FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION:
CURCUMIN MONOSULPHATE, ITS SALTS, PROCESS FOR SYNTHESIS THEREOF AND TREATMENT OF CANCER
2. APPLICANT:
(a) NAME : AVANCE PHYTOTHERAPIES PVT. LTD.
(b) NATIONALITY : AN INDIAN COMPANY
(c) ADDRESS : 204, CIRCLE-P BUILDING
PRAHLADNAGAR S. G. HIGHWAY AHMEDABAD-380054 GUJARAT, INDIA
3. PREAMBLE TO THE DESCRIPTION
COMPLETE
The following specification particularly describes the invention and the manner in which it is to be performed.

The present invention relates to curcumin monosulphate and its pharmaceutically acceptable salts and procedure for its synthesis thereof.
More particularly the present invention relates to a formulation containing curcumin monosulphate and its pharmaceutically acceptable salts having anticancer activity.
BACKGROUND OF INVENTION:
Based primarily on chemical, radiation and surgical therapy, cancer treatments have progressed only incrementally during more than thirty years of intensive research to discover the origins and devise improved therapies of neoplastic diseases.
Chemotherapy is the use of anticancer drugs to treat cancerous cells. Chemotherapy has been used for many years and is one of the most common treatments for cancer. While chemotherapy can be quite effective in treating certain cancers, chemotherapy drugs reach all parts of the body, not just the cancer cells. Because of this, there may be many side effects during treatment. There are over 50 chemotherapy drugs that are commonly used like carboplatin, cisplatin, cyclophosphamide, doxorubicin, etoposide, flourouracil, gemcitabine, irinotecan, methotrexate, paclitaxel, vincristine and vinblastin. Side effects may occur just after treatment (days or weeks) or they may occur later (months or years) after the chemotherapy has been given. Because anti-cancer drugs attempt to kill cancer cells, many of them will also attack and kill healthy cells. Side

effects are a direct result of the killing of healthy cells. Certain cells are more susceptible to damage than others, including bone marrow cells, cells in the digestive tract, reproductive system and hair follicles.
Almost all cancers of the large intestine and rectum (colorectal) are adenocarcinomas, which develop from the lining of the large intestine (colon) and rectum. Colorectal cancer usually begins as a buttonlike swelling on the surface of the intestinal or rectal lining or on a polyp. As the cancer grows, it begins to invade the wall of the intestine or rectum. Nearby lymph nodes also may be invaded. Because blood from the wall of the intestine and much of the rectum is carried to the liver, colorectal cancer usually spreads (metastasizes) to the liver soon after spreading to nearby lymph nodes. When cancer has spread to lymph nodes far from the colon or rectum, to the lining of the abdominal cavity, or to other organs, the cancer cannot be cured by surgery alone. Survival time is typically only about 7 months. Chemotherapy with fluorouracil (sometimes also with another drug) may be given after surgery as part of the treatment for colorectal cancer that has spread widely.
Metastatic colon cancer to the liver can be treated with chemotherapy in a variety of ways. One of the most common regimens is the systemic or intravenous administration of 5 - Fluorouracil (5 FU) and Leucovorin. This drug combination is given in the vein as an outpatient and is effective in killing metastatic cancer in 20 - 30% of patients and can potentially prolong life for approximately twelve months. Other systemic drug regimens include continuous infusion of 5 FU, Tomudex, Mitomycin

C, Irinothecan (CPT-11) and other experimental drugs. In addition, active research protocols are investigating the effectiveness of administering chemotherapy by mouth with the goal of making the treatment easier for the patient. The two most commonly used drugs are 5 FU and FUDR <5-fluoro-2'-deoxyuridine). Administering combinations of drugs can improve the chance of tumor shrinkage.
Current treatments of cancer and related diseases have limited effectiveness and numerous serious unintended effects. Current research strategies emphasize the search for effective therapeutic modes with less risk, including the use of natural products and biological agents. This change in emphasis has been stimulated by the fact that many of the consequences, to patients and their offspring/ of conventional cancer treatment, including new cancers, mutations and congenital defects, result from their actions on genetic material and mechanisms (Hong et al., J. Natl. Cancer Inst. Monogr. 17:49-53 (1995)). Efforts continue to discover the origins of cancer at the genetic level, and correspondingly new treatments, but such interventions also may have serious unanticipated effects.
The natural products are those prepared using plant products and not chemically synthesized. These products have least or no pronounced side effects, as compared to chemical moieties and these are relatively safe,
US5861415 discloses curcumin, bis-demethoxycurcumin and demethoxycurcumin and claims to have many bio-protectant properties. The composition is claimed to have anti ojdant, anti inflammatory, antibacterial, antifungal, antiparasitic, anti mutagen, anticancer and detox

