatives of curcumin of formula 5 and formula 5'as claimed in 3 wherein the temperature is in the range of -50°CtolOO°C. 15. The process for synthesis of hydrazino derivatives of curcumin of formula 5 and formula 5' as claimed in 14 wherein the most preferred temperature range is -10 °C to 30 °C. 22 16. The process for synthesis of hydrazino derivatives of curcumin of formula 5 and formula 5' as claimed in claims 3 to 15 wherein the analogues of curcumin are selected from: i. (IE, 6£)-l,7-bis-(4-hydroxy-3-methoxyphenyI)-l,6-heptadiene-3,5-dione; ii. (IE, 6£)-I,7-bis-(2,5-dimethoxyphenyl)-l,6-heptadiene-3,5-dione; iii. (IE, 6£)-l,7-bis-(4-nitrophenyl)-l,6-heptadiene-3,5-dione; iv, (\E, 6£)-l,7-bis-(2-nitrophenyl)-l,6-heptadiene-3,5-dione; v. (l£)6£)-l,7-bis-(4-methoxyphenyl)-l,6-heptadiene-3,5-dione; vi. (l£,6£)-l,7-bis-(3,4-dimethoxyphenyl)-l,6-heptadiene-3,5-dione; vii. (l^,6£)-l,7-bis-(2,4-dimethoxyphenyl)-l,6-heptadiene-3,5-dione; viii. (IE, 6E)-l, 7-bis-(4-benzyIoxy-3-methoxyphenyl)-l, 6-heptadiene-3, 5-Dione. Dated this 23rd day of July 2008 Saloni Rastogi OfS.Majumdar&Co. (Applicant's Agent) 23

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1568-MUM-2008-CORRESPONDENCE(18-5-2012).pdf

1568-MUM-2008-CORRESPONDENCE(2-3-2010).pdf

1568-MUM-2008-CORRESPONDENCE(2-8-2010).pdf

1568-MUM-2008-CORRESPONDENCE(24-9-2009).pdf

1568-MUM-2008-CORRESPONDENCE(3-4-2009).pdf

1568-mum-2008-correspondence.pdf

1568-mum-2008-description(complete).doc

1568-mum-2008-description(complete).pdf

1568-mum-2008-drawing.pdf

1568-MUM-2008-FORM 1(17-10-2008).pdf

1568-mum-2008-form 1(23-7-2008).pdf

1568-mum-2008-form 1.pdf

1568-MUM-2008-FORM 18 4-8-2008.pdf

1568-mum-2008-form 2(title page).pdf

1568-mum-2008-form 2.doc

1568-mum-2008-form 2.pdf

1568-mum-2008-form 3(23-7-2008).pdf

1568-MUM-2008-FORM 8(2-8-2010).pdf

1568-MUM-2008-POWER OF ATTORNEY(17-10-2008).pdf

1568-MUM-2008-POWER OF ATTORNEY(20-7-2012).pdf

1568-MUM-2008-REPLY TO EXAMINATION REPORT(25-1-2012).pdf

1568-MUM-2008-REPLY TO HEARING(20-7-2012).pdf

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