properties. The claimed composition does not give desired anticancer effect and thus is not up to mark.
US6673843 discloses various curcumin analogues and their extraction procedures. These analogues are claimed to be effective on carcinomas, myelomas and adenosarcomas of various tissues. Also it is claimed to cure various skin disorders. CN200610020156 discloses a pharmaceutical formulation containing curcumin, hawthorn general flavone and Gynostemma pentaphylla which is claimed to be anti tumour, blood fat lowering and for treatment of hepatic disease. These inventions do not give specific anti cancer activity and no efficiency.
The isolation of natural curcumin from the Curcuma longa rhizome is a difficult and costly procedure. No practical way has been found to effect separation of curcumin itself from two related demethoxy compounds with which it is found in nature. This difficulty of separation has led to several attempts to synthesize the compound, the most important of which has been aldol condensation of vanillin (3-methoxy-4-hydroxy benzaldehyde) and 2, 4-pentanedione. However, the yields of product from these syntheses have heretofore been very low, in large part because of the difficult and complicated procedures required for isolation and purification of the product. The problem related to curcumin is that the extract, crude product or any formulation prepared by any method does not enhance its bioavailability even after the claims to affect the water solubility.

Article on cancer research discloses curcumin in human and rat hepatocytes. The article discloses a method of synthesis of curcumin sulphate. Curcumin was dissolved in anhydrous 1, 4 dioxane and was incubated with sulphur trioxide N-triethylamine complex and maintained at 37°C for 2hours. The residue was reconstituted in acetonitrile: water (1:1) and separated by preparative HPLC using a Hypersil column. The process is complicated, [Christopher Ireson et al; Cancer Research 2001; 61:3:1058-1064]
Thus there is a need for an anticancer drug having least side effects, is effective and efficient on use.
OBJECT OF THE INVENTION:
The main object of the present invention is to provide molecule of curcumin monosulphate.
Another object of the invention is to provide with synthesis of curcumin monosulphate.
It is an additional object of the invention to provide a salt form of curcumin monosulphate and its synthesis.
It is yet an additional object of the invention to provide a formulation containing curcumin monosulphate or its salts and which has anticancer properties.
Yet another object of the invention is to provide curcumin monosulphate that has least side/ adverse effects.

It is yet another object of the invention to provide curcumin monosulphate effective for treatment of liver and colorectal cancer.
SUMMARY OF INVENTION:
The present invention deals with curcumin monosulphate and its salts. Curcumin monosulphate has anticancer property and is effective for treating liver and colon cancers. The salt form of the curcumin monosulphate has more water solubility. The process for synthesis of curcumin monosulphate and its salt forms, from curcumin, is disclosed herein. The process for synthesis of curcumin monosulphate and its salts mainly includes using curcumin as precursor and adding dry pyridine and chlorosulfonic acid to get the product.
DETAILED DESCRIPTION:
The nature of invention and the manner in which it is performed is clearly described in the specification. The invention has various components and they are clearly described in the following pages of the complete specification.
India has a rich history of using plants for medicinal purposes. Turmeric (Curcuma longa L.) is a medicinal plant extensively used in Ayurveda, Unani and Siddha medicine as home remedy for various diseases. Turmeric is obtained from the rhizomes of Curcuma longa L. of Zingiber aceae family.

Curcumin [1, 7-bis (4-hydroxy-3-methoxyphenyl)-l, 6 heptadiene-3, 5-dione) is a naturally occurring compound which is the main coloring principle found in the rhizomes of the plant Curcuma longa. This natural pigment is widely used as a coloring agent in foods and cosmetics and is reported to have various pharmaceutical uses. Among these is its action as a bile-secretion stimulating agent, as an anti-inflammatory agent and as an antioxidant.
The present invention discloses an anti cancer curcumin analogue known as curcumin monosulphate, and its pharmaceutically acceptable salts. Curcumin monosulphate is highly hygroscopic in nature and has melting point 260- 263 °C. The said curcumin monosulphate is effective against all sorts of cancers but preferably effective against liver and colorectal cancer. There is no detailed information on curcumin monosulphate molecule as such in scientific society.
The formulation of the curcumin monosulphate or its pharmaceutically acceptable salt form may be administered by a variety of routes, including oral, sublingual/ buccal, rectal, vaginal, transdermal, subcutaneous, intravenous, intravesical, intramuscular and intranasal routes. The curcumin monosulphate or its pharmaceutically acceptable salt form is preferably formulated prior to administration. Therefore, another embodiment of the present invention is a pharmaceutical composition or formulation comprising an effective amount of curcumin monosulphate or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, such as the diluents or excipients, is compatible with the

other ingredients of the formulation and not deleterious to the host or patient.
The curcumin monosulphate comprises from 0.1% to 99.9% by weight and preferably 10 to 60 % by weight of the formulation.
Pharmaceutical formulations may be prepared from the curcumin monosulphate or its pharmaceutically acceptable salt form by known procedures using known and readily available ingredients. In making any sort of the formulations, the active ingredient curcumin monosulphate will usually be admixed with a carrier or diluted by a carrier, or enclosed within a carrier, which may be in the form of a capsule, sachet, paper or other suitable container. When the carrier serves as a diluent, it may be a solid, semisolid or liquid material which acts as a vehicle, excipient or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), soft and hard gelatine capsules, sterile injectable solutions, sterile packaged powders and the like.
In the present invention to prepare curcumin monosulphate curcumin, pyridine, titanium dioxide, chlorosufonic acid, diethyl ether and dichloro methane are used.
In the present invention to prepare curcumin monosulphate 368± 20 mg, 1.0 mmol curcumin; 7± 20 ml pyridine; titanium dioxide; 0.7± 20 ml, 10 mmol chlorosufonic acid are used. The process of synthesis of curcumin

monosulphate and its pharmaceutically acceptable salts, from curcumin, is
disclosed below.
Synthesis of curcumin monosulphate:
a) Curcumin is dissolved in dry pyridine and cooled to 0°C
b) To this solution chlorosulfonic acid is added and the mixture is allowed to keep at room temperature overnight.
c) The mixture is evaporated under reduced pressure to get residue which is precipitated using diethyl ether or dichloro methane to obtain curcumin monosulphate.
d) The product is tranfered in a dry bottle immediately and kept in vacuum desiccator.
e) The product is then purified using TLC, eluted using dichloro methane: methanol (90:10 %).
To increase reaction rate, in mixture of curcumin and dry pyridine, titanium dioxide (TiO2) is added in step A as a catalyst. The synthesis of curcumin monosulphate is optionally conducted under nitrogen atmosphere in step B and at different temperature conditions.
Synthesis of curcumin monosulphate salt:
The process of synthesis of curcumin monosulphate salt is moreover same as of curcumin monosulphate. The mixture of curcumin and dry pyridine is concentrated under reduced pressure to afford a residue which is re-dissolved in water and pH is adjusted to 10 using potassium carbonate.

The solution is once again concentrated under reduced pressure to afford residue which on drying gives curcumin monosulphate salt,
The pharmaceutically acceptable salt of curcumin monosulphate is sodium and potassium salt.
The invention is illustrated more in detail in the following examples. The examples describe and demonstrate embodiments within the scope of the present invention. These examples are given solely for the purpose of illustration and are not to be construed as limitations of the present invention, as many variations thereof are possible without departing from the spirit and scope.
Example 1:
Synthesis of curcumin monosulphate:
A) 368 mg, 1.00 mmol Curcumin was dissolved in 10 ml dry pyridine and cooled to 0 °C.
B) To this solution 0.35 g, 3 mmol chlorosulfonic acid was added and the mixture was allowed to keep at room temperature for overnight.
C) Evaporation under reduced pressure afforded a residue which was precipitated using diethyl ether or dichloromethane to obtain 220 mg, 50 % w/w curcumin monosulphate as yellow solid.
D) The separated solid was filtered and packed in dry glass bottle and kept in vacuum desiccator.

E) The product was then purified using TLC using mobile phase of Dichloro methane: Methanol (90:10).
Example 2:
Synthesis of curcumin monosulphate:
A) 368 mg, 1.0 mmol curcumin with 0.05 g TiO2 was dissolved in 5 ml freshly distilled pyridine and the mixture is cooled to -16 °C.
B) 0.35 g, 3.0 mmol chlorosulfonic acid was added dropwise to the solution prepared, under Nitrogen atmosphere.
C) The reaction mixture was allowed to warm by stirring at room temperature.
D) After 3 hr the reaction mixture was filtered using whatman filter paper.
E) The filtrate was concentrated under reduced pressure, and
compound was re-precipitated using diethyl ether or dichloro
methane to obtain 260 mg, 60 %w/w curcumin monosulphate as
yellow solid.
F) The separated solid was filtered and packed in dry glass bottle and
kept in vacuum desiccator.
G) The product was then purified using TLC using mobile phase of
Dichloro methane: Methanol (90:10).
Example 3:
Synthesis of curcumin monosulphate:

A) 368 mg and 1.0 mmol curcumin was dissolved in 7 ml freshly distilled dry pyridine and cooled to 0 to -5 °C
B) To this solution 0.7 ml, 10 mmol chlorosulfonic acid was added dropwise with caution and the mixture was allowed to stand at room temperature overnight.
C) Solid separated was filtered and washed with dry diethyl ether during filtration to maintain anhydrous condition.
D) For the second crop, excess of pyridine was removed under reduced pressure, and the residue was triturated with dry diethyl ether or dichloro methane.
E) Solid separated was filtered and packed in dry glass bottle
immediately and stored in desiccator.
F) The product was then purified using TLC using mobile phase of
Dichloro methane: Methanol (90:10).
Example 4:
Synthesis of curcumin monosulphate:
A) 368 mg and 1.0 mmol curcumin with 50 mg Ti02 was dissolved in 7 ml freshly distilled dry pyridine and cooled to 0 to -5 °C.
B) To this solution 0.7 ml, 10 mmol chlorosulfonic acid was added dropwise with caution and the mixture was allowed to stand at room temperature overnight.
C) Solid separated was filtered and washed with dry diethyl ether during filtration to maintain anhydrous condition.

D) For the second crop, excess of pyridine was removed under reduced
pressure, and the residue was triturated with dry diethyl ether or
dichloro methane.
E) Solid separated was filtered and packed in dry glass bottle
immediately and stored in desiccator.
F) The product was then purified using TLC using mobile phase of
Dichloro methane: Methanol (90:10).
Example 5;
Synthesis of curcumin monosulphate salt:
A) 368 mg,1.0 mmol curcumin was dissolved in 10 ml dry pyridine and cooled to 0 °C.
B) To this solution 0.40 gm, 3.5 mmol chlorosulfonic acid was added and the mixture was allowed to keep at room temperature for overnight.
C) The mixture prepared was concentrated under reduced pressure to afford a residue which was re-dissolved in water and pH was adjusted to 10 with solid potassium carbonate.
D) The solution was then again concentrated under reduced pressure to afford residue which was then dried to obtain curcumin monosulphate salt.
E) The product was then purified using TLC using mobile phase of
Dichloro methane: Methanol (90:10).

Example 6:
Synthesis of curcumin monosulphate salt:
A) 368 mg, 1 mmol curcumin in 7 ml freshly distilled dry pyridine at 0 to -5 °C.
B) To the solution prepared above 0.3 ml, 4.28 mmol chlorosulfonic acid was added dropwise with caution.
C) The reaction was allowed to stir at room temperature for 14 hr.
D) The pH of the reaction mixture was increased to 9- 10 using 10ml 0.5M K2CO3 (potassium carbonate) and the reaction mixture was further stirred for 2 to 3 hr.
E) Excess of solvent was removed in vacuum.
F) Solid separated was collected and allowed to completely dry in
desiccator.
The pre-clinical tests, for anticancer activity of curcumin monosulphate, were conducted by MTT (3-(4/ 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) cell viability assay which is a colorimetric assay system. It measures the reduction of the tetrazolium component (MTT) into an insoluble blue/purple coloured formazan product by the mitochondria of viable cells. The absorbance of the complex is read spectrophotometrically and is directly proportional to the number of live viable cells. These cytotoxicity tests were conducted on liver and colonic cancer cell lines and the results obtained are included in table below.

Table 1: Percent cytotoxicity of curcumin monosulphate in Human Liver cancer cells

Concentration
((μg/ml) Percent Cytotoxicity (%)

SK-Hep-1 Hep-G2
0.1 7.8 22.6
1 2.4 19.3
10 2.2 23.0
100 16.0 36.6
250 37.6 ND
Table 2: Percent cytotoxicity of curcumin monosulphate in human colon cancer cells

Concentration
(Hg/ml) Percent Cytotoxicity (%)

Sw620 Colo205 HCT-15 Caco-2
0.1 30.5 30.5 12.4 7.2
1 29.2 20.8 14.1 1.6
10 30.2 27.7 14.0 0
100 31.2 34.8 39.1 2.9
250 64.3 58.4 59.8 36.6
Table 3: Percent cytotoxicity of curcumin monosulphate salt in Human Liver cancer cells

Concentration (μg/ml) Percent Cytotoxicity (%)

SK-Hep-1 Hep-G2

0.01
5.4 19.4
0.1 12.1 26.4
1 7.6 25.4
10 1.5 25.9
100 7.4 27.1
250 21.4 26.4
Table 4: Percent cytotoxicity of Curcumin monosulphate salt in human colon cancer cells

Concentration Percent Cytotoxicity (%)
(fig/ml) Sw620 Colo205 HCT29 HCT-15 Caco-2
0.001 6.8 15.8 3 14.6 49.5
0.01 4.1 15 44.8 26.0 41.6
0.1 16.6 9.8 49.5 35.3 47.5
1 9.2 18.8 46.4 31.2 52.4
10 23.5 11.9 59.5 30.2 57.3
100 11.9 9.2 64.5 26.8 64.7
250 26.4 13.2 72.3 28.3 89.9
The analysis of data obtained revealed that the test item/ curcumin monosulphate and its salt, demonstrated considerable cytotoxicity in both colon and liver cell lines. In colon cancer cell lines, Sw620 and HCT-15 more than 60% cell death was recorded at the concentration of 250 μg/ml

of curcumin monosulphate; while, in colon cancer cell lines, HCT 29 and Caco-2 more than 70% cell death was recorded at the concentration of 250 fig/ml of curcumin monosulphate salt. Similarly in liver cancer cell line, SK-Hep-1, curcumin monosulphate caused considerable cell death; while, curcumin monosulphate salt caused considerable cell death in Hep-G2 cell line.

We claim,
1. A method for synthesis of anticancer molecule curcumin
monosulphate and its pharmaceutically acceptable salt comprising:
a) dissolving curcumin in dry pyridine and cooling the solution to 0°C;
b) adding chlorosulfonic acid to the aforesaid solution and allowing the mixture to be kept at room temperature overnight;
c) evaporating the mixture under reduced pressure to get residue and precipitating the residue using diethyl ether or dichloro methane to obtain curcumin monosulphate;
d) transferring the product in a dry bottle immediately and keeping it in vacuum desiccator;
e) purifying the product.
2. A method for synthesis of anticancer molecule curcumin
monosulphate potassium salt comprising:
a) concentrating the mixture obtained in claim 1 step (B) under reduced pressure to afford residue;
b) redissolving the residue in water;
c) adjusting the pH of the solution to 10 by using potassium carbonate to obtain curcumin monosulphate potassium salt;
d) transferring the product in a dry bottle immediately and keeping it in vacuum desiccator;

e) purifying the product.
3. The method for synthesis of curcumin monosulphate as claimed in claim 1 step (A), wherein titanium oxide is optionally used.
4. The method for synthesis of curcumin monosulphate as claimed in claim 1 step (B), wherein nitrogen atmosphere is optionally used.
5. An anticancer formulation comprising curcumin monosulphate as claimed in claim 1 or curcumin monosulphate potassium salt as claimed in claim 2 and a pharmaceutically acceptable carrier.
6. The anticancer formulation as claimed in claim 5, wherein the pharmaceutically acceptable carrier are diluents and excipients.
7. The anticancer formulation of curcumin monosulphate and its pharmaceutically acceptable salts as claimed in claim 5, wherein said formulation is in form of either tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), soft and hard gelatine capsules, sterile injectable solutions or sterile packaged powders.
8. The anticancer formulation curcumin monosulphate and its pharmaceutically acceptable salts as claimed in claim 5, wherein the

said curcumin monosulphate and its potassium salt comprises from 10 to 60 % by weight of the formulation.
9. The anticancer formulation as claimed in claim 5, wherein the said formulation is effective against liver and colon cancer.
10. The method for synthesis of curcumin monosulphate and curcumin monosulphate potassium salt, which is substantially as herein described with reference to the foregoing description and the accompanying examples.